Commentary on Some Recent Theses Relevant to Combating Aging: December 2018

A Thromboresistant Cell-Derived Biomaterial Modification for Vascular Grafts

Nina Kristofik, PhD, Yale University

Cardiovascular diseases are responsible for as many as 17.3 million deaths per year worldwide. In cases where arterial bypass is necessary, the most common surgical treatment involves the use of the patient's own saphenous vein. Unfortunately, individuals requiring such grafts often do not possess sufficiently healthy vessels for bypass, or, if vascular harvest is possible, the extraction of a graft will result in additional morbidity at the donor site. Despite the great need for alternatives to autologous arterial grafts, none are clinically available. While some promising alternatives are currently being tested as dialysis access vessels, failure due to thrombosis remains an obstacle, even in this relatively low-stakes application. This thesis examines the use of thrombospondin-2 knockout extracellular matrix (TSP2 KO ECM) as a nonthrombogenic modification for the lumen of vascular grafts and proposes a mechanism for its unique thromboresistance.

Previous reports have shown that mice lacking TSP2 possess irregular collagen fibril morphology and present with a bleeding diathesis. In the current work, we investigated the contribution of TSP2 KO extracellular matrix (ECM) to these defects. Bone marrow transplants and endothelial denudation studies revealed that an ECM defect heavily contributed to the bleeding diathesis. Moreover, we report that decellularized TSP2 KO ECM produced by dermal fibroblasts in vitro had decreased von Willebrand Factor (vWF) binding compared to WT ECM. This finding was probed via immunofluorescence after whole blood flow over ECM to determine quantity of vWF adhesion, and via atomic force microscopy to determine vWF binding force to ECM to determine the quality of vWF adhesion (53.85 ± 8.44 pN on WT ECM compared to 16.47 ± 6.17 pN on KO ECM). This unique property presents an opportunity to use TSP2 KO ECM as a hemocompatible modification to the lumen of vascular grafts, potentially circumventing thrombosis using simple biological processes.

Herein, we probed the feasibility and efficacy of TSP2 KO ECM modification of decellularized vascular grafts in vitro and in vivo. Tensile testing via 1NSTRON and suture strength analysis of decellularized aortas with and without ECM modification showed that ECM modification did not alter graft mechanical properties. Scanning electron microscopy and immunofluorescent studies clearly indicated that TSP2 KO ECM readily modifies the lumen of decellularized vascular grafts. Moreover, exposure of platelets to unmodified and ECM modified decellularized aortas and subsequent analysis via SEM and fluorescent staining revealed significantly decreased platelet adhesion and activation on TSP2 KO ECM modified grafts compared to unmodified and Wild-Type ECM modified controls (1.77% ± 0.65% platelet coverage vs. 28.7% ± 8.02% and 22.6% ± 3.87%, respectively). In addition, implantation of TSP2 KO ECM coated vascular grafts in an infrarenal aortic interposition model resulted in increased endothelial and smooth muscle-like cell recruitment to the graft, and decreased the rate of graft failure.

Together, these data indicate that TSP2 plays a key role in the formation of collagen fibrils such that vWF is unable to adhere properly to them. This property may be leveraged to produce a nonthrombogenic vascular graft modification that has the potential to aid in the creation of the ideal arterial graft.

