ASMB 2025 Biennial Meeting Abstracts,November 16-19,2025,Baltimore,MD

Epigenetic Therapy Pitfalls: DNMT1 Inhibition Drives ECM Degradation in TNBC

Md Al Azim, Alicia Belcher, Leah Gutman, Julie S Di Martino

Department of Cell Biology & Anatomy, New York Medical College

ABSTRACT BODY: Epigenetic dysregulation is a hallmark of cancer, cooperating with genetic mutations to fuel disease progression. Aberrant DNA methylation patterns are especially prevalent in malignancies and are tightly linked to tumor aggressiveness. Given that DNA methylation is reversible, DNA methyltransferases (DNMTs) have emerged as promising therapeutic targets. DNMT inhibitors have already demonstrated clinical efficacy in hematologic cancers such as acute myeloid leukemia. DNMT1, the maintenance methyltransferase, is overexpressed in several solid tumors, including triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, where elevated DNMT1 levels predict poor prognosis. In this study, we evaluated the effects of GSK-3484862, a first-in-class DNMT1-selective inhibitor, on TNBC cell lines, with a focus on proliferation and invasive behavior. DNMT1 inhibition modestly reduced proliferation without inducing apoptosis. Unexpectedly, it markedly increased extracellular matrix (ECM) degradation in vitro and promoted tumor cell dissemination in vivo. Mechanistic studies revealed that GSK-3484862 enhanced the activity of the metalloproteases MMP-2 and MMP-9, enzymes critical for ECM remodeling. Strikingly, co-treatment with JNJ0966, a next-generation MMP9-specific inhibitor, abrogated the ECM degradation triggered by DNMT1 inhibition while further enhancing its antiproliferative effects. These findings highlight the complex consequences of epigenetic therapies in solid tumors and demonstrate the therapeutic potential of combining DNMT1 and MMP9 inhibitors to simultaneously suppress proliferation and invasion in TNBC.

Type V Collagen Regulates Matrix Integrity and Progenitor Cell Fate in TMJ Condylar Cartilage

Abdulaziz Alanazi¹, Prashant Chandrasekaran², Bryan Kwok², Lin Han¹

¹Drexel University, ²Children's Hospital of Philadelphia

ABSTRACT BODY: Type V Collagen Regulates Matrix Integrity and Progenitor Cell Fate in TMJ Condylar CartilageIntroduction: Temporomandibular joint (TMJ) disorders involve progressive degeneration of condylar cartilage, a unique hybrid tissue consisting of a fibrocartilage surface layer enriched in progenitor cells overlying a secondary hyaline layer contains mature chondrocytes. Our previous studies using the heterozygous type V collagen model (Col5a1+/−) showed that type V collagen regulates the extracellular matrix (ECM) organization and biomechanical properties of TMJ condylar cartilage. Distinct from other hyaline cartilage types, TMJ condylar cartilage harbors a population of progenitor cells, which are highly sensitive to their microenvironment and mechanical stimuli. Here, we studied the matrix and cell phenotypic changes in a cartilage-specific collagen V knockout model (Col5a1f/f/AcanCreER, or Col5a1-cKO) to elucidate how nearly complete ablation of collagen V impacts condylar cartilage ECM integrity, progenitor cell fate and activities during postnatal growth.Methods: In cartilage-specific Col5a1 knockout mice, ablation of Col5a1 gene was induced by i.p. tamoxifen induction at 3 weeks of age, and phenotypic changes of TMJ were analyzed at 5 and 8 weeks. We applied histology, immunostaining and microCT (μCT) were applied to assess condylar morphology and composition, RNAScope for spatial gene expressions, EdU assays for in situ cell proliferation, transmission electron microscopy (TEM) for collagen fibril structure and AFM-nanoindentation for tissue modulus. Single-cell RNA sequencing (scRNA-seq) was performed on 5-week-old TMJs to assess the cellular heterogeneity and transcriptional changes. Data were processed using the Seurat package in R, including dimensionality reduction, clustering, cell cycle analysis, and pathway enrichment to evaluate the effects on cell populations and signaling. Results: Deletion of Col5a1 was validated by RNAScope showing marked reduction in expression within the fibrous layer. At 5 weeks of age, Col5a1-cKO condyles exhibited fibrous layer hypercellularity and increased hyaline cartilage thickness. By 8 weeks of age, condylar cartilage underwent salient aberrant remodeling, in which the fibrous layer became hypocellular, and the hyaline layer demonstrated thinning and loss of organization. Notably, an abnormal overgrowth of the hyaline cartilage layer as well as subchondral bone was observed at the posterior end of the condyle, a region exposed to the highest occlusal loading, indicating disruption of mechanotransduction and altered loading-induced endochondral ossification. These changes can be attributed to the compromised matrix organization, mechanical properties, and in turn, altered mechanotransduction caused by collagen V deletion. In support, we found sparsely distributed, irregular, and thicker collagen fibrils in the matrix of Col5a1-cKO condylar cartilage via TEM, as well as a significant reduction in tissue modulus via AFM, underscoring the impairment of matrix integrity. Transcriptomic analysis via scRNA-seq found a selective expansion of progenitor cells predominantly arrested in the G1 phase of the cell cycle in Col5a1-cKO condyle, coupled with elevated EdU incorporation. These progenitors showed enrichment of mechanosensitive signaling pathways, including integrin, FGF, and RAS, while exhibiting suppression of differentiation-associated pathways such as Notch and TGF-β. These findings highlighted the pivotal role of collagen V in maintaining progenitor cell proliferation and mechanosensing by regulating the integrity of the matrix microniche. Conclusions: Type V collagen is essential for maintaining matrix integrity and regulating progenitor cell fate in TMJ cartilage. Its absence disrupts matrix assembly, mechanics and cell-matrix mechanosignaling, leading to dysregulated condylar postnatal growth. These findings also provides novel insights into the higher propensity of TMJ disorder among classic Ehlers-Danlos Syndrome (cEDS) patients caused by the mutation of COL5A1 gene.

NEO7: A Novel Blood-Based Biomarker for Organ-Specific Basement Membrane Remodeling in Systemic Sclerosis

Matej Andelic^1,2, Christoffer Tandrup Nielsen³, Louise Diederichsen³, Signe Holm Nielsen²

¹Department of Biomedical Sciences, University of Copenhagen

²Immunoscience, Nordic Bioscience, ³Center for Rheumatology and Spine Diseases, Rigshospitalet, University of Copenhagen

ABSTRACT BODY: Introduction: Systemic sclerosis (SSc) is a chronic connective tissue disease characterized by skin stiffness with high morbidity and mortality. Among the various organ systems affected, the lungs are commonly involved, with interstitial lung disease being one of the most frequent and serious complications. Its pathogenesis involves early microvascular damage, immune dysregulation, inflammation, and excessive extracellular matrix (ECM) accumulation, leading to tissue stiffness and fibrosis. ECM turnover can be assessed by measuring circulating ECM neoepitope fragments generated during ECM synthesis and degradation, reflecting immune cell-driven tissue remodeling. Collagen type XVIII, a multiplexin found primarily in basement membranes, has structural and signaling functions. Proteolytic cleavage by matrix metalloproteinases (MMPs), especially MMP-7, produces bioactive fragments like neostatin-7, which is a type XVIII collagen fragment, that may regulate angiogenesis and remodeling. We hypothesized that neostatin-7 could serve as a blood biomarker of basement membrane remodeling in SSc. The objective of this study was to develop and validate a competitive ELISA targeting neostatin-7, termed NEO7, and to explore its associations with clinical parameters of SSc patients.Methods: To assess assay specificity, the monoclonal antibody raised in mice was tested against elongated, truncated, non-sense peptides, and a non-sense coater. Technical validation included inter- and intra-assay variation, analyte stability, and interference from endogenous substances. The ELISA was applied to serum from 102 SSc patients and 42 healthy donors (HD). Group comparisons (SSc vs. HD and subgroups) used the Mann–Whitney U test. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic performance of the NEO7 assay, with area under the curve (AUC) values calculated.Results: The NEO7 monoclonal antibody demonstrated high specificity, with no detectable reactivity toward the elongated peptide, truncated peptide, non-sense standard peptide, or non-sense coater. Technical validation confirmed robust assay performance, including acceptable inter- and intra-assay variation, stable analyte detection, and minimal interference from endogenous serum components. The SSc group had a mean age 59 years (SD 12.7) with 23.5% males; HD had mean age 42.5 years (SD 14.1) with 52.4% males. NEO7 levels were significantly elevated in SSc patients compared to HD (p < 0.0001). Patients with cutaneous skin involvement exhibited significantly lower NEO7 levels compared to those without (p = 0.04). In contrast, NEO7 levels were significantly increased in patients with pulmonary impairment, as defined by reduced forced vital capacity (p = 0.02). Moreover, NEO7 levels were significantly lower in patients receiving anti-inflammatory treatment compared to those not on treatment (p = 0.03). ROC curve analysis yielded an AUC score of 0.73 (95% CI: 0.54–0.91), indicating moderate discriminative ability for identifying pulmonary involvement.Conclusion: These findings demonstrate successful development and validation of a novel ELISA assay with high specificity for the neostatin-7 fragment of collagen type XVIII. NEO7 can differentiate patients with SSc from HDs and may reflect organ-specific patterns of remodeling. Elevated NEO7 levels in patients with pulmonary involvement, alongside significantly lower levels in those with cutaneous manifestations, suggest its potential to reflect organ-specific patterns of basement membrane remodeling. Furthermore, its moderate discriminative power for detecting pulmonary impairment and its association with anti-inflammatory treatment support its relevance in disease management. Further studies are necessary to evaluate NEO7 across treatment modalities and disease severity to fully establish its utility as a biomarker of basement membrane remodeling in SSc.

Ex Vivo Culture of Mouse Femoral Head Cartilage Reduces F-actin and Dysregulates Chondrocyte Homeostasis

Mark Arranguez, Marin Herrick, Sofia Gonzalez-Nolde, Justin Parreno

University of Delaware

ABSTRACT BODY: Following injury, the inability of articular cartilage to self-repair leads to osteoarthritis (OA) progression. OA pathogenesis involves the dysregulation of matrix homeostasis favoring catabolism which leads to articular cartilage degradation. In a surgical OA model, gene set enrichment analysis and functional annotation of differentially expressed genes has shown the greatest changes in the actin cytoskeleton pathway. Previous studies have shown that actin reorganization is a potent regulator of the chondrocyte phenotype; however, these studies use isolated cells in vitro. Since actin organization within cells is context-specific, we aim to investigate the regulation of chondrocyte homeostasis by actin in the native tissue environment. In this study, we developed an ex vivo whole organ culturing methodology to elucidate the relationship between chondrocyte homeostasis and changes in actin polymerization. We found that culturing native mouse femoral head cartilage with serum/growth-factor free media results in mRNA level alterations that reflect cartilage catabolism, including a decrease in cartilage matrix and an increase in degradative enzyme mRNA. Coinciding with this shift in chondrocyte phenotype is a reduction of filamentous (F-)actin. To examine the contributions of F-actin, we next aimed to dysregulate F-actin stability through investigation of the Tropomyosins (Tpms), which bind and stabilize F-actin. We identified Tpm isoform 3.1 to be expressed in chondrocytes from various species. In mouse cartilage, we found that Tpm3.1 organizes along the cortex of chondrocytes. Tpm3.1 knockout reduces the amount of F-actin along the cortex, suggesting that F-actin is less stable. Currently, we are investigating if the culture of femoral heads from Tpm3.1 knockout mice will exacerbate chondrocyte matrix catabolism. Our ex vivo model will provide further insight into the regulation of OA pathogenesis via the actin cytoskeleton.

Iron Dysregulation Drives ECM Remodeling to Promote TNBC Invasion

Arun Asif¹, Kavya Nair¹, Ramon Bossardi Ramos¹, Elizabeth McDonough²

¹Dept. Molecular and Cellular Physiology, Albany Medical College

²Dept. Biomedical Engineering, Rensselaer Polytechnic Institute

ABSTRACT BODY: Metastatic triple-negative breast cancer (TNBC) is a major cause of mortality in women due to its aggressiveness and drug resistance. The divalent metal transporter 1 (DMT1) regulates TNBC invasion by modulating iron metabolism and extracellular matrix (ECM) remodeling, particularly collagen integrity, via endoplasmic reticulum (ER) stress. Using CRISPR, we generated DMT1 knockout (KO) and overexpression (OE) MDA-MB-231 cells, cultured in 2D and 3D models to assess phenotype and invasion. DMT1 KO spheroids exhibited reduced sphericity, increased volume, and lower cell density, with enhanced invasion in 3D Collagen1A and Matrigel matrices compared to compact OE spheroids and wild-type controls, corroborated by lung metastasis data. Ferro-Orange assays and transcriptomic analysis revealed that DMT1 KO increases the labile iron pool (LIP), inducing ER stress and activating the unfolded protein response (UPR). This disrupts oxidative folding anddisulfide bond formation, impairing Collagen 1A production and reducing ECM integrity, thus promoting epithelial-to-mesenchymal transition (EMT). Conversely, DMT1 OE cells maintained low LIP, reduced ER stress, and preserved collagen expression, slowing EMT. Transcriptomic data confirmed reduced collagen in DMT1 KO cells, linked to mesenchymal traits. Notably, 2D wound-healing assays showed faster migration in OE cells, highlighting 3D models’ relevance to in vivo conditions. TGF-β inhibition analysis further connected DMT1 KO to collagen loss and increased invasion. These findings demonstrate that DMT1 regulates TNBC invasion through ER stress-mediated collagen disruption, highlighting collagen as a key ECM component and potential therapeutic target.

An Integrated Framework for Characterizing the Extracellular Matrix Protein Interactions

Amanpreet Kaur Bains¹, Leanna Leverton¹, Ikram Isa¹, Sylvie Ricard-Blum²

¹University of Illinois Chicago, ²Université Claude Bernard Lyon

ABSTRACT BODY: The extracellular matrix (ECM) is a complex network of proteins and polysaccharides that provides structural support and organizes cells into tissues and organs in all multicellular organisms. Beyond its architectural role, the ECM mediates mechanical and biochemical signals through cell surface receptors, modulating key cellular processes such as proliferation, adhesion, survival, and differentiation. These functions are tightly regulated by ECM protein-protein interactions, which are critical during ECM protein biosynthesis and secretion, for the structural organization of the ECM, and to control the accessibility of functional protein domains that modulate ECM-mediated signaling functions (e.g., growth-factor and receptor binding). Despite their biological significance, our understanding of ECM protein interactions remains very limited. The ECM proteome, or matrisome, comprises over 1,000 proteins, with approximately 150 proteins expressed in any given tissue. However, current interaction databases such as MatrixDB, hu.MAP, or the protein data bank (PDB), capture only a limited subset of these components, highlighting a significant knowledge gap.Here, we present a comprehensive framework that integrates computational and experimental strategies for studying ECM protein interactions across molecular scales and biological contexts. We critically evaluate current tools and methodologies, including prediction-based protein sequence analysis, single-molecule techniques, high-throughput mass spectrometry-based approaches, and in-situ imaging. We also emphasize the need for improved assays, models, and data integration strategies tailored to the multimeric, multidomain, and insoluble nature of the ECM. By bridging computational predictions with experimental validation, our framework offers a flexible roadmap for systematically decoding the complexity of ECM interactions. Finally, we propose context-driven recommendations for constructing ECM interactome networks.

Cell Contractility Drives ECM Remodeling of Fibrillar Collagen and Matrix Function within Developing and Mature Hearts

Susanna Belt, Karanvir Saini, Dennis Discher

University of Pennsylvania

ABSTRACT BODY: Introduction: Tissue fibrosis causes almost half the deaths in the developed world, and cardiac fibrosis alone leads to heart failure in 6-million U.S. adults. Collagen-rich extracellular matrix (ECM) accumulates in fibrosis and both stiffens hearts and compromises beating. Current treatment efforts for progressive cardiac fibrosis primarily focus on ECM rather than the cell forces that conceivably regulate ECM levels. Cardiac myosin-II is one possible target based on recent studies, but the Rho kinases (ROCK) expressed in varying amounts by many cell-types including fibroblasts seem to also promote expression of ECM genes possibly via acto-myosin contractility. A new ROCK inhibitor (ROCKi) was FDA-approved recently for chronic Graft-versus-host disease (cGVHD) that affects many organs in recipients of stem-cell graft transplants. To visualize any effects of such contractility inhibitors on heart function and ECM collagen levels, we studied beating hearts from chick embryos. To track real-time kinetics of fibrillar collagen during perturbations with various myosin-II inhibitors, we used second harmonic imaging (SHG). Materials and Methods: For studying cellular contractility-based ECM effects and heart functions, we selected isolated embryonic chick hearts that beat autonomously (without any electrical stimulation) in contrast to mature hearts and comprises of different cell-types namely cardiomyocytes, fibroblasts as revealed by single-cell RNA sequencing. Acto-myosin based cellular contractility was perturbed by several small-molecule inhibitors namely ROCK-2 (Belumosudil) and Blebbistatin (a pan myosin-II inhibitor) whereas a pan matrix metalloproteinase inhibitor (MMPi) and collagenases were used to perturb ECM protein levels. The heart function, defined by beating strain magnitude, was accessed by means of wide-field time-lapse imaging during above mentioned perturbations in cellular-contractility and ECM protein levels whereas second harmonic imaging (SHG) was used to assess the real-time kinetics of fibrillar collagen levels in the tissues. Results and Discussion: Exposure of the heart tissues to ROCKi resulted in heart beating strain suppression similar to that of Blebbistatin treated hearts. Given that ROCK-2 is primarily expressed by fibroblastic cells in the heart and cardiomyocytes express extremely low levels, the slower rate of beating strain inhibition during ROCKi treatments (~30 mins) compared to blebbistatin treated hearts (~15 mins) likely results from cell-type specific distribution of ROCK-2 within the heart tissue while blebbistatin targets several myosin types abundant in most cell-types present in the heart. Considering the propagation of contractile wave among sparse immature cardiomyocytes within the heart relies on robust cell-ECM interactions, ROCK-2 inhibition likely promotes ECM catabolism and thus, disables contractile wave propagation in the heart. Such effects of altered ECM turnover on beating strain are evident during the presence of pan-MMPi when tissue function recovery is observed following drug-washouts. For various mature mice tissues and beating embryonic chick hearts, we find collagen-sensitive second harmonic generation (SHG) image intensity scales non-linearly versus tissue stiffness, aligning well with the results from cellularized gels of collagen. Chick hearts beating at ~5% strain maintain collagen levels until their contractile strain is suppressed by myosin-II inhibition and endogenous matrix metalloproteinases then degrade collagens within ~30-60 minutes based on SHG. Conclusions: Our results have potential to deepen the understanding of cell-based contractile forces on ECM in development, adaptation, and disease. The modulation of cellular forces during pathologies via targeting ROCK-pathway or other means might help target fibroblastic cells over other cell-types. Given that fibroblasts are primarily responsible for synthesis and degradation of a tissue ECM, such targeting of fibroblasts might help restore “normal” ECM and function of fibrotic hearts and other tissues.

Proteolytic Remodeling of Ocular Extracellular Matrix: Insights from the Vitreous, Zonule, and Aqueous Humor Degradome

Sumit Bhutada¹, Steven Bassnett², Suneel S. Apte¹

¹Lerner Research Institute, Cleveland Clinic, ²Ophthalmology & Visual Sciences, Washington University School of Medicine

ABSTRACT BODY: Purpose:The extracellular matrix (ECM) is a fundamental structural component of the ocular vitreous and zonule, yet the dynamics of its turnover remain poorly understood. Since the aqueous humor drains these tissues, it may harbor proteolytic fragments that reflect ongoing ECM remodeling. This study sought to systematically profile the proteolytic landscape (degradome) of the bovine vitreous, zonule, and aqueous humor, and compare these findings with the degradome of aqueous humor from patients undergoing cataract surgery. Our objective was to identify conserved proteolytic patterns relevant to using human aqueous humor to report ocular disorders. Methods:Proteins were isolated from bovine vitreous, zonule, and aqueous humor and subjected to Terminal Amine Isotopic Labeling of Substrates (TAILS) N-terminomics. N-terminal labeling was performed via reductive dimethylation, followed by trypsin digestion and enrichment for blocked N-termini. The resulting peptides were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Peptide positional annotation distinguished native from proteolytically generated N-termini, and cleavage patterns were compared with known protease degradomes to infer underlying mechanisms. In parallel, degradomics was performed on human aqueous humor samples to assess similarities with bovine tissue-derived degradomes.Results:A diverse repertoire of N-terminal peptides indicative of proteolysis was detected in both bovine vitreous and zonule samples. Zonule proteolytic fragments were predominantly derived from fibrillin-1 and latent TGFβ-binding protein 2, whereas vitreous degradome analysis revealed peptides primarily from proteoglycans, retinal, and circulating proteins. The degradome of the bovine aqueous humor showed overlap with those of the vitreous and zonule. Notably, several proteolytic products identified in human aqueous humor overlapped with bovine aqueous humor, suggesting conserved proteolytic processing across species. Multiple proteases and protease inhibitors were identified across all sample types.Conclusions:These findings demonstrate that both bovine and human ocular tissues undergo active proteolytic remodeling, with distinct ECM components subject to specific cleavage events. The presence of overlapping ECM-derived fragments in the aqueous humor, vitreous, and zonule highlights the interconnectedness of these compartments and suggests that aqueous humor can be used to report upstream ECM turnover. Proteolysis within the zonule may be relevant to pathologies such as ectopia lentis in Marfan syndrome, while ECM-derived fragments present in the aqueous humor could influence outflow resistance and anterior segment physiology. The proteolytic profiles identified here may provide valuable biomarkers for the study of ocular diseases associated with ECM dysregulation.

Uncovering the Role of the Extracellular Matrix in Intestinal Organoid Symmetry Breaking

Michael Blatchley, Fahima Jannat Koly, Yuman Guo, Michael R. Blatchley

Syracuse University

ABSTRACT BODY: Intestinal organoids hold unique promise as benchtop avatars of the mammalian gut, with applications ranging from the fundamental biology of development, homeostasis, and regeneration to disease modeling and cell therapy. Intestinal organoids are 3D, self-organized cellular constructs that mimic architectural, compositional, and functional features of their native counterparts. A key feature of intestinal organoids is their capacity to establish a biomimetic crypt-villus axis. To establish this biological complexity, nascent intestinal organoids undergo symmetry breaking, where intestinal stem cells differentiate into secretory progenitors through cell-to-cell heterogeneities in Yes-associated protein (YAP) nuclear localization. The sites of these differentiation events result in cellular shape changes, budding, and eventual crypt formation. While symmetry breaking and crypt formation are highly reproducible, they are also highly stochastic and heterogeneous, and upstream regulators of YAP are unknown in this context. In this work, we hypothesized that spatial heterogeneities in cell-extracellular matrix (ECM) interactions initiate these changes in YAP to kickstart symmetry breaking. Notably, the importance of the ECM in intestinal morphogenesis has been well established, but its specific role in symmetry breaking has not been studied. Motivated by these findings, we sought to address this significant gap in the field and study cell-ECM interactions of organoids cultured in Matrigel to understand how such interactions mediate YAP signaling and symmetry breaking.We first characterized cell-ECM interactions of organoids cultured in Matrigel and identified heterogeneities in nuclear YAP that were associated with heterogeneities in laminin-associated α6β4 integrin expression. Specifically, we noted high integrin α6β4 on cells with low nuclear YAP. Of note, α6β4 expression was not uniform around organoids, suggesting that these interactions may be associated with heterogeneities in YAP nuclear localization that result in symmetry breaking. Integrin inhibition experiments corroborated these findings, with altered organoid morphometrics, including reduced crypt formation, after anti-integrin β4 and anti-integrin α6 treatments. Interestingly, anti-integrin β1 and soluble RGD had little impact on organoid morphogenesis, suggesting that α6β4 interactions are crucial to crypt formation.To further investigate this phenomenon, we performed a series of small molecule inhibitor studies to assess the role of various cell-ECM interaction-mediated pathways on symmetry breaking, using crypt formation as a proxy readout. Because α6β4 integrins are a component of hemidesmosomes, we used a calcineurin inhibitor to disrupt hemidesmosomes and noted a reduction in crypt formation. Surprisingly, we saw similar results with an inhibitor of focal adhesion kinase (FAK), which lies downstream of other (non- α6β4) cell-ECM interactions and Rac1, which has been noted to play a role in crypt formation via negative regulation of α6β4 binding. In the FAK study, we did not anticipate a reduction in crypt formation, and in the Rac1 study, we hypothesized that Rac1 inhibition would lead to an increase in α6β4 mediated binding and increased crypt formation. The latter two studies indicate that a balance is needed between α6β4 interactions that regulate a reduction in nuclear YAP and other integrins that facilitate the cell-to-cell heterogeneity in YAP expression that is required for symmetry breaking.Ongoing studies are investigating more details related to cell-ECM mediated signaling pathways to understand how cell-ECM interactions regulate symmetry breaking. In parallel, we are designing synthetic ECMs to control integrin binding more precisely and, ultimately, develop an alternative to Matrigel that can be used to generate homogeneous, reproducible organoids for studies in fundamental mechanobiology, as well as disease modeling and translational research.

Remodeling the Tumor Extracellular Matrix via Hyaluronic Acid Depletion: Implications for Cancer Therapy and Fibrotic Disease

Alice Browne, Miranda Clements, Greta Forbes, Rosanda Kaplan

Pediatric Oncology Branch, Tumor Microenvironment Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health

ABSTRACT BODY: Aberrant extracellular matrix (ECM) accumulation is a hallmark of solid tumors, driving progression, immune exclusion, and treatment resistance. However, its complex architecture presents a challenge for comprehensive analysis. Among ECM components, hyaluronic acid (HA), a glycosaminoglycan involved in tissue hydration and structural integrity, has emerged as a potent regulator of tumor biology.Transcriptomic analyses across TCGA datasets revealed a consistent upregulation of hyaluronan synthases (HASes) in solid tumors (up to ~100-fold), which significantly correlated with poor overall survival. Immunohistochemical staining confirmed increased HA deposition. Hyaluronidases (HYALs), the enzymes responsible for HA degradation, were downregulated (3–5-fold) and associated with significant adverse outcomes. Notably, early HA accumulation was observed in the pre-metastatic lungs of osteosarcoma and breast cancer-bearing mice, preceding the detection of metastases and classical niche markers such as fibronectin, positioning HA as a potential initiator of metastatic ECM remodeling. Similarly, in radiation-induced fibrosis, HA levels rose from week 2 post-radiation, preceding classical fibrotic changes at weeks 12–16. These findings collectively identify disrupted HA homeostasis as a clinically significant and underexplored feature of the tumor and fibrotic stroma.To explore HA’s functional role, we developed genetically engineered mesenchymal stromal cells (GEMesys) expressing hyaluronidase for targeted HA depletion. Using in vivo second harmonic generation imaging, multiplex immunofluorescence, and biochemical assays, we tested GEMesys in syngeneic orthotopic murine models of osteosarcoma (F42010), rhabdomyosarcoma (M-3-9M), pancreatic cancer (Panc02), and spontaneous pancreatic ductal adenocarcinoma (KPC). GEMesys homed to tumor sites and initiated ECM remodeling within 24 hours, resulting in substantial HA degradation and a three-fold reduction in collagen fiber density and alterations in alignment. Collagen reorganisation appeared secondary to HA depletion, underscoring HA’s role in maintaining ECM structure. HA degradation also alleviated hypoxia, rapidly reducing Hypoxyprobe staining and HIF1α expression in these tumors. High HAS2/3 expression correlated with enrichment of glycolytic pathways and suppression of oxidative phosphorylation in TCGA datasets, and this was mirrored by downregulation of GLUT1 post-HA depletion in our models, implicating HA in modulating the Warburg effect. GEMesys further promoted vascular normalization (reduced CD31⁺ and CD105⁺ endothelial markers) and reprogrammed immune infiltration, enhancing CD4⁺CD44⁺ effector T cells while diminishing exhausted T cells and immunosuppressive myeloid populations.Therapeutically, GEMesys monotherapy reduced tumor burden (1.4-fold in osteosarcoma, 2-fold in rhabdomyosarcoma, 7.2-fold in Panc02). In the KPC model, GEMesys reduced HA/Col1 levels and extended overall survival, including one complete remission. Critically, GEMesys also sensitized tumors to chemotherapy, achieving a four-fold reduction in growth and stable disease in combination regimens. In a radiation-induced fibrosis model, GEMesys treatment significantly extended survival, suggesting broader therapeutic potential beyond oncology.Together, these findings position HA as a master regulator of ECM architecture, tumor metabolism, immune suppression, and metastatic priming. Targeted HA depletion remodels the tumor microenvironment and sensitizes it to therapy, unveiling new treatment vulnerabilities. This work underscores the value of matrix-focused interventions and provides compelling rationale for integrating anti-stromal approaches in the treatment of high-risk malignancies and fibrotic diseases.

Fibronectin Coating of Tissue Culture Polystyrene Improves Superficial Zone Chondrocyte Expansion

John Caputo¹, Thomas J. Manzoni¹, Issabella Ewine¹, Alvin W. Su²

¹University of Delaware, ²Nemours Children's Hospital

ABSTRACT BODY: Articular cartilage plays a critical role in proper joint function, by providing a lubricated surface for frictionless movement and load distribution during articulation. These properties are attained through zonal chondrocyte subpopulations in native articular cartilage via zone-specific protein production. In particular, the superficial zone chondrocytes (SZC) are responsible for the production of lubricin/proteoglycan-4 (PRG4) which lubricates the cartilage surface establishing a low coefficient of friction. Cartilage injuries present significant challenges due to the limited regenerative capacity of native chondrocytes. Passaged full-thickness chondrocytes are FDA approved for use in cartilage repair, though lack successful long-term repair capabilities due to the dedifferentiation of the chondrocytes throughout passaging. During passaging on tissue culture polystyrene, the actin cytoskeleton reorganizes cortical actin into stress fibers coinciding in an increase fibroblastic and contractile molecule expression, and a loss of chondrogenic expression during dedifferentiation. Furthermore, we and others have identified that the redifferentiation of full-thickness chondrocytes does not produce an adequate superficial zone. This is due in part to SZC poor attachment and proliferation on polystyrene compared to other zonal chondrocytes. We have found that passaging SZC on chondrocyte derived-decellularized extracellular matrix (CD-ECM: CELLvo ™ ChondroMatrix, StemBioSys) improves SZC attachment and proliferation, while limiting the effects of dedifferentiation. Examination of the CD-ECM matrix content shows a high abundance of fibronectin (FN). FN is an important matrix protein involved in cell adhesion and has been shown to regulate chondrocyte adhesion, proliferation, and differentiation. Additionally, SZCs have been shown to preferentially bind to FN due to a high expression of α5β1. It is currently unknown if passaging SZC on FN coated polystyrene will aid in overcoming the poor attachment, poor proliferation, and differentiation of SZC during culture. Here, we hypothesize: FN coating of tissue culture polystyrene will increase primary SZC attachment and proliferation, while limiting dedifferentiation throughout passaging. To test our hypothesis primary bovine SZC were seeded on uncoated polystyrene, FN coated polystyrene or CD-ECM. Cell attachment, expansion rate, and gene expression were characterized throughout passaging (Passage 0 (P0) and Passage 2 (P2)). Primary SZC had greater cell attachment when seeded on FN compared to polystyrene, and similar to that of SZC seeded on CD-ECM. Furthermore, SZC passaged on FN and CD-ECM reached confluency faster than SZC cultured on uncoated polystyrene. As compared to uncoated polystyrene, SZC on FN and CD-ECM culture flasks reached 70-90% confluency earlier. There were no differences in chondrogenic mRNA levels of aggrecan, collagen-II, and proteoglycan-4 between uncoated polystyrene, FN, and CD-ECM at P2. Additionally, both SZC passaged on FN and uncoated polystyrene had significantly increased expression differentiation markers Collagen-1 and Alpha-smooth muscle actin between P0 and P2. At P2, SZC passaged on polystyrene and FN had significantly higher Collagen-1 and Alpha-smooth muscle actin expression compared to SZC passaged on CD-ECM. Overall, our initial hypothesis was not fully supported. While passaging SZC on FN coated tissue culture polystyrene improves SZC expansion through increased primary cell attachment and decreased time to reach confluency, FN does not limit the effect of differentiation throughout passaging. Methodology to enrich and prevent the dedifferentiation of SZCs may help in achieving an adequate number of cells for bioengineering a native like superficial zone.

Macrophages Regulate Fibroblasts to Coordinate Fibrosis After Cardiac Pressure Overload

Upendra Chalise, Amrit Gaire, Steven Shen, Jop van Berlo

University of Minnesota -Twin Cities

ABSTRACT BODY: Cardiac hypertrophy is a compensatory response to left ventricular pressure overload. Reactive fibrosis develops in a hypertrophic heart where cardiac fibroblasts respond to hemodynamic alterations by activation and differentiation to a myofibroblasts phenotype responsible for excessive matrix deposition. Early in cardiac pressure overload monocytes infiltrate in the heart and direct inflammatory tissue niches. Our lab has previously shown that alterations in the macrophage populations can change the fate of fibrosis as resident macrophage depletion before cardiac pressure overload aggravated cardiac fibrosis. Here, we hypothesized that macrophages and fibroblasts regulate each other through cell-cell communication and control the fibrotic phenotype after transaortic constriction (TAC). As monocytes infiltrate to the heart early on in TAC, we focused our study on the earlier timepoints where we performed single cell transcriptomics assay at day (D) 0, 1, 3 and 5 after TAC to analyze how macrophages and fibroblasts are dynamically clustered after TAC. We then used the bioinformatics platform Nichenet to identify cellular communication cues between fibroblasts and macrophages, where one cell is taken as a sender based on ligand expression and the other cell is taken as a receiver based on cognate receptor expression. Spatial transcriptomics was performed in sham and TAC hearts to evaluate fibrotic niches, spatially. Immunofluorescence was performed to examine macrophages and fibroblasts in fibrotic niche, in sham and TAC hearts. Primary clustering analysis using uniform manifold approximation and projection (UMAP) shows that macrophages show eight distinct clusters (Resident macrophages, Ccr2 + macrophages, Ccr2- macrophages, Arg1high macrophages, interferon stimulated genes (ISG)high macrophages, proliferating macrophages, monocytes 1 and monocytes 2) and fibroblasts shows five distinct clusters (Naïve fibroblasts, Myofibroblasts, Wif1high fibroblasts, Spp1high fibroblasts and Cd248high fibroblasts). Based on gene expression data, macrophages as senders were examined for fibroblasts as receivers: five major ligand-receptor interactions identified were Tgfb1, Bmp2, Tnf, Il1b and IL10 secreted by macrophages to be received by Tgfbr1/2, Bmpr2/1a, Tnfrsf1a, Il1r1, and Il10rb receptors in fibroblasts, respectively. Tgfb1 is the key ligand identified from macrophages that directly signals to fibroblasts. Tgfb1 is a well-known myofibroblasts differentiating signal in vitro and in vivo. Similarly, with fibroblasts as senders and macrophages as receivers, Csf1, IL6, Hp, Bmp4, and Fgf2 were the major ligands secreted by fibroblasts to act on Csf1r, Il6ra, Cd163, Bmpr2, and Fgfr1/2 receptors on macrophages. These data suggest that macrophages directly regulate fibroblasts through Tgfb1 secretion and fibroblasts also dynamically regulate macrophage population through Csf1 secretion. Spatial transcriptomics identified fibrotic niches in TAC hearts using a fibrosis score calculated based on gene expression (Timp1, Postn, Col1a2, Col3a1, Ccn2, Ltbp2, Meox1, Lox, Tnc, and Fn1). Lyz2 expression showed myeloid cells accumulation in the fibrotic niches which were confirmed by immunofluorescence. Therefore, our overall data shows that the intricate interplay between macrophages and fibroblasts defines the development of cardiac fibrosis in response to cardiac pressure overload.

The MatriGO Project: Systematic Curation and Refinement of ECM-related Terms in Gene Ontology

Daiqing Chen¹, Pascale Gaudet², Chris Mungall³, Paul Thomas⁴

¹University of Illinois Chicago, ²SIB Swiss Institute of Bioinformatics, Centre Medical Universitaire, ³Lawrence Berkeley National Laboratory, ⁴University of Southern California

ABSTRACT BODY: Gene Ontology (GO) (http://www.geneontology.org/) is the oldest and most comprehensive tool to systematically categorize gene products using controlled vocabulary. GO terms are divided into three categories: Cellular Component (CC), Molecular Function (MF), and Biological Process (BP). Computational and experimental biologists have widely adopted GO due to its comprehensive data framework and representation of the current state of knowledge. However, due to the dynamic nature of biology and GO, new annotations are created at a rapid pace to represent new discoveries. These annotations can also become rapidly obsolete due to scientific advances, which causes inconsistencies. GO is also widely used to interpret omic datasets through enrichment analyses; but more comprehensively annotated parts of the GO will be more represented in such analyses, which causes biases in enrichment analyses.The extracellular matrix (ECM) is one of the categories that is still incompletely annotated in GO. This is due to two main reasons: ECM proteins remain largely understudied, and GO was initially built to capture proteins located within or in the vicinity of a cell. In 2012, we developed a comprehensive list of ECM proteins called the matrisome. The matrisome nomenclature is now broadly adopted to annotate omic datasets. In 2024, we initiated an effort in collaboration with the GO consortium to curate and update all ontologies and annotations pertaining to the ECM from GO and harmonize them with the matrisome classification. We first built a framework that could best represent the relationship between core matrisome proteins (collagen, proteoglycan, and glycoprotein) in the GO hierarchy format. Then, we reviewed all current CC terms related to the ECM and organized them across three aspects: protein localization, the secondary protein structure based on domain information, and protein assembly into supramolecular complexes. As of today, we have completed the re-annotation of all collagens and proteoglycans and obsoleted terms whose definitions were no longer accurate. We have now begun the reorganization of other ECM glycoproteins of the matrisome according to this framework. Once this is complete, our next focus will be to curate MF and BP terms related to ECM gene products to facilitate the investigations of the functions of ECM proteins. Our ultimate goal is to ensure that the representation of the ECM in GO uses a consistent language and reflects the current state of knowledge in ECM research.

