Journal of Circulating Biomarkers

The discovery of urinary extracellular biomarkers has been impeded by the lack of efficient methods for the isolation of extracellular vesicles (EVs: exosomes and microvesicles) and extracellular nucleic acid (RNA and DNA) from urine. Ultracentrifugation (UC), considered the gold standard for vesicle isolation from many biofluids, is efficacious but laborious, and, like most commercially available methods, is unable to isolate enough material from small volumes for protein or RNA-based biomarker discovery. We have developed a novel precipitation method for the isolation of EVs and nucleic acids from urine. The method, which is now commercially available, takes less than 30 min and does not require polyethylene glycol. Transmission electron microscopy and Nanosight particle analysis confirm that the method isolates intact vesicles with a similar size, shape, and number to UC. Immunoblot analysis of preparations made from a variety of urine samples demonstrates that the method isolates multiple vesicle protein markers more efficiently than other commercial kits, especially from more diluted samples. Bioanalyzer, quantitative reverse transcription polymerase chain reaction, and array analysis show that the method is extremely efficient at the isolation of extracellular miRNAs. The Ymir Genomics EV and Extracellular RNA Isolation Kits offer an efficient and rapid alternative to UC and other commercial kits.

Exosomes are ∼100 nanometre diameter vesicles secreted by mammalian cells. These emerging disease biomarkers carry nucleic acids, proteins and lipids specific to the parental cells that secrete them. Exosomes are typically isolated in bulk by ultracentrifugation, filtration or immunoaffinity precipitation for downstream proteomic, genomic, or lipidomic analysis. However, the structural properties and heterogeneity of isolated exosomes at the single vesicle level are not well characterized due to their small size. In this paper, by using high-resolution atomic force microscope imaging, we show the nanoscale morphology and structural heterogeneity in exosomes derived from U87 cells. Quantitative assessment of single exosomes reveals nanoscale variations in morphology, surface roughness and counts isolated by ultracentrifugation (UC) and immunoaffinity (IA) purification. Both methods produce intact globular, 30–120 nm sized vesicles when imaged under fluid and in air. However, IA exosomes had higher surface roughness and bimodal size population compared to UC exosomes. The study highlights the differences in size and surface topography of exosomes purified from a single cell type using different isolation methods.

Placental protein 13 (PP13), a glycan binding protein predominantly expressed in syncytiotrophoblast, dimeric in nature, lacks N-terminal signal peptide, bypasses the endoplasmic reticulum, and secretes into maternal circulation as exosomes or microvesicles. PP13 has jelly roll fold conformation with conserved carbohydrate recognition domain which specifically binds to β-galactosides of the glycan receptors during placentation. PP13 binds to glycosylated receptors on human erythrocytes and brings about hemagglutination by the property of lectin activity; other functions are immunoregulation and vasodilation during placentation and vascularization. The gene LGALS13 located on 19q13.2 comprising four exons expresses a 32-kDa protein with 139 amino acid residues, PP13. Impaired expression due to mutation in the gene leads to a nonfunctional truncated PP13. The low serum levels predict high risk for the onset of preeclampsia or obstetric complications. Hence, PP13 turned to be an early marker for risk assessment of preeclampsia. The recombinant PP13 and monoclonal antibodies availability help for replenishing PP13 in conditions with low serum levels and for detection and prevention of preeclampsia, respectively.

This study aimed to evaluate the effect of mesenchymal stem cells (MSCs)–derived exosomes in retina regeneration of experimentally induced diabetes mellitus (DM) in a rabbit model. Exosomes are extracellular vesicles that contain many microRNAs (micRNAs), mRNAs, and proteins from their cells of origin. DM was induced by intravenous (IV) injection of streptozotocin in rabbits. MSCs were isolated from adipose tissue of rabbits. Exosomes were extracted from MSCs by ultracentrifugation. Exosomes were injected by different routes (IV, subconjunctival (SC), and intraocular (IO)). Evaluation of the treatment was carried out by histopathological examination of retinal tissues and assessment of micRNA-222 expression level in retinal tissue by real-time polymerase chain reaction. Histologically, by 12 weeks following SC exosomal treatment, the cellular components of the retina were organized in well-defined layers, while IO exosomal injection showed well-defined retinal layers which were obviously similar to layers of the normal retina. However, the retina appeared after IV exosomal injection as irregular ganglionic layer with increased thickness. MicRNA-222 expression level was significantly reduced in diabetic controls when compared to each of healthy controls and other diabetic groups with IV, SC, and IO routes of injected exosomes (0.06 ± 0.02 vs. 0.51 ± 0.07, 0.28 ± 0.08, 0.48 ± 0.06, and 0.42 ± 0.11, respectively). We detected a significant negative correlation between serum glucose and retinal tissue micRNA-222 expression level (r = −0.749, p = 0.001). We can associate the increased expression of micRNA-222 with regenerative changes of retina following administration of MSCs-derived exosomes. The study demonstrates the potency of rabbit adipose tissue–derived MSCs exosomes in retinal repair. So, exosomes are considered as novel therapeutic vectors in MSCs-based therapy through its role in shuttling of many factors including micRNA-222.