Comment: The ideal replacement for failing tissue is an autologous bioidentical replacement—but although methods for the preparation of such grafts from a patient's own cells are already crossing into clinical viability, growing tissue is inherently a time-consuming process. Thus in cases where the damage poses an immediate threat to life, including most serious vascular defects, a more “off-the-shelf” solution is needed (in cases of chronic degeneration repair at the molecular level ^{11 –13} is of course more desirable, but also less generally applicable). Unfortunately all attempts to date to develop such a product have been unable to prevent the formation of thrombi (clots) which rapidly occlude the vessel, rendering it useless. First generated in the late 1990s, TSP2 knock-out mice exhibit a range of abnormalities which notably include remarkable resistance to thrombosis and accelerated wound healing. ¹⁴ The mechanism of thromboresistance has taken some time to decipher, but this thesis does largely complete the picture: TSP2 has an important function in the formation of the extracellular matrix, such that TSP2 KO ECM—while compositionally indistinguishable from wild-type matrix—is composed of abnormally short and disorganized collagen fibrils. This disorganization significantly interferes with the binding of von Willebrand Factor, the absence of which, in turn, almost totally inhibits platelet aggregation and the ensuing coagulation process. (TSP2 itself is not directly involved at this stage; reintroducing it to knockout matrix has no impact on subsequent thrombogenesis.) That alone would be a very promising finding, but TSP2 KO matrix has further desirable properties from a therapeutic perspective; its looser and weaker structure facilitates cellular migration, while the absence of TSP2 protein derepresses angiogenesis—both of which contribute to the improved rate of healing also seen in knockout mice, and could reasonably be expected to assist in integration with the host. The mechanical weakness of TSP2 KO ECM is certainly an obstacle to its use in vascular grafts, but this work neatly circumvents that limitation by employing it as a thin layer over a more robust biodegradable material. The excellent preliminary results thereby demonstrated certainly justify further development of this broadly applicable and potentially revolutionary technology.

Actomyosin Mediated Tension Orchestrates Thermogenic Programs in Adipocytes

Kevin Tharp, PhD, University of California, Berkeley

Innovative approaches to shift energy balance are urgently needed to combat metabolic disorders such as obesity and diabetes. One promising approach has been the expansion or activation of thermogenic adipose tissues to improve metabolic homeostasis. My doctoral studies presented in the following text have identified novel approaches to translate adipose-based metabolic therapeutics and the underlying mechanisms by which thermogenic adipocytes establish their therapeutically applicable metabolic capacity.

In chapter I, I present a novel biomaterial technology optimized to expand metabolically beneficial thermogenic adipose depots in vivo. This system enabled me to determine the degree of metabolic enhancement possible with the exogenous expansion of thermogenic adipose depots. To generate therapeutic adipose implants, I modified hyaluronic acid-based hydrogels to support the differentiation of white fat-derived multipotent stem cells (ADMSCs) into lipid accumulating uncoupling protein 1 (UCP1) expressing thermogenic adipocytes. Subcutaneous implantation of the synthetic tissues successfully attracted host vasculature and persisted for several weeks, and the implant recipients demonstrated elevated core body temperature during cold challenges, enhanced respiration rates, improved glucose homeostasis, and reduced weight gain demonstrating the therapeutic merit of this highly translatable approach.

In chapter II, I outline the experimentation leading to the discoveries presented in chapter III as well as thoroughly review pertinent tissue engineering strategies. Specifically, I sought to define the mechanism by which synthetic ECM components identified in chapter I could alter differentiation outcomes of preadipocytes to yield greater thermogenic capacity.

In chapter III, I demonstrate that actomyosin-based mechanical responses provide a critical differentiation cue for the development of thermogenic adipocytes. Since I had determined that the hydrogel optimization techniques described in chapter I were likely acting through cytoskeletal-mediated processes, I examined the role of cytoskeletal structure and tension in thermogenic adipose development. I identified that the muscle-like gene expression patterns of UCP1+ adipocytes are critical for the acute induction of oxidative metabolism and uncoupled respiration and regulate mechanosensitive transcriptional coactivators, YAP/TAZ, that control thermogenic gene expression.

This dissertation establishes the role of physical mechanics in the development and function of thermogenic adipocytes, which may engender future metabolic therapeutics.