Histamine-3 Receptor Activation Protects Against Cardiac Fibrosis Through Retention of Collagen in Cardiac Fibroblasts

Heather Connery¹, Anja Herrnreiter², Alexander Widiapradja¹, William Campbell²

¹West Virginia University, ²Medical College of Wisconsin

ABSTRACT BODY: A major contributor to diastolic dysfunction in heart failure is cardiac fibrosis. The histamine-3 receptor (H3R) was previously identified on cardiac synaptosomes, with its exogenous activation inhibiting norepinephrine release, and thus arrhythmias. Our laboratory extended these findings, identifying the H3R on cardiac fibroblasts in the mouse left ventricle (LV), and demonstrated that exogenous activation reduced cardiac fibrosis in an angiotensin II (Ang II)-infusion model. The present study aimed to determine the extent to which exogenous H3R activation improved diastolic function in the Ang II model of cardiac fibrosis and identify key cellular mechanisms by which the H3R opposes a profibrotic cardiac fibroblast phenotype. Eight-week-old male C57/B6 mice underwent 7-day saline or Ang II (1500 ng/kg/d) infusion via osmotic pump. Separate saline or Ang II mice also received subcutaneous injections of the H3R agonist imetit dihydrobromide (40 mg/kg/d). LV diastolic function was assessed via pressure-volume catheter. LV fibrosis was assessed using picrosirius red (PSR) staining. Primary cardiac fibroblasts were isolated from the LV of C57/B6 mice and treated with the profibrotic stimulus TGF-β1 (30 ng/ml), together with imetit (300 nM). Collagen synthesis, release, and retention were assessed to determine changes in cardiac fibroblast phenotype/function. TruPath assay was used to assess H3R-induced G-protein dissociation in Hrh3-transfected HEK293T cells. Presto-Tango was used to examine β-arrestin recruitment in Hrh3-transfected HTLA cells. The cAMP/PKA/CREB and p38 MAPK signaling pathways were tested in isolated cardiac fibroblasts. LV end-diastolic pressure was increased in Ang II mice (p<0.03 vs Saline); this was prevented by imetit (p<0.02 vs Ang II). There was a trend towards an increase in tau, a measure of LV stiffness in Ang II that was not observed in imetit groups. Collagen volume fraction, a measure of cardiac fibrosis, was increased in Ang II-treated mice (p<0.0001 vs Saline), which was normalized in imetit-treated mice (p<0.0001 vs Ang II). In cardiac fibroblasts, TGF-β1 upregulated col1a1(p<0.0001 vs Untreated) and col3a1 (p<0.01 vs Untreated) gene transcription, and imetit did not alter this upregulated transcription status. However, while TGF-β1 increased collagen I release (p<0.0001 vs Untreated) this was inhibited by imetit (p<0.0001 vs TGF-β1). Collagen I in the cell lysate was unchanged between untreated and TGF-β1-treated fibroblasts, however, imetit treatment significantly increased collagen I retention in the cell (p<0.01 vs TGF-β1). HSP47, a key molecular chaperone regulating collagen release, was increased in the LVs of Ang II-mice (p<0.05 vs Saline) while treatment with imetit decreased HSP47 levels. H3R activation induced G⍺i/o G-protein subunit dissociation in HEK293T cells, suggesting inhibition of cAMP signaling. Imetit was significantly more potent than histamine in inducing this dissociation. pGlo cAMP assay confirmed that activation of the H3R inhibited forskolin-induced cAMP. H3R activation also enhanced β-arrestin recruitment as assessed by Presto-Tango. However, cAMP inhibition in cardiac fibroblasts did not recapitulate the effects on collagen release seen with H3R activation, and pCREB was unchanged between groups in cardiac fibroblasts, suggesting that while imetit can inhibit the cAMP pathway, this was not the case in cardiac fibroblasts. Conversely, the ratio of pP38 MAPK to total P38, a non-canonical signaling pathway of G⍺i/o was increased by TGF-β1 (p<0.05 vs Untreated) and inhibited by imetit (p<0.01 vs TGF-β1). This indicates that H3R activation opposes a profibrotic cardiac fibroblast phenotype not by preventing collagen synthesis, but by preventing collagen release from the cell by inhibition of the collagen chaperone protein HSP47. This may be mediated by inhibition of p38.

Age-related Structural, Biochemical, and Mechanical Changes in the Mouse Intervertebral Discs

Chitra Dahia¹, Stephen Bogdan¹, Abdullah M Ali², Chitra L Dahia^1,3

¹Hospital for Special Surgery, ²Columbia University, ³Weill Cornell Medical College

ABSTRACT BODY: The intervertebral disc (IVD) forms a cartilaginous joint between the vertebrae in the spine. IVDs to provide flexibility to the spine and resist compressive forces in a healthy state. Each IVD has a proteoglycan-rich core made up of the nucleus pulposus (NP). NP is laterally surrounded by collagen-rich annulus fibrosus (AF). The extracellular matrix (ECM) is crucial for IVD hydration, structure, and function. However, following injury, or with natural aging, the IVD undergoes natural structural and molecular changes leading to loss of ECM and its degeneration, which is a leading cause of chronic back pain. Chronic back pain affects over 70% of adults, but there is no cure. Here, using the mouse as a model system and RNAseq, proteomics analysis, histochemical analysis, and atomic force microscopy, we analyze the structural, biochemical, and mechanical changes in lumbar IVDs from one week to over two years of age. Results from transcriptomics and proteomics analysis showed an age-related decline in ECM turnover in the NP and AF cells of mouse lumbar IVDs. Next, using an array of histochemical staining methods, we analyzed the structural changes in the lumbar IVD and biochemical composition of each region across the lifespan. Due to the high collagen and proteoglycan content of the IVD, safranin-O and fast green, or picrosirius red, are generally used to determine biochemical changes in the IVD. Here, using additional staining methods, including Masson’s trichrome and Movat pentachrome, we observed that, in addition to changes in collagen and proteoglycan content with age, the aging mouse IVDs become vascularized and have a high amount of fibrin(ogen) fibrinoid deposition. Next, we determined the clinical relevance of these findings using clinical samples obtained under an IRB-approved study, following informed consent, and validated the presence of fibrinoids in degenerated human IVDs. The proteomic data supported the presence of fibrin in the IVDs of aging mice. Moreover, atomic force microscopy determined tissue stiffness and revealed age-related changes in the viscoelastic properties of the different regions of the mouse IVDs. To our knowledge, this is the first study to describe the presence of fibrins in the IVDs, which was made possible by the histochemical staining approach.FUNDING SOURCE: The research was supported by NIAMS (R01AR077145), NIA (R01AG070079), and OD (1S10OD026763) of NIH.

Hypoxia-induced Stroma-driven Extracellular Matrix Remodeling Impairs Immune Infiltration and Identifies Targeting Vulnerabilities in High-grade Serous Carcinoma

Simona Plesselova¹, Pilar de la Puente^1,2,3

¹Cancer Biology and Immunotherapies Group, Sanford Research, Sioux Falls, SD, ² Department of Obstetrics and Gynecology, University of South Dakota Sanford School of Medicine, Sioux Falls, SD, ³ Department of Surgery, University of South Dakota Sanford School of Medicine, Sioux Falls, SD

ABSTRACT BODY: High-Grade Serous Carcinoma (HGSC) is the most common and lethal subtype of ovarian cancer where the presence of tumor-infiltrated lymphocytes (TILs) in the tumor microenvironment (TME) correlated with better outcomes. However, the majority of HGSC patients present immunologically cold tumors defined by immune exclusion, immunosuppressive cells, and immunotherapy resistance due to complex TME characterized by hypoxia. Hypoxia induces activation of cancer-associated fibroblasts (CAFs), which are key players in aberrant extracellular matrix (ECM) remodeling involving TGF-β signaling, converting it into a stiff physical barrier for TILs infiltration and drug diffusion into the TME. While the physiological oxygen levels in healthy ovaries are around 10% O2, current preclinical models compare hypoxic TME (1.5% O2) with hyperoxic culture models (21% O2), hindering the understanding of the role of physiological O2 levels and ECM dynamics in normal and tumorous ovaries in immune evasion. Our objective is to mechanistically investigate the immunologically cold TME and identify possible therapeutic targets in HGSC using physiologically relevant tumor-immuno-engineered patient-derived models. We bioengineered two human-based models either using HGSC plasma to create a 3D culture or decellularized human ovaries to study tumor-stroma-immune interactions and immune evasion. Both models were bioengineered to mimic hypoxia and aberrant ECM in the TME or physoxia and normal ECM in the healthy ovary. Specifically, a 3D model was bioengineered using human plasma from HGSC patients based on crosslinking of fibrinogen into fibrin. Patient-matched dissociated tumor biopsies were cultured in the presence of CAFs in the 3D model and exposed to autologous immune cells challenged to infiltrate into the 3D scaffold or as a multiculture. The O2 levels in the scaffolds were validated by an optical sensor, and the ECM remodeling characterized by second harmonic generation microscopy, immunohistochemistry, western blot, and atomic force microscopy. Immune infiltration, activation and cytotoxicity of the immune cells were evaluated by flow cytometry. Galunisertib (Gal), a TGF-β1 receptor inhibitor, was used to target TGF- β signaling and ECM remodeling. Additionally, human ovaries were decellularized (decellularized ECM, dECM) by exposure to low-dose detergents and recellularized with HGSC and CAFs and immune infiltration further validated. We identified that hypoxia triggered the activation of CAFs cocultured with HGSC cells, which correlated with an increased expression of TGF-β and collagen I, and a stiffer matrix compared to physoxia. This hypoxia-induced CAF-driven ECM remodeling caused an impaired immune infiltration compared to physoxia which was rescued by pre-treatment with Gal, confirming the role of TGF-β signaling in ECM remodeling and immune infiltration. Similar findings were observed in dECM from human ovaries with native-like ECM composition validating our 3D model as translationally relevant. Moreover, hypoxia enhanced the activation and cytotoxicity of CD8+ TILs, but also immunosuppressive T regs. Our data suggest that hypoxia acts as a friend and foe by enhancing CD8 activation and cytotoxicity to kill the tumor cells but also increases an immunosuppressive TME with aberrant ECM remodeling impairing immune infiltration. In conclusion, we have functionally characterized the role of physiologically relevant O2 levels and ECM dynamics in tumor-stroma-immune interactions recapitulated in biomimetic tunable human-relevant tumor-immune models. Our results can provide a paradigm-shifting understanding of hypoxia-driven ECM remodeling when compared to physoxia and its influence in immune evasion and cellular interactions, while providing targeting vulnerabilities to turn immunologically cold tumors into hot ones.

Novel Humanized X-Linked Alport Syndrome Missense Variants Induce ER Stress and Early Glomerular Dysfunction

Stephen Decker, Precious C. Opurum, Venisia Paula, Deborah Stuart

University of Utah

ABSTRACT BODY: Background: X-linked Alport Syndrome (XLAS) is caused by pathogenic COL4A5 variants and is characterized by progressive glomerular dysfunction and renal failure. While nonsense mutations are well characterized, the impact of disease-relevant missense variants on glomerular pathology and signaling remains poorly defined. We generated and characterized two humanized Col4a5 missense CRISPR/Cas9 knock-in mouse models—L1655R/Y and C1570S/Y— commonly reported in the Western United States to evaluate functional decline and molecular injury responses.Methods: Glomerular filtration rate (GFR) was longitudinally measured via transcutaneous FITC-sinistrin clearance from 3 months of age until 1 year of age. Transmission electron microscopy (TEM) was performed to examine ultrastructural changes in glomerular basement membrane (GBM) and podocytes. Whole kidney cortex was analyzed by qPCR to assess unfolded protein response (UPR), ER stress signaling, and TRPC5/Rac1 pathway activation.Results: Both Col4a5 missense models exhibited early GFR increases compared to WT controls, followed by a progressive decline in GFR. At 1 year, GFR was reduced to 594.2 ± 94.6 μL/min/100g in Col4a5C1570S/Y and 555.0 ± 78.0 μL/min/100g in Col4a5L1655R/Y versus 861.1 ± 152.4 μL/min/100g in WT and 752.7 ± 136.7 μL/min/100g in Col4a5+/Y littermates, indicating impaired renal function by 1 year. TEM revealed classic Alport features, including GBM thickening, lamellation, and podocyte foot process effacement in both models. qPCR revealed significant upregulation of UPR markers in both mutants, including HSPA5 (4.3- to 18.8-fold), DDIT3 (4.0- to 7.3-fold), and ATF4 (13.8- to 15.1-fold). TRPC5 and Rac1 expression were increased 3.7- to 13.7-fold and 3.0- to 3.1-fold, respectively, suggesting activation of stress-sensitive cytoskeletal signaling pathways.Conclusion: Humanized Col4a5 missense variants are sufficient to drive ER stress, TRPC5/Rac1 signaling, and glomerular injury in vivo. The L1655R variant produces a more severe phenotype than C1570S, highlighting allele-specific progression in XLAS. These models offer valuable tools for dissecting early disease mechanisms and support targeting ER and TRPC5 pathways as therapeutic strategies.

Fibulin-4 and Latent-Transforming Growth Factor Beta-Binding Protein-4 Interactions with Syndecan-2 and -3 are Required for Elastogenesis

Hana Hakami^*1; Neha Dinesh^*1; Valentin Nelea^1,2; Kerstin Tiedemann¹, Nathalie Lamarche-Vane¹; Sylvie Ricard-Blum³; Dieter P. Reinhardt^1,2

¹Faculty of Medicine and Health Sciences, Department of Anatomy and Cell Biology, McGill University, Montreal, QC H3A 0C7, Canada, ²Faculty of Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC H3A 0C7, Canada, ³Université de Lyon Institute of Molecular and Supramolecular Chemistry and Biochemistry, UMR 5246, Villeurbanne, France, ^*Co-First Authors

ABSTRACT BODY: Elastogenesis is a cell surface-located hierarchical process that requires the core components tropoelastin and fibrillins and several accessory proteins, including fibulin-4 (FBLN4) and latent TGF-β binding protein-4 (LTBP4). FBLN4 and LTBP4 interact with cells, but their cell receptors and associated molecular elastogenic mechanisms remain unknown. Using primary skin fibroblasts and several vascular smooth muscle cells as cell culture model system, we identified that these elastogenic cells bound strongly to FBLN4 multimers and LTBP4 monomers. We identified two cell interaction epitopes on FBLN4 located in cbEGF2-3 and the C-terminal domain, whereas FBLN4 multimerization sites were mapped to cbEGF4-5 and the C-terminal domain. We also determined a novel cell interaction site in the N-terminal half of LTBP4. Cell binding to FBLN4 and LTBP4 was strongly inhibited in the presence of heparin, heparan sulfate, or after enzymatic removal of heparan sulfate, suggesting heparan sulfate proteoglycans as relevant cell surface receptors. siRNA knockdown experiments identified syndecan (SDC)2 and SDC3 as cell receptors for FBNL4 and SDC3 for LTBP4. Direct protein interactions between FBLN4 and the recombinant ectodomains of SDC2 and SDC3, and between LTBP4 and SDC3 validated these results. Interaction of the elastogenic cells with FBLN4 and LTBP4 enhanced elastogenesis, whereas SDC2 and/or SDC3 knockdowns led to reduced elastic fiber formation. The cell interactions with FBLN4 and LTBP4 significantly enhanced focal adhesion formation, induced cell contraction, and led to activation of focal adhesion kinase (FAK), Erk1/2, and RhoA. Pharmacological inhibition of these effectors markedly attenuated elastic fiber formation, and siRNA knockdown of SDC2 and SDC3 led to reduced levels of pFAK, pERK, and active RhoA.

In summary, our findings unravel the syndecan-mediated mechanisms through which FBLN4 and LTBP4 drive elastogenesis. We demonstrate that FBLN4 and LTBP4 cell interactions through SDC2 and SDC3 promote elastogenesis by enhancing focal adhesion formation, leading to cell contractility through FAK, Erk1/2, and RhoA activation, establishing the significance of these pathways in elastogenesis. These data contribute significantly to our understanding of the molecular interactions governing elastogenesis and offer potential therapeutic targets for related pathologies characterized by altered elastin synthesis and assembly.

Fibronectin Drives Skeletal Development Through Chondrocyte Differentiation and Matrix Assembly

Neha E. H. Dinesh¹, Kerstin Tiedemann¹, Philippe M. Campeau^2*, Dieter P. Reinhardt ^1,3*

¹Faculty of Medicine and Health Sciences, Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada, ²Centre Hospitalier Universitaire Sainte-Justine Research Center, Montreal, QC, Canada, ³Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada

ABSTRACT BODY: Autosomal dominant mutations in fibronectin (FN) cause corner fracture-type spondylometaphyseal dysplasia (SMDCF), yet the mechanisms by which FN contributes to skeletal development remained unclear. To investigate this, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of SMDCF patients harboring FN mutations (p.Cys123Arg and p.Cys231Trp) and from unaffected control. Differentiation into mesenchymal stem cells (MSCs) and chondrocytes revealed that mutant FN impaired protein secretion, causing intracellular accumulation and reduced extracellular FN levels. Transmission electron microscopy of mutant MSCs revealed enlarged vesicles containing fibronectin aggregates with characteristics of both the endoplasmic reticulum and lysosomes, indicative of elevated cellular stress associated with mitochondrial dysfunction. Chondrogenic differentiation was impaired, with disrupted MSC condensation, decreased proliferation, and downregulation of more than 25 chondrogenic regulators, including TGFβ1 a key factor in chondrogenesis. Supplementation with exogenous FN or TGFβ1 partially rescued condensation defects and restored chondrogenic gene expression in mutant cells. To explore FN’s role in vivo, we created conditional FN knockout mouse models targeting cartilage-specific FN (cFNKO), plasma FN (pFNKO), and both isoforms (FNdKO). Histological and micro-CT analysis confirmed FN localization in resting and hypertrophic chondrocytes and trabecular bone. While cFNKO and pFNKO mice showed minor phenotypes, FNdKO mice exhibited marked postnatal skeletal abnormalities, including reduced body weight, shortened bones, lower trabecular bone volume, decreased bone mineral density, and increased marrow adiposity. Growth plate analysis in FNdKO mice revealed disorganized collagen type II and X expression, impaired hypertrophic chondrocyte apoptosis, and reduced bone formation markers. Mechanistic studies demonstrated reduced TGFβ1 and phospho-AKT signaling, implicating FN in the regulation of chondrocyte maturation and endochondral ossification. Together, these findings demonstrate FN as a critical regulator of chondrogenesis and skeletal development. FN deficiency disrupts chondrocyte function, matrix production, and cell fate transitions, suggesting therapeutic potential for FN or TGFβ1-regulated mechanisms in treating FN-related skeletal disorders.

Differentially Expressed Proteins Identified via Single Cell Proteomics Used as Spatial Omic Targets in Marfan Syndrome

Ashley Dinh¹, Lior Zilberberg¹, Divya Gupta¹, Anton Villamejor²

¹Department of Cardiology, Smidt Heart Institute, Cedars-Sinai Medical Center, ²Spatial Molecular Profiling Shared Resource and Samuel Oschin Cancer Center, Cedars-Sinai Medical Center

ABSTRACT BODY: Purpose: Marfan Syndrome (MFS), a congenital disorder induced by mutations in the fibrillin-1 gene (Fbn1), predisposes patients to aortic aneurysms/dissections that may result in sudden death. Currently, there are no treatments that have been successfully translated to humans to prevent aneurysms or ameliorate their progression as the linkage between the Fbn1 mutation and molecular pathogenesis of aortic aneurysms have yet to be fully described. Our group uses single cell proteomics with mass spectrometry (SCP-MS) in MFS mouse aortic root tissue to discover novel cell phenotypes and altered cell proportions in aneurysmal aorta. Our previously reported SCP-MS data produced seven subtypes of smooth muscle cells (SMCs) with clusters presenting a “modified” pattern expressing notable markers, LRP1, PRSS2, and TRPV1. DEPs in male MFS and WT cells suggested an increase of endothelial to mesenchymal transition (endMT) in MFS cells, which corroborates previous transcriptomic data revealing increased levels of Zeb1, an endMT regulator, in MFS endothelial cells (ECs). Additionally, “top markers” in both RNA transcript and protein levels identified Tpm4, Ace, and S1004A in MFS-enriched clusters. In the current project, we validate these SCP-MS phenotypes and markers in murine and human aortic tissue samples using spatial omic technology. Spatial insight into their expression in various cell types in the context of aortic tissue may elucidate how mutations in Fbn1 prompt adverse cell changes and matrix biology that ultimately contribute to aneurysm. Methods: Mouse and Human Tissues: Aortae from wild-type (WT) and Fbn1C1039G/+ mice, and MFS and unaffected patients were fixed in formalin and paraffin embedded. 5 um thick sections were collected for spatial omics experiments. Lunaphore COMET: All antibodies were individually validated using standard immunofluorescence techniques prior to assembly into the final multiplexed panel. Slides were baked and immersed in antigen retrieval before cyclic antibody staining using the Lunaphore COMET.Analysis: Multiplexed images were processed on Comet software to integrate imaging layers. Qupath’s InstanSeg extension was used to perform cell segmentation in regions of interest that included only the aortic region. Antibody intensities for each identified cell was then analyzed using python SCIMAP pipeline, in which cell phenotype is assigned using priori markers and unbiased clustering approaches. Results: We assembled a panel of 21 antibodies to target standard vascular cell markers (e.g., ACTA2, SMTN, PECAM1, COL1A1, VCL), indicators of metabolism (ATP5A1), TGFB (SMAD2) and inflammatory (NFKB) signaling, the endMT marker ZEB1, several immune cell markers (e.g., F480, CD68, CD4, CD8, CD11B, CD45), and multiple key cell markers of altered SMC phenotypes identified in our SCP-MS experiment (LRP1, PRSS2, S100A4, ACE, TPM4, TRPV1). Patterns of TPM4, ACE, S1004A and ACTA2 expression parsed SMCs into three subtypes: normal contractile, modSMC1, and modSMC2. As witnessed in the SCP-MS data, contractile SMCs were reduced and modified SMCs were increased in MFS relative to WT aorta. Additionally, LRP1 and PRSS2 expressions were higher in the modified SMCs. Cells expressing CD31, an EC marker, were assessed for ZEB1 and SMAD2 expression, corroborating the upregulation of EndMT cells in the MFS tissue. Conclusion: The use of spatial omics, informed by single cell proteomics and transcriptomics, allow for a visualization of how these novel DEPs and markers can contribute to the observed modified cell phenotypes and proportions in Marfan Syndrome. Ultimately, these discoveries better our understanding of how the Fbn1 mutation promotes an aberrant extracellular matrix and aortic aneurysm.

Machine Learning of Human Gene Expression Across Tissues Identifies a Matrix-Lamina Axis Confirmed by Live Imaging While Perturbing Tissue

Dennis Discher, Karan Saini

University of Pennsylvania

ABSTRACT BODY: Machine Learning (ML) based approaches to “big-data” have been widely applied to expression data, particularly for single cells, but the approach also applies to genes given enough samples. We apply ML to expression from 50 different tissues and more than 1,000 heathy humans to identify possible factors in a linkage from extracellular matrix to nuclear envelope components, and then we examine some of these within living tissues remains under real-time perturbations. First we show a “mechanoregulated” cluster of genes that include not only Collagen-I’s but also Piezo-1, Myosin-II (MYH9) and nuclear envelope genes Lamin-A, SUN2, and SYNE3. Closely related genes are missing: Lamin-B1, B2, SUN1, the other SYNEs, and Piezo2. The “mechanoregulated” genes also exhibit power-law scaling versus Collagen-I, which is the most abundant and mechanical load bearing protein in tissue extracellular matrix (ECM). Next, we investigated the effect of perturbing tissue mechanics - via either exogenous-collagenase based ECM softening or acto-myosin contractility inhibition in fresh human heart as well as other tissue samples – focusing on proteins of ECM and cytoskeleton to the lamina. We measured fibrillar collagen and myosin-based muscle striation level in “real-time” via label-free second harmonic generation (SHG) imaging and combined with assessments of collagen-I, F-actin, Lamin-A via immunostaining of intact tissues as well as many more proteins via mass-spectrometry (MS) of tissue extracts. Together with studies of beating embryonic chick hearts, we conclude that mechanical forces not only protect tissue fibrillar collagen network from degradation by collagenases including endogenous matrix metalloproteinases (MMPs) but also preserve muscle striation and nuclear morphology and lamin levels. Our ML approach thus identifies genes in pathways that can be literally seen in live human tissues.

Extracellular Matrix Dysregulation Induced By Platinum Chemotherapy Contributes to Treatment-Resistant Ovarian Cancer

Jessica Faragher, Kristin Calar, Pilar de la Puente

Sanford Research; USD Sanford School of Medicine

ABSTRACT BODY: One of the deadliest forms of gynecological cancers in women is ovarian carcinoma with high grade serous ovarian carcinoma (HGSC) being the most common subtype. Before surgery, 70% of patients are treated with platinum chemotherapy. Over 80% of these patients develop resistance to treatment within 1 year. Chemoresistance in HGSC has been associated with dysregulation of the tumor extracellular matrix (ECM). The ECM is a non-cellular 3D network of scaffold proteins secreted by cells that provides structural and biochemical support to the tissue landscape. Tumor ECM is biochemically distinct from healthy ECM, and ECM remodeling is associated with an increased risk of treatment failure and mortality in HGSC. Although the effects of different ECM compositions and structure have been well explored and linked to reduced therapeutic efficacy, chemotherapy-induced alterations in the ECM are still unknown. Therefore, elucidating the spatiotemporal dynamics of ECM in response to chemotherapy in HGSC is desperately needed. Our objective is to profile newly synthesized ECM in response to platinum-based treatment, the standard of care HGSC chemotherapy, utilizing biomimetic 3D co-culture models paired with metabolic labeling. In the absence of these approaches, the development of effective treatments to overcome chemotherapy resistance in HGSC will remain difficult. A biomimetic 3D hydrogel, termed 3D extracellular matrix (3D-ECM), was engineered by functionalization of collagen I and plasma-derived fibrinogen to ensure a biologically relevant viscoelastic and fibrillar microstructure. Within the 3D-ECM, we have developed a model resembling a HGSC tumor by co-culturing various HGSC cell lines or platinum sensitive/resistant patient derived xenografts lines (OV81 and OV81-CP40, respectively) with cancer-associated fibroblasts (CAFs). These 3D cultures were treated with cisplatin to assess the HGSC response to platinum-based therapy. Newly synthesized proteins, or “nascent ECMâ€, were identified via non-canonical bio-orthogonal metabolic labeling via L-azidohomoalanine (AHA) administration, combined with super resolution confocal microscopy and machine learning software analysis. Proteomic analysis of nascent ECM (enriched for AHA) was carried out to evaluate the differential composition of cisplatin-induced ECM protein, and targets were validated with immunofluorescence. Flow cytometry was performed to investigate epithelial-mesenchymal transition (EMT) in platinum exposed HGSC cell lines. Through this approach, we identified that cisplatin treatment significantly increases the secretion profile of HGSC cells. Cisplatin treatment of chemotherapy resistant cells increases the secretion profile of both cancer cells and CAFs, suggesting paracrine signaling between the cell types. With proteomic analysis, we were able to confirm that newly synthesized proteins, specifically core and associated ECM proteins, including those that are transient and low in abundance. Additionally, we found cisplatin treatment provokes EMT in HGSC lines, and that cisplatin resistant lines have a higher mesenchymal phenotype compared to cisplatin sensitive cells. In conclusion, these results suggest that platinum chemotherapy by itself dysregulates ECM and such changes could be linked to chemoresistance. Critically, our biomimetic tissue-engineered 3D-ECM model paired with metabolic labeling enables tracking of spatiotemporal ECM dynamics in response to treatment. These results are intended to have an important positive impact by providing evidence-based proof of principle assays for future therapeutic development to overcome chemotherapy-induced ECM changes, resulting in more effective treatment for ovarian cancer.

High Molecular Weight Hyaluronan is a Novel Therapeutic Agent Against Lung Epithelial Injury, Inflammation and Disease

Stavros Garantziotis, Vandy Stober, Jonas Ritter, Carol Trempus

National Institute of Environmental Health Sciences

ABSTRACT BODY: Introduction: Lung injury, whether from air pollution, infections or other noxious fumes, is detrimental to human health and contributes to increased morbidity and mortality from respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma. Hyaluronan (HA) plays a pivotal role in airway homeostasis, with its high molecular weight form (HMWHA) exhibiting protective properties against lung injury. We investigated the impact HMW HA on lung injury. Methods: We used a bench to bedside approach, utilizing differentiated air liquid interface cultures of primary murine and human tracheal epithelial cells, mouse models of disease and clinical studies. Non-infectious (pollution exposure, oxidative injury) and infectious (viral pneumonia) models were used to interrogate a broad aspect of HMWHA effects. Genetically modified mice were used to address the role of HA receptors (Cd44 and Rhamm, encoded by the gene Hmmr) and innate immunity (Myd88). Clinical studies addressed the role of inhaled HMWHA in human viral infection (COVID19) and disease exacerbation (COPD). Results: Oxidative injury significantly reduced epithelial integrity in both human and murine cultures. There was no effect of Cd44 or Rhamm on epithelial integrity. Transcriptomic analysis of both mouse and human cultures revealed upregulation of inflammatory pathways, epithelial – mesenchymal transition, oxidative stress response, and metabolic alterations. Cd44 deficient epithelia showed significantly reduced inflammatory levels, suggesting a Cd44 dependent mechanism in ozone – induced airway inflammation. Treatment with HMWHA effectively mitigated epithelial injury in a Myd88-dependent manner, suggesting that innate immunity mediates this effect. HMWHA inhalation mitigated lung inflammation and injury after viral pneumonia via inhibition of the transcription factor E2F1. In two clinical trials, inhaled HMWHA shorted the time to discharge from the hospital in patients with severe COVID19 and severe COPD exacerbation.Conclusions: HMWHA ameliorates epithelial injury through activation of the innate immune system and ameliorates inflammation after injury by inhibiting the activation of transcription factor E2F1. Together, these effects promote lung homeostasis. In clinical trials, HMWHA inhalation can shorten the time to recovery from severe viral pneumonia and severe COPD exacerbation. These findings support a role for HMWHA as a novel therapeutic for human lung disease.

Calreticulin (CALR), an Optimal agent for Tissue Regeneration of Corneal Wounds Acts by Accelerating Wound Closure and Attenuating Fibrosis-Hazing

Leslie I. Gold¹, Sarita Mishra¹, Justin Prater², David Culp²

¹New York University School of Medicine, ²Powered Research

ABSTRACT BODY: Healing injuries of the corneal (e.g., surgery/photo-refractive keratectomy, corneal transplant, infectious keratitis ulcerations, dry eye, and diabetic keratopathy) requires a delicate intrinsic program specific only to the eye for the prevention of blindness. Whereas angiogenesis is important to cutaneous healing, blood vessels emanating from the limbus due to deep corneal injury can obscure vision. Furthermore, collagen and other ECM proteins produced by myofibroblasts to reconstruct the wound must be highly regulated to revert to non-ECM producing stromal cells as continued ECM proteins produce fibers that obliterate the transparency of the corneal causing fibrosis, referred to as hazing. Following corneal injury that disrupts the epithelial basement membrane, TGF-β2 is released by corneal epithelial cells (CE) that reaches the stromal cells (keratocytes) below thereby inducing myofibroblast differentiation(α-smooth muscle cell actin (α-SMA) that secrete a disorganized ECM. Our past studies showed that Calreticulin (CALR) healed full-thickness excisional diabetic mouse wounds by abundant granulation tissue formation and not by inducing angiogenesis. Moreover, the wounds healed by a tissue regenerative process without the usual scar formation. Since these properties of CALR were consistent with optimal healing requirements for the cornea, the objective of this study was to analyze the rate and quality of CALR treatment in a rabbit vacuum corneal injury model involving the epithelium and 10% of the underlying stroma. The results show that the application of CALR to the injured cornea: 1) accelerated full wound closure by 96 hours post-injury, 3 days faster than the control buffer 2) accelerated delayed healing caused by corticosteroids (dexamethasone), routinely used to prevent post-injury inflammation and fibrosis, by 6 days, and 3) healed the wounds without vascularization or fibrosis/hazing. In fact, CALR appeared to lessen characteristics of the fibrotic response compared to buffer. In vitro, we show that CALR stimulated proliferation of human CE and keratocytes (stromal) by 1.5-fold and 1.4-fold, respectively. Using an in vitro scratch plate assay, CALR induced migratory wound closure by CE cells and keratocytes, by 72% and 85% compared to controls of 44% and 59%, respectively. These functions are crucial for re-epithelializing the wound to facilitate closure and for regeneration of underlying injured stromal tissue. As a marker of decreased fibrosis, CALR-treated corneal wounds showed decreased immunostaining for α-smooth muscle actin (α-SMA) and vimentin in the keratocytes (stromal tissue). Consistent with this response in vivo, CALR treatment of corneal cells in vitro, decreased the levels of the markers of fibrosis as determined by immunoblotting: TGF-β2 and vimentin human CE cells, and α-SMA in keratocytes. CALR has the potential to be a novel ophthalmic topical therapeutic to accelerate corneal healing from various injuries and potentially as an adjunct therapy to accelerate healing delayed by the routine use of anti-inflammatory topical corticosteroids. As dexamethasone is immunosuppressive, CALR alone would be useful to heal corneal ulcerations caused by viral infections.

Fibrillin-1 Mutation Affects Adhesome Complex Formation and Extracellular Matrix Signaling in Marfan Syndrome

Divya Gupta, Sean Escopete, Ashley Dinh, Liam McCarthy

Cedars-Sinai Medical Center

ABSTRACT BODY: Purpose: Marfan Syndrome (MFS) is one of the most common inherited connective tissue disorders with increased mortality from aortic complications. Characterized by a mutation in the fibrillin-1 gene, MFS affects matrisome integrity, specifically adhesome complex formation critical for intracellular signaling and tissue function. While there is a molecular understanding behind MFS pathophysiology, its cellular versus matrix contributions have not been investigated. To study cellular recognition of diseased surroundings and intracellular transmission, we interrogated the matrisome proteomic profiles of wildtype (WT) and MFS primary aortic smooth muscle cells (SMCs) repopulated onto decellularized MFS and WT matrices. Methods: WT and MFS mouse (Fbn1C1041G, N=3 each) SMC lines were pooled and expanded on 0.2% gelatin coating and high glucose DMEM/20% FBS conditions. Three days post 100% confluency, ECM were decellularized with detergent buffers. Decellularized ECM were blocked with 1% BSA and repopulated with individual WT or MFS cells and incubated for either 2 or 20 hours. Adhesome complexes were then crosslinked with di-tert-butyl peroxide, and isolated using dithiothreitol. Samples were prepared for mass spectrometry by S-trap digestion and analyzed by Data Independent Acquisition on an Orbitrap Ascend Tribrid. Results: Differentially expressed proteins (DEPs) driven by cell type were disproportionately higher in MFS relative to WT cells on either matrix at both timepoints. Interestingly though, regardless of cell type, focal adhesion DEPs were disproportionately greater on WT matrix at both timepoints compared to MFS matrix. Importantly, mitochondrial proteins were found to be significantly downregulated by both cell types on MFS matrix.Conclusion: Our data demonstrate that the fibrillin-1 mutation impacts adhesome complex formation acutely and after sufficient time for initial cell-mediated remodeling. Elucidating the direct link between matrix integrity and mitochondrial function in MFS could pinpoint potential therapeutic targets to combat impaired energy metabolism and cellular function consequent to the disease.

A Fibronectin-binding Peptide that Enables Real-time Imaging and Biochemical Analysis of Extracellular Matrix

Aaron Hamlin, Henry Resnikoff, Isabella Strumolo

Department of Molecular Biology, Princeton University

ABSTRACT BODY: Fibronectin (FN) forms a foundational extracellular matrix (ECM) that plays essential roles in cell adhesion, migration, and differentiation and enables the deposition and organization of other ECM components. FN fibrillogenesis is a dynamic assembly process that depends on cell-FN and FN-FN interactions to yield a fibrillar network. Visualizing the formation of this FN matrix in live-cell systems would further our understanding of this process in both biological and pathological contexts but remains a technical challenge. Using phage display, we identified a peptide called S2 that efficiently binds to FN in a solid phase assay. This peptide specifically binds to an amino-terminal fragment of FN containing the assembly and collagen-binding domains but not to other regions of FN. S2-phage, when added to cultured fibroblasts, are incorporated into cell-assembled FN fibrils. We created a GST-S2-GFP fusion protein, which allows us to use fluorescence microscopy and protein biochemistry to examine the localization and quantities of the fusion protein in different cell fractions. We found that the intact fusion protein, but not a cleaved S2-GFP protein, is incorporated into FN fibrils in cell culture. Similar results were obtained with a GST-S2-GFP containing an additional protein moiety fused to its C-terminus, indicating that this fusion protein can be used to target other molecules to the ECM. GST-S2-GFP’s association with FN fibrils is stable, remaining bound throughout matrix decellularization procedures and during subsequent storage in PBS for at least 10 days. GST-S2-GFP is present in the detergent-insoluble FN matrix fraction and its incorporation increases in cells treated with TGFb, a known stimulator of FN matrix assembly. The stability of the S2-FN interaction may allow its use in identifying sites of FN accumulation in fibrosis. Because the S2 fusion protein can be incorporated as fibrils are formed, we were able to use it to visualize FN fibrils, and their cell-mediated formation and rearrangement in real time. As such, the S2 peptide is a tool that enables real-time visualization of FN matrix assembly, providing significant advantages for studying FN matrix assembly and cell-matrix interactions.

