Identifying stable reference genes in polyethene glycol precipitated urinary extracellular vesicles for RT-qPCR-based gene expression studies in renal graft dysfunction patients.

BACKGROUND: Urinary extracellular vesicles (UEVs) hold RNA in their cargo and are potential sources of biomarkers for gene expression studies. The most used technique for gene-expression studies is quantitative polymerase chain reaction (qPCR). It is critical to use stable reference genes (RGs) as internal controls for normalising gene expression data, which aren't currently available for UEVs. METHODS: UEVs were precipitated from urine of graft dysfunction patients and healthy controls by Polyethylene glycol, Mn6000 (PEG6K). Vesicular characterisation confirmed the presence of UEVs. Gene expression levels of five commonly used RGs, i.e., Beta-2-Microglobulin (B2M), ribosomal-protein-L13a (RPL13A), Peptidylprolyl-Isomerase-A (PPIA), hydroxymethylbilane synthase (HMBS), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were quantified, and their stability was established through the RefFinder. The stability of identified RGs was validated by quantification of Perforin and granzyme B, signature molecules of renal graft dysfunction. RESULTS: Urine precipitated with 12% 6 K PEG yielded round and double-membraned UEVs of size ranging from 30 to 100 nm, as confirmed through transmission electron microscopy. Nanoparticle tracking analysis (59 ± 22 nm) and Dynamic-light-scattering (78 ± 56.5 nm) confirmed their size profile. Semi-quantitative Exocheck antibody array demonstrated the presence of EV protein markers in UEV. Using the comparative ΔCт method and RefFinder analysis, B2M (1.6) and RPL13A (1.8) genes emerged as the most stable reference genes. Validation of target gene expression in renal graft dysfunction patients confirmed the efficiency of B2M and RPL13A through significant upregulation compared to other RGs. CONCLUSIONS: Our study identified and validated B2M and RPL13A as optimal RGs for mRNA quantification studies in the UEVs of patients with renal graft dysfunction.

Nano-Messengers of the Heart: Promising Theranostic Candidates for Cardiovascular Maladies.

Till date, cardiovascular diseases remain a leading cause of morbidity and mortality across the globe. Several commonly used treatment methods are unable to offer safety from future complications and longevity to the patients. Therefore, better and more effective treatment measures are needed. A potential cutting-edge technology comprises stem cell-derived exosomes. These nanobodies secreted by cells are intended to transfer molecular cargo to other cells for the establishment of intercellular communication and homeostasis. They carry DNA, RNA, lipids, and proteins; many of these molecules are of diagnostic and therapeutic potential. Several stem cell exosomal derivatives have been found to mimic the cardioprotective attributes of their parent stem cells, thus holding the potential to act analogous to stem cell therapies. Their translational value remains high as they have minimal immunogenicity, toxicity, and teratogenicity. The current review highlights the potential of various stem cell exosomes in cardiac repair, emphasizing the recent advancements made in the development of cell-free therapeutics, particularly as biomarkers and as carriers of therapeutic molecules. With the use of genetic engineering and biomimetics, the field of exosome research for heart treatment is expected to solve various theranostic requirements in the field paving its way to the clinics.

Exosomal PTEN as a Predictive Marker of Aggressive Gliomas.

Background: Liquid biopsies have emerged as convenient alternative diagnostic methods to invasive biopsies, by evaluating disease-specific biomarkers and monitoring the disease risk noninvasively. Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) is a potent tumor suppressor, and its deletion/mutations are common in gliomas. Objective: Evaluate the feasibility of non-invasive detection of PTEN and its downstream genes in serum exosomes of glioma patients. Materials and methods: PTEN, Yes-associated-protein 1 (YAP1), and lysyl oxidase (LOX) transcript expression were monitored through polymerase chain reaction (PCR) in serum exosomes and their paired tumor tissues. The impact of PTEN and its axis genes expression on the overall survival (OS) was monitored. Results: Out of the 106 glioma serum samples evaluated, PTEN was retained/lost in 65.4%/34.6% of the tumor samples while it was retained/lost in 67.1%/32.9% of their paired exosomal fractions. PTEN expression in both tissue and paired exosomal fractions was observed in 48.11% of the samples. Sanger sequencing detected three mutations (Chr10: 89720791(A>G), Chr10:89720749(C>T), and Chr10:89720850(A>G). Both PTEN-responsive downstream genes (YAP1) and LOX axis were upregulated in the PTEN-deficient samples. PTEN loss was associated with poor survival in the glioma patients (hazard ratio (HR) 0.68, confidence interval (CI): 0.35-1.31, P = 0.28). The OS of the exosomal PTEN cohort coincided with the tumor-tissue PTEN devoid group (HR 1.08, CI: 0.49-2.36, P = 0.85). While, old age yielded the worst prognosis; gender, location, and grade were not prognostic of OS in the multivariate analysis. Conclusions: PTEN and its responsive genes YAP1 and LOX can be detected in serum exosomes and can serve as essential tools for the non-invasive evaluation/identification of aggressive gliomas.