Comment: The ready availability of energy-dense convenience foods, coupled with the relatively sedentary lifestyle of most modern humans, has somewhat inevitably led to an obesity “epidemic”—a major contributor to the incidence of chronic conditions including type 2 diabetes, ¹⁵ cardiovascular disease, and many types of cancer. While behavioral therapy can be effective in reducing body weight in many patients, its success (both initially and in the longer term) is highly dependent on psychological and circumstantial factors, some beyond the reasonable control of the individual affected. The persistence of the epidemic in the face of many decades of such efforts—coupled with the costs of care for obesity-linked disease—suggests that a more pragmatic approach, less dependent on individual resources and motivation, may offer a better return on investment. Metabolic uncoupling (in which the energy normally used to make ATP is instead released as heat, wasting energy that would otherwise ultimately be stored as fat) has been under consideration as a weight-control therapy since at least the 1930s, when the chemical uncoupler 2,4-dinitrophenol (DNP) was used widely as a “diet pill.” Unfortunately, while effective, DNP is also toxic at higher doses (which vary dramatically between users) and was banned within less than a decade of its introduction. This toxicity arises from its nonspecific action on all cell types, including those highly dependent on effectively coupled respiration, and is impractical to prevent even with the use of more advanced delivery mechanisms, thanks to the molecule's high solubility in biological membranes. Consequently, more recent efforts have focused on enhancing the activity of the brown/beige fat cells which naturally exhibit uncoupled metabolism. ¹⁶ Despite their shared phenotype, brown and beige adipocytes are quite distinct populations; the former arise from a progenitor shared with muscle cells, and are restricted to specific depots, whereas the latter share a precursor with conventional white adipocytes and arise within white adipose tissue in response to various forms of stimulation. The inducibility of beige adipogenesis appears to present a promising target for therapeutic intervention, but this approach has some flaws; in particular, the beige state is dependent on constant external stimulation (which must be assumed to carry side effects) and is inherently downregulated under obesity. Since 2009, the classical brown adipose tissue that makes up as much as 5% of a newborn baby's body mass has been known to exist in limited quantities in adults (albeit declining progressively with age)—hinting that the adult systemic milieu is not inherently hostile to brown adipocytes, which are far more phenotypically stable than their beige cousins. This exciting thesis demonstrates that physiologically competent brown fat can be expanded from a relatively noninvasive adipose biopsy and delivered in a biodegradable subcutaneous implant to temporarily increase the basal metabolic rate of a subject, thereby facilitating weight loss. Crucially, that therapy could reasonably be administered in primary or outpatient care—and while like any cell therapy its cost is likely to initially be high, the sheer size of the potential patient population should drive rapid economies of scale!

Age-Associated Changes in Intrathymic B Cell Population in Mice

Carolina Cantu, MS, The University of Texas Health Science Center at San Antonio

Autoimmune disease affects approximately 3% of the United States population and is the leading cause of death among young and middle-aged women. Susceptibility to autoimmune disease generally increases with age in both sexes; however, the mechanisms linking aging and increased susceptibility are incompletely understood. One hallmark of the aging immune system is atrophy of the thymus, the primary site of T lymphocyte generation. The thymus is the most rapidly aging tissue in the body, reaching its peak size at puberty, followed by lifelong progressive atrophy. We recently showed that in addition to size, critical thymic functions are lost with age, including expression of tissue-restricted self-antigen genes. Thymic tissue-restricted antigen expression allows clonal deletion of potentially autoreactive T cells, and loss of tissue-restricted antigen expression during aging represents a potential mechanism for age-associated increases in autoimmunity.

Until recently, only epithelial cells were known to express tissue-restricted antigens within the thymus, but it is now clear that immunoglobulin M–immunoglobulin D–intrathymic B lymphocytes can also be induced to express autoimmune regulator, a factor critical for tissue-restricted antigen expression, as well as downstream Aire-dependent tissue-restricted antigens. Here, we find that B cell frequency increases 5fold in the aged murine thymus, concomitant with a shift from an immunoglobulin M–immunoglobulin D–to an immunoglobulin M+ immunoglobulin D+ phenotype. In addition, immunofluorescence studies of these B cells have shown they reside at the corticomedullary junction of the young thymus, consistent with a role in negative selection. In the aged thymus, however, there is increased disorganization of B cell localization, with expansion of this population into the cortex occasionally observed.

Furthermore, other studies have shown that while thymic B cells can develop from multipotent progenitors intrathymically, they can also recirculate from the periphery in young mice. In ongoing studies, we aim to determine the relative contribution of recirculating and intrathymically derived B cells to age-associated B cell accumulation using adoptive transfer of age-matched and mismatched peripheral B cells into congenic young and aged hosts.