Collagen V-dependent Wound Healing: Microscale Stiffness Effect Through Traction and Adhesion Assembly

Sangyoon Han, Shaina P. Royer-Weeden, Sangyoon J. Han

Michigan Technological University

ABSTRACT BODY: Classical Ehlers-Danlos syndrome (cEDS) is a connective tissue disorder caused by a deficiency in collagen-V, leading to excessive tissue elasticity and poor wound healing. Collagen-V is known to modulate collagen fibril architecture, yet its biophysical role in regulating fibroblast behavior remains unclear. Here, we investigated how collagen-V alters traction force generation, adhesion dynamics, and migration patterns in fibroblasts.Using traction force microscopy, we observed that fibroblasts exerted significantly higher traction forces on collagen-V-coated substrates compared to collagen-I, despite identical substrate stiffness. This increase correlated with larger focal adhesion (FA) areas but lower FA density, suggesting that collagen-V promotes fewer but more mechanically robust adhesions. Adhesion turnover analysis further revealed accelerated assembly and disassembly kinetics on collagen-V, accompanied by a reduced nucleation ratio, indicative of enhanced contractility.Live-cell migration assays revealed distinct behavioral profiles: fibroblasts on collagen-I exhibited heterogeneous speeds, while those on collagen-V showed slower but more persistent movement. Although average speed and diffusion were greater on collagen-I, cells on collagen-V demonstrated increased directional persistence and a higher fraction of long-distance migration events (>200 μm in 24 hr), which may be critical for efficient wound closure.Unexpectedly, increasing substrate stiffness (12 kPa) on collagen-I did not enhance traction forces compared to softer gels (2.6 kPa) coated with either collagen-I or collagen-V, underscoring a nonlinear relationship between stiffness and matrix composition in regulating cell mechanics.These findings highlight the unique biophysical properties of collagen-V in enhancing adhesion strength and directional persistence, despite lower overall motility, offering new insights into the impaired wound healing observed in cEDS and the broader role of extracellular matrix composition in fibroblast mechanobiology.

Mechanical Overload Destabilizes F-actin Leading to Dysregulation of Chondrocyte Matrix Homeostasis

Marin Herrick, Mark Arranguez, Justin Parreno

University of Delaware

ABSTRACT BODY: Post-traumatic Osteoarthritis (PTOA) is a debilitating joint disease caused, in part, by aberrant mechanical overload onto affected cartilage surfaces following injury. While overload leads to articular cartilage degeneration, the underlying mechanotransduction signaling mechanisms by which overload alters chondrocyte homeostasis has not been fully elucidated. Previous studies using a surgical mouse PTOA model revealed the actin cytoskeleton to be one of the most down regulated pathways, coinciding with cortical filamentous (F-)actin loss. Alterations in F-actin have been linked to regulation of the chondrocyte phenotype; thus, mechanical overload-induced F-actin destabilization may play a pivotal role in cartilage matrix degeneration in PTOA. We aim to elucidate the relationship between disruptions in F-actin stability and chondrocyte homeostasis. We suspect that mechanical overload destabilizes cortical F-actin, which in turn dysregulates actin-mediated signaling and promotes chondrocyte dysfunction. As part of our investigation, we examine the regulation of cortical F-actin stability by a unique group of actin-stabilizing proteins, Tropomyosins (TPMs). We test the hypothesis that mechanical overload destabilizes cortical F-actin and cortical F-actin destabilization induces chondrocyte degeneration through modulating the activity of actin-associated transcription factors. We induced mechanical overload in two different ways: (1) by applying uniaxial strain on isolated chondrocytes seeded in two-dimensional monolayer on silicone substrates; and (2) by applying mechanical compression on native cartilage of osteochondral tissue. In both the 2D and 3D models, we observed a reduction of F-actin when applying strain &gt;25%. To examine the relationship between cortical F-actin loss and chondrocyte homeostasis, we destabilized cortical F-actin by inhibition of F-actin stabilizer, TPM3.1. We found that exposure of chondrocytes to TPM3.1 inhibitor, TR100, for 24 hours reduced cortical F-actin. In addition, TR100 reduced chondrogenic (Type II Collagen) and increased degradative enzyme (Matrix Metalloproteinase 3) mRNA levels. These alterations by TR100 were further confirmed at the protein level using WES capillary electrophoresis. Additionally, treatment with TR100 decreased nuclear staining for downstream actin-associated transcription factors: myocardin-related transcription factor-a (MRTF) and yes-associated protein (YAP). In a separate set of studies, we found that inhibition of MRTF or YAP specifically reduced Type II Collagen mRNA levels. Our results thus far support our hypothesis that overload leads to F-actin loss which is pivotal in inducing chondrocyte catabolism in part through downstream regulation of Type II Collagen by YAP and MRTF. Additional investigation aims to uncover the regulation of degradative enzyme mRNA levels by F-actin loss. A greater understanding on the regulation of F-actin stabilization by actin-binding Tpms may provide new insights into novel chondroprotective therapeutic targets against overload-induced OA.

Mapping Highly Compartmentalized Primary Fibroblasts Reveals Mitochondrial Dynamics are Governed by Cell-Matrix Interactions

Breanne Hewitt, Jacob Duggan, McKenzie Betts, Ryan Petrie

Drexel University Department of Biology

ABSTRACT BODY: Primary human fibroblasts readily migrate through homogenous 3D collagen matrices using low-pressure lamellipodial protrusions. Fibroblasts moving through covalently crosslinked cell-derived matrices (CDM) however, require substantial actomyosin contractility to help pull the nucleus through narrow openings in the matrix. This anteriorly concentrated contractility is connected to the nuclear envelope via the cytolinker protein plectin and vimentin filaments. As the nucleus is pulled forward it acts as a piston, pressurizing the anterior cytoplasm while maintaining a low-pressure zone in the rear of the cell. Thus, the nucleus separates the compartments and prevents the cytoplasm from freely moving between the front and back of the cell when the piston is active. Critically, it is not clear how organelles and endomembranes are distributed and exchanged between the two differentially pressurized cytoplasmic compartments. To address these questions, we first mapped and compared the subcellular organization of highly compartmentalized, pressurized fibroblasts in 3D CDM to uncompartmentalized, low-pressure fibroblasts on a 2D flat surface. In CDM we found the classical polarity machinery (PAR3/6) localized to the rear of the cell. In contrast, the position of the Golgi apparatus is not biased toward the direction of migration. Early endosomes and microtubules are enriched in the front, late endosomes are sequestered to the back, and lysosomes are clustered around the nucleus with no front-back bias. In contrast, all of these components were more evenly distributed in the cytoplasm of cells on 2D surfaces. Finally, we mapped the position of mitochondria in nuclear piston cells given their vital role in ATP production. When the piston is active, mitochondria are concentrated in front of the nucleus. Interestingly, the polarized mitochondrial networks were more fragmented in the perinuclear space compared to the leading edge. Imaging mitochondrial dynamics revealed the perinuclear mitochondria move much slower than the longer mitochondria at in the migratory protrusion. To test if activation of the nuclear piston triggered mitochondrial redistribution, we inhibited Rho-dependent kinase (ROCK) in cells migrating on 2D glass, 3D collagen, or 3D CDM. Upon ROCK inhibition, there is no change in the mitochondria in cells migrating on a 2D surface or in a collagen matrix however, in cells in CDM the polarized distribution of mitochondria is lost. Together, our results show there is a dramatic reorganization of the cytoplasmic components when the nuclear piston is active. The cytoplasmic organization of compartmentalized high-pressure fibroblasts differs greatly from low-pressure 2D cells. We speculate the polarized distribution of mitochondria is required to fuel the nuclear piston mechanism for effective migration through highly crosslinked matrices.

Collagen IV of Basement Membrane: Bromide-Mediated Sulfilimine Bonds Interlock the Quaternary Structure of NC1-Hexamer of Scaffolds Enabling Metazoan Evolution

Billy Hudson, Vadim Pedchenko, Tetyana Pedchenko, Monica Moran

Vanderbilt University Medical Center

ABSTRACT BODY: Collagen-IV (Col-IV) scaffolds, a primordial basement membrane component, enabled animal multicellularity, evolution and adaptation. These scaffolds provide tensile strength and tether macromolecules, forming supramolecular complexes that interact with cell-surface receptors and influence cell-behavior. Triple-helical Col-IV protomers, composed of three α-chains with a trimeric globular NC1-domain at the C-terminus, oligomerize, in the presence of chloride, forming a NC1-hexamer structure that connects adjoining protomers of Col-IVα121 and Col-IVα345 scaffolds. Up to six unique sulfilimine bonds (S=N) crosslink apposed methionine and hydroxylysine residues that covalently weld together the trimeric NC1-domains of the hexamer. The structural and biological functions of crosslinks remain obscure. We recently found evidence for a structural function whereby crosslinks, in the absence of chloride, stabilize the quaternary structure of the Col-IVα345 hexamer excised from the Col-IVα345 scaffold. Here, we extended our studies to the Col-IVα121 scaffold, which occurs ubiquitously across the animal kingdom. We found that the crosslink, independent of chloride, also stabilized the quaternary structure of the Col-IVα121 hexamer of bovine, mouse and a basal cnidarian, Nematostella vectinesis. Analyses of the NC1-hexamer crystal structure revealed the mechanism whereby sulfilimine bonds, assembled by peroxidasin and a bromide cofactor, fasten a clasp-motif forming a crosslink that interlocks the domain-swapping region of neighboring subunits of each NC1-trimer. The interlocked subunits reinforce hexamer quaternary structure, imposed by chloride conformational constraints. Collectively, our findings reveal that reinforcement of NC1-hexamer structure by covalent sulfilimine bonds is a critical event in scaffold assembly, enabling metazoan evolution. A knowledge of assembly mechanisms and function of scaffolds is prerequisite for development of therapies for collagen-IV diseases.

Uneven Light Scattering in Human Vitreous Correlates with Uneven Collagen Fiber Distribution in Mouse Models

Eileen Hwang¹, Denise J. Morgan¹, Brittany Coats²

¹Department of Ophthalmology & Visual Sciences, ²University of Utah

Department of Mechanical Engineering, University of Utah

ABSTRACT BODY: Background: Vitreous abnormalities have been associated with vision loss due to retinal detachment in aging and Stickler syndrome. Clinical examination of the vitreous in aging people and patients with Stickler syndrome reveals uneven light scattering, but the biochemical explanation is unknown. We hypothesized that uneven light scattering reflects an uneven distribution of collagen fibers, which could influence vitreous gel stiffness, force transmission to the retina, and retinal detachment.Methods: To investigate vitreous collagen fiber distribution, we used ex vivo mouse eyes. Our lab pioneered the use of confocal reflectance to image the vitreous, allowing imaging in a minimally perturbed state compared to prior vitreous imaging methods. We used 6-month-old wild-type C57BL6/J mice to model middle-age in humans, and compared them to 6-week-old mice. To model Stickler syndrome, we used 6-month-old Col9a1-/- knockout mice with signs of Stickler syndrome in the joints and ears.Results: We evaluated vitreous collagen fiber distribution in n = 8 (4 male, 4 female) Col9a1-/- knockout mice and n = 10 (3 male, 7 female) wild-type mice. There was greater unevenness in the collagen fiber distribution - a greater density of collagen fibers near the lens (0.86 +/- 0.39 um total fiber length/um2 area in knockout mice vs 0.41 +/- 0.21 um total fiber length/um2 area in the control mice, p = 0.007), and a trend towards lower density of collagen fibers near the retina (fraction of pixels occupied by fibers: 0.057 +/- 0.038 in knockout mice compared to 0.088 +/- 0.046 in wild-type mice, p = 0.21).We compared the vitreous collagen fiber distribution in middle-aged wild-type mice (n=10; 5 males, 5 females) and young wild-type mice (n=8; 5 males, 3 females). We observed clumping of fibers and empty spaces without fibers in the middle-aged mice. To quantify this, we divided each image into 16 tiles, counted the number of fibers in each tile, and calculated the coefficient of variation as a measure of unevenness within an image. Coefficient of variation was greater in the middle-aged mice (0.67 +/- 0.21) compared to young mice (0.34 +/- 0.09), p = 0.0008.Conclusions: Vitreous collagen fibers are distributed more unevenly in mouse models of aging and Stickler syndrome, indicating that uneven light scattering seen in humans with aging and Stickler syndrome are likely due to uneven collagen fiber distribution. We expect that uneven fiber distribution may cause uneven gel stiffness and eventually lead to retinal detachment.

Mapping Decellularization Process Conditions to Proteomic Profile and Cellular Function: Towards a Tunable Placental dECM Bioink

Dylan Jesner¹, Ryan Felix¹, Pieper Holeman¹, Peter Hu²

¹Fischell Department of Bioengineering, ²University of Maryland

Department of Anesthesiology, University of Maryland School of Medicine

ABSTRACT BODY: Major challenges within tissue engineering are the development of biomaterials that recapitulate niche tissue microenvironments and promote functional cellular activity and the vascularization of engineered tissue constructs for the delivery of nutrients. Tissue-derived decellularized extracellular matrix (dECM), comprised of endogenous structural proteins, glycosaminoglycans (GAGs) and signaling factors, has shown promise at producing physiologically relevant microenvironment. Presently, xenogeneic dECM is the most abundantly utilized due to availability, however, it is limited in translational use due to concerns of immune response to xenogeneic material. Human dECM would be more desirable for clinical translation, yet it is generally difficult to source at scales for biomaterial production, and it is poorly characterized as a biomaterial. Placenta is an excellent candidate for dECM production, as it is a commonly collected large organ typically discarded as medical waste; placental pro-angiogenic compounds (VEGF, sulfated GAGs) may also help vascularization of tissue constructs. Placental dECM has demonstrated increased vascularization and proliferation of fibroblasts in wound healing in vitro models, as well as increasing rate of wound healing in vivo.While the various applications of placental dECM are promising, the decellularization process has yet to be rigorously optimized. Decellularization is a multi-stage process that uses multiple agents in series, which must be optimized for the end-use of the dECM. The process design has not yet been mapped to dECM material character, resulting in time dedicated to reperforming process optimization or adoption of protocols developed for dECM with a different end-use. Agents commonly considered include detergents, acids/bases, enzymes, and mechanical disruption methods. Decellularization methods are selected based on the tissue source and the intended use of the dECM. Detergents vary in their potency, with more effective cell lysis coming at the cost of matrix disruption, exemplified by two common detergents: sodium dodecyl sulfate (SDS) is able to penetrate the matrix and thus denatures solubilized proteins while being the superior cell lysis detergent, and Triton X-100, which has minimal effect on matrix integrity, is generally ineffective at fully removing cellular material.This work aims to evaluate a series of decellularization protocols to characterize the process and map process components to material character metrics. We will consider 5 detergents (SDS+Triton X-100, Sodium Deoxycholate (SD), CHAPS, Triton X-100), freeze-thaw cycles, osmotic pressure, and exposure time to detergents. The resulting dECM will be screened by protein yield via a BSA assay and by DNA content via a PicoGreen assay. The samples will then be characterized in greater depth using Q Exactive Mass Spectrometry, the 1,9 dimethylmethylene blue (DMMB) assay, and the metabolic activity of fibroblasts cultured on the placental dECM substrate. The former two measurements will analyze the protein and GAG total yield, respectively, as well as elucidating the proteomic profile of the placental dECM product. The cell viability-based measurement will allow direct correlation of process and dECM makeup to cellular metabolic activity. After optimization of the decellularization process, the parameter sets which were most effective will be optimized for use in a bioink for an extrusion bioprinting application. A heatmap will be produced visualizing Spearman Rank-Ordered Correlation of the process parameters and characterization metrics, to guide understanding of the causal relationships in decellularization. We hope that this map can be used to accelerate future work in tissue engineering by providing a roadmap to selecting decellularization process conditions for various applications of placental tissue.

Whole Organ Ocular Lens Culture to Investigate the Regulation of Myofibroblast Differentiation by the Actin Cytoskeleton Ex Vivo

Rylee King, Grace Emin, Christian Le, Justin Parreno

University of Delaware

ABSTRACT BODY: During lens fibrosis, epithelial cells differentiate into myofibroblasts in the process of epithelial-to-mesenchymal transition (EMT). In early EMT, there is a loss of filamentous (F-)actin in the cell cortex and at cell-cell junctions, accompanied by an increase in F-actin rich stress fibers. Contractility is further enhanced by the presence of αSMA in F-actin stress fibers that exert force on the surrounding extracellular matrix, leading to matrix stiffening that could further exacerbate EMT. While F-actin reorganization is known to occur in early EMT, there remains a limited understanding on actin associated molecules required for F-actin reorganization and whether molecules can be targeted to suppress EMT progression. To determine the potential actin associated molecules involved in early lens EMT, we curated a list of 50 actin associated molecules known to be involved with stress fiber formation. We evaluated the expression of these molecules in an in vivo surgical model of lens fibrosis using a publicly available RNA sequencing dataset (LIRTS). We found that 43 molecules were upregulated within 48 hours of EMT. Within 6 hours of surgery, we found 12 stress fiber-associated molecules to be upregulated. The top upregulated gene was Transgelin (Tagln), an actin bundling protein thought to be critical for stress fiber formation. To examine the regulation of stress fiber formation and EMT by Tagln, we developed an ex vivo culture model to study lens EMT progression. By ex vivo culture, we observed an upregulation of Tagln within 6 hours of incubation, consistent with the findings in the ex vivo cataract model. Concomitantly, ex vivo culture led to stress fiber formation. We found that TAGLN co-localized with F-actin in stress fibers. Next, we examined stress fiber formation and EMT progression in a TAGLN1 mouse. We found that TAGLN knockout delayed the formation of stress fibers F-actin regulators may be important in driving EMT and myofibroblast differentiation. The development of new ex vivo models will enable a better understanding of these mechanisms and could lead to the development of new strategies to target stress fiber formation and slow fibrosis.

Investigating Progesterone Resistance and Fibrogenesis in Endometriosis Using Mechanosensitive and Long-Term Ex Vivo Lesion Platforms

Danielle Klunk, Daniel Abebayehu

University of Virginia

ABSTRACT BODY: Endometriosis is an inflammatory disease affecting ~10% of women characterized by the presence of fibroblast-rich endometrial lesions outside the uterus. These lesions vary widely in size, color, and location, yet maintain common characteristics of elevated inflammation, fibrogenesis, and progesterone resistance. Despite its prevalence, the etiology of endometriosis remains poorly understood due to its heterogeneity and the lack of robust human-relevant models. Current in vitro models have been able to replicate stromal and epithelial compartments, but lose native, patient-specific structures as well as more complex immune compartments. Additionally, the acquisition of progesterone resistance- a hormone critical to regulating the endometrial environment through stromal cell differentiation (decidualization), anti-inflammatory signaling, and controlled matrix remodeling- remains a key mechanistic gap in the understanding of endometriosis. Within this work, we show preliminary data supporting that ectopic stiffnesses and extracellular matrix (ECM) changes alter progesterone responses and introduce a hydrogel ex vivo model to extend these findings to human tissue.In this work, primary uterine fibroblasts (UFs) were cultured on polyacrylamide gels to mimic physiological stiffnesses of eutopic (4kPa) and ectopic substrates (100kPa) coated with either 10mg/mL collagen type I or fibronectin. Cells were treated for 72 hours with a cocktail of 10nM E2, 0.5mM cAMP, and 1uM P4 to induce decidualization, and flow cytometry confirms changes in FOXO1, progesterone receptors (PGR), and phosphorylated protein kinase B (pAkt) expression to determine differentiation. We next developed a matrix metalloproteinase (MMP)-degradable PEG hydrogel functionalized with RGD peptides to long-term culture of human endometriosis lesion explants. To confirm biocompatibility of the gel, UFs were added to hydrogels during crosslinking and seeded on 96-well plates. At 24- and 72-hour timepoints, a Live/Dead cell imaging kit (Thermofisher) was used to compare viability to 2-dimensional culture. Fluorescent imaging (Leica) was performed after 15-minute incubation to determine percent viability for each timepoint and condition (gel or no gel). This same method was used on precision-cut lesions from surgical samples embedded in PEG hydrogels. Explants were cultured for up to 7 days, with viability assessed at time points of 24h, 48h, 72h, and 7d. Retention of stromal, epithelial, and immune compartments will be assessed via immunofluorescence.Our results show that stiffness affects responses to both ECM proteins and progesterone. On 4kPa soft gels, cells seeded on collagen and fibronectin-coated hydrogels responded positively to progesterone treatment, as marked by increase FOXO1, PGR, and pAkt. However, cells on fibronectin show stunted responses, with significant decreases in PGR when compared to collagen. Meanwhile, cells on stiffer gels lose responsiveness to both progesterone treatment and ECM proteins, suggesting that stiffnesses rather than ECM composition of ectopic locations may contribute to acquired resistance in lesions. The PEG explant model exhibits biocompatibility and prolonged explant viability when compared to free-floating controls. UFs when cultured in hydrogel showed no significant differences when compared to traditional 2-dimensional culture, allowing us to apply this model to patient tissues. Viability assessed immediately after acquiring tissue slices showed moderate loss, with tissues averaging to only 70% viable. Free-floating tissue cultures continue to lose viability, dropping to 20% after 24 hours which is maintained across the 7-day timepoint. In contrast, PEG-embedded hydrogels maintain 70% viability across the 7-days. While morphology has yet to be assessed at this time, these results indicate that embedded lesions could be a promising route to investigating patient and lesion-specific patterns to treatments across several days.

Blood-Brain Barrier Model Using Extracellular Matrix for Evaluating Nanoplastic Toxicity

Mako Kobayashi¹, Yuji Zhao¹, Naoto Washihira¹, Sho Fujii²

¹Department of Materials Processing, Graduate School of Engineering, Tohoku University, ²Department of Science, Faculty of Science, Yamagata University

ABSTRACT BODY: Introduction:Nanoplastics (NPs) have been shown to cross biological barriers such as the blood-brain barrier (BBB), potentially affecting the central nervous system (1). Although the exact impact of NPs on BBB integrity is not yet fully understood, studying these effects is important for evaluating their neurotoxic potential. In vitro BBB models are essential tools for such investigations, but conventional models based on collagen alone often lack the complexity of the native brain extracellular matrix, which can limit their physiological relevance. Decellularized extracellular matrix (dECM), which is derived from biological tissues by removing cellular components while preserving key components such as laminin, glycosaminoglycans, and growth factors, offers a more biomimetic environment to support endothelial cell function and barrier formation. In this study, we developed a collagen/dECM hybrid BBB model to better mimic the in vivo microenvironment and examined its suitability for assessing the effects of NPs on BBB integrity.Materials and Methods:Decellularized extracellular matrix (dECM) was prepared from porcine aorta and brain tissues by applying high hydrostatic pressure treatment to remove cellular components while preserving the extracellular matrix. The tissues were then freeze-dried, pulverized, and solubilized in a pepsin–hydrochloric acid solution. The resulting dECM solution was pH-adjusted and mixed with 1 wt% collagen solution at a volume ratio of 1:3 on ice. This collagen/dECM hybrid gel was applied to Transwell inserts and incubated at 37°C for 24 hours to form a coating on the membrane. Human brain pericytes were seeded on the underside of the Transwell membrane, while human brain microvascular endothelial cells (hCMEC/D3) were cultured on the upper side. The effect of dECM on blood-brain barrier (BBB) maturation was assessed by monitoring endothelial cell morphology via phase-contrast microscopy, measuring transendothelial electrical resistance (TEER), and evaluating tight junction formation through ZO-1 immunofluorescence, Claudin-5 Western blotting, and permeability assays using FITC-dextran and sodium fluorescein (Na-F). Nanoplastic (NP) model particles were generated by applying advanced oxidation processes (AOP) to low-density polyethylene (LDPE), simulating environmental degradation. The particles were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), and dynamic light scattering (DLS). The impact of NP exposure on BBB integrity was then evaluated using the developed BBB model.Results and Discussion: TEER measurements demonstrated that the incorporation of dECM into the collagen matrix (collagen + d-Aorta and collagen + d-Brain) led to a substantial increase in barrier integrity at 72 hours post-seeding compared to collagen-only or uncoated models. Immunofluorescence staining for ZO-1 showed clear localization of tight junctions, and endothelial cells exhibited more uniform monolayer coverage on dECM-containing membranes. Western blot analysis further confirmed up-regulation of Claudin-5 in the presence of dECM. These findings indicate that dECM provides essential biochemical cues that support rapid and robust BBB maturation.To investigate their biological impact, the dECM-based BBB model was exposed to NPs measuring 50–200 nm and showing signs of surface oxidation. A notable decrease in TEER values was observed following exposure, indicating a loss of barrier integrity. This was accompanied by reduced ZO-1 expression as seen in immunostaining, suggesting disruption of tight junctions. Taken together, these results highlight the utility of dECM-based tissue-engineered BBB models as a robust platform for evaluating NP-induced biological effects on barrier integrity.Reference:(1) Cell Reports 2023: 42, 112346

Effects of Macrophage Polarization on Aneurysmal Smooth Muscle Cell Behavior in an in vitro Non-Contact Coculture System

Taylor Krajewski, Anand Ramamurthi

Lehigh University

ABSTRACT BODY: Abdominal Aortic Aneurysms (AAA) are localized, rupture-prone expansions of the aorta that involve progressive enzymatic degradation of elastic fibers in the media layer of the aorta wall resulting in loss of vessel elasticity1. Ongoing research in our lab seeks to develop regenerative platforms to restore the integrity of elastic matrix within AAAs since these extracellular matrix (ECM) components do not repair in adults. When screening regenerative therapeutics on cells associated with AAAs, i.e. aneurysmal smooth muscle cells (SMCs), SMCs dedifferentiate into fibroblast-like phenotypes when cultured long term2. Thus, their responses to therapeutics tend to not be evocative of their responses in native tissue, therefore slowing down the therapeutic’s path to clinical application. Cell-cell communication through secreted molecules and membrane receptors has been shown to have a drastic impact on essential cell processes3. With this knowledge, we have developed an in vitro coculture system using both aneurysmal SMCs and polarized macrophages that presents more physiologically relevant environment. This culture method will allow us to understand the impact that macrophage polarization has on aneurysmal SMC behavior and, in future experiments, provide a screening process for AAA-specific therapeutics that better translates to in vivo and clinical application.To generate this coculture system, human monocytes were differentiated into unpolarized macrophages (M0) using phorbol 12-myristate 13-acetate (PMA) followed by polarization into pro-inflammatory (M1) or reparative (M2) phenotypes using LPS + IFN-γ or IL-4, respectively. Successful polarization was determined using IF staining and flow cytometry both immediately following polarization and 7 days post-polarization. Macrophages were seeded on transwell inserts and added to SMC cultures to create a non-contact coculture system. After 5 days culture, supernatant and cell lysates were collected for analyses including BCA assay, cytokine array, and PCR. Macrophage differentiation was verified via cellular adhesion to culture surface seen after 24 hours of exposure to PMA. Through IF and flow cytometry, it was determined that in the M0 group ~49% of cells were positive for CD11b marker; in the M1 group, ~53% of cells were positive for CD86 (a known M1 marker); and in the M2 group, ~3% of cells were positive for CD206 (a known M2 marker). When cultured for an addition 7 days post polarization, phenotypic differentiation was maintained at ~71% in M1 cells but decreased to control levels in M2 cells. A BCA assay on SMC lysates post coculture showed that SMCs exposed to any macrophage group trended to decrease in population with the largest decrease seen in SMC-M1 cocultures. Cytokine arrays conducted on coculture supernatant showed increased expression of secreted proteins associated with ECM breakdown (MMP1, TIMP-1) and immune cell infiltration (CCL5, CXCL10, IL-8) as well as a decrease in TIMP-2, an inhibitor of ECM degradation from MMPs. Larger differences were seen in samples from SMC-M1 cocultures than in SMC-M2 cocultures when compared to SMC-only controls. PCR analysis of SMC lysates post coculture showed a trend towards increased MMP gene expression, especially MMP9, when SMCs were exposed to all macrophage groups. A decrease in COL3A1 gene expression was also seen in SMCs exposed to all macrophage groups. Ongoing studies aim to investigate a broader array of gene expression, observe functional differences in experimental groups (migration and contraction), and analyze proteomic expression.We have successfully differentiated human macrophages from monocytes and generated 3 distinct polarizations: unpolarized, pro-inflammatory, and reparative. We have shown that polarization is maintained after 7 days with no additional cytokine stimulation for M1 macrophages. From the coculture experiments, we can confirm that the addition of polarized macrophages affects SMC behavior towards ECM degradation and enhanced immune response. These changes are also seen in AAA; therefore, this culture system can provide a more physiologically relevant environment for future testing. In future experiments, we will be comparing the drug response of SMCs with and without the addition of macrophages. Specifically, we will analyze differences in SMC tropoelastin synthesis when treated with a previously studied pro-elastic matrix regenerative drug: afatinib4.1. Sakalihasan, N. Lancet, 2005. 2. Chang, S. PLoS One, 2014. 3. Su, J. Sig Transduct Target Ther, 2024. 4. Dayal, S. Tissue Eng Part A, 2024.

CCN3-Derived Peptide BLR-200 Attenuates Bleomycin-Induced Fibrosis by Preventing Activation of Engrailed-1/COL8A1-Expressing Universal Fibroblasts

Andrew Leask¹, Alex Peidl², Richard J Stratton³, M Nadeem Aslam⁴

¹University of Saskatchewan, ²Western University, ³University College London, ⁴University of Michigan

ABSTRACT BODY: Background:Scleroderma is an autoimmune connective tissue disease characterized by fibrosis of the skin and internal organs. We have shown that this fibrosis is induced/maintained by an autocrine proadhesive signaling loop mediated by YAP and the YAP target CCN2. The CCN family of matricellular proteins have subtle pro-adhesive effects in vitro. Collagen-lineage (universal) fibroblast-specific expression of CCN2 is required for fibrosis in variety of in vivo models. Anti-CCN2 therapies have failed clinically, likely due to functional redundancy in the CCN family. Conversely, the CCN family member CCN3 is inherently antifibrotic. We have identified a CCN3-based peptide that mimics the antifibrotic action of CCN3. We are developing this peptide, BLR-200, as our lead drug. If BLR-200 has antifibrotic properties in models of scleroderma, and its mechanism of action is unclear.Methods: Functional screens were used to initially identify BLR-200. We used the bleomycin-induced model of scleroderma, lineage tracing, spatial transcriptomics, proteomic and RNAseq/scRNAseq analyses to evaluate the anti-fibrotic properties of BLR-200. Results: In vitro, BLR-200 impairs, but does not block adhesion of primary human foreskin fibroblasts to type I collagen. In vivo, BLR-200 blocks bleomycin-induced increases in skin thickness, matrix production and myofibroblast differentiation. BLR-200 acts by impairing the activation of the pro-fibrotic engrailed/collagen8A1-positive subset of collagen-lineage universal fibroblasts and promoting the induction of a pro-regenerative engrailed/sfrp2-positive subset. This activity is accompanied by reduced expression of focal adhesion, wnt, oxidative phosphorylation and matrix-associated clusters, and ï ¡-smooth muscle actin, Smad3, YAP1, PLOD2, tenascin-C, CCN1 and CCN2 mRNA expression. Discussion/conclusion: BLR-200 is the only drug in development that targets both CCN1 and CCN2. BLR-200 could be a novel treatment to block scleroderma skin fibrosis.

Nascent Extracellular Glycoprotein Profiling During Critical Lung Stages: A Nascent Glycoproteome Atlas for Studying Lung Rejuvenation

Qingyang Li¹, Qingyang Li¹, Brian L Frey², Kentaro Noda³

¹Department of Biomedical Engineering, Carnegie Mellon University,

²Department of Chemistry, University of Wisconsin, ³Division of Lung Transplant and Lung Failure, Department of Cardiothoracic Surgery, University of Pittsburgh

ABSTRACT BODY: Lung is the organ responsible for gas exchange, and its aging is an inevitable physiological process, which is accompanied by gradual functional decline and decreased regeneration capacity. Thorough understanding of the dynamic changes in the extracellular matrix (ECM) and cell-ECM interactions during lung development, regeneration and aging will help us to better leverage the molecular mechanisms of those processes to boost the study of lung regeneration and rejuvenation. In this study, we present an innovative strategy to chemoselectively label and identify newly synthesized ECM (newsECM) during different physiological stages in mouse and rat lungs by in vivo administration of N-azido-acetylgalactosamine tetraacetyl (Ac4GalNAz) for two to three days, which can incorporate the azide-taged monosaccharide onto newsECM via glycosylation. The extracellular proteins were subsequently enriched with stepwise extraction, and azide-tagged newsECM were purified by affinity pulldown and analyzed with liquid chromatography coupled tandem mass spectrometry (LC-MS/MS). Comparative analysis of the newsECM in aged and young lung tissues revealed increased synthesis of 80 proteins and decreased synthesis of 256 proteins in aged tissues. Notably, we observed increased synthesis of typical age-related proteins in aged lung tissue. Furthermore, our approach was able to detect differentially synthesized proteins that were previously undetectable using traditional bulk proteomics, perhaps due to low abundance or background signal interference. We will continue to compare the newsECM of adult lung, neonatal lung, and post-pneumonectomy alveolar formation in order to identify key extracellular proteins that can be used as biomarkers or therapeutic targets to enhance alveolar regeneration. In summary, our data set of newsECM provides a valuable resource for elucidating the molecular mechanisms controlling lung development, regeneration, and aging, and may inform future strategies to promote lung regeneration.

Decorin Promotes Nascent Proteoglycan Retention in Cartilage Matrix by Strengthening Collagen II-Aggrecan Integration

Thomas Li, Gabriela Canziani, Yuchen Liu, Neil Patel

Drexel University

ABSTRACT BODY: Introduction: Osteoarthritis (OA), the most prevalent musculoskeletal disease, is hallmarked by the irreversible breakdown of articular cartilage. Regenerative strategies aim to restore the extracellular matrix (ECM) of cartilage, a hydrated composite primarily composed of a type II collagen fibrillar network and the large proteoglycan aggrecan. However, the molecular regulation of cartilage ECM assembly, turnover, and degeneration remains poorly understood. Our recent findings suggest that decorin may play a crucial role in mediating the assembly and turnover of ECM in both normal and diseased states. This study thus aims to investigate the mechanisms by which decorin regulates the turnover of ECM constituents and its potential as a therapeutic target in cartilage degeneration. Methods: Cartilage explants from 3-week-old wild-type (WT) and decorin-null (Dcn−/−) littermates were cultured and metabolically labeled with azide-modified analogs to track nascent GAG and protein synthesis. Click-labeling with DBCO-488 was used to visualize incorporation, followed by inflammatory IL-1β stimulation with or without exogenous decorin added at defined timepoints. Confocal imaging and custom-built MATLAB imaging analysis were applied to track the spatial distributions of nascent GAGs and proteins. Fluorescent signals in explants and media were assessed to quantify the retention of nascent GAGs/proteins. Surface plasmon resonance (SPR) was applied to quantify the binding kinetics and affinity among decorin, aggrecan and collagen II, with kinetic parameters predicted by ClampXP. Finally, AFM-nanoindentation was applied to quantify the effects of decorin on the modulus of cartilage collagen II fibril networks. Bulk RNA sequencing was applied to knee cartilage of 3-month-old mice to assess changes in transcriptional profiles. Results: Click-labeling and spatial mapping showed that loss of decorin led to accelerated release of nascent GAGs under both normal and IL-1β conditions, without altering their preferred localization in the pericellular matrix. In contrast, loss of decorin did not significantly affect the retention of nascent proteins. These results suggest that decorin is crucial in stabilizing the nascent aggrecan, but not collagen II, during cartilage matrix turnover. Conversely, supplementing WT cartilage explants with exogenous decorin significantly attenuated IL-1β-induced nascent GAG loss, supporting a therapeutic potential of decorin. Molecular assays elucidated the biophysical mechanisms by which decorin regulates cartilage matrix. SPR showed similar binding affinities among decorin-aggrecan, decorin-collagen II, aggrecan-collagen II and aggrecan-aggrecan. When supplemented as a coupler, decorin enhanced the binding affinities of aggrecan-collagen II and aggrecan-aggrecan by 2-3 folds, suggesting that decorin forms supramolecular networks to augment collagen II-aggrecan integration. Additionally, AFM-nanoindentation showed that infiltration of decorin stiffened the collagen II fibril network of WT cartilage, indicating that decorin reinforces the confinement effect of collagen II network that resists the osmotic pressure-driven diffusive loss of aggrecan. In contrast, RNA-seq revealed no significant gene expression changes in Dcn−/− chondrocytes compared to WT, supporting the primary role of decorin is to regulate matrix integrity, rather than cell signaling. Conclusions: This study highlights a crucial role of decorin in regulating cartilage matrix turnover and integrity. In cartilage, decorin increases the retention of nascent aggrecan under both normal and degenerative conditions by augmenting the integration of aggrecan and collagen, and reinforcing the collagen II fibril network. In turn, a supraphysiologic level of decorin delays the loss of nascent aggrecan from degrading cartilage, supporting decorin as a molecular therapeutic for preserving degenerating cartilage.

Mouse Alport Podocytes Are Susceptible to AAV9 Transduction in Vivo

Meei-Hua Lin¹, Kohei Omachi^1,2, Joshua F. Begin¹, Jennifer L. Richardson¹

¹Washington University School of Medicine, ²RIKEN Center for Biosystems Dynamics Research (BDR)

ABSTRACT BODY: Alport syndrome features a defective glomerular basement membrane (GBM) due to variants in COL4A3, COL4A4, and COL4A5. The most severe forms, which lack the GBM’s collagen α3α4α5(IV) network, progress from hematuria in early childhood to proteinuria, chronic kidney disease, and kidney failure by the age of ~30. As a monogenic disease without specific treatments, and with podocytes being the only glomerular cells that synthesize collagen α3α4α5(IV), the ability to efficiently deliver genes to Alport podocytes could open up new possibilities for treatment. As a proof-of-concept study, we investigated whether podocytes in mouse models of X-linked Alport syndrome were susceptible to transduction by adeno-associated virus (AAV)9. AAV9 delivered intravenously to control mice failed to transduce podocytes, but heterozygous female and hemizygous male Alport mouse podocytes showed a range of transduction from 1.8% to as high as 33% when urinary albumin/creatinine ratios were greater than 0.59 mg/mg. We conclude that the Alport GBM becomes leaky to AAV9, allowing it to reach and transduce a substantial subset of podocytes. Interestingly, mesangial cells of control and young Alport mice were moderately susceptible to transduction, demonstrating that gene delivery to mesangial cells in mice is a viable approach to investigate mesangial cell biology.