Cardioprotective effect of the secretome of Sca-1+ and Sca-1- cells in heart failure: not equal, but equally important?

AIMS: Both progenitor and differentiated cells were previously shown to secrete cardioprotective substances, but so far there has been no direct comparison of the paracrine effects of the two cell types on heart failure. The study sought to compare the paracrine effect of selected progenitors and the corresponding non-progenitor mononuclear cardiac cells on the cardiac function of transgenic heart failure mice. In addition, we aimed to further enhance the paracrine effect of the cells via pretreatment with the heart failure mediator aldosterone. METHODS AND RESULTS: Transgenic heart failure mice were injected with the supernatant of murine cardiac stem cell antigen-1 positive (Sca-1+) and negative (Sca-1-) cells with or without aldosterone pretreatment. Cardiac function was determined using small animal magnetic resonance imaging. In addition, heart failure markers were determined using enzyme-linked immunosorbent assay, RT-PCR, and bead-based multiplexing assay. While only the secretome of aldosterone pretreated Sca-1+ cells led to a significant improvement in cardiac function, N-terminal pro brain natriuretic peptide plasma levels were significantly lower and galectin-1 levels significantly higher in mice that were treated with either kind of secretome compared with untreated controls. CONCLUSION: In this first direct comparison of the paracrine effects of progenitor cells and a heterogeneous population of mononuclear cardiac cells the supernatants of both cell types showed cardioprotective properties which might be of great relevance for endogenous repair. During heart failure raised aldosterone levels might further increase the paracrine effect of progenitor cells.

Global secretome analysis of resident cardiac progenitor cells from wild-type and transgenic heart failure mice: Why ambience matters.

Resident cardiac progenitor cells (CPCs) have gained attention in cardiac regenerative medicine primarily due to their paracrine activity. In our current study we determined the role of pathological conditions such as heart failure on the autocrine-paracrine action of stem cell antigen-1 (Sca-1) expressing CPC. This comparative secretome profiling of Sca-1+ cells derived from transgenic heart failure (αMHC-cyclin-T1/Gαq overexpression [Cyc] cells) versus healthy (wild-type [Wt] cells) mice, achieved via mass-spectrometric quantification, enabled the identification of over 700 proteins. Our results demonstrate that the heart failure milieu caused a 2-fold enrichment of extracellular matrix proteins (ECM) like biglycan, versican, collagen XII, and angiogenic factors like heparan sulfate proteoglycan 2, plasminogen activator inhibitor 1 in the secretome. We further elucidated the direct influence of the secretome on the functional behavior of Sca-1 + cells via in vitro tube forming assay. Secreted factors present in the diseased milieu induced tube formation in Cyc cells (1.7-fold; p < 0.01) when compared with Wt cells after 24 hr of exposure. The presence of conditioned media moderately increased the proliferation of Cyc cells but had a more pronounced effect on Wt cells. Overall, these findings revealed global modifications in the secretory activity of adult Sca-1 + cells in the heart failure milieu. The secretion of ECM proteins and angiogenic factors, which are crucial for cardiac remodeling and recovery, was notably enriched in the supernatant of Cyc cells. Thus, during heart failure the microenvironment of Sca-1 + cells might favor angiogenesis and proliferation suggesting their potential to recover the damaged heart.

Pathophysiological aldosterone levels modify the secretory activity of cardiac progenitor cells.

Cardiac progenitor cells (CPCs) trigger regenerative processes via paracrine mechanisms in response to changes in their environment. In the present study we explored alterations in the secretory activity of CPCs induced by raised aldosterone levels symptomatic for heart failure. The cytokine profile of the supernatant of CPCs that were treated with the mineralocorticoid showed an induction of interleukin-6 secretion. Mass spectrometric analyses revealed an increase in the abundance of secreted proteins associated with regeneration and cell migration like gelsolin and galectin-1. Differential regulation of proteins associated with the extracellular matrix further points to an activation of cell migration. In response to supernatant, migration and proliferation were induced in CPCs, indicating a potential role of paracrine factors in the activation of CPCs from other regions of the heart or extra-cardiac sources. Changes in the secretory activity of CPCs might aim to compensate for the detrimental actions of aldosterone in heart failure.