Circulating tumour cells (CTCs) are important indicators of metastatic cancer and may provide critical information for individualized treatment. As CTCs are usually very rare, the techniques to obtain information from very small numbers of cells are crucial. Here, we propose a method to perform a single cell quantitative reverse transcription polymerase chain reaction (qPCR) analysis of rare tumour cells. We utilized a microfluidic immunomagnetic assay to separate cancer cells from blood. A combination of detailed immunofluorescence and laser microdissection enabled the precise selection of individual cells. Cancer cells that were spiked into blood were successfully separated and picked up for a single cell PCR analysis. The breast cancer cell lines MCF7, SKBR3 and MDAMB231 were tested with 10 different genes. The result of the single cell analysis matched the results from a few thousand cells. Some markers (e.g., ER, HER2) that are commonly used for cancer identification showed relatively large deviations in expression levels. However, others (e.g., GRB7) showed deviations that are small enough to supplement single cell disease profiling.

The tumour microenvironment is characterized by pro-inflammatory profiles, including interleukin-1β (IL-1β). This profile is generated by infiltrating macrophages following interactions with tumours or their components. The objectives of this study were to identify whether tumour exosomes could induce macrophage IL-1β production and the mechanism involved. Exosomes were isolated from ovarian cancer patients and ovarian tumour cells by chromatography and ultracentrifugation. Specific exosomal proteins were defined by mass spectrometry (MS) and confirmed by Western immunoblotting. Using macrophage-like THP-1 cells, induction of IL-1β release was investigated by ELISA. RGD peptides were used to block fibronectin binding by THP-1 α5β1 integrin. Exosomes isolated from ovarian cancer patients and from ovarian cancer cells were demonstrated, by MS and immunoblotting, to express fibronectin. Incubation of THP-1 cells with these exosomes induced pro-inflammatory cytokines, in particular IL-1β. Blocking of THP-1 binding of exosomal fibronectin with RGD peptides abrogated exosome-mediated IL-1β production and down-stream phosphorylation of Akt and c-Jun. Although cancer patients generally exhibit increased levels of IL-1β, the underlying mechanism is unclear. Here, tumour-derived exosomes are demonstrated to induce pro-inflammatory cytokine in macrophages including IL-1β, whose induction is mediated by fibronectin.

Retrospective analysis of patient tumour samples is a cornerstone of clinical research. CTC biomarker characterization offers a non-invasive method to analyse patient samples. However, current CTC technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. We sought to investigate CTC recovery from frozen, archived patient PBMC pellets. Matched samples from both mCRPC patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed “fresh” via Epic CTC Platform or from “frozen” PBMC pellets. Samples were analysed for CTC enumeration and biomarker characterization via immunofluorescent (IF) biomarkers, fluorescence in-situ hybridization (FISH) and CTC morphology. In the frozen patient PMBC samples, the median CTC recovery was 18%, compared to the freshly processed blood. However, abundance and localization of cytokeratin (CK) and androgen receptor (AR) protein, as measured by IF, were largely concordant between the fresh and frozen CTCs. Furthermore, a FISH analysis of PTEN loss showed high concordance in fresh vs. frozen. The observed data indicate that CTC biomarker characterization from frozen archival samples is feasible and representative of prospectively collected samples.