LC3/GABARAP-independent Autophagy of Misfolded Type I Procollagen in Mouse Osteoblasts

Elena Makareeva¹, Shakib Omari¹, Anna M. Roberts-Pilgrim¹, Laura Gorrell^1,2, Bella Radant^1,3, Muthulakshmi Sellamani¹, Edward L. Mertz¹, Basma Khoury⁴, Kenneth Kozloff⁴, and Sergey Leikin¹

¹Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, Bethesda, MD, USA, ²Rensselaer Polytechnic Institute, Troy, NY, USA, ³Marshall University, Huntington, WV, USA, ⁴University of Michigan, Ann Arbor, MI, USA

ABSTRACT BODY: Type I procollagen is a precursor of type I collagen, a large triple helical protein forming the structural scaffold of bone, tendons, and other tissues. Because of challenges to properly fold the triple helix at body temperature, over 10% of type I procollagen is misfolded and rerouted from the endoplasmic reticulum (ER) to lysosomes for degradation. The process of cargo delivery to lysosomes for removing misfolded proteins and damaged organelles is called autophagy and plays a key role in maintaining cell homeostasis. Secretory cargo like procollagen can be rerouted from the ER to lysosomes via two distinct autophagic pathways referred to as micro- and macro-autophagy. In ER microautophagy (microreticulophagy), lysosomes engulf ER regions akin to phagocytes. In ER macroautophagy (macroreticulophagy. aka ER-phagy), ER regions containing the cargo are surrounded by phagophore membranes, fragmented, isolated inside autophagosomes, and delivered to lysosomes upon autophagosome-lysosome fusion.Bone synthesis is expected to be dependent on autophagy, but osteoblast-specific macroautophagy knockouts in mice have produced only mild bone defects. To reconcile these observations, we compared how hypomorphic expression and a conditional knockout (cKO) of ATG5 – a protein required for autophagosome formation and therefore ER macroautophagy – affected Col1a2G610C /+ versus wild-type Col1a2+/+ osteoblasts in vivo and in vitro. The Gly610-to-Cys substitution (G610C) in the triple helical region of the COL1A2/α2(I) chain in type I procollagen increases procollagen misfolding, causing its accumulation in the ER, cellular stress, and osteoblast malfunction. Because autophagy reroutes misfolded procollagen from the ER to lysosomes, disruption of procollagen autophagy should significantly increase osteoblast malfunction and bone pathology in Col1a2G610C/+ mice. Nonetheless, we found only minor effects of the atg5 cKO on osteoblast function and bone formation in the Col1a2G610C/+ mice, like in Col1a2+/+ controls. The atg5 cKO did not reduce the autophagy flux of misfolded G610C or wild-type procollagen in primary osteoblast cultures, even though the LC3 and GABARAP lipidation required for autophagosome formation and ER macroautophagy was disrupted. Procollagen autophagy flux measurement and imaging of fluorescently tagged procollagen, SEC31, LC3, and LAMP1 in cultures of primary cKO osteoblasts demonstrated that procollagen was efficiently delivered to lysosomes without LC3 via ER exit site (ERES) microautophagy. These observations indicate that LC3/GABARAP-independent ERES microautophagy is the primary pathway of misfolded procollagen degradation in osteoblasts both in culture and in vivo. In conclusion, macroautophagy cKO affects bone by disrupting osteoblast metabolism and transition to osteocytes without inhibiting procollagen recycling. Beyond addressing the relative roles of micro- and macro-autophagy in osteoblast function and bone pathology, our findings support recent evidence for ERES microautophagy as a crucial protein quality control and recycling pathway in highly secretory cells, like osteoblasts.

Zone-Specific Tuning of Hyaluronic Acid-Based Hydrogels to Generate Bioengineered Cartilage with Native-Like Zonal Properties

Thomas Manzoni, Aditya Natu, John Caputo, Yinzhi Fang

University of Delaware

ABSTRACT BODY: Forming mechanically competent tissues that resemble the structure and function of native articular cartilage remains a critical challenge in developing functional bioengineered repair cartilage. Native cartilage owes its compressibility and low-friction surface to its depth-dependent zonal architecture. Deep zone chondrocytes (DZC) produce Collagen type X (COLX), essential for transmitting compressive loads to subchondral bone, while superficial zone chondrocytes (SZC) secrete lubricating proteins such as Proteoglycan-4 (PRG4). The bulk matrix is rich in Aggrecan (ACAN) and Collagen type II (COL2), with ACAN most concentrated in the middle and deep zones, contributing to biomechanical integrity. While passaged chondrocytes are clinically approved, they lose chondrogenic and zone-specific expression during expansion, resulting in repair tissue lacking appropriate matrix composition. Our prior work shows that passaged full-thickness (FT) and zonal chondrocytes can be redifferentiated to express chondrogenic and zone-specific markers. However, a persistent limitation lies in the inability to recapitulate native zonal cellular organization, hindering restoration of zone-specific functional properties in engineered cartilage. To address this, we utilized a novel hyaluronic acid (HA)-based hydrogel platform designed to support spatiotemporal control over matrix properties, enabling precise recreation of zone-specific microenvironments during 3D cell culture. This system leverages bioorthogonal tetrazine ligation with slow and fast-reacting dienophiles: norbornene (Nb) and trans-cyclooctene (TCO). This strategy allows sequential hydrogel tuning, without harming cells, to mimic the mechanics and microenvironment of distinct cartilage zones. Here, we test the hypothesis that tunable HA-based hydrogels can be used as an artificial matrix to generate zone-specific microenvironmental cues to produce bioengineered cartilage with zonal properties. Tetrazine-modified HA (HA-Tz), a norbornene-tagged degradable crosslinker (SMR-bisNb), and a TCO-conjugated adhesive peptide (RGD-TCO) were synthesized. Separately, FT chondrocytes were harvested to model deep and middle zones, while SZC were isolated to model the superficial zone, enabling targeted evaluation of zone-specific redifferentiation and matrix deposition. After two passages, chondrocytes were homogeneously mixed with the hydrogel precursors to generate 3D constructs and cultured in chondrogenic media supplemented with TGFβ3. Initially, FT chondrocytes were encapsulated in soft hydrogels formed via slow TZ/Nb crosslinking at a molar ratio of 2/1, allowing uniform seeding and early matrix development. Immunostaining after 21 days revealed robust accumulation of ACAN and COL2, with minimal Collagen type I. To promote the establishment of the deeper zone, the cellular construct was stiffened using a bisTCO crosslinker via interfacial bioorthogonal crosslinking. The dynamic stiffening significantly enhanced the deposition of chondrogenic matrix proteins and increased the expression of deep and middle zone-specific markers, COLX and Cartilage Intermediate Layer Protein (CILP), respectively. Similar experiments were conducted using SZC to identify optimal conditions for superficial zone generation. Notably, the softest, non-stiffened condition had the highest PRG4 accumulation after 21 days, consistent with a superficial zone phenotype. Ultimately, in vitro stiffening enhanced COLX and CILP expression, while soft, unstiffened hydrogels promoted PRG4 production by SZC. Our ongoing work integrates these zone-specific approaches into a unified, spatially patterned hydrogel scaffold, where region-specific cues directing localized chondrocyte redifferentiation. This platform enables in vitro recreation of cartilage zonal architecture within a single construct and offers a promising strategy to engineer cartilage with native-like structure and function for cartilage repair.

Investigating the Role of Extracellular CCN1 in the Dysregulation of Hematopoietic Lineage Commitment in the Aging Bone Marrow Niche

Piper Wilburn ¹, Wendy Deras ², Lindsay Wathen ², Melissa Kacena ¹, Maegan Capitano ², Milos Marinkovic ¹

¹Department of Orthopaedic Surgery, Indiana University School of Medicine and Richard L. Roudebush VA Medical Center, Indianapolis, Indiana, Department of Microbiology & Immunology, Indiana University School of Medicine

ABSTRACT BODY: Declining hematopoietic function in aging adults is the most significant risk factor for hematologic malignancies such as acute myeloid leukemia (AML). The bone marrow (BM) niche, comprising mesenchymal stem cells (MSCs), cytokines, and the extracellular matrix (ECM), plays a central role in regulating hematopoiesis. Aging perturbs this regulatory network, contributing to functional decline of hematopoietic stem and progenitor cells (HSPCs) and promoting a lineage bias toward myelopoiesis at the expense of lymphopoiesis, a hallmark of pre-malignant hematopoietic aging. Leveraging advances in ECM-based modeling of the BM microenvironment, our studies show that ECM produced by elderly human BM stromal cells is enriched in adipogenic cues and depleted in osteogenic signals critical for HSPC support. In particular, we identified that CCN1, a matricellular component highly expressed in young ECM and required for osteogenic MSC function, is absent from aged ECM. Restoration of CCN1 in elderly ECM improves MSC phenotype, increases expression of hematopoietic-supportive factors such as Runx2, suppresses PPARγ, and partially reverses the myeloid bias observed in aging-associated hematopoiesis. Critically, our recent findings indicate that ECM-derived CCN1 also regulates MSC-mediated support of HSPCs and mitigates age-related myeloid skewing. Using matched human stromal cells and HSPCs from human umbilical cord blood, we show that aging-related ECM remodeling effects expression of hematopoietic regulators, HSPC differentiation bias, and colony formation. Finally, we show that targeted recovery of specific matrix components (e.g., CCN1) to the elderly ECM, rescues the function of cultured MSCs and HSPCs. We conclude that age-associated remodeling of the BM matrix promotes malignant hematopoiesis and may suggest ECM-targeted strategies to prevent or delay myeloid malignancies in the elderly.

Mast Cell Chymase contributes to ECM Remodeling in Thoracic Aortic Aneurysm in Marfan syndrome

Daniel Martin¹, Sumit Bhutada¹, Frank Cikach², Emidio Germano da Silva²

¹Department of Biomedical Engineering, Cleveland Clinic Research,

²Department of Thoracic and Cardiovascular Surgery, Cleveland Clinic Heart and Vascular Institute

ABSTRACT BODY: Dysregulated proteolytic remodeling is implicated in thoracic aortic aneurysms (TAAs) but proteolytic landscapes (degradomes) of aneurysmal and normal aorta, and their mechanisms are undefined. Although proteome-wide analyses of aortic tissue are available, the mechanisms of matrix degrading enzymes in aortic remodeling is lacking. The objective of this work was to define the major proteases present in TAAs, their substrate attribution and precise cleavage sites. For this purpose, the mass spectrometry-based N-terminomics strategy Terminal Amine Isotopic Labeling of Substrates (TAILS) was applied to Marfan syndrome TAAs (n=5) and non-diseased aorta (TAs, n=4) as a forward degradomics application to define substrate and protease degradomes. The secreted serine protease mast cell chymase (CMA1) was specifically investigated due to its selective detection in TAAs and cleavage sites generated by CMA1 were resolved using reverse degradomics, i.e., digestion of aortic tissue with this protease, followed by TAILS. Cleavage of the proteoglycans BGN, PRELP, HSPG2, OGN, and VCAN by CMA1 was further investigated using Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS). Our approach experimentally annotated 3,210 proteolytically derived peptides (substrate degradome) and 124 proteases (protease degradome) in the aorta (TAA and donor combined). Reverse degradomics elucidated &gt; 150 CMA1 cleavage sites in 77 substrates (38 ECM proteins), of which, many occurred in the disease degradomes. ATOMS validated proteolysis of 10 aortic proteoglycans confirming for the first time the substrate affinity and cleavage site location, many of which matched those in the aortic disease degradome. In conclusion, mast cell derived CMA1 is active in aortic remodeling in Marfan syndrome, and cleaved peptides from CMA1 digested substrates offer potential biomarkers for monitoring aortic remodeling.

Remodeling of The Maternal Mitral Valve During Pregnancy: A Recapitulation of Fetal Development?

Meghan Martin¹, Olivia Day², Sarah Wells², Joshua Hutcheson¹

¹Florida International University, ²Dalhouise University

ABSTRACT BODY: In the mammalian heart, the mitral valve is found separating the left atrium from the left ventricle and is responsible for ensuring unidirectional blood flow. These structures are composed predominantly of ECM such as fibrous collagen, elastin and glycosaminoglycans. Remarkably, pregnancy induces adaptive maternal mitral valve remodeling such as increased leaflet area, changes in ECM composition with subsequent changes to mechanical properties and remarkably, an increase in chordae attachments. The only other time during life that these positive adaptations are observed is during fetal development. The fetal and maternal mitral valves are developing and remodeling at the same time, in the same closed system, just on either side of the placenta. Therefore, it is hypothesized that the adaptive positive remodeling observed in the maternal valve is simply a reactivation of fetal developmental mechanisms. Further, it is unknown whether these changes reverse in the postpartum period. The goal of this study was to compare anatomical and matrix remodeling of the mitral valve during pregnancy and fetal development as well as examine the reversibility of these changes postpartum. We examined the anatomical, structural, and compositional changes that occur simultaneously in the maternal and developing fetal mitral valve during pregnancy, gestation, and postpartum using a bovine model. Hearts were obtained from a local abattoir, and pregnancy/gestational duration were calculated from fetal crown-rump length. Hearts from previously pregnant cattle were also collected and defined as the postpartum group. Leaflet area was measured and number of chordae tendineae attachments were counted. Sircol™ Soluble and Insoluble Collagen assays determined the concentration of immature and mature fibrous collagen content respectively. Collagen crimp wavelength (distance between each peak) was visualized using picrosirius red staining and polarized light microscopy. Finally, elastin content was measured using the Fastin™ Elastin assay. Current work is being done to establish a murine model of this adaptive remodeling and if available data will also be presented alongside the bovine model. Leaflet area and chordae attachments increased in both maternal valves and fetal valves during gestation. Strikingly, leaflet area did not regress back to pre-pregnancy levels postpartum. In both the maternal and fetal valves, insoluble collagen accumulated faster than leaflets enlarged, resulting in increased concentrations of mature collagen. Paradoxically, the amount of immature collagen remained unchanged in both maternal and fetal leaflets, suggesting that collagen crosslinking is upregulated over pregnancy/gestation. Collagen crimp wavelength was lost in early pregnancy before being re-established at a greater wavelength in late gestation but increased continuously in the fetal valves – suggesting that initial passive tissue stretch during pregnancy may trigger this remodeling. Elastin content increased in fetal leaflets throughout development but was decreased in maternal leaflets throughout pregnancy. However, postpartum elastin content was restored to similar levels as pre-pregnancy while collagen content was unchanged. Observed together, these observations suggest that maternal mitral valve remodeling involves reactivation of developmental mechanisms. Initial passive stretch may serve to trigger the remodeling events in pregnancy, which mirror what occurs in the fetal mitral valve. Further, the maternal leaflet size does not reverse back to pre-pregnancy levels and is accompanied by a second remodeling event where elastin content is increased - potentially to increase elasticity in the larger maternal leaflets. Understanding the physiological mechanisms of this adaptive regeneration could contribute to the development of alternative treatments for pathological remodeling whereas, currently the only treatment for severe valve disease is total replacement.

Defining Matricellular Spatial Heterogeneity in the Maturing Abdominal Adhesion

Alexander Mathewson¹, Matthew Byrne², Abigail Loszko², Nicole M. Wilson²

¹Department of Pathology & Laboratory Medicine, University of Rochester Medical Center, ²Department of Surgery, University of Rochester Medical Center

ABSTRACT BODY: Abdominal adhesions are matrix-dense bands of tissue that form following damage to the peritoneum, affecting over 79% of abdominal surgery patients and resulting in an annual clinical burden exceeding $1.3 billion. Adhesiolysis (surgical removal) is the only treatment available for adhesion-related complications, but reoperation comes with a high risk of morbidity and recurrent adhesion formation, highlighting the critical need for novel therapeutic interventions. Mesothelial cells (MCs) and sub-mesothelial fibroblasts activate in response to peritoneal damage and play a key role in restoring homeostasis by migrating to the wound site to deposit extracellular matrix (ECM) and reestablish the mesothelial barrier. However, persistence of these mechanisms results in the formation of mature adhesions, containing regions of both dense connective tissue and of loose, highly vascularized matrix, with the mechanisms that facilitate adhesion maturation and expansion being poorly defined. To identify spatial alterations in fibroblast gene expression that drive post-surgical adhesion heterogeneity, we generated and analyzed high resolution Visium HD spatial transcriptomic data from a cecal abrasion mouse model of visceral-to-parietal adhesion formation across the adhesiogenic timeline (post-operative days (POD) 1, 3, 5, and 7). Myeloid cells, defined by Lyz2 and S100a8/9, are the predominant cell type in the adhesive interface at POD 1 and 3, and are accompanied by a small population of Eln positive, Sfrp2 positive fibroblasts. Monocle3 pseudotime analysis shows that the Eln+/Sfrp2+ population expands by POD5 and results in a subset of fibroblasts that defines the adhesion periphery. These peripheral fibroblasts begin expressing the matricellular proteins Periostin (Postn), Thrombospondin 2 (Thbs2), and Tenascin-C (Tnc) as early as POD5, with the population increasing in size by POD7. In addition to matricellular signaling, Cellchat analysis predicts that the Postn+/Tnc+ population plays a central role in pro-fibrotic cell-cell communication, serving as the primary sender in TGFb, MIF, and PDGF signaling networks. We propose that the establishment of a matricellular-rich microenvironment at the adhesion periphery maintains fibroblast activation and adhesion expansion and prevents resolution of the peritoneal wound healing response. As such, targeted disruption of this matricellular signaling may provide an avenue for the prevention or amelioration of abdominal adhesions.

Single Cell Spatial Profiling of the Matrisome in Malignant Brain Cancer

Joseph McCarty, Arpan De, Santiago Forero, Ali Pirani

MD Anderson Cancer Center

ABSTRACT BODY: The human brain contains a rich milieu of extracellular matrix (ECM) components that promote normal physiology and are often dysregulated in pathologies including the malignant cancer glioblastoma (GBM). Here, we have used in situ single-cell spatial transcriptomic platforms to map the expression patterns of nearly 400 ECM-related genes in multiple GBM samples (IDH1 wild type). Our analysis identifies at least four different GBM cell populations with unique ECM expression profiles that show spatial enrichment in distinct intratumor regions. Spatial mapping also demonstrates largely non-overlapping expression signatures of various ECM components in stromal cell types, particularly in GBM vascular endothelial cells and reactive microglia/macrophages. Comparisons of GBM versus lower-grade II and III astrocytoma samples (IDH1 R132H) identifies differential expression of key ECM components, including elevated levels of select ECM glycoproteins (IGFBP2 and MGP) and ECM-affiliated proteins (ANXA1 and ANXA2). In addition, we detect spatially enriched expression of COL8A1 (collagen), LUM (proteoglycan), and POSTN (ECM glycoprotein) in perivascular stromal cells in GBM but not in lower grade tumors. Computational analysis of putative ligand-receptor interactions reveals novel communication networks between cancer cells and stromal components, particularly in regions of GBM microvascular proliferation and pseudopalisading necrosis. In summary, this comprehensive spatial map provides new insights into ECM control of tumor initiation and progression and identifies potential therapeutic targets within the GBM microenvironment.

Regulation of the Extracellular Matrix by Hyperglycemia May Contribute to Behavioral Dysregulation

Adriana Mendez¹, Mohammad Jodeiri Farshbaf², Jake Tetenman³, Zim Kahn⁴

¹Department of Neuroscience, Icahn School of Medicine at Mount Sinai,

²Department of Psychiatry, Icahn School of Medicine at Mount Sinai, ³Washington University,

⁴Hunter College

ABSTRACT BODY: Diabetes is highly comorbid with neuropsychiatric disorders such as depression. Yet, little research has been focused on understanding the biological mechanisms by which these two disorders are linked. The habenulo-interpeduncular pathway in the midbrain stands out as a promising circuit to study diabetes-induced changes to mood-related behavior because this circuit has been shown to regulate both blood glucose and behavior. In this study we aim to investigate how diabetes affects the organization and function of the extracellular matrix (ECM) in the habenulo-interpeduncular pathway to influence the development of mood-related neuropsychiatric disorders. Adult male mice were treated with either saline or streptozocin (50 mg/kg for 5 consecutive days), a pancreatic beta cell toxin that induces the development of hyperglycemia, a hallmark symptom of diabetes, in rodents. Building on evidence for the role of the medial habenula (mHb) in the expression of aversive memories, we found that hyperglycemic mice demonstrate impairments in both fear extinction and cue discrimination. Specifically, hyperglycemic mice were unable to extinguish aversive memories nor distinguish between a fear-associated cue and a neutral cue following cued fear conditioning. Consistent with the known role of the mHb in reward processing and aversion, we also found that hyperglycemic mice display an anhedonia-like phenotype in a two-bottle choice task where mice were given the option of water or saccharin, a zero-calorie sweetener. To further understand how hyperglycemia may be altering the function of the mHb to promote these behavioral phenotypes, we conducted targeted purification of polysomal mRNA (TRAP-Seq) in cholinergic neurons of the mHb to examine changes to the expression of mRNA in hyperglycemic and normoglycemic mice. We found that several pathways involved in the regulation of the ECM including activation of matrix metalloproteinases, collagen biosynthesis, and ECM organization are upregulated in mice with diabetes. Specifically, we found that the expression of MMP-2 and MMP-9 were amongst the genes that were most significantly altered in the hyperglycemic mice. Following these findings, we used biotinylated lectins to stain and visualize the ECM in the mHb and interpeduncular nucleus (IPN) of hyperglycemic male mice. We found that hyperglycemia induced changes to the structural organization of the ECM, with a significant reduction to the total area and length of the ECM in the interpeduncular nucleus (IPN), but not the medial habenula (mHb), following chronic hyperglycemia. In line with this observation, we found that there is a highly dynamic response to both acute and chronic hyperglycemia, as we observe a complete reversal in the expression levels of MMP-2 and MMP-9 when transitioning from acute to chronic hyperglycemia. Together, these findings identify the ECM as a possible mechanism by which diabetes is able to elicit changes to the central nervous system and influence the development of neuropsychiatric disorders. Understanding how diabetes interacts with the ECM to elicit changes in neuronal function will be imperative as cases of diabetes increase worldwide.

Recombinant Human Type I Collagen using CHO Cells

Kazunori Mizuno, Tomonori Ueno, Yuki Taga, Haruki Yokoyama

Nippi Research Institute of Biomatrix

ABSTRACT BODY: There is a growing demand for alternative testing methods to animal experiments in the development of pharmaceutical drugs. It remains challenging to replicate cell culture systems that accurately mimic in vivo tissues.Currently, collagen, one of the most abundant proteins in the human body, is sourced from animal tissues or several cell types. While it is possible to prepare collagen from cultured cells, the yield is typically low, making it difficult to obtain enough amount for cell culture experiments. Also, obtaining collagen with minimal lot-to-lot variation through pepsin treatment is difficult, even when derived from the same original tissue. We previously discovered that introducing the p180 (RRBP1) and Splicing Factor SF3b4 genes into CHO-S host cells significantly boosts their protein synthesis in the endoplasmic reticulum. We have applied this technology to recombinant protein synthesis using CHO cells. Utilizing this enhanced CHO cell protein synthesis system, we attempted to synthesize several types of collagen, including Type I collagen. We aimed to synthesize a heterotrimeric molecule comprising two α1 and one α2 chain. Through optimization of culture conditions, we successfully obtained Type I collagen molecules with post-translational modifications similar to those found in native tissues. The ratio of the α1 and α2 chain were analyzed by MS using stable isotope. This CHO-cell derived Type I collagen notably exhibits a significantly lower proportion of β-chains and higher-order cross-linked structures compared to its tissue-derived counterparts. Observations of temperature-dependent changes in the circular dichroism spectrum revealed that its denaturation temperature was also close to that of Type I collagen in native tissues. We plan to conduct further physicochemical and cell-based analyses of this CHO-cell derived Type I collagen.

DOE-Guided ECM Optimization Induces Colonic Fibroblast Quiescence In Vitro

Zahra Mohammadalizadeh¹, Kaithlin Fogg², Ana Maria Porras¹

¹University of Florida, ²Oregon State University

ABSTRACT BODY: Intestinal fibrosis is a frequent and clinically significant complication in Crohn’s disease (CD), yet the molecular mechanisms initiating and perpetuating this process remain incompletely understood. Fibroblasts, a key mesenchymal cell population in the intestinal wall, are central to ECM deposition and fibrosis and are activated by both inflammatory and matrix-derived cues. However, in vitro studies of fibroblast activation are hampered by the tendency of these cells to adopt an activated phenotype on stiff substrates such as tissue culture polystyrene (TCPS), which does not recapitulate the physiological microenvironment of the intestinal stroma. To address this, we aimed to develop a protocol for inducing quiescence in CCD-18 colonic fibroblasts through the use of natural ECM protein coatings. Prior studies suggest that ECM coatings and vitamin D supplementation can attenuate pro-fibrotic activation in fibroblasts from various tissues, but the optimal conditions for achieving a true quiescent state in intestinal fibroblasts remain undefined. Our preliminary experiments demonstrated that individual application of collagen I, collagen III, laminin, vitamin D, and fibroblast growth factor (FGF) could modestly modulate fibroblast activation, but none fully reverted cells to a resting state. Therefore, we employed a Box-Behnken design of experiments (DOE) to systematically investigate the combined effects of these five factors at three concentrations each, generating 42 unique experimental conditions. CCD-18co fibroblasts were cultured under these conditions for five days, and responses were assessed by quantifying α-SMA intensity, cell viability, proliferation, and fibronectin secretion. Our optimization strategy targeted minimal α-SMA, proliferation, and fibronectin secretion, with maximal viability as hallmarks of quiescence. DOE analysis revealed significant interactions between collagen III, laminin, vitamin D, and FGF in regulating α-SMA expression. Laminin exhibited a quadratic positive effect, collagen I a negative effect, and FGF a negative linear effect on α-SMA. Notably, the lowest α-SMA expression was achieved with higher collagen III and vitamin D, while minimum proliferation occurred at low concentrations of both, indicating that reduced proliferation alone is not a reliable marker of fibroblast quiescence. Validation experiments confirmed that optimizing for minimal proliferation did not yield a quiescent phenotype, as α-SMA remained high, whereas excluding proliferation from the optimization produced a robust reduction in α-SMA (to one third of control), maximized viability, and significantly decreased fibronectin secretion. These findings suggest an inverse relationship between α-SMA expression and proliferation in vitro and support literature indicating that proliferation is more closely linked to inflammatory signaling than to fibroblast activation. Furthermore, cells treated with the predicted protocol exhibited reduced expression of the fibroblast activation markers FAP and vinculin, confirming the development of a quiescent phenotype. Collectively, our DOE-driven approach enabled the identification of microenvironmental conditions that reliably induce a quiescent phenotype in colonic fibroblasts, providing a valuable in vitro model for dissecting the mechanisms of intestinal fibrosis and informing future anti-fibrotic strategies in CD.

Enhanced Collagen Purity Profiling of Commercial Collagens Using Liquid Chromatography Mass Spectrometry Based Proteomics

Jiyoung Moon, Aabha Shakari, Jamie Dale, Ashleigh Dale

School of Medical Sciences, Faculty of Medicine and Health, University of Sydney

ABSTRACT BODY: High-purity collagen is critical for the development of reliable biomaterials used in clinical and tissue engineering applications. Conventional quantification techniques such as SDS-PAGE and hydroxyproline-based assays are widely employed to assess collagen quality. However, these methods often lack the sensitivity and resolution required to detect low-abundance impurities or to verify the structural integrity of specific collagen subtypes. In this study, we expanded the application of liquid chromatography–mass spectrometry (LC-MS), technique known for its high sensitivity and specificity but traditionally reserved for final product quality control, to evaluate the purity of commercial collagen products. Commercial Collagen I and IV samples were dissolved in 0.1 M acetic acid, and protein identification and purity were evaluated using SDS-PAGE, Western blot, and LC-MS. Label-free quantification was performed using MaxQuant, Proteome Discoverer 2.3, and MASCOT software. iBAQ and riBAQ values were calculated to determine the relative abundance of total proteins and collagen subtypes. SDS-PAGE results showed subtype-specific bands for both Collagen I and IV. For Collagen I, LC-MS identified two major subunits: Collagen alpha-1(I) chain and (47.6%) Collagen alpha-2(I) chain (52.4%) , confirming the expected heterotrimeric composition. LC-MS analysis of Collagen IV revealed batch-to-batch variability in purity, with values of 68.6%, 73.9%, and 87%. Low-abundance proteins such as non-collagen ECM proteins, undetected by SDS-PAGE or Western blot, were clearly identified via LC–MS. Notably, collagen viscosity was positively correlated with purity, where higher purity collagen samples exhibited increased viscosity.To date, publicly available data on LC-MS-based analysis of commercial collagen products remains limited. Our findings demonstrate that LC-MS enables the detection of impurities not identified by SDS-PAGE or Western blot, offering a more precise and comprehensive assessment of collagen purity. Despite its complexity, LC-MS provides detailed molecular insights that support the selection of reliable biomaterials for clinical and bioengineering applications.

Quantitative Analysis of Elastin Disruption and Compensatory Matrix Remodeling in Abdominal Aortic Aneurysms

Francesca Morrell, Anand Ramamurthi

Lehigh University

ABSTRACT BODY: Abdominal aortic aneurysm (AAAs) represent a progressive pathology marked by chronic extracellular matrix (ECM) degradation and structural failure of the aorta wall. Central to this pathophysiology is the fragmentation and loss of elastin fibers in the medial layer. This breakdown is driven by chronic upregulation of matrix metalloproteinases (MMPs) locally in the aorta wall, besides disorganized compensatory collagen deposition. Naturally poor and aberrant elastic matrix regeneration and repair leads to biomechanical instability and eventual aortic rupture. Despite its central role in AAA pathophysiology and outcomes, methods for quantitative morphometric assessment of the elastic matrix in human AAA tissues remain underdeveloped. The proteolytic degradation of elastic fibers generates distinctive morphological changes that can be visualized and quantified through specialized histological techniques. To address critical gaps in understanding elastin’s structural deterioration patterns, we applied an image-based computational morphometry workflow developed in our laboratory to quantify structural parameters of elastin in paraffin-embedded human AAA tissue sections. Tissues were obtained from 31 patients undergoing open aneurysm repair with IRB approval. The modified Hart’s stain ( 1 volume of Wiegert’s iron resorcin fuchsin and 9 volume of 1% hydrochloric acid in 70% ethanol) proved essential for this analysis, as it selectively highlights elastin fibers in dark purple while providing excellent contrast against surrounding tissue components. This staining specificity is crucial for accurate automated image analysis and reliable morphometric quantification. Whole slide imaging was performed via Leica digital pathology systems, and comprehensive image analysis was conducted using ImagePro software. The Hart’s stain enabled precise pixel based selection of elastin deposits, allowing for automated object recognition and morphometric parameter computation across entire tissue sections. Morphometric metrics included elastin area, minimum and maximum fiber diameter, perimeter length, tortuosity, and aspect ratio computed across full-section regions of interest. Our analysis revealed significant correlations between patient demographics and elastin fiber characteristics. Male predominance (81%) and smoking history (84%) were associated with increased elastin fragmentation. Critically, patients with concurrent aortic dissection demonstrated significantly higher percent elastin area, indicating more extensive fiber fragmentation. Additionally, increased variability in maximum diameter and perimeter length suggested progressive structural compromise in the elastic matrix. These quantitative findings provide novel insights into stage-wise elastic fiber changes correlating with morphological and physiological aspects of AAAs. The morphometric approach enables prediction of elastic fiber fate and AAA rupture risk while identifying targeted outcomes for regenerative therapies. Our data demonstrate that elastin distribution follows predictable patterns linked to disease severity, with tortuosity changes reflecting biomechanical adaptation to wall stress redistribution. This study establishes morphometric analysis as a valuable tool for understanding ECM remodeling in AAA pathophysiology, bridging the gap between molecular mechanisms of elastin degradation and clinical manifestations. Future applications include development of elastin-targeted therapeutics and improved risk stratification based on matrix integrity assessment. FUNDING SOURCE (optional): NIH

Bioengineering Temporomandibular Joint (TMJ) in vitro models for cell-matrix and mechanobiology investigations

Priti Mulimani, Dakshina Acharya, David Reed

University of Illinois Chicago

ABSTRACT BODY: Mechanical loads regulate the homeostatic and allostatic signaling controlling temporomandibular joint (TMJ) heath and disease. Allostatic signaling is promoted through mechanical overloading, initiating both the remodeling and degeneration of the extracellular matrix (ECM) during arthropathies such as osteoarthritis (OA). Changes in the composition and organization of the ECM have a multifaceted role on cellular mechanotransduction, impacting the mechanical microenvironment surrounding the cell, the cytoskeletal architecture, and the transcription of key differentiation markers. There is an important gap in knowledge related to understanding how these cell-matrix derived signals control cell behaviours deterministic of TMJ pathobiology such as migration, proliferation, differentiation, and function. To test this knowledge gap, new in vitro model systems that recapitulate this higher-order organization of the healthy and diseased ECM are needed. Here we evaluate complementary 2-dimensional (2D) and 3-dimensional (3D) in vitro model systems with tuneable substrate stiffness and in-built mechanical loading functionality. To determine the impact of substrate stiffness on cytoskeletal remodelling and TMJ chondrocyte differentiation, we fabricated 2D substrates using a flexible polydimethyl siloxane (PDMS) made from a mixture of Sylgard-527 and Sylgard-184 on glass coverslips and cured at 65°C. Altering the percentage of the PDMS mixture changes the stiffness, yielding substrates with a 0.5, 7.7, or 16 kPa micromodulus. Substrates were then coated with 5ug/mL fibronectin and seeded with immortalized mouse mandibular fibrochondrocytes (iMFCs). After 24 hours, cells were fixed, stained for F-actin/DAPI, and imaged with immunofluorescent (Leica DMI6000 B, 40X). F-actin intensity and cytoskeletal fiber properties were quantified using FIJI image analysis software. For the 3D in vitro culture system, tissue constructs were fabricated containing iMFCs mixed with 4 mg/ml type I rat tail using the Mantarray system (Curi Bio). The resultant 3D constructs were then cultured with and without recombinant TGFβ1 at 10ng/mL. After 24 hours, the constructs were fixed, cryosectioned, stained with Alcian blue, Herovici polychrome, and Picrosirius Red, and imaged (Leica DMI2000; 5X and 20X). The 2D in vitro cell culture system demonstrates that the iMFCs are responsive to substrate stiffness, with the 7.7 kPa substrate resulting in an increase in F-actin intensity and upregulation of the TGFβ1 gene when compared to the 0.5 kPa or 16 kPa stiffness substrates (p<0.05). The 3D in vitro culture system resulted in a cell-collagen scaffold with viable cells embedded in a heterogeneous ECM. Following TGFβ1 treatment, this ECM was altered in proteoglycan content as measured by Alcian blue, collagen remodeling as measured through the Herovici Polychrome, and matrix organization as measured by picrosirius red circular polarizing microscopy. These data illustrate that TGFβ1stimulates changes in the higher-order organization and composition of the ECM. Together, these two complementary preclinical in vitro systems provide a pipeline permitting mechanistic interrogation of mechanical positive autoregulation of TGFβ1 in matrix and cytoskeletal remodeling and cell differentiation in TMJ cells. The long-term outcomes from this work will advance our understanding of how feedback and feedforward autoregulatory signaling loops initiate matrix remodeling during TMJ pathophysiology and advance the translational pipeline for new therapeutic interventions focused on the matrix.