The Other Side of the RAAS: Aldosterone Improves Migration of Cardiac Progenitor Cells.

Stem cell therapy is a promising new option for patients suffering from heart failure. Though many clinical studies show encouraging results, little is known about the signals which cause stem cells to home to diseased but not to healthy hearts. We hypothesized that aldosterone as one of the main players of heart failure functions as an attractant for progenitor cells and stimulates their migration. Stem cell antigen-1 (Sca-1) positive cells were isolated from the hearts of wild type FVB mice via magnetic cell sorting. The migration rate of the cells was determined using aldosterone as an attractant in a modified Boyden chamber (n = 5). Aldosterone led to a dose dependent increase in migration rate and this effect could be prevented by adding its blocker eplerenone. The mineralocorticoid receptor could be detected on Sca-1+ cells via western blot and immunofluorescence. Therefore, aldosterone seems to play a role in stem cell migration and there the effect is most likely mediated by the mineralocorticoid receptor.

Brain derived neurotrophic factor contributes to the cardiogenic potential of adult resident progenitor cells in failing murine heart.

AIMS: Resident cardiac progenitor cells show homing properties when injected into the injured but not to the healthy myocardium. The molecular background behind this difference in behavior needs to be studied to elucidate how adult progenitor cells can restore cardiac function of the damaged myocardium. Since the brain derived neurotrophic factor (BDNF) moderates cardioprotection in injured hearts, we focused on delineating its regulatory role in the damaged myocardium. METHODS AND RESULTS: Comparative gene expression profiling of freshly isolated undifferentiated Sca-1 progenitor cells derived either from heart failure transgenic αMHC-CyclinT1/Gαq overexpressing mice or wildtype littermates revealed transcriptional variations. Bdnf expression was up regulated 5-fold during heart failure which was verified by qRT-PCR and confirmed at protein level. The migratory capacity of Sca-1 cells from transgenic hearts was improved by 15% in the presence of 25 ng/ml BDNF. Furthermore, BDNF-mediated effects on Sca-1 cells were studied via pulsed Stable Isotope Labeling of Amino acids in Cell Culture (pSILAC) proteomics approach. After BDNF treatment significant differences between newly synthesized proteins in Sca-1 cells from control and transgenic hearts were observed for CDK1, SRRT, HDGF, and MAP2K3 which are known to regulate cell cycle, survival and differentiation. Moreover BDNF repressed the proliferation of Sca-1 cells from transgenic hearts. CONCLUSION: Comparative profiling of resident Sca-1 cells revealed elevated BDNF levels in the failing heart. Exogenous BDNF (i) stimulated migration, which might improve the homing ability of Sca-1 cells derived from the failing heart and (ii) repressed the cell cycle progression suggesting its potency to ameliorate heart failure.

OMICS-based exploration of the molecular phenotype of resident cardiac progenitor cells from adult murine heart.

Resident cardiac progenitor cells have emerged as a potential source of adult stem cells for regeneration of damaged myocardium. Sca-1 cells, expressing Stem cell antigen-1 as a cell surface marker, are multipotent cells that were shown to differentiate into different cell types i.e. cardiomyocytes. Previous studies have reported that Sca-1 positive cells are able to home to the injured heart. However, the mechanism of improving cardiac function is still unclear. In the current study, we have profiled the proteome and transcriptome of Sca-1 positive cells in comparison with other endogenous heart cell types to unravel the molecular phenotype of the progenitor cells. Among the 861 proteins identified with high confidence in total, 331 non-redundant proteins were overrepresented in Sca-1 positive cells. Highly abundant candidates were mostly associated with cell growth and proliferation, cell migration and cytoskeletal organization. Transcriptional profiling disclosed significant expression of surface antigens such as CD31, CD36, CD38, CD66a, CD102, and CD202B. Growth factors like KITL, JAG2, PDGFB and VEGFC showed a higher expression in Sca-1 progenitor cells than in Sca-1 negative cells. Selective candidates were validated by Western blotting. These global findings provide a basis for the study of their capability to participate in the cardiac regeneration process.