Investigation of the involvement of extracellular vesicles (EVs) in parasite biology has burgeoned in recent years. Human infecting protozoan parasites, such as Trypanosoma cruzi, Lesihmania sp. and Trichomonas vaginalis, have all demonstrated the utilization of EVs as virulence factors in order to activate or hamper host immunity. Novel findings have provided evidence that the deployment of EVs by Plasmodium sp. has a major impact in disease outcomes and serves as an integral part in controlling stage switching in its life cycle. Clinical studies have highlighted elevated levels of EVs in patients with severe malaria disease and EVs have been linked to increased sequestration of infected red blood cells to the endothelium, causing obstruction of blood flow. It has also been found that EVs produced during malaria disease activate innate immunity. Intriguingly, recent discoveries indicate that Plasmodium sp. “highjack” the erythrocyte microvesiculation system in order to cross-communicate. Both the transfer of DNA and parasite density regulation has been suggested as key mechanisms of EVs in malaria biology.

Aim: To evaluate the relevance of endothelial-derived apoptotic microparticles (EMPs) with inflammatory cytokine outcomes in patients with ischaemic chronic heart failure (CHF).

Methods: A total of 154 patients with moderate-to-severe CHF were enrolled in the study. Flow cytometry analysis was used for quantifying the number of EMPs. All-cause mortality, CHF-related death, and CHD-readmission rates were examined.

Results: During a median follow-up of 2.18 years, 21 participants died and 106 subjects were hospitalized repetitively. Medians of circulating EMPs in survivor and non-survivor patient cohorts were 0.286 n/mL (95% CI = 0.271–0.309 n/mL) and 0.673 n/mL (95% CI = 0.65–0.74 n/mL) (P<0.001). There was a significantly lower concentration of sRANKL, OPG, TNF-alpha, sFAS, and sFAS ligand in the survivor patients when compared with those who met composed endpoints. The sFAS/sFAS ligand ratio in the non-survivor patient cohort was significantly higher than in the survivor cohort (P<0.001). In multivariate model EPMs, NYHA class, NT-pro-BNP, TNF-alpha, sFAS/sFAS ligand ratio, and OPG remained statistically significant for the cumulative endpoint: all-cause mortality, CHF-related death, and CHF-related readmission.

Conclusion: Increased apoptotic circulating EMPs, OPG, and FAS-sFAS ligand ratio independently predicted cumulative survival in CHF patients.

Mesenchymal stem cell (MSC) has just been approved as the first “off-the-shelf” stem cell pharmaceutical drug with an anticipation of more approvals following completion of numerous rigorous clinical trials. Despite this progress, the rationale for MSC therapeutic efficacy remains tenuous and is increasingly rationalized on a secretion rather than differentiation mechanism. Recent studies identifying exosome as the secreted agent mediating MSC therapeutic efficacy could potentially reduce a cell-based drug to a safer biologic-based alternative. Here we review the development of MSC exosome as a potential first-in-class therapeutic, and the unique challenges in the manufacture and regulatory oversight of this new class of therapeutics.

Essentially, all cells release extracellular vesicles (EVs) that end up in biofluids, including blood, and the contents of these EVs can provide a window into the status of the cells from which they are released. This is particularly interesting in cancer, since these EVs allow for ‘ex-vivo’ analysis of the properties of the tumours without the need for biopsy. Gene mutations, rearrangements, amplifications, and epigenetic changes in the transcriptome can be monitored in circulating EVs. In this study, we used two human tumour cell lines derived from an epidermoid carcinoma and a medulloblastoma, which had amplification for the epidermal growth factor receptor (EGFR) and c-Myc genes, respectively. Cells were implanted subcutaneously into immunocompromised mice, and levels of gene amplification in both groups of subcutaneous tumours were quantified. We then determined if elevated levels of transcripts for the human EGFR and c-Myc were represented in circulating EVs in tumour-bearing mice. The expression levels of both human EGFR (h-EGFR) and human c-Myc (h-c-Myc) mRNAs in circulating EVs correlated well with their amplified status in the tumours. This data provides further support to the idea that circulating EVs are a potential platform for tumour biomarkers.