Physiological Loading via Voluntary Wheel Running (VWR) maintains Tendon Homeostasis by Spatially-Distinct Cellular Processes

Samantha Muscat, Elsa Lecaj, Nolan Sparks, Anne E.C. Nichols

University of Rochester

ABSTRACT BODY: Tendons are composed of a unique and hierarchal network of collagen fibrils and other critical extracellular matrix components that allow these tissues to withstand extreme loads to enable successful locomotion within the musculoskeletal system. Previous work has shown that in response to physiological loading, tendon cells produce collagen and other important matrix proteins to maintain the tendon’s structural integrity and functional properties (i.e. homeostasis). However, the precise cellular processes that allow tendon cells to construct the tendon’s intricate extracellular matrix to maintain homeostasis in response to physiological load are unknown. In the wild, mice consistently run long distances, which suggest that normal cage activity in a laboratory setting limits their natural activity levels resulting in an underloaded Achilles tendon. Other models of loading such as forced treadmill running (FTR) induce a confounding stress response and cause tendon degeneration, which suggest that FTR does not model physiological load, but rather overuse. Therefore, in the present study, we evaluated voluntary wheel running (VWR) as a model of physiological load in mouse Achilles tendons to investigate how tendon cells maintain homeostasis in response to physiological load. VWR mice ran for 8 weeks (10 weeks old, male C57BL/6J, n=10) for a total of 854 ± 83.52 km, while underloaded control mice had wheels locked to prevent running. Functional changes were evaluated by uniaxial tensile testing, which assessed mechanical properties at the midsubstance. Tendons from VWR mice exhibited a ~17% increase in peak load (p=0.0178), a ~55% increase in peak stress (p=0.0009) and a ~44% increase in elastic modulus (p=0.0058), compared to underloaded controls. No significant differences were observed in cross sectional area or stiffness between VWR and underloaded control tendons, suggesting that there were no large structural changes or hypertrophy with physiological loading. These data suggest that VWR resulted in stronger tendons compared to underloaded controls. To investigate transcriptional changes underlying the increased mechanical properties observed at the midsubstance, bulk-RNA sequencing was performed. VWR Achilles tendons demonstrated a modest transcriptional response with 46 differentially expressed genes (22 down- and 24 upregulated), compared to underloaded controls. Genes upregulated with VWR include those with known roles in collagen remodeling (Lum [p=0.0028], Fap [p=0.0008], P4ha3 [p=0.0003]), suggesting that VWR promoted a matrix-producing, anabolic response. A collagen hybridizing peptide (CHP), which specifically binds to unfolded collagen chains, was then used to validate and assess the spatial location of collagen remodeling between VWR and underloaded control tendons. Significant collagen remodeling was observed in the calcaneal insertion of VWR tendons compared to underloaded controls. Interestingly, no collagen remodeling was present at the midsubstance of VWR tendons where mechanical properties were increased. This suggests that processes other than collagen remodeling were responsible for the increased mechanical properties at the midsubstance. Taken together, our data suggests that physiological loading via 8 weeks of VWR results in mechanically superior tendons compared to underloaded controls. Interestingly this appears to occur in a spatially-dependent manner as we observed increases in mechanical properties at the midsubstance, with significant collagen remodeling restricted to the calcaneal insertion region. Therefore, we hypothesize that physiological loading via VWR maintains tendon homeostasis by spatially-distinct cellular processes. To investigate these spatially-distinct cellular responses to physiological load we have leveraged spatial RNA-sequencing (Visium HD, 10X Genomics) which can define transcriptional signatures in native tissue location with single cell resolution. We are currently analyzing this data set to comprehensively assess the cellular contributions to tendon homeostasis in response to physiological load between the midsubstance and calcaneal insertion area. Ultimately, understanding the fundamental spatially-distinct cellular processes that allow tendons to maintain homeostasis in response to physiological load can help identify therapeutic targets to preserve tendon health.

Mapping the Collagen Landscape: Tissue-Specific Chain Expression and Modifications in Mice

Ayush Nigam^#1, Ashutosh Joshi^#1, Maxwell C McCabe², Kirk Hansen^*2, Trayambak Basak^*,1,

¹School of Biosciences and Bioengineering, IIT Mandi, Himachal Pradesh,

²School of Medicine, University of Colorado Anschutz Medical Campus

^*Corresponding authors ^#equal contributions

ABSTRACT BODY: Collagen, an abundant protein of the extracellular matrix (ECM), is essential for maintaining the structural integrity and function of all tissues. Collagen is decorated with numerous post-translational modifications (PTMs), including prolyl and lysyl hydroxylations, as well as lysyl-O-glycosylations. PTMs investigated included 4-hydroxyproline, 3-hydroxyproline, hydroxylysine (Hyl), galactosyl-Hyl, and glucosylgalactosyl-Hyl. However, the relative distribution of collagen chains and the variation of PTMs across different tissues remain unknown. In this study, we have evaluated the distribution of different collagens and respective PTMs in mouse tissues by analyzing LC-MS/MS data.

Aim: We aim to test our hypothesis that collagen PTMs are dynamic and highly tissue-specific. Therefore, LC-MS/MS data of sECM (soluble ECM) and iECM (insoluble ECM) across 25 mice organs were analyzed to identify and quantify site-specific collagen PTMs.

Methods: Using an in-house developed analytical pipeline, we profiled collagen chains and PTMs across 25 mouse organs (data from Hansen Lab). PTMs investigated included 4-hydroxyproline, 3-hydroxyproline, Hydroxylysine, galactosyl-Hyl, and glucosylgalactosyl-Hyl. We have used MyriMatch for database search (<1%FDR), and IDPicker for site-specific PTM identification. Furthermore, we identified and quantified the collagen PTMs in both soluble (sECM) and insoluble (iECM) ECM fractions across 12 tissues. followed by Skyline and ThermoFisher XCalibur for calculating the occupancies.

Results: We observed significant variation in the relative abundance of collagen chains across tissues. Based on the number of spectral counts from the database search (FDR less than 1%), we found 39 different collagen chains across 25 mouse tissues in both sECM and iECM. However, the top five abundant chains were COL1A1, COL3A1, COL1A2, COL4A2, and COL2A1, with COL1A1 being the predominant one. COL1A1 contained almost 15-20 3-Hyp PTM sites and Hyl PTM sites (~10-15), and some G-Hyl and GG-Hyl PTMs across tissues, though exceptions were observed in tissues such as the brain and liver. PTM analysis showed distinct occupancy patterns that differed across organs and collagen chains within a single organ. Our findings advance our knowledge of ECM dynamics in tissue-specific contexts, offering explanations for how collagen PTM variations contribute to adrenal cortex maintenance and function. We identified significant differences in collagen types and PTM signatures between the two fractions, suggesting functional specializations in the extracellular matrix (ECM). These results set the stage for subsequent research to explore the role of ECM and collagen modification in specific tissue-related diseases, offering potential biomarkers for tissue health and dysfunction. This study provides the first comprehensive analysis of collagen PTMs for 25 mouse tissues altogether.

Keywords:

Collagen, Post-Translational Modification, Extracellular Matrix, Mass Spectrometry

Stromal and Immune Subpopulations Differentiate Between Regenerative and Fibrotic Responses to Biomaterials

Jeremy Ortmann¹, Colleen Roosa¹, Donald Griffin¹,², Daniel Abebayehu¹

¹University of Virginia, Department of Biomedical Engineering, ²University of Virginia, Department of Chemical Engineering

ABSTRACT BODY: Tissue damage due to injury, disease, or congenital defect continues to pose critical clinical challenges to human health. Biomaterial-based therapies show promise to heal this damage, but their success depends on two crucial determinants: immune response and fibrosis. In tissue repair, inflammation and fibroblast activation are tightly controlled to guide remodeling. However, if the body rejects an implanted biomaterial, a fibrotic capsule forms around the implant. Investigating immune-stromal crosstalk can lead to the refinement or development of biomaterial therapies to avoid fibrotic outcomes. Thus, we examined cell subpopulations present in subcutaneously implanted biomaterials that either elicit a fibrotic or regenerative response and used single cell RNA sequencing (scRNA-seq) to identify the enriched fibroblast and immune cell subpopulations. We used microporous annealed particle (MAP) hydrogels that have been shown to evade fibrosis and enhance tissue regeneration, comparing this system to a traditional fibrotic bulk nanoporous (NP) hydrogel of the same chemistry. MAP and NP gels were fabricated from four-arm PEG-Maleimide with an RGD peptide and MMP-2 degradable crosslinker. After performing subcutaneous MAP and NP implantation in mice, implants were harvested at days 7 and 21, and cells were collected from the harvested implants via a Liberase digestion. Cell viability was confirmed to be >85% for each sample. The cells from each sample underwent automated single cell barcoding and library preparation using the 10X Genomics Chromium Platform and then underwent sequencing. With the gene-barcode matrices, we used PCA and UMAP dimensionality reduction along with clustering approaches to identify all cell subpopulations present at the implant site, focusing on immune and stromal cells. Subsequent analysis included fibroblast and macrophage sub-clustering, differential gene expression analysis, gene set enrichment analysis, and trajectory analysis. Our dimensionality reduction revealed several distinct subpopulations of cells, including macrophages, fibroblasts, mast cells, and T cells. However, the dominant cell populations in our dataset were fibroblasts and macrophages. Macrophage and fibroblast sub-clustering unveiled distinct subpopulations unique to our fibrotic and regenerative biomaterials. Among our regenerative MAP hydrogels, the fibroblast compartment at day 7. One fibroblast subpopulation was enriched for IL-11, and a second subpopulation was enriched in IL-11 receptor alpha. This points to a potential paracrine signaling axis among early fibroblasts that promotes regeneration. Macrophage populations upregulated in NP implants displayed increased gene expression related to immune defense; for example, one cluster expressed many genes triggered by interferon exposure, and another expressed high levels of receptors typically associated with natural killer cells. In contrast, one subpopulation of macrophages upregulated in MAP displayed genes related to mechanosensing, including binding receptors for collagen and cytoskeletal reorganization genes including SDC1. Macrophages have been shown to be mechanosensitive, and they can combine these mechanical cues with other signaling molecules to orchestrate reparative healing responses. These findings point to possible pathways to improve biomaterial design. Future work will involve deciphering cell-cell crosstalk using CellChat, validation via immunofluorescence, and analyzing upregulated pathways using knockout studies. Together, our findings will provide insight into the dynamic cell-cell relationships that influence either regeneration or fibrosis in biomaterial implants. In summary, our work revealed distinct macrophage and fibroblast subpopulations that point to important immune and stromal coordination for fostering regenerative outcomes of biomaterial implants.

Sequence Dependencies of Collagen Persistence Length Determined via All-atom Molecular Dynamics Simulation

George Pantelopulos, Robert B. Best

National Institutes of Health / NIDDK

ABSTRACT BODY: The mechanics of collagen fibers are often regarded as the principal determinant of tissue structure and mechanical function. Recent advances in Atomic Force Microscopy (AFM) and image analysis have made it possible to characterize the persistence length of subdomains of collagen triple helices. Using all-atom molecular dynamics simulation, we have characterized the local persistence length along the length of monomeric, full-length tropocollagen. Using these characterizations of local persistence length, we have parameterized predictive models for determining persistence length based on sequence

Macrophage Stromelysin-2 (MMP10) Moderates Lung Fibrosis

Tanyalak Parimon, Tyler C. Vandivort, Timothy P. Birkland, Ying Wang

Cedars-Sinai Medical Center

ABSTRACT BODY: Depending on their state of activation, macrophages can either promote or lessen tissue injury, inflammation, fibrosis, and scarring. Among the factors that control macrophage activation, MMP10 (stromelysin-2) functions in a cell–autonomous manner to promote their differentiation to alternatively activated (M2) cells and their ability to degrade collagenous scars in skin. Given these findings, we assessed whether MMP10 impacts acute lung injury, inflammation, and fibrosis in response to bleomycin toxicity. In human idiopathic pulmonary fibrosis, we found that MMP10 was prominently expressed by macrophages proximal to fibrotic areas. In mouse models, our data indicate that MMP10 serves beneficial functions in response to lung fibrosis. Intratracheal instillation of bleomycin mediated a robust increase in Mmp10 expression in the whole lung that was associated with increased mortality and morbidity (weight loss) in Mmp10–/– mice. Although endpoints of acute lung injury did not differ between wildtype and Mmp10–/– mice, macrophage influx and expression of several pro-inflammatory mediators, such as CCL2, were elevated in Mmp10–/– lungs compared to wildtype. In addition, collagen deposition was elevated in Mmp10–/– lungs; however, this phenotype was not accompanied by a parallel or consistent increase in endpoints of collagen production. Rather, we found reduced expression of MMP13, a macrophage-derived metallocollagenase, in Mmp10–/– mice but no difference in the expression of other putative collagenases. These results indicate that MMP10 serves a beneficial role in fibrosis by controlling the collagenolytic potential of macrophages.

Dual Roles of Syndecan-4 in Regulating Fibrosis

Mona Elisabeth Pedersen¹, Nina Therese Solberg¹, Marianne Lunde², Cathrine Rein Carlson²

¹Nofima AS, Raw Materials and Optimization, ²Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo

ABSTRACT BODY: Wooden Breast (WB) is a myopathy affecting the skeletal breast muscle (Pectoralis major) in broiler chickens and is characterized by muscle fiber damage and varying degrees of fibrosis, ECM remodeling and inflammation. Several key factors such as pro-inflammatory cytokines like TGF-β1 and IL-1β, drive fibrosis in WB myopathy. We have previously shown that the expression of syndecan-4 (SDC4), a transmembrane proteoglycan, was increased in WB poultry skeletal muscle tissue. Furthermore, the ectodomain shedding of SDC4 by matrix metalloproteinases (MMPs) differed in the skeletal muscle satellite cells from isolated affected chickens compared with normal. While SDC4 has been previously implicated as a key driver for regulating myofibroblast activity in mechanically induced fibrosis in cardiac tissue, its specific role and shedding activity in chicken fibroblasts in relation to WB myopathy remain poorly understood. In this study overexpression of SDC4 in vitro using chicken fibroblasts was used to mimic the previously detected increased SDC4 levels in WB, and to further investigate fibrotic markers and syndecans at the gene and protein levels. Furthermore, we used blocking peptides derived from the SDC4 ectodomain and investigated their effect on SDC4 shedding and fibrotic markers. Additionally, TGF-β1 treatment, the main trigger of myofibroblasts, fibrosis, and cytokines, was used to investigate the connection between SDC4 shedding and fibrosis. Our data showed that overexpression of SDC4 in chicken fibroblasts reduced the gene and protein expression of fibrotic markers such as collagen I, collagen III and MMP-2. At the same time, we observed an increase in the gene expression of TGFB1 and IL1B. SDC4 overexpression also modulated intracellular proteins connected to fibrosis-relevant signaling pathways, with increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt. Moreover, we could observe a decreased production of ribosomal protein S6 and β-catenin. SDC4 overexpression induced shedding of a 15 kDa SDC4 fragment, while a 20 kDa fragment was produced at similar level regardless of overexpression. Modulation of ectodomain shedding using blocking peptides targeting various ectodomain regions (amino acids 76-106 and 100-130) significantly reduced the production of the 20 kDa endogenous fragment. However, it did not affect the expression level of the 15 kDa fragment. This contrasts with what we have seen with the same blocking peptides in chicken skeletal muscle satellite cells where the expression level of the 15 kDa fragment was reduced. When we stimulated the cells with TGF-β1, an increase in gene expression of fibrotic marker was observed as expected. No change in SDC4 gene expression was observed, while SDC4 fragment of 20 kDa was increased. Taken together, our results reveal a complex role of SDC4 and shedding in modulating fibrotic responses in chicken fibroblasts, suggesting a potential dual function where the full-length SDC4, produced by overexpression, may act as an anti-fibrotic regulator, while the TGF-β1 induced shed ectodomain fragments could promote pro-fibrotic and inflammatory processes.

Vascular Ehlers-Danlos Syndrome Donor Cell Derived Matrix Elucidates Role of COL3A1 Mutations in ECM Mechanics

William Polacheck, Ryan N Stack, Madeline Singletary, Elizabeth Doherty

UNC Chapel Hill

ABSTRACT BODY: Vascular Ehlers-Danlos Syndrome (vEDS) is a rare disease caused by autosomal dominant mutations in the COL3A1 gene, which lead to a reduction of collagen III, a major component of the extracellular matrix (ECM) of blood vessel walls. Clinically, vEDS patients are susceptible to aneurysm and arterial rupture, and there are no FDA approved treatments and limited diagnostic and therapeutic approaches. The effects of reduced collagen III on overall ECM composition and mechanical properties of vessel ECM, and the response of cells resident in the vascular wall are not well characterized. To address this lack of understanding, we developed a protocol to generate fibrous, collagen-rich ECM (cell-derived-matrix, CDM) from donor-derived fibroblasts in vitro and have analyzed the biochemical and biophysical properties of the resulting CDM. We then generated CDM vEDS patient and healthy donors to characterize changes in composition, structure, and mechanical properties with respect to healthy cells. More recently, we have further adapted the protocol to generate hydrogels from donor-derived CDM, enabling, for the first time, vEDS patient-specific vascular organs-on-chip. We found that the class of COL3A1 mutation (i.e. heterozygous null vs. glycine substitution) has an impact on the rate of production, the Young’s Modulus, and the time-dependent mechanical properties of CDM. Specifically, we found that vEDS CDM is significantly different from healthy CDM in both its stiffness and relaxation response to applied stress, and that specific COL3A1 mutations contribute to these differences. We report on the mechanotransduction of these signals by vascular endothelial cells, resulting in decreased migration and wound healing responses, suggesting a possible early mechanism for aneurysm formation. Overall, these results show that collagen III plays a key role in ECM mechanical properties and suggest pathological matrix assembly and resulting mechanics may be critical drivers of vEDS progression.

Genomic Profiling and Functional Validation of Bacteroides Enzymes Driving Extracellular Matrix Degradation

Ana Maria Porras, Marcos Javith, Isabela Yamhure, Ana Maria Porras

University of Florida

ABSTRACT BODY: Intestinal homeostasis is contingent on the dynamic remodeling of the extracellular matrix (ECM). Likewise, gut homeostasis depends on the symbiotic relationship between the microbiota and its host. Gut microbiota dysbiosis has been associated with the exacerbation and progression of gastrointestinal diseases characterized by ECM remodeling, such as inflammatory bowel disease (IBD) and colorectal cancer. Nonetheless, the interactions between commensal gut microbes and host colonic ECM are poorly understood. The studies described here focus on the Bacteroides genus, a prominent bacterial genus in the human gut, with an emphasis on species whose abundances have correlations with IBD and colorectal cancer. Bacteroides genomes are known to encode an assortment of enzymes, including proteases and carbohydrate degrading enzymes (CAZymes). Thus, we hypothesized that members of the Bacteroides genus can alter the host microenvironment through enzymatic degradation of the ECM. To test our hypothesis, we delved into comparative functional genomics and substrate-based degradation assays in vitro. Through a bioinformatic analysis, the genomes of 14 Bacteroides species were annotated, the encoded enzymes were functionally classified and used to predict ECM-specific substrate interactions. Among all identified CAZymes families, glycoside hydrolases (GH) genes were the most frequently identified family among all species; meanwhile, glycotransferases (GT), carbohydrate-binding modules (CBM), and polysaccharide lyases (PL) were also identified at varying less frequent proportions among strains. Further, specific subfamilies within GT, CBM, and PL were predicted to interact with the following ECM components: chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronan, collagen, and decorin. Next, we will similarly profile the abundance of peptidase encoding genes and categorize the enzymes by their catalytic residue family: serine, cysteine, aspartic, or metallopeptidase. This future analysis will identify the potential peptidases capable of hydrolyzing ECM proteins, glycoproteins, and proteoglycans.Experimentally, we characterized the degradation of individual host ECM components by bacterial culture supernatants obtained from strains belonging to three Bacteroides species: B. fragilis, B. ovatus, and B. thetaiotaomicron. We observed preferential degradation of glycosaminoglycans (hyaluronic acid and chondroitin sulfate) by B. ovatus and B. thetaiotaomicron. Meanwhile, crude supernatants from four strains of B. fragilis degraded gelatin, collagen I, collagen IV, and elastin substrates. Sterile filtering of these B. fragilis supernatants eliminated their ability to degrade gelatin or collagen I. Thus, these results suggest that some of these proteases are secreted extracellularly, while others may remain bound to the bacterial membrane. Next, we sought to identify the specific protease classes responsible for the detected ECM degradation. We concluded that cysteine proteases, serine proteases, and metalloproteases were present after observing decreases in activity with the addition of select protease inhibitors. Lastly, we assessed the impact of pH fluctuations on ECM degradation ability. Changes in the pH during culture had a significant effect on elastin and glycosaminoglycan (chondroitin sulfate) degradation by B. fragilis or B. theta, respectively. These results are consistent with bacterial growth and behavior adaptations during environmental changes. Overall, we concluded that Bacteroides species encode and can produce enzymes capable of degrading multiple ECM glycosaminoglycans, proteins, and glycoproteins. Additionally, our results suggest that the synthesis of these enzymes is likely impacted by environmental conditions sensed during bacterial growth. Given the vast and diverse microbial community in the human gut, our work highlights critical microbe-ECM interactions that can impact tissue remodeling and host health.

Pseudoachondroplasia (MT-COMP) Mouse Model of Progressive Chronic Pain Associated with Matrix Abnormalities and Joint Degeneration

Karen Posey¹, Nicole Meyers¹, Jinbin Tian², Frankie Chiu³

¹Department of Orthopedic Surgery, McGovern Medical School

²Department of Integrative Biology and Pharmacology, ³McGovern Medical School

Department of Pediatrics, McGovern Medical School

ABSTRACT BODY: Background: Pseudoachondroplasia (PSACH), a disproportionate dwarfing condition, is caused by mutations in Cartilage Oligomeric Matrix Protein (COMP), a matricellular protein that contributes to cartilage health. PSACH is associated with lifelong joint pain, joint degeneration and laxity and requiring multiple early joint replacements to address joint pain. Joint pain in PSACH is the most substantial feature and it begins early and is a life-long issue. Understanding the pain and joint degeneration mechanisms is critical for developing effective therapies. This study examines pain, joint degeneration and tendon properties in a mouse model of PSACH (MT-COMP). Methods: Pressure Application Measurement (PAM) was used to evaluate hyperalgesia or increased sensitivity to pain. Conditioned Place Preference (CPP) was used to assess ongoing pain. Electrophysiological recordings from nociceptors isolated from ganglia associated with the hind limbs assayed hyperexcitability and spontaneous activity that can drive pain. Collagen Hybridizing Peptide (CHP) was used to determine whether there was subchondral bone collagen remodeling, associated with joint degeneration pain. Raman spectroscopy was used to assess matrix composition of tendons in C57 controls and MT-COMP mice. Results: Hyperalgesia was observed in MT-COMP mice at all ages evaluated (8, 12, 20 weeks) consistent with persistent inflammation and previous indirect pain assessments including grooming and voluntary running. Nociceptors from MT-COMP mice displayed increased spontaneous activity and hyperexcitability at 20 weeks but not at 9 weeks. Moreover, CPP analysis showed that MT-COMP mice spent more time in the chamber associated with pain relief than controls, indicating ongoing pain. Interestingly, CHP signal was elevated in MT-COMP subchondral bone at the knee joint at 20 and 36 weeks, suggesting remodeling consistent with mechanical stress and degeneration. Taken together these findings indicate that the MT-COMP mouse model is a valuable resource for studying PSACH pain. Raman spectroscopy showed increases in collagen crosslinking and significantly higher Advanced Glycation End Products (AGE) accumulation and matrix stiffening in the MT-COMP mice. Nevertheless, mechanical testing of MT-COMP tendons revealed increased compliance and stretching compared to controls, but failed at significantly lower force. These results suggest reduced mechanical strength despite biochemical stiffening, consistent with PSACH joint laxity. Conclusion: MT-COMP mice experience pain early in life, their pain progresses with age to become chronic, and joints become hypersensitive to mechanical stimuli. Collagen remodeling in MT-COMP mice is consistent with increased mechanical stress and joint degeneration, further supporting the presence of ongoing matrix damage and repair processes that may promote pain. The CHP results suggest that PSACH pathology extends beyond cartilage to include subchondral bone, that may be contributing to joint instability and pain. Raman spectroscopy is a powerful tool for non-invasive matrix assessment, revealing increased matrix stiffening and AGE accumulation in MT-COMP tendons. These biochemical changes, while indicative of stiffening, paradoxically coincide with increased tendon compliance and reduced mechanical strength, suggesting that disorganized collagen networks may underlie joint laxity in PSACH.

YAP/TAZ Transcriptional Co-factors Maintain Extracellular Matrix Homeostasis and Control Fibrotic Scar Formation in Mouse Dermal Fibroblasts

Taihao Quan, Alexandre Ermilov, Zhaoping Qin, Zhaolin Zhang

University of Michigan

ABSTRACT BODY: The collagen-rich dermal extracellular matrix (ECM) provides essential mechanical support and forms a microenvironment critical for dermal cell function. Dermal fibroblasts, the primary cells responsible for synthesizing and maintaining dermal ECM, play a central role in this process. In this study, we explore the roles of two highly homologous transcriptional co-factors, YAP and TAZ, in dermal ECM maturation during early postnatal development, post-wound scar formation, and the fibrotic response to the antineoplastic agent bleomycin. We generated conditional Cre-LoxP deletion to specifically ablate Yap/Taz in dermal fibroblasts. The deletion of Yap/Taz in dermal fibroblasts three days postnatally resulted in substantial reductions in dermal collagen fibril production, ECM deposition, and ECM organization. Both dermal collagen density and type I collagen gene expression were significantly decreased, by 67% (p< 0.035) and 88% (p< 0.001), respectively. Spatial RNA sequencing of the dermis revealed marked downregulation of major collagen genes, including COL1A1 (76% reduction, p< 0.0001), COL1A2 (73% reduction, p< 0.0001), and COL3A1 (58% reduction, p< 0.0004), alongside a negative enrichment of the Yap/Taz signaling pathway. Investigating fibrotic scar formation following incisional tail wounding, we found that YAP/TAZ knockout in fibroblasts significantly attenuated scar formation at 4, 8, and 12 weeks post-injury, with dermal thickness reduced by 50% (p<0.01) at 12 weeks compared to age- and sex-matched littermate controls. Similarly, in the bleomycin-induced skin fibrosis model, Yap/Taz knockout in fibroblasts significantly reduced fibrotic response (33% reduction, p<0.01). Collectively, our findings highlight the critical role of YAP/TAZ in fibroblasts in the regulation of dermal ECM homeostasis and fibrotic scar formation and suggest that the YAP/TAZ signaling pathway in fibroblasts represents a promising therapeutic target for the treatment of fibrotic skin disorders.

The Absent in Melanoma 2 (AIM2) Inflammasome is Required for Lamellipodia Formation and Fibroblast Migration

Ive-Anwuli Ralph-Uyalor, Carol M Artlett, Ryan Petrie

Drexel University

ABSTRACT BODY: Fibroblast migration is a vital component of wound healing, where fibroblasts move to the wound site and synthesize and incorporate matrix proteins such as collagen and fibronectin into the wound matrix. Primary human fibroblasts exhibit migratory plasticity when moving through different 3D environments. Specifically, tropomyosin 1.6 (Tpm 1.6) helps to activate the actomyosin machinery responsible for pulling the nucleus forward through the narrow openings in covalently crosslinked 3D ECMs, enabling dynamic switching of cells from low-pressure lamellipodia to high-pressure lobopodia-based migration. To determine how Tpm 1.6 controls migratory plasticity, we first identified Tpm 1.6 interacting proteins in human dermal fibroblasts. Our preliminary findings revealed an interaction between Tpm 1.6 and absent in melanoma 2 (AIM2), a cytosolic DNA sensor and an inflammasome-forming protein involved in wound healing and fibrosis. To investigate the potential role of AIM2 in cell migration, we generated AIM2-deficient mouse embryonic fibroblasts (MEFs). Live cell imaging revealed that AIM2-deficient MEFs crawled across 2D surfaces much slower than their wild-type counterparts. Morphological and cytoskeletal analysis of AIM2-KO MEFs revealed disrupted lamellipodia at the leading edge and reduced lamellipodial actin, suggesting a role for AIM2 in actin remodeling and cell migration. In human dermal fibroblasts migrating on 2D surfaces, a small amount of AIM2 was enriched at the cell’s leading edge, with the bulk of the protein targeted to the nuclear envelope and the perinuclear region, with occasionally filamentous distribution in the cytoplasm. In contrast, AIM2 became more uniformly distributed within the cytoplasm in both 3D collagen and cell-derived matrix (CDM), suggesting that AIM2 localization is highly responsive to the physical environment. Finally, to functionally evaluate AIM2’s role we transiently knocked down AIM2 in primary human fibroblasts and compared their 2D migration to control cells. Immunofluorescence staining with the lamellipodia marker cortactin revealed a significant reduction in lamellipodia formation in AIM2-deficient cells. Preliminary data suggest reduced motility and lamellipodia formation in fibroblasts with reduced AIM2 expression. Collectively, these findings show that AIM2 is responsive to the cell’s physical matrix environment and has an unexpected role governing the fibroblast migration. Future studies will explore how AIM2 mechanistically integrates physical cues to regulate migratory plasticity during tissue remodeling and fibrosis.

ABSTRACT TITLE: Multi-Omics Analysis of Matrisome Alterations in Glioblastoma

Shanmathi Ramasubramanian¹, Shanmathi Ramasubramanian¹, Sergei Shalygin², Sonali Sunsunwal², Aditya Mishra²

¹University of Georgia, ²Complex Carbohydrate Research Center UGA

ABSTRACT BODY: Glioblastoma (GBM) is the most aggressive form of brain cancer, with a low survival rate of 12 to 15 months after diagnosis and treatment. The therapeutic resistance and heterogeneity of GBM are primarily attributed to a highly specialized tumor microenvironment (TME). While the secreted factors and cellular composition of the GBM TME have been thoroughly characterized, the compositional heterogeneity and disease relevance of the GBM extracellular matrix (ECM), an essential component of the GBM TME, remains poorly defined. Understanding ECM adaptations is crucial because these changes directly influence tumor invasion, drug resistance, and tumor infiltration of immune cells. We hypothesized that characterizing the GBM ECM using a multi-omics approach will identify ECM adaptations that facilitate tumor progression and immune evasion. To test this hypothesis, we performed liquid chromatography-mass spectrometry (LC-MS) based proteomics analysis of mutationally distinct human GBM tumors (n = 13) and non-tumor brain cortex control tissues (n = 3). Results were compared to RNA-Seq data from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) public-domain data to validate differentially expressed human matrisome proteins and transcripts. Results indicated a significant upregulation of collagens and ECM glycoproteins along with other matrisome-associated proteins in GBM when compared to non-tumor brain cortex tissues. Network analysis revealed increased connectivity and enhanced positive correlations among matrisome proteins in GBM, suggesting coordinated matrisome remodeling in GBM tissues. Weighted gene co-expression network analysis (WGCNA) of transcriptomic data revealed functional clustering of glycosyltransferase, ECM glycoprotein, proteoglycan, and inflammatory/immune response transcripts. Taken together, these results suggest that ECM adaptations and glycosylation in GBM could potentially promote tumor progression and immune evasion. This study provides a template for developing integrated multi-omics approaches to identify pathologically relevant changes in the GBM matrisome composition and potential therapeutic targets that could help overcome current treatment limitations.

Metabolic Regulation of Stem Cell Fate through Modulation of the Extracellular Matrix

Zeenat Rashida, Abby Krall, William Tu, Lisa Kalies

University of California, Los Angeles

ABSTRACT BODY: The various functions of stem cells during development, regeneration and in homeostasis are determined by their trajectory towards differentiation, self-renewal, or quiescence, and disrupting these paths can lead to a variety of diseases. While transcription factors and chromatin regulators are known to regulate pluripotency and cell fate, increasing evidence shows that extrinsic factors like the nutrient environment and the extracellular matrix (ECM) can regulate stem cell functions. The ECM is a complex network of proteins that surrounds the cells, provides structural support and transduces mechanical and biochemical signals to influence cell behavior, including proliferation, migration, and differentiation. Despite its importance, the role of ECM in regulating stem cell function remains inadequately explored. Our work aims to address this gap by investigating how nutrient-driven changes in metabolism influence ECM composition and stem cell reprogramming. Specifically, we focus on Vitamin C (VitC), an essential nutrient that enhances pluripotency induction and is commonly used in iPSC culture media. Using metabolomic approaches, we show that VitC rapidly enhances glucose flux towards Pentose phosphate pathway (PPP) and Hexosamine biosynthesis pathway (HBP) early in reprogramming. Further, VitC addition leads to depletion of intracellular Col1 and ColIV and enhances ECM deposition. VitC derived ECM can regulate cell physiology and enhance reprogramming efficiency. Altogether, we propose that VitC modulates ECM deposition through its effect on cellular metabolism, particularly by enhancing glycosylation pathways necessary for glycosaminoglycan and collagen production. This, in turn, alters ECM composition and promotes iPSC generation. These findings provide novel insights into how metabolism and ECM interact to regulate stem cell fate transitions. Our work also advances the understanding of ECM’s role in stem cell function, with broad implications for improving stem cell therapies, tissue engineering, and regenerative medicine, as well as addressing diseases linked to ECM dysregulation.

Impact of N-Linked Glycans on the Dual Short Fibulin/LTBP-4 Axes Regulating Elastogenesis

Dieter Reinhardt¹, Heena Kumra², Chae Syng Lee¹, Daniel B. Williamson³

¹McGill University, ²Harvard Medical School, ³University of Georgia

ABSTRACT BODY: Elastic fibers are essential components of the extracellular matrix, providing elasticity to dynamic tissues such as blood vessels, lungs, skin, and the bladder. Their proper assembly depends on the coordinated action of accessory proteins, including fibulin-4, fibulin-5, and the long and short isoforms of latent TGFβ binding protein-4 (LTBP-4L and LTBP-4S). In this study, we delineate two distinct molecular axes—LTBP-4L/fibulin-4 and LTBP-4S/fibulin-5, with both shared and isoform-specific roles in elastic fiber formation. We further uncover the critical regulatory role of N-linked glycans in these interactions. Glycoproteomic profiling identified specific N-linked glycans on each of the involved proteins. Biophysical analyses demonstrated that N-linked glycans on LTBP-4L are required for fibulin-4-mediated conformational extension of LTBP-4L, a structural change essential for its function and incorporation into elastic fibers. In contrast, glycans on fibulin-4 played an inhibitory role; their enzymatic or recombinant removal significantly enhanced fibulin-4’s interaction with tropoelastin and promoted elastic fiber formation. Fibulin-5 exhibited a robust interaction with LTBP-4S, inducing a strong conformational extension that enhanced LTBP-4S binding to fibronectin, increased its extracellular deposition, and doubled elastic fiber formation. However, the removal of N-linked glycans from fibulin-5, but not from LTBP-4S, drastically reduced this interaction by nearly tenfold and abolished the ability of fibulin-5 to induce the conformational shift in LTBP-4S. Interestingly, while fibulin-5–extended LTBP-4S alone did not drive tropoelastin aggregation in vitro, the co-presence of LTBP-4L and fibulin-4 led to a dramatic increase in elastic fiber-like structures, revealing a synergistic mechanism dependent on fibulin-5 glycans. These findings highlight critical and distinct roles for N-linked glycans in regulating protein conformation, interaction dynamics, and functional outcomes in elastic fiber formation. Specifically, glycans on LTBP-4L and fibulin-5 are indispensable for conformational activation of LTBP-4 isoforms and downstream assembly events. Together, the two axes, LTBP-4L/fibulin-4 and LTBP-4S/fibulin-5, operate through independent and cooperative mechanisms to orchestrate elastic fiber assembly. This study provides new mechanistic insight into how glycosylation modulates the activity of accessory elastogenic proteins to drive elastic fiber biogenesis and tissue elasticity, offering potential targets for therapeutic strategies in connective tissue disorders.

The Relationship Between Alterations in Cellular Structure and Gene Expression During Chondrocyte Dedifferentiation

Stephanie Richardson-Solorzano, Justin Parreno

University of Delaware

ABSTRACT BODY: Monolayer expanded chondrocytes are an FDA approved cell source for cartilage repair. However, when removed from their native extracellular matrix and cultured in vitro, articular chondrocytes progressively lose their phenotype in the process of dedifferentiation. This phenotypic shift is marked by alterations in gene expression that coincides with changes in cell and nuclear morphology, and cytoskeletal remodeling. The mechanisms driving dedifferentiation - particularly the relationship between gene expression, cell size and shape, cytoskeletal organization, nuclear architecture and gene regulation within the first days of dedifferentiation is not fully understood. Furthermore, the contribution of extracellular factors, such as serum, to dedifferentiation has not been fully elucidated. To gain a better understanding of dedifferentiation in vitro, we investigated the regulation of chondrocyte morphology, actin cytoskeletal organization, nuclear morphology, in relation to gene expression over the first five days of chondrocytes cultured in Ham’s F12 medium containing 0.5, 5, or 20% fetal bovine serum (FBS). We found increased fibroblastic molecules mRNA levels as COL1 increased by day 3 in 20%FBS, and TNC and αSMA levels increased by day 5 in 20% FBS. Exposure of cells to low serum 0.5%FBS, prevented the upregulation of these genes.. The expression of select chondrogenic molecules such as CHAD, ADS, GSN showed earlier alterations with decreased mRNA levels within 3 days. These alterations were independent of serum concentration. as they occurred in cells exposed to 0.5%FBSWhile cell morphology was not altered by day 3, at day 5 in culture there were increases in cell size as well as a reduction in cell circularity, irrespective of serum concentration. Nuclear morphology was altered earlier at day 3 as cells exposed to 20%FBS, but not 0.5%FBS had larger nuclei that were less wrinkled. At day 3, there were alterations in cytoskeletal networks in cells exposed to 20%FBS as cells contained stress fibers and larger focal adhesions, as compared to those in 0.5%FBS. Our data revealed the upregulation of fibroblastic molecules mRNA levels are dependent on serum. The alterations to fibroblastic molecule expression coincide with changes to nuclear morphology and the actin cytoskeleton, but not cell morphology. On the other hand, the downregulation of several specific chondrogenic mRNA levels occurs independent of serum and do not coincide with alterations to cell morphology, nuclear morphology, or actin reorganization. Understanding the relationships between cell structure and transcriptional responses during dedifferentiation may lead to novel insights into suppressing chondrocyte dedifferentiation and/or promoting redifferentiation for cell-based therapies to repair cartilage.

Integrated Single Cell and Spatial Transcriptomics Analysis Reveals Distinct Senescent Cell Phenotypes that Regulate Fibrosis

Alexandra N. Rindone¹, Sushma Nagaraj², Kavita Krishnan¹, Joscelyn C. Mejías³, Anna Cho¹, Anna Ruta¹, Frank Haoning Yu¹, Christopher Cherry⁴, Elana J. Fertig², Jennifer H. Elisseeff¹

¹Johns Hopkins University, Baltimore, MD; ²University of Maryland Baltimore, Baltimore, MD; ³Georgia Institute of Technology and Emory University, Atlanta, GA; ⁴C M Cherry Consulting, Baltimore, MD

ABSTRACT BODY:

Background: Fibrotic conditions contribute to 35.4% of mortality worldwide, but there remains a lack of effective therapeutics to halt and reverse fibrosis [1]. Senescent cells (SnCs) are growth arrested cells expressing a senescence-associated secretory phenotype (SASP) that have been recently linked to fibrosis in aging and various diseases. However, SnCs are heterogeneous in vivo, and their phenotypes and functions (senotypes) in fibrosis remain poorly understood, hindering the development of senescence-targeting therapies. This heterogeneity also makes it difficult to identify SnCs using a single marker, and thus, it remains challenging to characterize fibrosis-associated senotypes. In this study, we establish a new computational pipeline that incorporates transfer learning and non-negative matrix factorization algorithms to identify and profile SnCs in single-cell and spatial transcriptomics datasets. Applying this pipeline to a murine fibrosis model enriched in SnCs, we develop a spatial taxonomy of senotypes that are associated with distinct aspects of fibrotic tissue development and remodeling.