Lastly, using autoimmune regulator green fluorescent protein-reporter mice and next-generation ribonucleic acid sequencing, we show that the expression of autoimmune regulator, as well autoimmune regulator-dependent and autoimmune regulator-independent tissue-restricted antigens, declines with age in intrathymic B cells. Our findings indicate that age-associated changes in intrathymic B cells inhibit their ability to express tissue-restricted antigens and negatively select autoreactive T cells, revealing a novel potential mechanism linking aging with increased susceptibility to autoimmune disease.

Comment: To those seeking a comprehensive solution to the problem of age-related disease, autoimmune conditions—with their incidence predominantly among the young—may seem like an odd topic of interest. Yet the fact that most such conditions do increase in prevalence with age marks them out as a necessary part of any comprehensive rejuvenation program; their representation among causes of death will only increase as other pathologies are resolved, and it would be imprudent to wait until then to address them! Thankfully, methods for the regeneration of immune function are already under intensive development. Gross restoration of thymic size and cellularity can be achieved via surprisingly straightforward means in various model systems ¹⁷ —and although this achievement yielded frustratingly little improvement in immunocompetence versus novel antigens, it nonetheless thereby uncovered a major role for fibrosis of the peripheral lymph nodes (home for much of our lives to the majority of the naive lymphocytes produced largely in childhood) in age-related immune dysfunction. We now know that restoration of that peripheral support function will be critical to the success of long-term immune rejuvenation; fortunately, the engineering of transplantable lymph nodes is progressing quite rapidly. Meanwhile, this work reveals that the B cell population within the thymus remains essentially constant in size with age despite atrophy of the host tissue; since each type of antigen-presenting cell in the organ is predisposed to handle different sets of tissue-restricted antigens, this leads to skewing of the self-antigen panel displayed to passing T cells. While B cell-intrinsic factors and alterations to the stromal microenvironment are also implicated in the decline of efficacious thymic negative selection, ¹⁸ the simple reversal of glandular atrophy will be an important step toward restoring normal selection.

Endoplasmic Reticulum Stress Is Transmissible Among Cells of the Central Nervous System

Neil Sprenkle, MS, West Virginia University

Improper protein folding and trafficking are common pathological events observed in various cell types in neurodegenerative diseases. If the protein quality control mechanisms of the endoplasmic reticulum (ER) fail to re-establish proteostasis, misfolded proteins accumulate within the lumen of the ER and perturb normal cellular processes. While low-level stimulation of the unfolded protein response (UPR) is considered a beneficial physiological response to transient protein misfolding stress, or ER stress, sustained UPR activation resulting from prolonged ER stress can promote neurotoxicity. The cell-autonomous mechanisms of the UPR have been extensively characterized in the context of neuropathology. Nevertheless, there still remain unanswered questions regarding the cell-extrinsic role of the UPR under normal physiology, and how this mechanism is compromised in diseased states. To address this, we evaluated whether transferring conditioned media from ER-stressed astrocytes to different cell types could modulate their functional characteristics. Our results indicate that ER-stressed astrocytes secrete a mediator(s) which regulates both inflammatory and ER stress responses in other astrocytes and neurons in vitro. Initial exposure to this stress factor(s) confers resistance against subsequent ER stress to neurons by engaging the adaptive signals of the neuronal UPR. However, persistent exposure to this unidentified mediator(s) suppresses the initial protective effect and becomes cytotoxic. Overall, these findings provide insight into the cell-nonautonomous influence of ER stress on cells of the central nervous system. Further understanding the molecular mechanisms underlying this mode of intercellular communication would present novel therapeutic opportunities to treat neurodegenerative diseases.