Growth and differentiation factor-15 (GDF-15) has been implicated in fibrosis, inflammation, and ventricular remodeling. The role of GDF-15 in the regulation of cardiac remodeling in idiopathic dilated cardiomyopathy (DCM) remains poorly defined. This study attempts to analyze the molecular interactions between GDF-15 and markers of fibrosis as well as its positive correlations with worsening functional capacity. The study population consisted of 24 DCM patients and 8 control subjects. All DCM patients had normal coronary angiographic studies. Plasma levels of GDF-15, matrix metalloproteinase-2 (MMP2), MMP3, MMP9, tissue inhibitor of MMP 1 (TIMP1), and soluble suppression of tumorigenicity-2 protein (sST2) were determined by enzyme-linked immunosorbent assays. Brain Natriuretic Peptide (BNP) was measured as per core laboratory protocol assay at Scott and White Memorial Hospital core laboratory. Correlation analysis was performed between GDF-15 and each of the MMPs—MMP2, MMP3, MMP9, and TIMP as well as New York Heart Association (NYHA) class and echocardiographic parameters (left ventricular ejection fraction (LVEF) and left ventricular internal dimension in diastole (LVIDd)). LVEF and LVIDd were obtained by two-dimensional echocardiography. The protocol was approved by Scott and White Memorial Hospital Institutional Review Board (S&W IRB). Correlation analysis of control versus all DCM patients showed a strong correlation of GDF-15 with TIMP1 (r = 0.83, p < 0.0001) and weaker correlation with MMP3 (r = 0.41, p = 0.011) and MMP2 (r = 0.47, p = 0.003). MMP9 showed poor correlation with GDF-15 (r = 0.3036, p = 0.046). GDF-15 correlated negatively with MMP2/TIMP1 ratio (r = −0.47, p = 0.006). sST2 correlated strongly with GDF-15 (r = 0.7, p < 0.0001). GDF-15 correlated negatively with LVEF (r = −0.49, p = 0.004) and positively with LVIDd (r = 0.58, p = 0.0006). GDF-15 showed significant positive correlation with NYHA functional class (r = 0.71, p < 0.00001) and BNP (r = 0.86, p < 0.00001). Significant associations of GDF-15 with MMPs, sST2, LVIDd, LVEF, and NYHA class reported here for the first time in nonischemic dilated hearts may open up new avenues of investigations to better understand molecular mechanisms controlling cardiac remodeling. This study is limited by its small size and needs validation in larger populations.

Less than 100 nm in size and spherical in form - exosomes – vesicles expelled and taken up by cells, have ignited a new-found fascination. One which is derived from the sheer variety of exosomal content, ranging from microRNAs to transcription factors, capable of affecting a multitude of processes and pathways simultaneously within a target cell. Initially dismissed in 1983 as a waste disposal mechanism, today they form an entire field of research, being documented thus far in invertebrates, mammals, pathogens and potentially some plants. Many studies have suggested these spherical enigmas may possess a function, being implicated in processes ranging from animal behaviour to viral infection. This review will evaluate the evidence for the role of exosomes in physiology and pathophysiology, as well as their potential for application in the diagnosis and treatment of disease.

Alcoholic liver disease (ALD) progresses from steatosis to alcoholic hepatitis to fibrosis and cirrhosis. Liver biopsy is considered as the gold standard method for diagnosis of liver cirrhosis and provides useful information about damaging process which is an invasive procedure with complications. Existing biomarkers in clinical practice have narrow applicability due to lack of specificity and lack of sensitivity. The objective of this article is to identify proteomic biomarker candidates for alcoholic liver cirrhosis by differential expression analysis between alcoholic liver cirrhotic and healthy subjects. Blood samples were collected from 20 subjects (10 alcoholic liver cirrhosis and 10 healthy) from R. L. Jalapa Hospital and Research Centre, Kolar, Karnataka, India. Differential protein analysis was carried out by two-dimensional electrophoresis after albumin depletion, followed by liquid chromatography–mass spectrometry. The image analysis found 46 spots in cirrhotic gel and 69 spots in healthy gel, of which 14 spots were identified with significant altered expression levels. Based on the protein score and clinical significance, among 14 spots, a total of 28 protein biomarker candidates were identified: 13 with increased expression and 15 with decreased expression were categorized in alcoholic liver cirrhosis compared to healthy subjects. Protein biomarker candidates identified by “-omics” approach based on differential expression between alcoholic liver cirrhotic subjects and healthy subjects may give better insights for diagnosis of ALD. Prioritization of candidates identified is a prerequisite for validation regimen. Biomarker candidates require verification that demonstrates the differential expression will remain detectable by assay to be used for validation.