Methods: To study fibrosis senotypes, we used a biomaterial-mediated method to induce fibrosis by implanting synthetic polycaprolactone particles into a murine traumatic muscle injury. We have previously shown that this model reproducibly induces fibrosis with a high senescence burden at 6 weeks following injury [2]. To conduct scRNA-seq, we isolated the cells from the fibrotic muscle implants, enriched for CD45+ immune cells, and sequenced the cells using 10x Chromium 3’ (n=3 mice). To conduct spatial transcriptomics, we applied the VisiumHD platform to FFPE sections of the fibrotic muscle implants (n=2 mice) following manufacturer protocols. All datasets were processed using a standardized pipeline in Seurat. To identify SnCs in the datasets, we adapted a transfer learning approach, projectR [3], to label SnCs using an in vivo-derived senescence signature, SenSig, that can capture heterogeneous SnC subpopulations [4]. Cells (scRNA-seq) or pixels (VisiumHD, 8 μm bin) with a projection weight >0 and adjusted p-value <0.01 were considered senescent. We conducted differential expression analysis and non-negative matrix factorization, CoGAPS [3], on select subpopulations within the scRNA-seq dataset to identify biological processes associated with distinct senotypes. Select sequencing results were validated using protein-based multiplex immunofluorescent staining and colocalization analysis in HALO software.

Results and Discussion: Using scRNA-seq and transfer learning, we identified multiple clusters that were enriched in SnCs (>30% SnCs per cluster): activated fibroblasts (ActF), myofibroblasts (MyoF), and perivascular cells (PV). We validated the presence of fibroblast (F) and PV SnCs by co-staining p16 (senescence marker) with CD31 (endothelial cells), CD11b (myeloid cells), and PDGFRb (F and PV cells). The majority of p16+ cells were positive for PDGFRb (62.0±16.6% PDGFRb+ of p16+; n=4) and negative for CD31 and CD11b (91.1±3.76% CD31- and 68.4±6.02% CD11b- of p16+; n=3).

Using differential expression analysis between SnCs and non-SnCs in each cluster, we identified distinct SASP genes associated with each SnC subtype (p_adj<0.05), illustrating their phenotypic differences. ActF SnCs upregulated diverse genes related to inflammation (Cxcl12, Ccl8, Saa3), ECM degradation (Mmp2/3, Ctsk, Adamts7), and fibrocartilage ECM (Col11a1, Fmod, Cilp). By contrast, MyoF SnCs upregulated fibrosis-associated ECM genes, including several types of fibrillar and non-fibrillar collagens and other glycoproteins and fibrosis regulators (Col5/6/12/15a1, Tnc, Spp1, Timp1). PV SnCs upregulated several vascular associated ligands and ECM molecules (Angpt1/2, Col4a1/2, Col18a1, Igfbp7), which suggests their function may be specific to vascular remodeling.

As clustering was insufficient to fully capture the heterogeneity of fibroblast SnC gene expression patterns, we conducted CoGAPS on the fibroblast clusters to identify gene patterns related to specific senotypes that extended beyond discrete clusters. We identified four patterns upregulated in SnCs (p_adj<0.05) that were expressed by distinct fibroblast subpopulations: Immune Signaling & ECM Degradation ActF (C2, C3, C4a/b, Cxcl12, Mmp3/8, p_adj=2e-8), Cartilage Development ActF (Scx, Chad, Col11a1, Fmod, Comp; p_adj=3e-305), Fibrosis MyoF (Col4/6/7/8/12a1, Notch1/3, Runx2, Tead4, p_adj=1e-118) and Cell Adhesion & Signaling MyoF (Cdh13/23, Itga5/6/10, Gjb2/3/4, Fgf1, p_adj=4e-15).

Next, we spatially mapped these senotypes using transfer learning of SenSig and the CoGAPS patterns to the paired VisiumHD and H&E datasets. The Fibrosis and Cell Adhesion & Signaling MyoF SnCs were spatially restricted to regions around the implant particles in proximity to Spp1-high myeloid cells—immune subpopulations previously linked to fibrosis. By contrast, Immune Signaling & ECM Degradation ActF resided in immune active zones consisting of loosely organized ECM, lymphocytes, and myeloid cells. Cartilage Development ActF were located in regions surrounding the fibrotic and immune signaling zones and colocalized with aligned ECM fibrils. PV SnCs were detected in regions with blood vessels, but they were not spatially confined to specific zones relative to the implant particles.

Conclusion: Taken together, our results identify heterogeneous fibroblast and perivascular senotypes associated with distinct SASP factors, biological processes, and spatial niches related to fibrosis, immune signaling, ECM degradation, fibrocartilage development, and vascular remodeling. These findings provide a taxonomy of senotypes in fibrosis that will inform the development of cell-type specific senolytics to treat a wide range of fibrotic diseases.

References (PMID): [1] 37974206, [2] 32295900, [3] 31121116, [4] 37079217

FUNDING SOURCE: NIH U54AG079779 (JHE), R01AG082965 (JHE), DP1AR076959 (JHE), K99AG081564 (JCM)

Role of Macrophage–Adipocyte Communication in Obesity-Driven Inflammation

Julieta Rios-Vergara, Evangelia Bellas

Temple University

ABSTRACT: Obesity is a growing public health crisis linked to several diseases such as type 2 diabetes, cardiovascular disease, and cancer, characterized by excessive adipose tissue (AT) expansion, without appropriate angiogenesis, leading to hypoxia that disrupts homeostasis by inducing cellular stress, impairing adipocyte function, and promoting AT structural remodeling. Here, hypoxia-inducible factor 1 alpha (HIF1α), stabilized under low oxygen conditions, activates transcriptional programs driving fibrosis, inflammation, and extracellular matrix (ECM) remodeling. These changes alter adipocyte morphology and function, triggering mechanosensitive signaling pathways that perpetuate the inflammatory process. At once, adipose tissue macrophages (ATMs) accumulate in AT and polarize toward a proinflammatory (M1) phenotype. As a result, ATMs contribute to systemic insulin resistance and further AT dysfunction that reinforces this inflammatory loop, linking mechanical stress to immune activation.

We hypothesize that under hypoxic conditions, HIF1α accumulation in adipocytes upregulates fibrotic mediators, such as lysyl oxidase (LOX) and fibronectin 1 (FN1), thereby enhancing ECM crosslinking and matrix stiffness. This disrupts adipocyte morphology and mechanical balance, marked by increased actin alpha 2 smooth muscle (ACTA2) expression. The altered matrix impairs adipocyte function, resulting in a dysfunctional adipokine profile that promotes the secretion of monocyte chemoattractant protein 1 (MCP1) and interleukin 6 (IL6), driving ATM infiltration and M1 polarization. The resulting proinflammatory environment, enriched in tumor necrosis factor alpha (TNFα), further exacerbates AT inflammation and fibrosis.

Methods

AT constructs were generated by encapsulating human adipocytes at a density of 8×10⁶ cells/mL within a 2 mg/mL type I collagen hydrogel. Constructs were cultured either alone or co-cultured with macrophages at adipocyte: ATM ratios of 10:1 or 1:1, modeling lean and obese tissue conditions, respectively. Cultures were maintained in adipocyte maintenance media, with or without 1 mM dimethyloxalylglycine (DMOG), to simulate hypoxic conditions. After 7 days, conditioned media were collected for lipolysis assays, and constructs were harvested for quantitative polymerase chain reaction (qPCR), confocal microscopy, and glucose uptake assays to assess structural, metabolic, and inflammatory responses.

Results

Our findings demonstrate that hypoxia alone is a major driver of early adipocyte dysfunction, inducing a contractile phenotype, ECM remodeling, and metabolic impairment. However, co-culture with ATMs at a physiological 10:1 adipocyte: ATM ratio did not exacerbate these effects, although TNFα, an ATM-specific cytokine, was exclusively detected in ATM-containing groups, the overall impact of ATMs on adipocyte dysfunction remained low, likely due to the limited ATM's density. In obesity, ATM numbers increase, approaching a 1:1 ratio through monocyte infiltration and proliferation. This elevated ATM burden may be necessary to trigger the inflammation and metabolic dysfunction of advanced AT remodeling. Future studies using a 1:1 ratio will help determine whether increased ATM abundance is sufficient to initiate pathogenic changes.

Conclusion

These findings indicate that hypoxia alone is sufficient to activate ECM remodeling pathways and alter adipocyte function, while ATM-driven inflammation may require higher cell density or a more advanced pathological state. Understanding the conditions under which ATMs transition from homeostatic to pro-inflammatory states is critical to explaining the mechanisms underlying obesity-associated tissue remodeling and insulin resistance. Targeting early immune interactions may provide a valuable therapeutic window to reduce chronic inflammation, preserve AT function, and reduce the burden of obesity comorbidities.

FUNDING: NSF CAREER Award and Lipedema Foundation Collaborative Research Award (to EB).

Uncovering the Immunoregulatory Role of NIDOGEN-1 in Tumor Progression and Its Modulation by Proteolysis

Juan Carlos Rodríguez-Manzaneque, Rita Caracuel-Peramos, Francisco Javier Rodríguez-Baena, Carlos Peris-Torres

(GENYO) Center for Genomics and Oncological Research

ABSTRACT BODY: The extracellular matrix (ECM) plays an active role in tumor progression by shaping immune cell infiltration and vascular remodeling. In our recent work, we identify the basement membrane glycoprotein NIDOGEN-1 (NID1) as a previously unrecognized immunomodulatory ECM component that restrains tumor growth and promotes an anti-tumor immune response. We previously characterized NID1 as a substrate of the secreted protease ADAMTS1, raising the possibility that proteolysis regulates its function in the tumor microenvironment.Using melanoma and mammary tumor models in Adamts1-knockout mice, we found that the absence of ADAMTS1 led to accumulation of full-length NID1 in the tumor vasculature, correlating with impaired tumor growth and enhanced infiltration of macrophages and other immune effectors. Notably, ectopic expression of full-length NID1 in wild-type mice recapitulated the anti-tumor effects observed in Adamts1-deficient tumors, indicating that intact NID1 is a key effector of the observed immune response. In contrast, proteolytically processed NID1 fragments lacked this immunomodulatory and tumor-suppressive function.Mechanistically, we demonstrated that full-length NID1 promotes M1-like macrophage polarization in vitro, primarily through signaling mediated by αvβ3 integrin. RNA-seq from our tumor models, projected onto transcriptomic data from large human melanoma cohorts, revealed a novel immune gene signature associated with M1 macrophages and favorable patient prognosis, underscoring the translational relevance of this ECM-immune axis.Building on this framework, our ongoing work in uveal melanoma -a rare aggressive cancer type- has further highlighted the context-dependent roles of ECM proteolysis. Importantly, our previous studies demonstrated a pro-tumorigenic role for ADAMTS1 during early uveal melanoma development. Current and ongoing results from this model reveal significant alterations in ECM-related pathways, and we are now investigating the potential involvement of NID1 in regulating macrophage dynamics in this distinct tumor context.Overall, our findings position NID1 as a novel ECM-derived immunomodulator, whose function is tightly regulated by extracellular proteolysis. These insights open new avenues for targeting ECM components and protease-substrate interactions in the development of new immunotherapies and biomarker discovery.

Age-Dependent Remodeling of Extracellular Matrix in Brain Meninges

Liudmila Romanova¹, Kristy Urquhart¹, Alia Alia¹, Scott Muller²

¹Rush University Medical Center, ²Banner Neurodegenerative Disease Research Center

ABSTRACT BODY: Meninges are multi-layered membranes that envelope the brain. Dura is a highly vascularized outer layer while the arachnoid and pia (leptomeninges) are largely avascular inner layers. The arachnoid layer forms an epithelial tissue, which creates a tight barrier that strictly limits the movement of macromolecules to and from the cerebrospinal fluid (CSF) and brain. It is currently known that the meninges are critical for brain clearance and immunity, and play an active role in prevention of age-related neurodegenerative diseases. Despite this demonstrated importance, little is known about the most abundant group of proteins at the core of the meningeal structure: the proteins of extracellular matrix (ECM). In addition to providing a structural scaffold, ECM is a regulator of vascular and immune functions, tissue repair, and water homeostasis in meninges and brain. Both dura and leptomeninges have a well-developed ECM that includes structural proteins, vasculature associated ECM, and proteolytic enzymes. While it is known that ECM remodels with age, changes in the meningeal ECM in aging have not been systematically investigated. It remains unknown how these changes affect the key functions of dura and leptomeninges and contribute to brain aging and neurodegeneration. In our studies, we have observed that aging is associated with gross morphological changes in structural ECM of the dura in rat and non-human primates (NHP). Follow up proteomic studies of NHP, rat and human meninges identified a high number of ECM proteins that differentially expressed in aged meningeal tissues. We also observed age-dependent shifts in the solubility of the structural ECM. Differences between species were also noted, which underscored the difference in anatomy and meningeal physiology between rodent and primate translational models and humans. These observations provide a basis for our hypothesis that morphological and structural changes in ECM collectively affect dura and leptomeninges in aging and potentially contribute to neurodegenerative diseases.

The Receptor for Hyaluronan-Mediated Motility (RHAMM) Activates NFkB and the NLRP3 Inflammasome and Is a Critical Component in the Development of Bronchopulmonary Dysplasia

Rashmin Savani^1,2, Jenny I-Chen Chen³, Jie Liao², Christopher Longoria²

¹University of Florida, ²University of Texas Southwestern Medical Center,

³Kaohsiung University

ABSTRACT BODY: Background: Bronchopulmonary Dysplasia (BPD) is a chronic lung disease in preterm neonates with respiratory failure treated with ventilator and oxygen therapy. Interleukin 1 beta (IL1ß), a critical determinant of BPD, is induced by TLR signaling and NFkB and NLRP3 inflammasome activation. Endogenous danger-associated molecular patterns (DAMPs), such as low molecular weight hyaluronan (LMW HA), promote the production of IL1ß in macrophages. The Receptor for HA-Mediated Motility (RHAMM) determines inflammatory and fibrotic outcomes in lung injury models. AZM-152 is a peptide derived from RHAMM that blocks inflammation and fibrosis in several injury models. Exposure to oxygen in the first five days of life in mice results in inflammation and a lack of alveolar development, essential features of BPD.Objective: To determine the role of RHAMM in the mouse neonatal hyperoxia model of BPD and the activation of the NLRP3 inflammasome.Methods: Neonatal wild-type (WT) and RHAMM KO mice were exposed to 95% oxygen for 5 days and harvested on postnatal day 7 (PN7) and PN14. Lung IL1ß mRNA and protein, inflammation, and lung histology were examined. WT mice exposed to hyperoxia were treated with AZM-152, and a dose response was determined. LPS, a TLR4 agonist, was used to activate the NLRP3 inflammasome in cultured human macrophages in the presence and absence of AZM-152. Bone marrow-derived macrophages from WT and RHAMM KO mice were also examined with LPS stimulation. Akt and IkB phosphorylation and IL1ß production were determined. Results: WT mice exposed to neonatal hyperoxia had increased lung IL1ß mRNA and protein, lung neutrophils and macrophages, and decreased alveolarization. In contrast, RHAMM KO mice exposed to neonatal hyperoxia did not have increased IL1ß mRNA and protein and did not develop inflammation. However, alveolarization was worse in RHAMM KO mice exposed to hyperoxia. AZM-152 blocked IL1ß and inflammation in a dose-dependent manner and improved alveolarization in WT hyperoxia-exposed mice. LPS stimulation of cultured human macrophages increased Akt and IkB phosphorylation and IL1ß production. Treatment with AZM-152 blocked these LPS-stimulated responses. RHAMM KO BMDM failed to phosphorylate Akt and IkB or produce IL1ß.Conclusions: We conclude that RHAMM is a critical component in the development of BPD, the activation of NFkB, and the production of IL1ß, key features in the activation of the NLRP3 inflammasome. AZM-152 is a potent inhibitor of this pathway and a potential therapeutic agent to prevent BPD.

Fibrillin-1 Regulates Extracellular Matrix Cues and Modulates Adipose Tissue Differentiation and Metabolism

Iram Fatima Siddiqui, Kerstin Tiedemann, Muthu L. Muthu, Ling Li

Faculty of Medicine and Health Sciences, McGill University

ABSTRACT BODY: INTRODUCTION: Fibrillin-1 is an extracellular matrix protein organized in microfibrils that frequentlyintersect with basement membranes. Mutations in fibrillin-1 lead to Marfan syndrome (MFS), characterizedby either a lipodystrophic or overweight body status, among other clinical symptoms. This study reveals anovel dual role of fibrillin-1 in controlling adipogenesis at both the initial and late stages of adipose tissuedevelopment.METHODS AND RESULTS: To investigate the role of fibrillin-1 during early adipogenesis, we usedadipose-derived and bone marrow-derived mesenchymal stem cells, which were induced to differentiateinto adipocytes. The presence of a recombinant fibrillin-1 fragment containing an integrin-binding RGDmotif (Fbn1-RGD) significantly inhibited adipocyte differentiation, as demonstrated by Oil Red O andperilipin staining. It also resulted in downregulation of key adipogenic markers (Adipoq, Cebpa, Pparg,Glut4). In contrast, a control fragment harboring a non-functional RGA motif (Fbn1-RGA) showed noinhibitory effect, indicating the specific involvement of integrin receptors. To further study the signalingpathways involved, we used the adipogenic 3T3-L1 and C3H10T1/2 cell lines. Cell-binding assaysdemonstrated reduced adhesion to Fbn1-RGA compared to Fbn1-RGD, further implicating integrininvolvement in this interaction. Fbn1-RGD treatment reduced phosphorylation of the integrin-associatedkinases Fak and Src, as well as the downstream insulin signaling mediators Pi3k and Akt. Selectivepharmacological inhibition of Fak, Src, and Pi3k restored adipogenesis even in the presence of Fbn1-RGD,indicating that fibrillin-1 inhibits early adipogenesis via the integrin–Fak/Src–Pi3k/Akt axis. To identify thespecific integrins involved, we performed siRNA-mediated knockdown of known fibrillin-1 receptors—α5β1, αvβ3, and αvβ6, which are present during adipogenesis. Only knockdown of αvβ3 restored adipocytedifferentiation in Fbn1–RGD–treated cells, establishing αvβ3 as a key mediator of fibrillin-1's inhibitoryeffect on adipogenesis.To understand the role of fibrillin-1 during the late stages of adipogenesis, we generated an adipocytespecificFbn1 knockout mouse (Fbn1-AKO) by crossing floxed Fbn1 with Adipoq-Cre mice. The progenymice exhibited a ~25% reduction in body weight and fat mass by 30 weeks of age, as measured by dualenergyX-ray absorptiometry. Histological analysis showed adipocyte hypotrophy in ~90% of whiteadipocytes. Gene expression analysis of adipose tissue revealed a ~75–80% reduction in adipogenicmarkers. Despite maintaining normal glucose clearance, Fbn1-AKO mice exhibited severe insulinresistance and dyslipidemia, with a 2-fold increase in circulating triglycerides and a 1.2-fold reduction inhigh-density lipoprotein (HDL) levels. Single-nucleus RNA sequencing of adipose tissue demonstrated adecreased proportion of mature adipocytes and progenitor cells in Fbn1-AKO mice. Transcriptomic datahighlighted extensive ECM remodeling and elevated stress response pathways. Notably, genes involved ininsulin signaling (Pik3r1, Igf1) were downregulated in Fbn1-AKO mice. Consistently, Western blot analysisfurther confirmed significant reduction in phosphorylation of integrin pathway mediators Fak and Src, aswell as Pi3k and Akt, key effectors in insulin signaling.CONCLUSION: Together, these findings reveal that fibrillin-1 plays a biphasic role in adipogenesis,inhibiting adipocyte differentiation in the initial stages while supporting metabolic homeostasis in matureadipocytes. Fibrillin-1 regulates adipose tissue function via the integrin αvβ3–FAK/Src–Pi3K/Akt signalingaxis. Targeting this axis may offer therapeutic potential for adipose dysfunction in Marfan syndrome andrelated conditions.

Collagen Post-Translational Modifications are Altered in Pulmonary Fibrosis Including Within ECM Receptor Binding Sites

Juliane Merl-Pham^1#, Larissa Rothkirch^2#, Leonhard Binzenhöfer^2#, Elisabeth Hennen², Naftali Kaminski³, Rudolf Hatz⁴, Juergen Behr⁵, Stefanie M. Hauck¹, Anne Hilgendorff², Oliver Eickelberg^1,6,7, Claudia A. Staab-Weijnitz^2,8*

¹Metabolomics and Proteomics Core, Helmholtz Zentrum München, ²Institute of Lung Health and Immunity, and Comprehensive Pneumology Center with the CPC-M bioArchive, Ludwig-Maximilians-Universität München and Helmholtz Zentrum München, Munich, Germany, Member of the German Center for Lung Research (DZL), ³Pulmonary, Critical Care and Sleep Medicine, Yale School of Medicine, ⁴Thoraxchirurgisches Zentrum, Klinik für Allgemeine-, Viszeral-, Transplantations-, Gefäß- und Thoraxchirurgie, LMU Klinikum, Ludwig-Maximilians-Universität München, ⁵Department of Medicine V, LMU University Hospital, Comprehensive Pneumology Center, LMU Munich, German Center for Lung Research (DZL), ⁶Pulmonary, Allergy, Critical Care, and Sleep Medicine, University of Pittsburgh, ⁷Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease, University of Pittsburgh School of Medicine, ⁸Department of Pediatrics, and Division of Pulmonary Sciences and Critical Care Medicine, School of Medicine, University of Colorado Anschutz Medical Campus

^#These authors contributed equally.

ABSTRACT BODY: Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease characterized by increased deposition and crosslinking of fibrillar collagen in the alveolar interstitium hampering gas exchange. We have previously discovered upregulation of FK506-binding protein 10 (FKBP10) and collagen prolyl-3-hydroxylase 1 (P3H1) in IPF (1,2). Both proteins catalyze collagen post-translational modification (PTM): P3H1 has been described to introduce collagen prolyl-3-hydroxylations in fibrillar collagens while FKBP10 has been reported to facilitate dimerization and activity of collagen lysyl hydroxylase 2 (LH2). This study therefore aimed to identify whether collagen PTMs were altered in IPF. The function of 3-hydroxyproline (3-HyP) is little understood, but it has been reported that 3-hydroxylation of the remaining prolines (“P”) within the GPVI binding motif GPOGPO (O=4-HyP) attenuates GPVI binding and platelet aggregation which may affect disease progression in IPF. Therefore, we quantified prolyl hydroxylations in GPOGPO sites within fibrillar collagen chains. While we found few alterations in GPOGPO sites within type I (COL1A1, COL1A2) collagen, we identified three GPOGPO sites in type V collagen (COL5A1) with significantly increased prolyl-3-hydroxylation in IPF. To clarify whether these sites were P3H1-dependent, we silenced P3H1 in primary human lung fibroblasts (phLF). Quantification of collagen PTMs in the resulting insoluble ECM revealed that prolyl-3-hydroxylation of the COL5A1 GPOGPO sites harboring the 3-HyP sites P992/P995 and P1019/P1022 were indeed partially P3H1-dependent. As to FKBP10 and hydroxylysines (HyKs), we identified 11 HyKs with significantly increased hydroxylation frequencies in IPF. Silencing FKBP10 in phLF followed by HyK quantification in the generated ECM revealed that 3 out of 8 quantifiable sites were indeed FKBP10-dependent (COL1A1 K781 and K934; COL1A2 K846). In summary, we describe for the first time site-specific changes in collagen PTMs in IPF, including in ECM binding receptor motifs, and identify responsible enzymes. These findings suggest that collagen PTMs could serve as markers of disease-driving collagen and warrant their detailed spatial and functional analysis in IPF and other progressive fibrotic lung diseases to inform future ECM-targeting therapies. The results of this study have been published recently (4).

References:

(1) Staab-Weijnitz CA et al, Am J Respir Crit Care Med 2015

(2) Schiller HB et al, Am J Respir Crit Care Med 2017

(3) Merl-Pham J et al, Matrix Biol Plus 2019

(4) Merl-Pham J et al, Am J Respir Crit Care Med 2025; doi: 10.1164/rccm.202505-1276RL

A Novel Collagen Hybridizing Peptide for Activating LAIR-1 Inhibitory Pathway at Damaged Collagen Sites

Regan Stephenson, Bridget Glass, Kyle Dunlap, Mallory Longacre

3Helix, Inc

ABSTRACT BODY: Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), an inhibitory collagen receptor expressed on a variety of immune cells, plays a crucial role in maintaining immune homeostasis. In inflammatory diseases characterized by collagen damage, immune regulation via LAIR-1 is impaired due to loss of LAIR-1 binding sites in damaged collagen. Collagen hybridizing peptides (CHP) are peptide sequences that mimic the Gly-X-Y repeats found in collagen which enable specific binding to the damaged collagen triple helix. Building off CHP technology, we developed bioACTIVE CHPs, an innovative peptide platform to enrich areas of damaged collagen with cell binding sites tailored for desired cellular responses. Here we present FLAIR2, a novel bioACTIVE CHP with a LAIR-1 binding motif to reinstate immune regulation to the collagen triple helix at sites of collagen damage. We hypothesized that this peptide would bind to damaged collagen and inhibit immune cell activation at those sites. In vitro testing confirmed FLAIR2 binds to damaged collagen and promotes LAIR-1 binding. At the cellular level, FLAIR2 exhibited immunomodulatory effects by attenuating secretion of IL-2 and interferon gamma in activated T cells. In macrophages, FLAIR2 exposure reduced expression of M1 macrophage marker transcripts for CCR7 and CD86 and the secretion of inflammatory cytokines IL-6 and IL-1β. Furthermore, in an ex vivo human psoriasis model FLAIR2 treatment demonstrated broad anti-inflammatory effects and reduced key pathogenic cytokines at levels comparable to betamethasone, the current standard for psoriasis treatment. Lastly, in an in vivo model of dry eye disease, topical treatment with FLAIR2 prevented tear volume loss and development of severe corneal damage characteristic of dry eye disease. These findings establish FLAIR2 as a novel anti-inflammatory therapeutic capable of simultaneously targeting damaged collagen and activating LAIR-1-mediated immune cell inhibition. This platform presents a revolutionary collagen-focused approach for topical treatment of inflammatory diseases.

Cardiac Remodeling and Molecular Alterations in Ageing Female Rats

Thea Stole¹, Andreas Romaine², Marianne Lunde¹, Kristin Jenssen,¹

¹Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, ²Institute for Surgical Research, Oslo University Hospital and University of Oslo

BODY: The incidence of heart disease has a different age-distribution for women and men, with women being at greater risk of developing cardiac disease in the second half of their lives. This increase in incidence rate is associated with reduced estrogen levels after menopause. Estrogen has been found to protect the heart by limiting the development of hypertrophy and fibrosis. However, the molecular mechanisms for these effects remain unknown. To better understand the protective effects of estrogen and its involvement in the age-dependence of cardiac disease in females, we employed female rats of 9, 16, and 24 months of age. In vivo cardiac structure and function were evaluated by echocardiography. Serial blood sampling was performed during the five-day estrus cycle to determine changes in estrogen levels and its cycling by ELISA. Levels of fibrosis were assessed in excised tissue with Masson`s trichrome, and alterations in signaling networks and gene levels were quantified with immunoblotting and qPCR. To determine whether alterations were sex-specific, age-matched male rats were used for comparison. Our results indicate that females and males displayed similar intraventricular septal and left ventricular posterior wall thickness at 9 months of age, normalized to tibia length. At 16 months, however, sex differences were more prominent, where females displayed thinner intraventricular septal walls compared to males. This was due to the female cardiac structure remaining largely unchanged from 9 to 16 months, whereas males displayed a significant increase in wall thicknesses. Mean serum estrogen levels over five days trended towards being lower in rats from 9 to 16 months of age. This was also reflected in the cyclicity over a five-day period, where the amplitude of the cycle trended towards being reduced. On a molecular level, estrogen receptor β, pSer473-Akt, and p-p44/42-MAPK levels were also reduced with age in both sexes. Our data indicate age and sex dependent structural changes within the heart, accompanied by alterations in estrogen levels and cyclicity, and molecular alterations

Elucidating The Role of Hemicentin-1 in the Retina

Conor Sugden¹, Richard B. Tunnicliffe¹, Anil Kumar Ganga², Alana Stevenson Harris¹

¹Manchester Cell-Matrix Centre, Division of Cell Matrix Biology and Regenerative Medicine, University of Manchester, ²Department of Biology, Duke University

ABSTRACT BODY: Basement membranes (BMs) are specialized extracellular matrix (ECM) sheets with diverse roles such as providing structural support, regulating cell behaviour, and facilitating tissue connections. In tissues such as the eye, lung, and kidney, adjacent BMs interconnect through a specialized adhesion complex consisting of collagen IV, hemicentin and fibulin-1. Hemicentin-1 (HMCN1) is a key structural and organisational component of these BM-BM linkages, yet its precise interplay with other BM constituents remains poorly understood. Variants in the HMCN1 gene have been linked to age-related macular degeneration (AMD) the most common cause of severe visual impairment although the underlying molecular mechanisms remain unclear and the association with the tissue connection has not yet been established.To understand HMCN1 expression in the developing eye, we analysed publicly available human single-cell RNA sequencing datasets. HMCN1 expression peaked around the predicted time of BM-BM linkage at Bruch's membrane, supporting its possible involvement in this tissue connection. Immunofluorescence of eye tissue from wild-type and a collagen IV knockout mouse (Col4a5), using Airyscan confocal microscopy, revealed that HMCN1 was interlaced within collagen IV sheets at the BM-BM linkage, and punctate HMCN1 patterning decorated the retinal synaptic ribbon, suggesting an additional role in photoreceptor cell maintenance. Intriguingly, its patterning was perturbed at the BM-BM linkage in Col4a5 knockout mice.To investigate the molecular function of HMNC1, we determined the crystal structure of its von Willebrand factor A (vWA) domain at 1.7 Å resolution. This domain is hypothesized to bind collagen IV. To probe these interactions, we generated mStayGold-tagged HMCN1-vWA constructs for overexpression in human retinal pigment epithelial cells- the cells that make the BM-BM linkage at Bruch’s membrane.Complementing these studies, we identified human variants in HMCN1, including 30 missense variants from ClinVar (4 linked to AMD) and 18 conserved variants within the HMCN1-vWA domain from the 100,000 Genomes Project. We are now modelling how these human variants affect protein structure. Collectively, this research contributes to a greater understanding of the role of HMCN1 in tissue connections and in in retinal health and disease.

Astrocytes Increase Fibronectin Matrix Assembly and Upregulate Fibronectin-Binding Proteins in Response to Local Stimuli

Yu Sun, Jean E. Schwarzbauer

Department of Molecular Biology, Princeton University

ABSTRACT BODY: Tumor growth and invasion depend on support by the extracellular matrix (ECM) and in breast tumors the ECM is rich in fibronectin (FN) and collagens. Breast tumor cells metastasize to the brain where the ECM is different, primarily containing laminins, tenascins, and hyaluronan. Astrocytes play essential roles in the brain supporting neuronal connections and communication. They synthesize FN and we have shown that primary cortical astrocytes assemble their FN into a fibrillar matrix. Whether breast tumor cells stimulate astrocyte FN matrix production to support metastatic tumor formation remains to be shown. To understand the importance of ECM in affecting astrocyte FN matrix assembly, we compared cells on laminin, FN or a non-ECM substrate and found that both laminin and FN substrates enhance FN assembly by astrocytes. Providing exogenous FN in their culture medium further increased assembly more than two-fold. This result indicates that an increase in available FN from blood that accompanies tumor cell invasion could stimulate FN matrix formation by astrocytes. RNA-sequencing analysis shows that astrocytes have several FN-binding β1 integrins – α5, αv, and α3. α5β1 integrin co-localizes with nascent FN fibrils and paxillin focal adhesions, while integrin αv forms focal complexes adjacent to but not overlapping with FN fibrils. Therefore, astrocytes appear to follow similar FN assembly principles as fibroblasts. Breast tumor cells secrete transforming growth factor beta (TGFβ) and we recently showed that increases in FN matrix in lung fibrosis are accompanied by upregulation of FN-binding ECM proteins induced by TGFβ. In astrocytes, we found that along with an increase in FN matrix assembly, TGFβ also stimulates expression of the FN-binding proteins tenascin-C, thrombospondin-1, and insulin-like growth factor binding protein 3 (IGFBP3) detected by qPCR and immunoblotting. Thus tumor cells may stimulate ECM production to support their invasion into brain tissue. In addition, TGFβ also upregulates IGF1 in astrocytes suggesting that IGF1-IGFBP3 may form an autocrine signal to increase astrocyte numbers with concomitant increases in ECM production. Altogether, we have identified several mechanisms to increase ECM production by astrocytes to provide a microenvironment that is conducive to tumor growth in the brain.

Exosomes Are Specialized Vehicles To Induce Fibronectin Assembly

Bong Hwan Sung^1,2, Merlyn Emmanuel^1,2,#, Metti K. Gari³, Jorge F. Guerrero⁴, Maria Virumbrales-Muñoz^5,6, David Inman³, Evan Krystofiak^1,7, Alan C. Rapraeger⁸, Suzanne M. Ponik^3,6, Alissa M. Weaver^1,2,9

¹Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, ² Vanderbilt Center for Extracellular Vesicle Research, Vanderbilt University School of Medicine, ³Department of Cell and Regenerative Biology, University of Wisconsin-Madison School of Medicine and Public Health, ⁴ McArdle Laboratory for Cancer Research, University of Wisconsin-Madison School of Medicine and Public Health, ⁵Department of Obstetrics and Gynecology, University of Wisconsin-Madison School of Medicine and Public Health, ⁶Carbone Cancer Center, University of Wisconsin-Madison School of Medicine and Public Health, ⁷Cell Imaging Shared Resource, Vanderbilt University School of Medicine, ⁸ Department of Human Oncology, University of Wisconsin-Madison School of Medicine and Public Health, ⁹Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, ^# Current location: Faculty of Pharmaceutical Sciences, University of British Columbia

ABSTRACT BODY: Fibronectin is a key stromal matrix molecule whose assembly into fibrils is thought to require cells. In contrast, we find that small exosome-type extracellular vesicles (EVs) are critical initiators of fibronectin assembly. Fibroblasts engineered to be deficient in exosome secretion showed greatly reduced assembly of fibronectin and other stromal matrix molecules in 2D, 3D, and in vivo environments, and led to reduced tumor growth and lung metastasis by breast cancer cells. Furthermore, transgenic mice with defects in exosome secretion had greatly reduced lung fibrosis after treatment with bleomycin. In a direct test of exosome function, we find that the addition of purified small EVs to purified soluble fibronectin in a cell-free system is sufficient to induce fibronectin assembly. The EV-induced fibronectin assembly requires the presence of fibronectin-binding integrins and Syndecan-1 in the EVs. We propose a new model in which secreted exosomes directly drive stromal matrix assembly and tissue fibrosis.

FUNDING SOURCE:

NIH grants R01CA206458, R01CA249864, R01CA249424, R50CA283661

AHA fellowship 19POST34370067

Investigating Macrophage-Mediated IL-1R1 Signaling in Pulmonary Fibrosis

Hayley Sussman

University of Virginia, Daniel Abebayehu, University of Virginia

ABSTRACT BODY: Introduction: Idiopathic pulmonary fibrosis (IPF) is a fatal disease marked by the dense buildup of scar tissue in the lungs with no known cause or lasting cure. The prevalence of this disease continues to rise globally, with up to 45.1 cases per 100,000 persons and a poor prognosis with a 3–4-year median survival time following diagnosis. Fibrosis is a downstream response commonly triggered by dysregulated wound healing and chronic inflammation which leads to excessive amounts of extracellular matrix (ECM) deposition and collagen secretion. Common histopathological indicators of IPF are marked by areas of active fibrogenesis known as fibroblastic foci which comprise concentrated regions of myofibroblasts and newly synthesized ECM. Various immune and inflammatory markers are implicated to be highly expressed in these fibrotic regions, including IL-1β and its receptor IL-1R1. However, the mechanistic role of IL-1R1 in lung fibrosis remains unclear. We seek to understand the pathological role of IL-1R1 signaling in IPF progression and strive to reveal more biomolecular insight into IL-1R1 as a therapeutic target for IPF. Materials and Methods: Human tissue samples of IPF lungs were sectioned and stained with fluorescent antibodies for α-smooth muscle actin (αSMA) and IL-1R1 to assess the extent of fibrosis. Non-fibrotic regions within IPF lungs were also identified through histological staining and fluorescent imaging. To investigate IL-1's role in fibrosis in vivo, we utilized a bleomycin-induced lung injury model in C57BL6/J wildtype (WT) mice and IL-1R1 knockout (KO) mice. Bleomycin is a chemotherapeutic known to trigger epithelial injury and consequential fibrosis in the lungs. Mice were treated with a single dose of bleomycin via oropharyngeal delivery (1 U/kg), while control mice received saline instead. To investigate whether IL-1R1 induces fibrosis, mouse lungs were harvested for histology 21 days following bleomycin administration. Histological stains included Hematoxylin & Eosin, Picrosirius Red, and Masson’s Trichrome to visually assess the fibrotic response. To assess IL-1β secretion levels following bleomycin injury, a saline bronchoalveolar lavage (BAL) was performed on mice harvested for 7 days post lung injury. Supernatant from the BAL was collected and measured for mouse IL-1β with an ELISA. To identify which innate immune cell populations secrete Il-1β, lungs were harvested from fluorescent reporter ASC-Citrine mice 7 days post lung injury and digested into a single-cell suspension for imaging flow cytometry. Results and Discussion: In human IPF tissue, regions of IL-1R1 expression were adjacent to αSMA+ foci in contrast with non-fibrotic tissue regions. IL-1R1's elevated presence in human IPF tissue indicates its potential critical role as a key inflammatory signaling axis in lung fibrosis. The levels of secreted IL-1β, a ligand for IL-1R1, were the highest in mice 4 days following lung injury. Imaging flow cytometry revealed that macrophages are the most abundant cells responsible for secreting mature IL-1β at 7 days post-bleomycin. We next sought to determine whether IL-1β/IL-1R1 signaling is necessary for lung fibrosis. As expected, we observed intensified fibrosis at 21 days via histology in WT mice treated with bleomycin. IL-1R1 KO mice treated with bleomycin, however, had a reduced fibrotic response. Altogether, these findings indicate that macrophage-mediated IL-1R1 signaling is necessary for a fibrotic response in the lungs. Conclusion: Pulmonary fibrosis is clearly reduced in the absence of IL-1R1, and macrophages are a critical cell source for IL-1β secretion. These findings highlight that IL-1R1 could be a potential therapeutic target for IPF treatment. Our ongoing research involves investigating the therapeutic role of Anakinra, an FDA-approved IL-1R1 inhibitor, to explore whether we can reverse lung fibrosis in vivo.