Comment: Most neurodegenerative diseases feature the initial appearance of misfolded protein aggregates in one or a small number of distinct loci, followed by their spread through the central nervous system (CNS) along a typically predictable course that correlates with clinical progression. The mechanism of this cell-to-cell transmission is hotly contested, with several possibilities under investigation; however, it has been generally assumed in the field that the key factor is transport of the toxic species per se. This dissertation approaches the problem from a different perspective, discovering that endoplasmic reticulum stress can itself propagate intercellularly—absent the influence of any specific disease-associated protein driver! That finding is particularly exciting given the recent discovery that senescent astrocytes play a critically important role in neurodegeneration. Senescence and activation of the unfolded protein response go hand in hand; excess production of senescence-associated secretory phenotype factors places a heavy load on the cell's protein folding machinery, while high UPR activity increases oxidative stress (amelioration of which can be protective against neurodegeneration ¹⁹ ) and signals through p21 to promote stress-induced senescence. Thus it now seems plausible that the age-related accumulation of senescent cells may not merely exacerbate neurodegeneration, but contribute directly to its initiation—by inhibiting proper protein folding in nearby cells, creating the conditions for primary aggregation to occur. The argument for therapeutic clearance of senescent cells in advance of symptomatic disease, as we have recommended since the turn of the century, is growing steadily more incontrovertible in this context just as in others. ²⁰

Evaluation of Early Tumor Angiogenesis Using Ultrasound Acoustic Angiography

Sarah Shelton, PhD, The University of North Carolina at Chapel Hill

Cancer angiogenesis is a feature of tumor growth that produces disorganized and dysfunctional vascular networks. Acoustic angiography is a unique implementation of contrast-enhanced ultrasound that allows us to visualize microvasculature with high resolution and contrast, including blood vessels as small as 100 to 150 micrometers. These angiography images can be analyzed to evaluate the morphology of the blood vessels for the purpose of detecting and diagnosing tumors.

This thesis describes the implementation, advantages, and disadvantages of acoustic angiography and evaluates tumor vasculature in a preclinical cancer model. Measurements of tortuosity and vascular density in tumor regions were significantly higher than those of control regions, including in the smallest palpable tumors (2–3 mm). Additionally, abnormal tortuosity extended beyond the margin of tumors, as distal tissue separated from the tumor by at least 4 mm exhibited higher tortuosity than healthy individuals. Vascular tortuosity was negatively correlated to distance from the tumor margin using linear regression.

Analysis of full images to detect tumors was performed using a reader study approach to assess visual interpretations, and quantitative analysis combined tortuosity with spatial relationships between vessels using a density-based clustering approach. Visual assessment using a reader study design resulted in an area under the receiver operating characteristic (ROC) curve of approximately 0.8, and the ROC curve was significantly correlated with tumor diameter, indicating that larger tumors were detected more accurately using this approach. Quantitative analysis of the same images used a density-based clustering algorithm to combine vessels in an image into clusters based on their tortuosity (using 2 metrics), radius, and proximity to one another. In tumors, highly tortuous vessels were closely packed, forming large clusters in the analysis, while control images lacked such patterns and formed much smaller clusters. Therefore, maximum cluster size was used to detect tumors, achieving an area under the ROC curve of 0.96.

Finally, superharmonic molecular imaging was used to image targeted microbubbles with higher contrast to tissue ratios than conventional molecular imaging. These molecular images were combined with vascular acoustic angiography images to begin to relate the expression of endothelial markers of angiogenesis with vascular features such as tortuosity.

Comment: While the early detection of cancer (especially the development of clinical symptoms) is widely agreed to be critical to successful diagnosis and treatment, it has thus far proven impossible to achieve in the general case. There has been good progress in developing tests to confirm or deny the presence of specific types of neoplasia, but it is unlikely to ever be practical to conduct precautionary screening using a battery of dozens of different assays! Fortunately, the vast majority of cancers do share certain key features—most fundamentally of all, the simple presence of a deviation from normal tissue organization. Detecting such anomalies in arbitrary tissue in vivo is still far beyond the reach of contemporary imaging methods. However, indirect detection via the impact of a growing tumor on the vasculature—a phenotype largely independent of cell of origin—is considerably closer to clinical applicability, as illustrated by the excellent sensitivity and specificity achieved in this work. Although ultrasound-based imaging techniques are physically incapable of sufficient tissue penetrance to be suitable for full-body scans, MRI scans achieving comparable resolution at arbitrary depths have already been demonstrated (the vasculature is of course a particularly easy target for contrast enhancement). If the same quantitative analysis can be applied to MRI angiographs, it would constitute the most credible candidate to date for near-universal cancer detection.