This study shows that fibroblasts migrating into a collagen matrix release numerous microvesicles into the surrounding medium. By spreading in regions of the matrix far distant from cells of origin, microvesicles carry metalloproteinase 9 (MMP-9) to act upon the collagen fibrils. As a result, the collagen matrix is gradually transformed from a laminar to a fibrillar type of architecture. As shown by western blots and gelatin zymography, MMP-9 is secreted as a 92 kDa precursor and activated upon release of 82 kDa product into the culture medium. Activation is more efficient under three-dimensional than in two-dimensional culturing conditions. While MMP-9 labeling is associated with intraluminal vesicles clustered inside the microvesicles, the microvesicle’s integrin β1 marker is bound to the outer membrane. The intraluminal vesicles are recruited from the cortical cytoplasm and eventually released following uploading inside the microvesicle. Here, we propose that fusion of the intraluminal vesicles with the outer microvesicle’s membrane could work as a mechanism controlling the extent to which MMP-9 is first activated and then released extracellularly.

The recently discovered klotho proteins have roles in a diverse range of metabolic processes with the oldest protein, α-klotho, implicated in various cellular pathways in energy, glucose, and phosphate metabolism. Circulating soluble klotho (sKl), derived from membrane α-klotho cleavage, not only has effects on ion channels and insulin signaling pathways, but is inversely associated with mortality. Effects of physical exercise on sKl have not been well studied. The effect of a single high-intensity standardized exercise on sKl and serum phosphate (sPi) levels in healthy adults was investigated. A standard Bruce protocol treadmill exercise was undertaken by 10 fasting healthy volunteers. sKl, sPi, and blood glucose levels were measured in samples collected 1-week prior, immediately pre (T _pre), 0 (T _post), 30 (T ₃₀), 240 (T ₂₄₀) min, and 1-week after exercise. Median (interquartile range) age of participants was 47.5 (44–51) years; five (50%) were male. All study participants achieved at least 90% predicted maximum heart rate (MHR). sKl increased acutely after exercise (T _pre median 448 pg/mL vs. T _post median 576 pg/mL; p < 0.01). There was a nonsignificant sPi decline at T ₃₀ (T _pre 0.94 ± 0.12 mmol/L vs. T ₃₀ 0.83 ± 0.22 mmol/L). Exercise led to a reduction in blood glucose by T ₂₄₀ with median glucose levels at T _pre, T _post, T ₃₀, and T ₂₄₀ of 6.0, 6.5, 6.3, and 5.7 mmol/L, respectively. In conclusion, a single high-intensity exercise session is associated with a transient increase in sKl, a delayed reduction in blood glucose, and a nonsignificant decrease in sPi levels in healthy adults. The evaluation of long-term effects of cardiovascular fitness programs on sKl and sPi in healthy individuals and disease cohorts are required to identify potential lifestyle modifications to help improve chronic disease management and long-term outcomes.

The role of pattern of circulating endothelial cell-derived microparticles, platelet-derived microparticles (PMPs), and monocyte-derived microparticles (MMPs) in metabolic syndrome (MetS) patients with chronic heart failure (CHF) is not still understood. The aim of the study was to investigate a pattern of circulating microparticles (MPs) in MetS patients with CHF in relation to neurohumoral and inflammatory activation. The study retrospectively involved 101 patients with MetS and 35 healthy volunteers. Biomarkers were measured at baseline of the study. The results of the study have shown that numerous circulating PMPs- and MMPs in subjects with MetS (with or without CHF) insufficiently distinguished from level obtained in healthy volunteers. We found elevated level of CD31+/annexin V+ MPs in association with lower level of CD62E+ MPs. Therefore, we found that biomarkers of biomechanical stress serum N-terminal brain natriuretic peptide and inflammation (high-sensitive C-reactive protein ,osteoprotegerin) remain statistically significant predictors for decreased CD62E+ to CD31+/annexin V+ ratio in MetS patients with CHF. In conclusion, decreased CD62E+ to CD31+/annexin V+ ratio reflected that impaired immune phenotype of MPs may be discussed as a surrogate marker of CHF development in MetS population.