Investigating Components Involved in Alveolar Basement Membrane Fusion

Matilda J. Thuringer¹, Alana Stevenson Harris¹, Conor J. Sugden¹, Gema Bolas¹, Erin Plosa, David R Sherwood and Rachel Lennon^1,2

¹Manchester Cell-Matrix Centre, Division of Cell-Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, The University of Manchester, ²Department of Paediatric Nephrology, Royal Manchester Children’s Hospital, Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre

ABSTRACT BODY: Basement membranes (BMs) are dynamic, specialised sheet-like layers of extracellular matrix that are critical for tissue architecture, development and function. While they predominantly act as barriers between tissues, adjacent BMs can atypically fuse, forming stable intercellular connections, structures which are observed across multiple species. A specialised matrix adhesion system which fuses adjacent BMs was discovered in C. elegans, and vertebrate orthologs of these components are also involved in BM-BM linkages, suggesting this adhesion complex is conserved. In the lung, at the alveolar-capillary barrier, alveolar type I and endothelial BMs fuse, forming a thin BM which facilitates efficient gas exchange. While it is known collagen-IV α3α4α5 is abundant at the alveolar-capillary barrier; the impact this heterotrimer, along with other key components, has on the development and integrity of this critical BM-BM linkage is yet to be determined. To address this, we elucidated components of the BM-BM linkage at the alveolar-capillary barrier across various developmental stages, up to adulthood. We analysed publicly available scRNA-seq data which revealed developmental patterning of proposed BM-BM linkage components. Additionally, we used high-resolution imaging techniques, including insights from a Col4a2-mScarlet tagged reporter mouse, to characterise the spatial and temporal distribution of key BM proteins in wildtype and Col4a5^(-/Y) mice. This approach has identified potential components involved in the formation and maintenance of the alveolar basement membrane, which will inform future research into lung development and respiratory disease.

Production of Full-Length Recombinant Laminins With spERt Technology

Tomonori Ueno, Hiromi Nagatsuka, Haruka Takahashi, Kazunori Mizuno

Nippi Research Institute of Biomatrix

ABSTRACT BODY: Laminins are essential basement membrane proteins that contribute to extracellular matrix structure and influence cellular behavior through interactions with cell surface receptors. The diversity of laminin isoforms, resulting from various combinations of α, β, and γ chains, leads to the formation of extracellular matrices with different biological properties. In metabolic tissues such as the pancreas, adipose, muscle, and liver, specific laminin isoforms are expressed in distinct temporal and spatial patterns, suggesting their role in supporting the development and function of particular metabolic cell types.

We have established a high expression production system for human recombinant laminin using spERt technology and analyzed its functional properties. spERt technology is a recombinant protein production technology using CHO cells developed at Nippi, in which high expression is achieved by activating the translation process in the cells. spERt technology was used to establish CHO cell lines stably expressing laminin-111, -511 and -521. After studying the conditions of the culture process, culture conditions were determined that resulted in titers of approximately 1 g/L in 4 days. Purification conditions were subsequently determined using the purification platform of the spERt expression system. These purified products were gelled at 37°C and returned to sol at 4°C. Their properties as temperature-sensitive gels were similar to those of laminin extracted from mouse Engelbreth-Holm-Swarm (EHS) tumors, indicating that they have similar aggregation capacity to naturally occurring laminin. When the gel strength between isoforms was examined, the gel strength was strongest for laminin-111, followed by laminin-511, indicating differences in gel strength between the isoforms.

Matrigel, a basement membrane extract containing laminin-111 as the main component, was extracted and purified from mouse EHS tumors and is widely used in tissue engineering and cancer research as a 3D cell culture substrate but is not clinically applicable and suffers from large lot-to-lot differences. The recombinant laminin developed in this study suggests the potential for multiple applications, including 3D culture substrates as an alternative to Matrigel and for regenerative medicine.

Remodeling of the Meningeal Extracellular Matrix in Aging

Kristy Urquhart¹, Alia O Alia¹, Hui Chen², Lasanthi Jayathilaka²

¹Rush Medical Center, Department of Neurological Sciences, ²University of Illinois at Chicago, Mass Spectrometry Core

ABSTRACT BODY: The meninges, historically viewed as passive protective coverings of the brain and spinal cord, have recently emerged as dynamic participants in central nervous system (CNS) homeostasis. Composed of three anatomically and molecularly distinct layers: the collagen-rich dura mater, the fluid-regulating arachnoid mater, and the glia-associated pia mater, this highly specialized tissue is rich with extracellular matrix (ECM). Meningeal ECM supports immune surveillance, mechanical stability, cerebrospinal fluid (CSF) regulation, and solute clearance. Despite these essential functions, the meningeal ECM remains one of the least characterized connective tissues in the body. While the ECM of parenchymal brain tissue has been studied in aging and disease, the meningeal matrix has been largely overlooked, even as increasing evidence implicates its dysfunction in age-related neurodegenerative diseases. Here, we present the first study that investigated protein components of ECM of the meninges in aging in the translational species, Fischer rat (Rattus norvegicus F344) and Cynomolgus macaque (Macaca fascicularis). We used our new optimized protein fractionation protocol tailored for the unique biochemical heterogeneity and insolubility of meningeal ECM. Our approach enabled the extraction of structural and regulatory matrix components across the meninges. Analysis of the resulting fractions with liquid chromatography-tandem mass spectrometry (LC-MS/MS) generated high-confidence proteomic datasets encompassing over 1,800 proteins per species. Our analyses revealed conserved and species-specific signatures of age-associated ECM remodeling, including shifts in collagen subtypes, matricellular proteins, fibrosis-associated signaling, and markers of chronic inflammation. These findings reposition the meningeal ECM as an actively regulated, aging-sensitive tissue with relevance to CNS disorders in aging. By generating the first cross-species data of the composition of the aging meningeal ECM and introducing a novel biochemical workflow tailored to its unique structure, we established a platform for future mechanistic and translational research. This approach enables deeper investigation into the matrix's role in CNS pathology and opens new avenues for biomarker discovery and matrix-targeted interventions in age-related brain disorders.

A Human Thrombospondin-4 Gene Variant That Alters Sarcolemmal Stability and Predisposes to Dilated Cardiomyopathy

Davy Vanhoutte¹, Godelieve RF Claes^2,3, Apollonia TJ Helderman-Van den Enden³, Yasuhide Kuwabara¹

¹Department of Pediatrics, University of Cincinnati, Cincinnati Children's Hospital Medical Center, ²Reference Network for Rare, Low Prevalence and Complex Diseases of the Heart (ERN GUARD-Heart), ³Department of Clinical Genetics, Maastricht University Medical Center

ABSTRACT BODY: The genetic underpinnings of dilated cardiomyopathy (DCM) have been increasingly understood to involve both common and rare gene variants. However, the mechanisms by which these variants drive disease progression and clinical outcomes remain unclear. Here, we identify a rare human Thrombospondin-4 (THBS4) variant (NM_003248.6:c.2149G&gt;A; p.Asp717Asn, rs759241386) in two siblings diagnosed with DCM and/or ventricular tachycardia (a 52-year-old male and a 63-year-old female). The p.D717N missense change alters a highly conserved aspartic acid within the C-terminal type 3 repeat of Thbs4, a domain implicated in cardioprotection via integrin trafficking and cell membrane stabilization. This variant is exceptionally rare (minor allele frequency = 0.0003103% in gnomAD v4.1), supporting its potential pathogenicity. Notably, comprehensive genetic testing of other known cardiac disease-associated genes showed no relevant variants. To evaluate the in vivo impact of this variant, we generated gene targeted mice harboring this variant at the syntenic site of D719N in mouse Thbs4. Both heterozygous and homozygous Thbs4D719N mice were viable and displayed normal cardiac structure and function at 2 and 18 months of age. However, following 12 weeks of pressure overload induced by transverse aortic constriction (TAC), homozygous Thbs4D719N mice exhibited significantly exacerbated cardiac hypertrophy, fibrosis, left ventricular dilation, impaired contractility, and pulmonary congestion compared to heterozygous and wild-type control mice. Mechanistically, we show that pressure overload stimulation in Thbs4D719N mice impairs sarcolemmal integrity, leading to exacerbated cardiac decompensation. Together, these findings support a role for the THBS4D717N variant in causing or predisposing to DCM in affected patients through a mechanism that likely involves cardiomyocyte membrane instability. These results also suggest the importance of rare variant functional analysis to stratify risk and make lifestyle choices, with the promise of future new therapeutic approaches.

MatriSpheres: A 3D Self-Assembly Platform Integrating Decellularized Matrix to Recapitulate Tumor Heterogeneity and Immune Regulatory Phenotype

Michael J. Buckenmeyer¹, Elizabeth A. Brooks¹, Madison S. Taylor¹, Liping Yang², Ronald J. Holewinski³, Thomas J. Meyer⁴, Mélissa Galloux⁵, Marcial Garmendia-Cedillos⁶, Thomas J. Pohida⁶, Thorkell Andresson³, Brad St. Croix², Matthew T. Wolf¹

¹Cancer Biomaterials Engineering Laboratory, Cancer Innovation Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA., ²Tumor Angiogenesis Unit, Mouse Cancer Genetics Program, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA, ³Protein Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, 21701, USA., ⁴CCR Collaborative Bioinformatics Resource, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA., ⁵Independent Bioinformatician, Marseille, Provence-Alpes-Côte d'Azur, France, ⁶Instrumentation Development and Engineering Application Solutions, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, USA.

ABSTRACT BODY:

Introduction: The tumor microenvironment is a three-dimensional (3D) assembly of both cancer and stromal cells supported by an extracellular matrix (ECM) scaffold that contributes to growth and progression. The TME also encourages protection from immune surveillance by reprogramming and suppressing immune function, hampering effects of cancer immunotherapies. In vitro cell culture models are invaluable tools for investigating the TME, however, analyzing the impact of stromal elements such as ECM remains a challenge. Traditional in vitro models lack matrix or utilize purified ECM molecules (Collagen I or Matrigel) that fail to recapitulate the complexity of ECM within tissues. The immunoregulatory role of ECM is unknown.

Methods & Results: We established ECM-rich 3D “MatriSpheres”, a hydrogel-free self-assembly platform, to decouple cancer cell-ECM interactions. Mouse and human colorectal cancer (CRC) MatriSpheres actively incorporated microgram quantities of decellularized small intestine submucosa (SIS) ECM. We decellularized CRC tumors for proteomics in parallel with scRNAseq and found that ECM molecules within tumors are expressed by stromal fibroblasts, but also macrophages and cancer cells, supporting the need for decellularized tissue matrix complexity. SIS was used as a homologous ECM source, which proteomically-mimicked CRC tumor ECM (76 shared proteins) to a greater extent than traditional formulations like Matrigel (38 shared) or commercial Collagen I (8 shared).

We developed the MatriSphere method of supplementing cancer cell lines in non-adherent conditions with pepsin-solubilized SIS-ECM. At sub-gelation concentrations determined by rheology, the matrix was organized and concentrated by cancer cells into intercellular stroma-like regions within 5 days - displaying morphological similarity to clinical pathology.

MatriSpheres stimulated ECM-dependent transcriptional and cytokine secretion profiles associated with malignancy, lipid metabolism, and immunoregulation. The chemokines Cxcl1, Cxcl8, Cxcl10, and Cxcl11 were all upregulated (both gene and secreted protein) greater than 2-fold in MatriSpheres compared to cells-alone spheroids, suggesting the ECM alters the immune regulatory phenotype of cancer cells. The transcriptional programs were compared to scRNA sequencing demonstrating that MatriSpheres (34.2%) enhanced correlation with in vivo tumor cells over traditional ECM-poor spheroids (18.6%).

We investigated the role of ECM-dependent tumor immune regulation on T cell function using both 2D cultures and 3D MatriSpheres. MC38-OVA cancer cells were readily killed by OVA-specific OT-I transgenic T cells (12.7% apoptosis at 24 hr), which was reduced to 10.1% when co-cultured with decellularized ECM in 2D conditions (p < 0.0065). Similarly, the 3D MatriSphere model inhibited cell killing with ECM compared to cells-alone spheroids, with an increase in cell viability from 72.9% to 80.6% at 48 hr. Flow cytometry analysis showed that T cells exposed to SIS ECM had reduced CD25 expression (to 13.8% from 19.5% in cells alone), a marker of activation but increased effector memory phenotypes shown by CD44⁺/CD62L^- expression (up to 62.0% from 58.9%), with ECM MatriSpheres relative to cells alone. T cells showed a reduction in the exhaustion marker LAG3 with ECM (down to 77.9% from 84.4%).

Summary & Conclusion: We describe the MatriSphere platform, a facile approach of decellularized ECM-cancer cell co-assembly that enables tumor-specific tissue morphogenesis, promoting cell-ECM communication to improve fidelity for high-throughput disease modeling applications. Using MatriSpheres, we find that tissue-specific matrix regulates chemokine production and regulates cytotoxic T cell function in vitro towards an effector memory phenotype. These results show that complex decellularized ECM can be used to model cancer immunology-in-a-dish, and to decouple the role of ECM in the TME to better design cancer immunotherapy in the future.

FUNDING SOURCE (optional): This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, CCR, CIL.

Distinct Molecular and Structural Traits of Permanent versus Transient Cartilage in Early Development

Jiaqi Xiang¹, Bryan Kwok¹, Meghan E. Kupratis², Mingyue Fan¹

¹Drexel University, ²University of Pennsylvania

ABSTRACT BODY: INTRODUCTION: Hyaline cartilage is a family of cartilaginous tissues characterized by an extracellular matrix of porous collagen II fibril networks entrapping densely packed proteoglycans. Endochondral ossification starts with the formation of transient epiphyseal cartilage (EC), providing the template that remodels into bone. In contrast, articular cartilage (AC), which initiates at the cavitation sites defining future joint surfaces, is not replaced by bone and remains as hyaline cartilage throughout life, endowing joints with load transmission and energy dissipation functions. These two hyaline cartilages thus follow distinct developmental trajectories and have different functions in vivo. However, due to their similar composition, there have been few attempts to delineate differences in their matrix structure-function relationships or assembly/remodeling paths. This study aims to distinguish the molecular, structural and mechanical traits of these two cartilages during the initial phase of matrix formation in both small (murine) and large (porcine) animal models. METHODS: Knee joints were harvested from wild-type (WT) C57BL/6J mice at embryonic day 15.5 (E15.5) and postnatal day 0 (P0), and from Yorkshire pigs at E42, E84 and P0. RNAscope in situ hybridization was applied to visualize the spatial expression profiles of Col2a1 and Acan on P0 murine joint sections. Histology was applied to evaluate joint morphology and sulfated glycosaminoglycans (sGAGs). Immunofluorescence staining was performed to assess the spatial distributions of collagen VI, perlecan and collagen V. TEM was applied to quantify collagen fibril nanostructure in P0 murine joints. AFM-nanoindentation was applied to 10 μm-thick, unfixed cryo-sections to quantify the tissue micromodulus. RESULTS: In P0 murine knee joint, we observed higher expressions of Col2a1 and Acan in epiphyseal chondrocytes compared to articular chondrocytes. Histology also showed stronger sGAG staining in EC for both animal models. Furthermore, these two tissues exhibited differential distributions of other key matrix molecules. Collagen VI, a pericellular matrix (PCM) biomarker, was present only in AC, while another PCM biomarker, perlecan, was in both tissues. Surprisingly, collagen V, the initiator of collagen I fibrillogenesis, was found only in developing AC, suggesting it may be involved in the initial templating of the permanent cartilage matrix. This differential localization of collagens V and VI in AC but not EC suggests these two units constitute unique cellular microniches during their initial establishment. Comparing the two species, murine AC did not develop collagen V/VI-exclusive PCM domains until P0, while porcine AC already showed distinct PCM domains defined by localization of collagens V and VI at E84, illustrating differences in the timeline of matrix elaboration and specialization across species. In murine joints, EC exhibited much thinner fibrils compared to AC at P0 via TEM, which could potentially serve as a template to enable efficient remodeling to leave space for angiogenesis and bone formation. Indeed, we found the modulus of EC was moderately higher than AC at E15.5 but similar at P0. The higher modulus at E15.5 could be attributed to the higher Acan expression and sGAG content in EC, and this effect was likely offset by the much thinner collagen fibrils in EC at P0. In contrast, in porcine joints, the modulus of EC was similar to AC at E42, but higher at E84 and P0, again underscoring differential development paths and traits of EC versus AC in small versus large animals. CONCLUSION: Our study identifies early molecular, structural, and mechanical distinctions between the matrices of permanent versus transient hyaline cartilage, providing a foundation for the development of new osteoarthritis treatments and cartilage regeneration approaches.

Effect of Ionic Strength on Flexibility and Network formation of Collagen IV

William Yost, Aratrika Pan, Sahanna Shanker, Alexa Hayes

The Ohio State University

ABSTRACT BODY: Collagen type IV is an influential component of the basement membrane underlying the endothelial and epithelial cells. It has a profound effect on the structure and function of the basement membranes through its network lattice. The ionic composition of the body fluids and interstitial fluids traversing through the basement membrane can affect the mechanical properties of the underlying Col IV network. The purpose of this study is to understand the change in flexibility (persistence length, LP) of Col IV molecules as a function of ionic strength. Toward this goal, we obtained atomic force microscopy (AFM) images of Col IV under low and high molarity phosphate buffer saline (PBS). We utilized the 2D-wormlike chain model (2D-WLC) to track the contour of individual molecules in AFM images and determine LP. The thickness (height) of individual molecules was also ascertained from AFM topographical images. Our results elucidate how both the height and LP of Col IV molecules increased with ionic concentration. In addition, the Col IV network morphology was different across the two ionic strengths. Our results provide novel insights on the influence of ionic strength on Col IV and can be used to examine the properties of different Col IV heterotrimers in health and disease.

Dual-Cellular 3D Bioprinting of Corneal Stroma and Endothelium Using Collagen I and IV Bioinks

Jingjing You¹, Yunong Yuan², Gerard Sutton^3,4, Jingjing You^2,3

¹School of Medical Science, University of Sydney, ²School of Medical Sciences, Sydney Biomanufacturing Incubator, University of Sydney, ³Save Sight Institute, University of Sydney, ⁴Vision Eye Institute

ABSTRACT BODY: Corneal transplantation is the most commonly performed form of transplantation using donor tissue and remains the gold standard for restoring vision in patients with corneal blindness. However, a global shortage of donor corneas has left an estimated 12 million individuals awaiting transplantation. Bioengineering human corneas offers a promising alternative. The human cornea consists of three major layers—epithelium, stroma, and endothelium—with the stroma forming the bulk of the tissue. Current 3D bioprinting approaches either print corneal layers in flat geometries or struggle to achieve high transparency and multicellular viability in curved, multilayered constructs.To address these limitations, we leveraged our previously developed neutral pH collagen I (Col-I) and collagen IV (Col-IV) bioinks, which can dissolve up to 12 mg/mL of collagen without precipitation and allow for cell encapsulation. This study aimed to dual-print human corneal stroma and endothelium using extrusion-based 3D bioprinting with Col-I and Col-IV bioinks. Throughout the culture period, we assessed cell viability and proliferation, and performed immunostaining to verify cell identity and morphology. Confocal microscopy was used to evaluate the 3D architecture, curvature, and surface smoothness. Optical clarity was measured using light transmission assays. Scanning electron microscopy (SEM) was performed to visualize microstructural features and collagen fibre orientation. Mechanical properties were evaluated using compression testing to monitor changes in stiffness over time.Our results demonstrated successful fabrication of a dual-layered, cell-laden corneal construct that maintained &gt;80% cell viability. Endothelial cells formed a confluent monolayer with characteristic hexagonal morphology, while stromal cells were evenly distributed throughout the Col-I layer. Immunostaining confirmed cell-specific marker expression: ZO-1 and Naâ º/Kâ º ATPase for endothelial cells, and Keratocan and Lumican for stromal cells, appropriately localized to each layer. The constructs preserved curvature and surface smoothness, and optical clarity remained high (&gt;80%) throughout the culture period. No delamination was observed between the printed layers. SEM revealed distinct fibre orientations between Col-I and Col-IV layers. Mechanical testing showed stable Young’s modulus over 21 days, but the overall stiffness remained significantly lower than that of native corneal tissue.This study addresses a critical challenge in corneal bioengineering: replicating the native curvature, multilayered architecture, and cellular composition of the human cornea while preserving optical clarity. Through the integration of Col-I and Col-IV bioinks and extrusion-based 3D printing, we successfully fabricated a dual-layer construct that supports high cell viability, spatial localisation, and morphological fidelity. The printed tissues closely mimic the stromal–endothelial interface and maintain curvature and transparency over time, offering a promising platform for drug testing and disease modelling. However, the overall mechanical strength remains lower than that of native cornea, indicating the need for further optimisation of bioink formulations and crosslinking strategies to enable potential applications in corneal transplantation.

FUNDING SOURCES: NHMRC Idea Grant, MRFF Frontier Health and Medical Research Initiative.

Th2-like exTregs Contribute to Th2 Expansion in ECM-treated Muscle Wound

Haoning Yu¹, Jordan Garcia², Anna Ruta¹, Jennifer Elisseeff¹

¹Johns Hopkins School of Medicine, ²Vanderbilt University Medical Center

ABSTRACT BODY: Treatment of chronic wounds following trauma, infection, or disease remains an unmanageable clinical challenge with few innovations in regenerative medicine have reached the clinical standard of care. Decellularized extracellular matrix (ECM) scaffolds have emerged as promising biomaterials that not only provide a three-dimensional framework for structural support and tissue organization, but also actively modulate the immune response to promote tissue regeneration while minimizing inflammation. Notably, ECM scaffolds promote a type 2, pro-regenerative immune environment characterized by increased recruitment of T helper 2 (Th2) cells and eosinophils. However, the mechanisms by which ECM scaffolds shape T cell phenotypes and clonal dynamics remain poorly understood. This study aims to define the transcriptional states and clonal relationships of T cells in ECM-modulated muscle wounds using integrated single-cell RNA and T cell receptor (scRNA/TCR) sequencing. To investigate the underlying T cell dynamics, we performed 5′ paired scRNA/TCR sequencing of ECM-treated muscle wounds. ECM scaffolds were implanted into a murine model of volumetric muscle loss (VML), and quadriceps muscles were harvested at 1 week post-injury. CD3⁺ T cells were isolated from wounded tissue using fluorescence-activated cell sorting (FACS) following enzymatic digestion and staining for live CD45⁺CD11b⁻CD3⁺ cells. Single-cell libraries were prepared using the 10x Genomics Chromium 5′ V(D)J kit, and sequencing data were processed using Cell Ranger and downstream analysis was performed using Seurat and scRepertoire. To validate lineage relationships in vivo, we used a triple-reporter mouse model (Foxp3eGFP-Cre/eGFP-Cre Rosa26YFP/YFP IL13TdTomaot/WT) to simultaneously fate-map regulatory T cells and identify IL-13–producing Th2 cells by flow cytometry.Using scRNA-seq, we found that ECM scaffolds promoted expansion of both regulatory T cells (Tregs) and Th2 cells 1 week following injury and scaffold implantation. Notably, both subsets exhibited elevated expression of immune checkpoint receptors LAG3 and CTLA4, suggesting they may arise from a common precursor population and acquire features of exhaustion or regulation during the healing process. To assess their lineage relationships, we integrated transcriptional profiles with paired αβ T cell receptor (TCR) sequences and applied RNA velocity analysis to infer differentiation trajectories. We identified Treg clones that were transcriptionally aligned with multiple effector T helper subsets, including Th1, Th2, and Th17 cells, suggesting considerable lineage plasticity. Among these, Tregs and Th2 cells shared the greatest number of clonotypes across all ECM-treated biological replicates. Furthermore, RNA velocity vectors revealed a clear directional flow from Tregs toward a highly polarized Th2 state. These findings suggest that both checkpoint-high Tregs and Th2 cells originate from a common Treg precursor population that transitions toward a Th2 phenotype in the ECM-modulated environment. To validate this transition in vivo, we studied the fate of Tregs using a triple-reporter mouse model that enables tracking of IL-13 expression—a Th2-specific cytokine—specifically in Treg lineage cells. We found that ECM treatment led to an approximately eightfold increase in IL-13–producing ex-Tregs compared with saline controls, confirming that a subset of Tregs adopts a Th2 phenotype and contributes to IL-13 production in ECM-treated muscle wounds.Together, these findings uncover a novel immunological mechanism by which Th2 cells involved in ECM-driven tissue repair may arise from Tregs. The co-expression of checkpoint molecules on these cells suggests a potential therapeutic opportunity to augment regenerative outcomes through immune checkpoint inhibition.

Perlecan-Conjugated Laminin Fragment as a Next-Generation Culture Substrate For Human Pluripotent Stem Cells

Kohei Minobe¹, Yasuhiro Shimizu^1,2, Kakeru Sugiyama¹, Taiko Kunieda¹, Philipp Boder³, Aisha Amari³, William Hadlington-Booth³, Alex Sim³, Yukimasa Taniguchi^1,2, Kiyotoshi Sekiguchi^1,2

¹Matrixome Inc., ²Institute for Protein Research, Osaka University, ³Amsbio LLC, Cambridge MA USA

Introduction: Cells in our body need to attach to the basement membrane, which mainly consists of laminin, collagen IV, nidogen and heparan sulfate (HS) proteoglycan (e.g. perlecan). Laminin and perlecan are key components to provide essential cues for stem cell maintenance and function. Laminin binds integrins to regulate cell survival, proliferation, and differentiation, while perlecan captures growth factors via HS chains attached to its N-terminal perlecan domain I (PDI), modulating growth factors availability to cell surface receptors.

Aim: Given the cooperative signalling between integrins and growth factor receptors, a culture substrate integrating both laminin’s adhesive properties and perlecan’s growth factor-binding capacity is highly desirable.

Methods: We engineered a recombinant laminin E8 fragment conjugated with perlecanʼs HS-rich PDI domain, which was produced in CHO-S cells and purified via a series of chromatographic steps.

Results: The resulting chimeric protein, named perLAM, fully retains both the integrin-binding activity of laminin and the growth factor-binding activity of perlecan.

Conclusion: This study highlights the use of perLAM as a “next-generation culture substrate” for stem cell culture & differentiation in regenerative medicine & tissue engineering using ES/iPS cells.

Sex-specific Differences in Cellular Response to Matrix Stiffness and Composition

Aude Angelini¹, Stefano Serpelloni^2,3, Federica Banche-Niclot^2,3, Francesca Taraballi^2,3, Alexander Saltzman^4,5, Anna Malovannaya^4,5, George E. Taffet^1,6, Katarzyna A. Cieslik¹.

¹ Department of Medicine, Houston Methodist Hospital and Houston Methodist Research Institute, ²Center for Musculoskeletal Regeneration, Houston Methodist Academic Institute, Houston Methodist, ³Orthopedics and Sports Medicine, Houston Methodist Hospital, Houston Methodist, ⁴Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, ⁵Mass Spectrometry Proteomics Core, Baylor College of Medicine, ⁶Section of Geriatrics and Palliative Medicine, Department of Medicine, Houston Methodist Hospital

ABSTRACT BODY: Left ventricular stiffening represents a main feature of cardiac aging that can lead to diastolic dysfunction and further to heart failure with preserved ejection fraction (HFpEF). Unlike men, older women have a higher incidence and prevalence of HFpEF.

Changes affecting myocardial extracellular matrix (ECM) viscoelastic properties are suspected to play a critical role in sex-specific cardiac aging phenotype and HFpEF progression. Yet, the underlying mechanisms remain unclear.

In this study, we aim to decipher the respective influence of ECM stiffness and biochemical composition on cardiac aging, using intervention-free young (3 months-old) and old (21-24 months-old) C57BL/6J male and female mice.

First, while Young’s Modulus measurements showed an increased stiffness in old (25-31 KPa) versus young heart (8-12 KPa) but no significant difference between male and female hearts, additional findings from echocardiography parameters, immunostaining of degraded/total collagens and hydroxyproline assay (mature/crosslinked collagens) all suggested that the old female cardiac ECM is intrinsically more prone to crosslinking and remodeling than the old male cardiac ECM. Proteomics analysis also notably highlighted an enrichment of collagens and integrins in old female and old male hearts/ECM respectively.

Then, to evaluate the part of ECM changes driven by cardiac fibroblasts (CFs), the main ECM producers and safekeepers, mouse hearts from young and old mice (both sexes) were enzymatically digested, and CFs (CD45^negCD31^negPDGFRa⁺) isolated, sorted and analyzed by mass spectrometry. Proteomics analysis identified pathways and proteins that are respectively enriched in old female CFs (fibronectin formation; collagens; prolyl-4-hydroxylases) and in old male CFs (ECM glycoproteins, integrin signaling; Vitronectin)

At the ECM-CF interface, integrins α and β subunits dimerize, orchestrating ECM “sensing” and CF signaling response. Our proteomics dataset had consistently highlighted differences in integrins, and notably integrin αV and β1. Integrin αVβ1 dimer can mediate TGF-β-dependent fibroblast activation, a process that we previously reported to be impaired in old male CFs. Ex vivo integrin activation status and ECM binding capacities were assayed by flow cytometry, using an antibody targeting IntαVβ1 dimer and a probe specific to an ECM ligand of IntαVβ1 (Fibronectin (FN)). We identified two distinct CF populations with active integrins that can else dimerize (Int+) and bind to ECM ligand (Int+FN+) or dimerize but not bind to ECM ligand (Int+FN^neg). Intriguingly, while there was no difference among the groups regarding the Int+FN+ population, an Int+FN^neg subpopulation of old male CFs was resistant to chemically induced integrin inhibition. This observation suggests that part of old male CFs may be able to maintain a set of “locked-in” activated integrin dimers in-situ.

To study the ECM-to-integrin response, CFs were then cultured on different collagen-made supports that mimic the stiffness of young or old cardiac ECM; else 2D hydrogels or 3D scaffolds.

On a 2D young-like (8KPa) hydrogel, IntαV expression was lower in old male CFs, but not in old female CFs. Yet, coating hydrogels with Vitronectin alongside collagen had no influence on integrin expression. In a 3D environment similarly, there was no significant change in the level of integrins in response to stiffness. However, female CFs, exhibited a higher expression of procollagen 1, regardless their age or scaffold stiffness. These observations aligned with our in vivo results and suggested that female CFs are more prone to initiate an intensive ECM remodeling.

In conclusion, our study suggests that ECM stiffness has a stronger impact on CFs phenotype than composition. Old male CFs maintain a “lock-in” integrin activation status, while old female CFs preserve a more flexible integrin response but a higher trend to initiate ECM remodeling.

FUNDING SOURCE (optional): This work was supported by the NIH Grants AG080925, AG059599 and the Medallion Foundation.

Automated Semi-manual Quantification of Collagen and Elastic Fiber Ultrastructure in EM Using Custom Fiji Macros: A Proof of Concept

Apoorva Bhandari MSc, MD¹, Sara Tufa², Nobuyo Mizuno¹, Lynn Y. Sakai PhD^1,3, Sherene Shalhub MD, MPH¹, Douglas R. Keene^2,3

^¹Division of Vascular Surgery, Oregon Health & Science University, Portland, ²Shriners Children’s Hospitals, ³Molecular and Medical Genetics, Oregon Health & Science University

Background: Quantitative analysis of extracellular matrix (ECM) ultrastructure in electron microscopy (EM) is limited by inconsistent manual workflows and the absence of reproducible, open-source tools. Automated segmentation methods often fail in noisy or irregular images. To address this gap, we developed two Fiji/ImageJ (v1.54p) macros for semi-manual, standardized quantification of collagen fibril diameters and elastic fiber fragmentation in EM images of human dermis.

Methods: Both tools were written in the ImageJ IJ1 macro language. The Collagen Diameter Macro enables user-guided wand selection of fiber cross-sections, automatically reporting area-equivalent diameter (EqDiam), best-fit ellipse axes (Major/Minor), and quality metrics including AxisRatio and EqDiff_% to identify true cross-sections (EqDiff_% ≤10%). A quadrant tag and on-image overlays (ROI outline and axes) provide immediate validation.

The Elastic Fiber Fragmentation Macro quantifies grayscale heterogeneity within freehand Regions of Interest (ROIs) after fixed 8-bit mapping (MAP_MIN/MAX). Pixels are tri-classified as Dark, Mid, or Light using default thresholds (LOW_BIN=90, HIGH_BIN=200). Derived indices include %Light (primary fragmentation metric), Frag_Mode_%, Frag_Extremes_%, and Frag_Entropy3 (0–1). Both macros append results to standardized tables with metadata fields (macro version, operator, calibration, quality-control flags) to ensure reproducibility.

Results: Pilot testing across representative EM images confirmed reliable performance under variable contrast conditions. Quality-control thresholds (AxisRatio ≤1.15, EqDiff_% ≤10%, ClipLow/High <2%) yielded ≥90% valid ROIs. Inter- and intra-operator trials demonstrated <5% variation in EqDiam and fragmentation indices, meeting predefined reproducibility targets. All outputs were directly exportable for downstream statistical analysis.

Conclusion: This dual-macro framework establishes a reproducible, transparent approach for ECM morphometric analysis from EM images. The macros provide a validated, scalable proof-of-concept for ultrastructural phenotyping of collagen and elastic fibers in connective tissue disease research.

Quantitative EM Analysis of Dermal Collagen and Elastic Fibers Using Custom Fiji Macros: Distinct Extracellular Matrix Architecture Revealed in Vascular Ehlers–Danlos Syndrome

Apoorva Bhandari MSc, MD¹, Nobuyo Mizuno¹, Sara Tufa², Douglas R. Keene^2,3, Lynn Y. Sakai PhD^1,3, Sherene Shalhub MD, MPH¹

¹Division of Vascular Surgery, Oregon Health & Science University, Portland, ²Shriners Children’s Hospital, ³Molecular and Medical Genetics, Oregon Health & Science University,

Background: The ultrastructural abnormalities of the extracellular matrix (ECM) in vascular Ehlers–Danlos syndrome (vEDS) reflect intrinsic substrate vulnerability that may parallel vascular wall fragility. While ultrastructural scoring systems have been described previously, quantitative assessment has been limited by the lack of standardized tools. We developed validated Fiji/ImageJ macros for semi-manual, standardized quantification of collagen fibril diameters and elastic fiber fragmentation in transmission electron microscopy (TEM) images of human dermis. This proof-of-concept and validation study applies these tools to a small cohort of vEDS and control skin biopsies to test feasibility for quantitative ECM assessment, with the goal of correlating ultrastructural changes with clinical severity and integrating tissue-level data into the Aortic and Arterial Vulnerability Spectrum (AAVS) framework for biologically informed risk stratification.

Methods: TEM images from GR008 (26-year-old male, COL3A1 c.1347+1G>A, splice donor), GR003 (48-year-old male, COL3A1 c.852+1G>A, p.Ala280*, null variant), and age- and sex- matched controls were analyzed. Collagen fibril diameters were measured using vEDS_Wand_VALIDATE_Diameter (EqDiam, AxisRatio, EqDiff_%). Elastic fiber fragmentation was assessed using Elastin_ROI_darklight (%Light). Analyses were performed separately for papillary and reticular dermis with standardized quality-control thresholds (EqDiff_% ≤10, AxisRatio ≤1.15).

Results: Across over 6,000 collagen fibrils analyzed, papillary dermis in GR008 exhibited a left-shifted distribution with smaller mean diameters (0.050 ± 0.007 µm) compared with GR003 (0.058 ± 0.009 µm) and controls (0.074 ± 0.009 µm). In the reticular dermis, GR008 (n=3767) exhibited larger and more heterogeneous fibrils (0.079 ± 0.015 µm) than C003 (n=916, 0.059 ± 0.008 µm) but smaller fibrils than GR003 (n=653, 0.112 ± 0.010 µm), indicating altered organization across dermal layers. Elastin analysis (49 total fibers) revealed increased fragmentation and grayscale heterogeneity in GR008 (%Light 12.7 ± 12.4) compared with GR003 (22.1 ± 11.3) and controls (8.3 ± 4.6). The null-variant GR003 displayed intermediate collagen and elastin morphology, suggesting partial preservation of matrix architecture.

Conclusions: This proof-of-concept validation study demonstrates the feasibility of custom Fiji macros for reproducible quantitative EM analysis of dermal ECM ultrastructure. Variant-specific collagen and elastin signatures in vEDS reflect differential substrate vulnerability and support expansion to larger cohorts for clinical correlation and integration into the AAVS framework for biologically informed risk stratification.