Interactions Between Amyloid-Beta and Microglial Cells

Lisa Gouwens, PhD, University of Missouri–Saint Louis

Alzheimer's disease (AD), the most common cause of dementia, is a neurodegenerative condition characterized by loss of memory and intellectual abilities. Intracellular plaques of aggregated amyloid-beta (Aβ) protein are a well-known pathology associated with AD. Although symptoms usually appear late in life, the accumulation of Aβ begins decades earlier and causes activation of microglia, the brain's immune cells. The ensuing inflammation contributes significantly to neurodegeneration. Determination of the particular form of Aβ that causes the most damage in the brain is one of the major questions in the AD field. My research focused on the interactions of microglia with monomers, protofibrils, and fibrils of Aβ. I found that protofibrils, not monomers or fibrils, bind to microglial surfaces, and I confirmed earlier reports that protofibrils elicit a proinflammatory response from microglia. These results were consistent regardless of changing conditions such as temperature, incubation time, and Aβ concentrations.

Another aspect of my research was to investigate how microglia internalize different forms of Aβ. The distinction between monomers and protofibrils may have physiological significance in AD, yet there are few reports in the literature in which these two forms of Aβ are examined separately. Monomers and protofibrils were carefully separated by size exclusion chromatography before cell treatments, which sets apart this work from research done in other labs. Multiple conditions and strategies, including a novel quantitation method for internalized Aβ, demonstrated that microglia favor internalization of protofibrils over monomers. Further experiments determined that microglia are capable of internalizing protofibrils in high amounts without degradation. A significant amount of Aβ protofibrils remain in the cytoplasm and are not routed to lysosomes, contradicting reports in the literature.

A third research objective involves the study of microvesicles released from microglia. Microvesicles may have a role in AD by transporting Aβ within the brain. I conducted experiments in which microglia were stimulated to produce microvesicles, and carried out assays to both confirm the presence of and visualize microvesicles. The studies described here contribute to the understanding of the interactions between microglia and Aβ, potentially leading to a possible treatment or cure for AD.

Comment: Microglia make up 10%–15% of the cells in the adult brain and serve a role analogous to that of neutrophils outside the CNS—primarily that of taking up and degrading foreign material and cellular debris—as well as acting as antigen-presenting cells. The former behavior is of course highly relevant to neurodegenerative disease, where an accumulation of recalcitrant molecular waste is strongly implicated in pathogenesis; however, the involvement of microglia in the progression of Alzheimer's and similar conditions has for some time been highly controversial, with studies finding both beneficial and detrimental roles. ²¹ A possible resolution to the contradiction was identified last year by a paper in Cell which employed single-cell techniques to characterize a specific subpopulation of disease-associated microglia (DAM) ²² ; these cells colocalize very tightly with aggregates, avidly phagocytose misfolded protein, and were initially thought to have a protective role. However, very recent work suggests DAM themselves can exhibit either anti- or pro-inflammatory phenotypes, and thus their true in vivo effect remains somewhat unclear. (It would be unsurprising to find that the accumulation of senescent astrocytes and/or lipofuscin in the aging CNS was partly responsible for a bias toward proinflammatory DAM activity; still, this has yet to be confirmed.) This thesis contributes a valuable piece to the puzzle, demonstrating that oligomeric amyloid-betaAβ, in particular, can accumulate in microglia without being effectively degraded—a finding which helps to explain the limited benefit observed in clinical trials of antiamyloid immunotherapies, since at least one pool of pathologically relevant waste is sequestered away from antibody surveillance. Dealing with these intracellular deposits will require a different approach. While the upregulation of microglial lysosomal biogenesis or activity is already under investigation as one option, that tactic will clearly be an uphill struggle—the proinflammatory state induced by aggregates actually represses innate lysosomal activity (this oddity appears to relate to the secondary role of microglia as antigen-presenting cells; processing of engulfed material for presentation occurs optimally at higher lysosomal pH than its degradation, and inflammatory signaling favors the former). Our preferred strategy of degrading the toxic species with a specific engineered protease seemingly remains the pragmatic choice, albeit with the new nuance of requiring cytosolic rather than lysosomal function (which at least simplifies the process of targeting the therapeutic protein to its destination).