Collagen I Promotes Bladder Cancer Growth through GCN2-Dependent Integrated Stress Response

Mark E ALONZO, Ethan J SUBEL, David F CHANG, Khanh M CHAU, Grace L WONG, Beth GAO, Stephen QR WONG, Yung Hsing HUANG, Sung Yun JUNG, Fotis NIKOLOS, Keith S CHAN

Houston Methodist Research Institute

ABSTRACT:

Bladder cancer (BLCA) is the sixth most common cancer in the United States and has a high recurrence rate despite recent therapies. Excess extracellular matrix Collagen I (Col I) is associated with poor survival and drives pro-tumorigenic processes, including immune evasion and metastasis. Col I also restricts nutrient and oxygen availability, causing metabolic stress that activates the integrated stress response (ISR), which suppresses global mRNA translation and promotes cell survival. How Col I triggers ISR in BLCA remains unclear. In this study, we show that Col I directly activates the ISR to promote BLCA tumorigenesis. Col I rapidly suppresses protein translation in vitro while increasing spheroid size and tumor growth in vivo. Mechanistically, Col I binds and activates DDR1 phosphorylation at tyrosine 513, leading to intracellular amino acid depletion via downregulation of the transporter LAT1. Amino acid deprivation activates the GCN2–EIF2α axis, triggering the ISR. Pharmacological inhibition of GCN2 blocks ISR activation, restores translation, and reduces Col I–driven spheroid and tumor growth. Clinically, TCGA data show high ISR gene expression and GCN2 signaling correlate with elevated Col I and poor survival. These findings reveal a mechanism by which Col I activates the ISR to drive BLCA progression and identify the LAT1–GCN2–ISR axis as a potential therapeutic target.

Aging-Associated Extracellular Matrix Remodeling in Healthy Primary Breast Adipose Tissue

Mackenzie L. Hawes¹, Katherine L. Herbert¹, Delia Carlino¹, Bruce A. Bunnell², Bridgette M. Collins-Burow¹, Van T. Hoang^1,3, Jorge A. Belgodere^1,3,4, Elizabeth C. Martin^1,3, Matthew E. Burow^1,3

¹Department of Medicine, Section of Hematology and Oncology, Tulane University School of Medicine, ²Department of Microbiology, Immunology and Genetics, University of North Texas Health Science Center, ³Tulane Cancer Center, Tulane University, ⁴Department of Biological and Agricultural Engineering, Louisiana State University and Agricultural Center

ABSTRACT BODY: The extracellular matrix (ECM) is a dynamic regulator of breast tissue homeostasis and disease. Across the lifespan, the composition of the breast is restructured, driving shifts in ECM architecture and deposition. In the aged breast, ECM is replaced by expanding white adipose tissue (WAT) through the process of involution. In aged mouse mammary tissues, this process is associated with sparse deposition of thin and curvy collagen fibers as well as an environment rich in fibrillin and poor in collagen types I, V, and XV. While the characteristics of the aged matrisome have been studied in vivo, tissues from mice are physiologically distinct from human tissue. There has yet to be an analysis of the aged human breast adipose tissue, highlighting the need for primary models of ECM and breast aging. To address this need and to identify potential biomarkers of abnormal breast aging, we performed protein profiling of healthy breast adipose tissue from aged (>50) and young (<40) women, combined with analyses of ECM and tissue architecture. Contrary to existing findings, we saw no difference in total collagen composition via Masson’s trichrome staining and rheological measures of tissue stiffness between the aged and young tissues. However, proteomic analysis revealed a decline in collagen types I, IV, V, and XV with age, consistent with prior analyses in mouse mammary tissue. In the aged breast adipose there was additionally a decrease in POSTN expression, a matricellular protein required for the wound healing response and collagen synthesis. Taken together these data demonstrate that the ECM in the breast adipose is age-specific and that shifts collagen content and ECM regulators, such as POSTN, may dictate differential susceptibility to breast disease with age.

β2 Laminins in Optic Chiasm Development and Retinal Ganglion Cell Axon Routing

Alanis Hernández-Arce, Madeline Turo, Adam N. Robinson, Skylyn McNamara, Danny Yeo, and Reyna I. Martínez-De Luna

Department of Ophthalmology & Visual Sciences, Upstate Medical University, Syracuse, NY

ABSTRACT BODY:

Retinal ganglion cells (RGCs) are the output neurons of the retina. RGCs collect visual information and transmit it to the brain through their axons that collectively form the optic nerves and tracts. During formation of the visual pathway, RGC axons are selectively routed to the correct side of the brain. The selective routing of RGC axons is directed by radial glia and midline neurons of the optic chiasm. We found that genetic deletion of β2 laminins increases the proportion of RGC axons routed to the ipsilateral tract. Here we asked if genetic deletion of β2 laminins disrupt the organization of the radial glia and midline neurons in the optic chiasm and if the laminin receptor Dystroglycan (Dag) mediates these effects. Loss of β2 laminins resulted in a fragmented pial basement membrane (PBM) and radial glia basal processes that were undulated and, in some cases, terminated early before contacting the PBM. The Lamb2^-/- radial glia also had poorly elaborated endfeet. The expression of β-Dag and β1-integrin was reduced in the radial glia, likely contributing to the basal process and endfoot defects. Conditional deletion of Dag specifically in radial glia resulted in similar defects as in the Lamb2^-/- optic chiasm. Many of the basal processes terminated early and did not contact the PBM and the endfeet lacked their typical morphology. In addition to the defects in the radial glia we found that the Lamb2^-/- chiasm has a reduced number of midline neurons. Our results suggest that β2 laminins organize the cells of the optic chiasm required for normal RGC axon routing.

FUNDING SOURCES: National Institutes of Health, National Eye Institute Grant R01 EY034653, Research to Prevent Blindness Career Development Award and departmental unrestricted grant, Fight for Sight Grant-in-Aid.

Designing a Focused Ultrasound Crosslinkable Scaffold for Tissue Regeneration and Chemotherapeutic Drug Delivery

Malik Hickman, Estelle He, Remington Martinez, Natasha Claxton, Richard Price, Matthew DeWitt, Chris Highley

University of Virginia Department of Biomedical Engineering, University of Virginia Department of Chemical Engineering

ABSTRACT BODY:

Hydrogel biomaterials can be designed to replicate critical features of the extracellular matrix that support tissue regeneration, and these materials have been used to support regeneration in tumor cavities^1,2. Recent work from our group involves the development of highly porous hydrogel scaffolds that support vascularization³. We have demonstrated that these scaffolds can be engineered for minimally-invasive delivery by injection, stabilization by non-invasive focused ultrasound (FUS), and controlled release of anticancer drugs⁴. This material system is a granular hydrogel that, compared to traditional continuous hydrogels, facilitates injectable delivery and stability upon injection that can be enhanced by additional interparticle crosslinking⁵. Additionally, the hydrogel microparticle components of our material are stabilized by hydrogel nanofibers, which allow for increased microporosity within the scaffold to support tissue regrowth.

A key innovation is the design of the system to support crosslinking by a spatiotemporally controlled FUS stimulus as opposed to traditional external stimuli, such as light in photoinitiated crosslinking⁶, which may not be able to sufficiently penetrate the tissue site to trigger hydrogel crosslinking in clinical applications^6,7. FUS applies a non-invasive, focused beam of ultrasound waves to a target site, depositing energy that can be used to ablate cancerous tissue⁸ or induce crosslinking⁴. Additionally, the physical response of a hydrogel to FUS sonication can be designed to support controlled release of entrapped payloads, such as chemotherapeutic drugs⁹.

Here, we present the development of a fiber enhanced microporous (FEMP) scaffold designed from norbornene-modified hyaluronic acid (NorHA) microgels and electrospun NorHA nanofibers with porosity specified by sacrificial gelatin microgels. This new material formulation leverages native extracellular matrix-derived materials and is designed with high porosity to support dynamic cellular behaviors that facilitate regeneration. Additionally, we assess ultrasound-responsive payload delivery towards applications in regenerating tissue and delivering chemotherapeutics in cancer therapies. To enable crosslinking of the NorHA components, a radical initiator, potassium persulfate (KPS), and a multi-armed poly(ethylene glycol) (PEG)-thiol are included in a precursor solution, allowing thiol-end reactions under FUS sonication. A thermoresponsive polymer, poly(di(ethylene glycol) methyl ether methacrylate (PDEGMA), is also included as an acoustic absorber to support FUS-initiated crosslinking by increasing the viscosity of the precursor to slow convective flows upon sonication-induced heating. Fluorescently-labeled dextrans are used as a model for drug payloads with higher

Mechano-Immune Regulation of LRRC15+ Cancer-associated Fibroblasts

Yiyue Jia^1,2, Xiongfeng Chen^1,2, Shunheng Liu², Zhuan Zhou^1,2, Alex C. Kim^1,2, Huocong Huang^1,2,3

¹Department of Surgery, University of Texas Southwestern Medical Center, ²Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, ³Department of Immunology, University of Texas Southwestern Medical Center

ABSTRACT BODY:

Cancer-associated fibroblasts (CAFs) have been attractive targets for improving cancer therapies due to their various functions, including the synthesis and modification of the extracellular matrix, complex signaling with cancer cells, and interactions with infiltrating immune cells. However, the diversity and plasticity of CAFs present significant challenges in developing effective CAF-targeted treatment. Here, we integrated single-cell RNA sequencing (scRNA-seq) data from 15 types of human cancers and genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDAC) to construct a comprehensive fibroblast molecular atlas. The analysis unexpectedly revealed a distinct myofibroblastic CAF (myCAF) population marked by leucine-rich-repeat-containing protein 15 (LRRC15+), which is evolutionarily conserved across most cancer types. Spatial niche analysis from human PDAC tissues demonstrated that LRRC15+ myCAFs predominantly localize to extracellular matrix (ECM)-dense, immune-excluded regions. Trajectory analysis revealed that resident fibroblast progenitors differentiate into LRRC15+ myCAFs through a TGFβ-driven signaling axis. Notably, mechanotransduction pathways were significantly upregulated in LRRC15+ myCAFs, with PIEZO2 emerging as the most differentially expressed mechanosensor among all cell surface mechanosensors. In vitro TGFβ stimulation induced upregulation of PIEZO2 and LRRC15 in pancreatic stellate cells (PSCs). Furthermore, TGFβ-induced PIEZO2 can be activated in PSCs while being cultured in a plate recapitulating the stiffness of pancreatic tumor. Strikingly, in mouse models of pancreatic cancer, we found CAF-specific PIEZO2 knockout resulted in a reduction of LRRC15+ myCAF differentiation and significant increase of CD8+ T cell infiltration. Collectively, our findings establish that PIEZO2-mediated mechanotransduction is essential for the formation and the immunosuppressive function of LRRC15+ myCAFs, serving as a potential therapeutic target to reprogram the fibrotic tumor microenvironment and enhance immunotherapy efficacy in cancer.

BIGH3 Is a Mediator of TGFβ-Induced Collagen Formation in Fibrosis and Pancreatic Cancer and a Potential Therapeutic Target

Morten A. Karsdal, Kim Henriksen, Cecilie Bager, Khaled E. Mohamed, Diana J. Leeming, Mark Alexander Skarsfeldt, Rasmus S. Pedersen, Nicholas Willumsen

Nordic Bioscience

Background: Fibroblast-driven deposition of extracellular matrix, particularly type III collagen, is associated with poor outcomes in patients with liver fibrosis, fibrotic solid tumors such as hepatocellular carcinoma and pancreatic cancer, and other fibrotic diseases. The pro-peptide of type III collagen (PRO-C3) serves as a serum biomarker of active collagen formation and is elevated and prognostic in patients with liver fibrosis and cancer. Understanding the mechanisms driving elevated PRO-C3 could reveal novel therapeutic targets for fibrosis and cancer.

Methods: We performed a genome-wide association study (GWAS) in the PERF cohort (n = 4,968) to identify genetic variants associated with circulating PRO-C3 levels. Findings were validated through analyses of serum samples from patients with pancreatic ductal adenocarcinoma and liver fibrosis. Functional studies were conducted in fibroblast models to assess the effect of candidate genes on PRO-C3 production, and an anti-BIGH3 antibody was used to determine causal relationships. Expression analyses were performed to characterize the cellular sources of BIGH3 and its regulation by TGFβ.

Results: GWAS identified variants in the gene TGFBI (BIGH3/βigH3) as significantly associated with elevated circulating PRO-C3 levels. Serum analyses confirmed strong correlations between PRO-C3 and BIGH3 in patients with pancreatic cancer and liver fibrosis. In vitro, BIGH3 induced PRO-C3 production in a dose-dependent manner, which was blocked by an anti-BIGH3 antibody. BIGH3 expression was detected in macrophages and fibroblasts, particularly under TGFβ stimulation, suggesting that BIGH3 acts as a downstream mediator of TGFβ-induced fibrosis. In a TGFβ-driven in vitro fibrosis model, inhibition of BIGH3 reduced PRO-C3 levels.

Conclusion: BIGH3 is a key mediator of TGFβ-induced fibrosis and contributes to elevated PRO-C3 levels in fibrotic diseases and cancer. Targeting BIGH3 may represent a novel therapeutic strategy for pathological fibrosis and cancer, particularly in patients exhibiting high PRO-C3 levels.

Cytokine-Induced Thy-1 Loss in Fibroblasts Is Mediated by DNA Methylation

Mathew Kibet, Daniel Abebayehu*

Department of Biomedical Engineering, University of Virginia.

*Corresponding author

ABSTRACT BODY:

Fibrosis is a maladaptive wound-healing response characterized by excessive extracellular matrix deposition and tissue stiffening, ultimately leading to organ dysfunction. Our previous work has demonstrated that inflammatory cytokines, particularly interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), promote the emergence of Thy-1–negative fibroblasts, a hallmark of the pro-fibrotic phenotype. Thy-1 (CD90) is a glycophosphatidylinositol (GPI)-anchored surface glycoprotein that serves as a rigidity-sensing molecule regulating stiffness-dependent activation of fibroblasts. Evidence has shown that Thy-1 expression is significantly reduced in fibrosis across multiple organ systems, including the lungs, liver, kidneys, skin, and heart. In healthy tissue, Thy-1 is highly expressed on the surface of fibroblasts, where it acts. Loss of Thy-1 expression shifts fibroblasts toward a contractile, myofibroblast-like state that drives fibrosis progression. However, the actual molecular mechanism underlying Thy-1 loss remains unknown. Previous work by others has implicated DNA methylation as well as shedding of Thy-1 onto extracellular vesicles (EV) as the mechanisms of Thy-1 loss. Our previous work has shown that Thy-1 loss happens at the transcriptional level, indicating that DNA methylation is a more likely mechanism. Therefore, we hypothesize that cytokine-induced Thy-1 loss in fibrosis is driven by epigenetic silencing through DNA methylation.

Methods and Results: We sought to determine the mechanism underlying cytokine-induced Thy-1 loss in fibroblasts by investigating whether DNA methylation or shedding onto EVs mediates this effect. Previous studies have implicated methylation of the Thy-1 promoter; however, it remains unclear whether this mechanism is involved with cytokine-induced Thy-1 silencing. To test this, we treated human fibroblasts with zebularine, a DNA methyltransferase (DNMT) inhibitor and vehicle (DMSO) for 48 hours, followed by stimulation with IL-1β and TNFα (100 ng/mL each). After 72 hours, we assessed Thy-1 expression via flow cytometry. We observed that zebularine treatment was able to inhibit cytokine-induced loss of Thy-1, indicating that Thy-1 downregulation occurs through DNA methylation-dependent. We next examined whether Thy-1 is shed onto EVs. We collected EVs from fibroblast cultures treated with IL-1β and TNFα and performed nanoparticle tracking (NTA) to get the concentration of EVs. We then analyzed them using small particle flow cytometry to detect Thy-1 expression on their surface. Interestingly, we did not detect Thy-1 on EVs following cytokine stimulation, suggesting that cytokine-induced Thy-1 loss does not occur through vesicle shedding. Collectively, our findings demonstrate that cytokine-induced Thy-1 loss in fibroblasts is mediated by DNA methylation rather than extracellular vesicle release, highlighting epigenetic regulation as a key mechanism driving Thy-1 silencing in fibrosis.

Future Directions: To further elucidate epigenetic regulation, we are currently conducting ongoing studies using long-read sequencing technologies (Oxford Nanopore and PacBio HiFi) to map methylation patterns across the Thy-1 promoter and enhancer regions at single-base resolution. By integrating molecular and epigenetic analyses, this study seeks to uncover the key regulatory pathways responsible for this transcriptional repression of Thy-1. Ultimately, these insights will provide a mechanistic framework for targeting cytokine-induced Thy-1 loss in fibrotic diseases.

Mechanial Cues as Functional Markers for 3D in Vitro Models

Kyle McGuire, Malin Becker, Massimilliano Berardi, Elizaveta Loseva, Svetlana Pasteuning, Giulia Pilia

Optics11 Life

ABSTRACT BODY:

Introduction

Cellular behavior is dictated by its environment - a combination of soluble and immobilized signaling molecules, matrix composition and architecture, as well as physical properties of both cells themselves, and their surrounding matrix (1). Amongst the latter, viscoelastic properties of the microenvironment have been shown to play a central role in health and disease (1). For example, matrix viscoelasticity promotes liver cancer progression (2), altered matrix stiffness leads to fibroblast activation, excessive ECM deposition, and reduced treatment efficacy in fibrosis (4) in many organs.

Despite the importance of such cues, the field lacks a feasible method to measure these quantities in a way that is (i) compatible with the variety of 3D in vitro models, (ii) localized, i.e. providing mechanical cues perceived at the cell scale, and (iii) amenable to large scale screening, for both biomaterial optimization and preclinical drug research. Here, we show how the Pavone nanoindentation platform can be leveraged at every step of a bottom-up tissue engineering and screening workflow, from native tissue testing to model engineering and in-vitro functional screening, to provide design guidelines for better in vitro models and functional markers of disease and treatment efficacy.

Materials and Methods

We present a workflow based on the Pavone nanoindenter. This starts with the characterization of the native tissue to be replicated in vitro. The ability to mechanically map properties in space provides valuable information in terms of absolute viscoelastic values, their distribution and variability. These represent the target values to obtain for instance via 3D bioprinting. In this step, the nanoindentation assay can be used for QC of both mechanical parameters and morphology, via analysis of topography and correlation with imaging. Once these targets have been reached, nanoindentation can be used to assess stability of the constructs in time, cellular action (for instance in the form of ECM deposition or remodeling), as well as to evaluate the mechanical effect of administered drugs.

Conclusions

Mechanical properties play a central role in many diseases such as fibrosis and bears relevance at the patient level, as exemplified by the use of elastography methods to detect liver scarring or gut fibrosis (4). The ability to characterize the mechanical properties at multiple scales and levels of model complexity, with the same device, represent a fundamental step forward towards more physiological models, with higher predictive power thanks to more comprehensive readouts.

The compatibility of the approach with standard formats such as 96 or 384 wellplates, as well as the label-free and non-destructive nature of the assay make it a powerful addition to preclinical workflows and model development.

Active Mechanosensing in Lymph Node Fibroblastic Reticular Cells as a Therapeutic Lever to Restore Immune Tolerance In Autoimmune Diabetes

Marvin Mendoza^1,2, Leonor N. Teles,^2,3 Alice A. Tomei^1,2,3

¹Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, ²Diabetes Research Institute, Miller School of Medicine, University of Miami, ³Department of Biomedical Engineering, University of Miami, Miami, Florida

ABSTRACT BODY:

Type 1 diabetes (T1D) is an autoimmune disease that affects 1.84 million people in the US and emerges when immune tolerance fails in central and peripheral lymphoid organs, promoting autoreactive T cell destruction of pancreatic β-cells. There are no treatments for T1D prevention, in part because T1D pathophysiology mechanisms are not completely understood. Identifying new mechanisms of immune dysregulation could lead to the identification of new targets for T1D interventions. Within lymph nodes, fibroblastic reticular cells (FRCs), immunologically specialized myofibroblasts, contribute to peripheral tolerance by orchestrating T-cell recruitment and trafficking along their conduit network, expressing and presenting self-antigens to autoreactive T cells in presence of low co-stimulation and immune inhibition favoring regulation over activation, and producing immunoregulatory cytokines and extracellular matrix (ECM). Biomechanically, FRCs form a contractile reticular cell scaffold with secreted ECM that enables them to integrate chemical and physical signals in LNs. During acute immune responses, CLEC-2⁺ dendritic cells engage surface glycoprotein podoplanin (gp38) on FRCs to transiently suppress contractility, permitting network relaxation and lymph node expansion without disrupting conduit architecture; as CLEC-2 wanes during resolution, the LN network remodeling is reversible, with FRC contractility and ECM patterns returning to baseline which is critical to maintain immune homeostasis. Although the impact of FRC contractility in acute responses is well characterized, how chronic inflammation and T1D alter FRC contractility affecting immunity and tolerance remains poorly defined. Fibroblasts are highly adaptable cells that respond to their microenvironment. In inflammatory, cancer, and fibrotic diseases, they can become persistently activated and drive pathogenic processes. This involves a dynamic, mechanobiological feedback loop where altered ECM deposition and stiffness impact fibroblast cytoskeleton, contractility, and gene expression, further exacerbating the disease. Therefore, we hypothesize that similar mechanisms drive FRC dysregulation in T1D. To test our hypothesis, we isolated primary FRCs from the skin-draining lymph nodes of 12 wk-old NOD (T1D-prone), and NOR and B6 (T1D-resistant) mice and profiled them using a multimodal approach: traction force microscopy (TFM), flow cytometry, immunofluorescence, and bulk RNA-seq. TFM revealed that NOD-derived FRCs exhibit aberrant contractility relative to NOR/B6, passage-matched controls. Flow cytometry and immunofluorescence demonstrated reduced gp38, a canonical FRC marker and mechanosensor in NOD-derived FRCs. Transcriptomic profiling of NOD- and NOR-derived FRCs cultured on NOD versus NOR decellularized lymph node sections also corroborate T1D gp38 downregulation, along with other mechanosensitive and regulatory pathways, in NOR FRCs seeded in NOD sections and gp38 expression and FRC mechanosensitive and regulatory pathways rescue in NOD FRCs seeded in NOR sections. These results suggest microenvironment (ECM)-dependent reprogramming of the FRC transcriptome, indicating that the diseased tissue environment may reshape FRC identity and function and these changes can be reversed by normal microenvironments. Together, these findings suggest that autoimmune T1D is associated with differential FRC identity and contractility which can be corrected by exogenous interventions. Future works will delineate this mechano-immune axis and test whether mechanomechanism-guided drugs and biomaterials can normalize FRC biomechanics and restore immunoregulatory function in autoimmunity. Establishing a stromal, non–T cell–intrinsic therapeutic entry point could enable programmable, antigen-specific strategies to prevent or delay T1D progression.

FUNDING SOURCE (optional): McKnight Doctoral Fellowship – Florida Education Fund (Mendoza); Diabetes Research Institute Foundation (Tomei)

Targeting the Matricellular TSP-1/CD47 Axis With TAX2: a First-in-Class Peptide for Cancer Therapy

Aurélie Moniot¹, Marion Etiennot¹, Aubéri Henry¹, Mariem Ghoula¹, Sonia Poli², Stéphane Dedieu³, Albin Jeanne¹

¹Apmonia Therapeutics, ²Poli Consulting, ³MEDyC UMR CNRS URCA 7369, Université Reims Champagne-Ardenne

ABSTRACT BODY:

Thrombospondin-1 (TSP-1) is a matricellular glycoprotein with a pivotal role in the tumor microenvironment, influencing angiogenesis, immune regulation, tissue remodeling, and cell adhesion. In many cancers, TSP-1 is overexpressed and correlates with poor prognosis. Among its receptors, CD47 is a key driver of tumor progression through angiogenesis and immune evasion. While most therapeutic strategies have focused on blocking the interaction between CD47 and its macrophage counter-receptor SIRPα (‘don’t eat me’ signal), these approaches are compromised by serious hematological and immunotoxic adverse effects. Recent evidence highlights the TSP-1/CD47 axis as an alternative and promising target.

We developed TAX2, a 12-amino acid cyclic peptide acting as an orthosteric antagonist that selectively inhibits the TSP-1/CD47 interaction. TAX2’s pharmacological profile was characterized through cross-species binding assays, selectivity assessment versus the CD47/SIRPα axis, in vivo efficacy and biodistribution studies in tumor-bearing mice. Complementary investigations included peptide stability and pharmacokinetic (PK) profiling after single i.v. administration, off-target and cytokine release assays, toxicology studies in rats and dogs, and human PK prediction by allometric scaling.

Computational and experimental data confirmed that TAX2 selectively binds to TSP-1 from human, rat, mouse, and dog origins and disrupts the TSP-1/CD47 interaction without interfering with the CD47/SIRPα axis. In vivo efficacy studies identified a pharmacologically active dose (PAD) of 30 mg/kg in mice, with significant antitumor effects observed in several cancer models. In a highly aggressive ovarian cancer model, TAX2 improved mouse survival and demonstrated combination potential with PARP inhibitors. TAX2 PK profile in rodents and dogs showed rapid plasma clearance (1-4 h) with dose-proportional exposure, while biodistribution studies indicated preferential accumulation in tumors and organs with high TSP-1 expression, supporting its targeted mechanism of action. Importantly, TAX2 exhibited no significant off-target binding or functional activity across a 44-target panel and did not induce uncontrolled cytokine release in human whole blood, contrasting with safety concerns reported for CD47-blocking agents. Toxicology studies further supported the safety of TAX2, which was well tolerated with NOAEL values of 400 mg/kg in rats and 100 mg/kg in dogs. Based on allometric scaling, the estimated PAD in humans was projected within the range of 9 to 33 mg/kg.

TAX2 is a first-in-class peptide targeting the TSP-1/CD47 interaction, offering a novel approach to modulate the tumor microenvironment. Robust preclinical data strongly support its development, with Phase 1/2a trials in advanced solid tumors upcoming.

eGFP-Laminin Knock-in Mice for Visualizing Basement Membrane Dynamics

Kohei Omachi¹, Hiroko Sasaki¹, Takaya Abe², Hiroki Machida¹, Hiroshi Kiyonari², Hironobu Fujiwara¹

¹Laboratory for Tissue Microenvironment, RIKEN Center for Biosystems Dynamics Research, ²Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research

ABSTRACT BODY:

The basement membrane (BM) is a specialized extracellular matrix (ECM) that underlies epithelial cells and surrounds parenchymal cells. Laminins are central BM components that regulate adhesion, polarity, and differentiation, whereas type IV collagen provides mechanical stability. Although traditionally regarded as a static scaffold, growing evidence indicates that the BM is highly dynamic, undergoing continuous remodeling and turnover. These properties suggest that the BM actively regulates organogenesis, yet the underlying mechanisms remain poorly understood.

To investigate how BM dynamics influence cell behavior, we focused on laminin, which interacts with cells via integrin receptors and initiates BM assembly. To visualize endogenous laminin, we systematically screened eGFP insertion sites in laminin-α3 and laminin-α5 chains. Using site-directed mutagenesis, we generated five eGFP-tagged human cDNA constructs for each isoform. Co-expression with their respective β and γ chains in cultured cells demonstrated that eGFP-tagged laminins were secreted as αβγ heterotrimers. Guided by these findings, we generated Lama3A-eGFP and Lama5-eGFP knock-in mice using CRISPR/Cas9 genome editing. Immunofluorescence and live imaging confirmed proper BM localization of the tagged proteins. Importantly, homozygous knock-in mice for both Lama3 and Lama5 were viable and fertile, demonstrating that the tagged laminins retained functionality.

These knock-in models provide powerful tools to study BM dynamics and to elucidate their roles in tissue architecture and development.

MatriSpace: A Spatial Transcriptomics Toolbox to Explore the Extracellular Matrix in Health and Disease

Ayomide Oshinjo¹, Annalena Dittmann¹, Petar Petrov¹, Daiqing Chen², Alexandra Naba², Valerio Izzi¹

¹Faculty of Biochemistry and Molecular Medicine, University of Oulu, ²Department of Physiology and Biophysics, University of Illinois Chicago

ABSTRACT BODY:

The matrisome, the system formed by the extracellular matrix (ECM) and its associated molecules, orchestrates crucial aspects of tissue organization in health and disease through complex spatiotemporal dynamics. Most of our understanding of matrisome composition currently comes from traditional bulk sequencing approaches, which fail to capture the spatial heterogeneity of ECM components, limiting our insight into their region-specific functions and disease-related alterations. MatriSPACE is a user-friendly web application, built in R Shiny, designed to analyze and visualize matrisome spatial patterns across tissues. It provides comprehensive spatial correlation analyses and region-specific ECM interaction mapping for both user-generated data and a curated collection of over 200 pre-processed 10x Visium spatial transcriptomics datasets from healthy and diseased tissues. The analysis of these datasets reveals tissue-wide matrisome organization through interactive visualizations and correlation maps. Using breast cancer as a demonstration case, the tool rapidly identifies spatial relationships between ECM components and maps their distribution across functional and user-defined regions. The interface enables smooth exploration of matrisome expression patterns from tissue-level organization down to specific gene pairs of interest, with automated generation of publication-ready visualizations. MatriSPACE represents a significant advance in exploring the systems biology of the matrisome, making spatial transcriptomics analysis accessible to the wider matrix biology community. By enabling systematic investigation of ECM spatial patterns across diverse tissue contexts, our tool opens new avenues for understanding matrix organization in development, homeostasis, and disease.

FUNDING SOURCE (optional): V. Izzi: Cancer Foundation Finland, DigiHealth-project - a strategic profiling project at the University of Oulu - and the Infotech Institute of the University of Oulu. A. Naba: NIH/NHGRI Human Biomolecular Atlas Program (HuBMAP) - Grant U01HG012680, and NIH/NCI Innovative Molecular Analysis Technologies Program - Grant R21CA261642

Investigating an Ex Vivo Lung Model Using Precision Cut Lung Slices Embedded in Hydrogel to Prolong Tissue Viability

Gabriela Ros¹, Daniel Abebayehu¹

¹University of Virginia, Department of Biomedical Engineering

ABSTRACT BODY:

Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive scarring of the lung interstitium and loss of the alveoli. Each year, it is estimated that between 30,000 to 50,000 new cases of IPF are diagnosed in the United States with no known cause or cure. Currently methodology for studying IPF involves mouse models that can provide insight into disease development, morphology, and potential therapeutic avenues, but fails to replicate key anatomical and clinical features as presented in humans. Precision cut lung slices (PCLS) have been a new approach that addresses the drawbacks of mouse models, however PCLS approaches display short-term viability of 5-7 days. Our work seeks to develop a new PCLS approach whereby we embed PCLS in a hydrogel to prolong tissue viability and enable the detection of tissue remodeling in PCLS. We hypothesize that by embedding PCLS in a substrate that more closely reflects the native microenvironment of lung tissue, we can prolong viability out to 21 days. Within this work we outline the fabrication of a matrix metalloproteinase 2 (MMP2)- degradable 4-arm polyethylene glycol-vinylsulfone (PEG-VS) hydrogel functionalized with RGD peptides to capture both the native extracellular matrix (ECM) and biochemical environments of lung tissue.

We first needed to determine the optimal orientation of the slice with respect to the hydrogel. To determine the optimal orientation, we decided to measure slice contractility because it morphologically reflects the stability and viability of the slice as shrinkage can be indicative of tissue death. The first orientation involved placing 300 um PCLS on top of hydrogel within the well of a 96-well plate and the other orientation included embedding the slice between two layers of gel with DMEM on top. The control group was placing the PCLS in the well with only DMEM. Across the orientations, PCLS that were embedded within the gel maintained 94.11% out to day 21of the initial area measured on day 0. From there a histological pilot study comparing the tissue structure of PCLS on day 0 versus day 21 allowed us to determine that we were able to maintain the native structure of the tissue compared to the freshly sliced lung sample.

We then moved on to measure the viability of PCLS using the two-layer embedding model comparing tissue in hydrogel versus no hydrogel. LIVE/DEAD staining was used to quantify the number of viable cells across time points of 3 days, 7 days, 14 days, and 21 days. Currently we are testing the application of a fibrotic cocktail to induce fibrosis in the two-layer gel embedded model. Altogether, we have demonstrated the ability to have a PCLS lung model that prolongs survival and allows us to test therapeutic options in a patient specific way.

Transcriptional Control of ECM Factors in the Development and Morphogenesis of the Drosophila Compound Eye

Scott J Neal

Department of Neuroscience & Physiology, SUNY Upstate Medical University

ABSTRACT BODY:

The Drosophila adult compound eye comprises ~750 ommatidia (simple eyes) whose spatial and angular patterning is critical for vision. This geometry is first established during larval stages of development in a precursor tissue called the eye imaginal disc. The “eye disc” is a bilayer epithelial vesicle in which the columnar retina develops in one layer and the apically-opposed squamous cell layer, the peripodial epithelium or PE, serves primarily to support retinal development. Genetic analyses revealed a phenotype – Retinal Displacement or RDis – in which retinal tissue becomes mislocalized to the PE side of the larval eye disc and that results in congenital malformation of the adult compound eye. While cells proliferate at vastly different rates in the PE and retinal layers of the eye disc, the boundary between these domains remains fixed at the posterior edge of the wildtype eye disc throughout larval development. In contrast, PE cells and posterior retinal neurons appear to progressively slide over PE-deposited Collagen IV ECM in RDis. Genetic screens have identified Yki (YAP1 / TAZ/WWTR1) and Mitf (MITF) as transcription regulators whose activities are required in the PE to prevent RDis. In fact, loss from the eye disc PE of Yki, its cofactor Hth (MEIS1-3), Mitf, laminin α-chain (LanA) or β-PS Integrin (Mys) can each induce the RDis phenotype, whereas overexpression of Mys can partially rescue the RDis phenotype induced through loss of Yki. These results have led to the hypothesis that Yki and Mitf regulate the expression of “ECM Factors” in the developing PE to promote normal morphogenesis of the fly compound eye. Current results will be presented.

Characterizing the Pancreatic Ductal Adenocarcinoma Microenvironment Using Decellularized Xenograft Tumor Extracellular Matrix

Maryam Younis¹, Madison A. Stampley², Sara Adair^3,4,5, Todd W. Bauer^3,4,5, Lakeshia J. Taite^2,4,5

¹University of Virginia Department of Computer Science, ²University of Virginia Department of Chemical Engineering, ³University of Virginia School of Medicine Surgical Oncology Division, ⁴University of Virginia Comprehensive Cancer Center, ⁵University of Virginia Center for Systems Analysis of Stress-adapted Cancer Organelles

ABSTRACT BODY:

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most lethal cancer at over 400k deaths per year worldwide and has a current 5-year survival rate of less than 4%. This cancer is characterized by highly fibrotic stroma and resistance to common chemotherapeutics. Tissue stiffness is associated with tumor malignancy, and research suggests that the increase in stiffness is due to altered mechanotransduction via extracellular matrix (ECM)-mediated signaling pathways. Activation of these signaling pathways associated with tumor stiffness are implicated as an initiator of epithelial-to-mesenchymal transition (EMT), which is characterized by carcinoma cells acquiring a mesenchymal phenotype, leading to the progression of invasion and metastasis. In this study, we utilize a xenograft tumor model of PDAC to interrogate ECM properties in order to inform the design of in vitro PDAC models. Human tumor segments were grafted onto the pancreata of mice, harvested after significant tumor growth, decellularized, and characterized. Successful decellularization of PDAC tumors was confirmed by multiple assays, including MBAS (for cytotoxic SDS quantification), BCA (for protein concentration), PicoGreen (for dsDNA detection), and mass spectrometry. These demonstrated effective removal of cellular debris, DNA, and cytotoxic agents while preserving the extracellular matrix. To characterize stroma composition, decellularized were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the proteins that are abundant in the TME. Human proteins accounted for ~60% of total proteins in all samples. Collagens involved in neo-fiber formation (Col1a2) and fibrillogenesis (Col6 family) were among the most abundant ECM proteins in the dECM samples. The abundance of Col6 collagens, known for their role in ECM remodeling, are critical for understanding tissue regeneration and tumor progression in PDAC models. Laminins, essential for integrin binding and basement membrane formation, were detected in all samples, along with glycoproteins contributing to elastic fiber development. With this knowledge, we can expand our synthetic model to include other functional peptides that are physiologically relevant and derived from the most abundant proteins present in the stroma. This will allow us to design a more complex hydrogel microenvironment to better replicate the native TME.

FUNDING SOURCE (optional): This research was supported by state funding within the University of Virginia Comprehensive Cancer Center and the Systems Analysis of Stress-Adapted Cancer Organelles (SASCO) Center, an NCI-funded U54 Cancer Systems Biology Consortium research center at UVA. Services and products in support of the research project were generated by the VCU Massey Comprehensive Cancer Center Proteomics Shared Resource, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059.

Mechanosensitive Control of LASP1 Regulates Fibroblast Contractility

Tom Whalley, Jason Wong, Adam Reid and Christoph Ballestrem

Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester

ABSTRACT BODY:

Fibrosis results in increased ECM deposition and tissue stiffness that disrupts tissue function. In response to an increase in stiffness, fibroblasts become activated and differentiate into contractile myofibroblasts. In this project, Telomerase Immortalised Fibroblasts (TIFs) were grown on PAA hydrogels that replicate healthy (1 and 4kPa) and diseased (50kPa) tissue stiffnesses. Mass spectrometry analysis showed significant differences in protein expression on soft and stiff gels. One protein identified as significantly increased on 50kPa gels was Lim and SH3 Domain Protein 1 (LASP1). Further analysis showed that LASP1 localises preferentially to adhesions and stress fibres in response to stiff conditions in a Rho dependent manner. Furthermore, siRNA knockdown of LASP1 led to reduced collagen gel contraction, indicating a role for LASP1 in fibroblast contractility.