Upregulated miR-200c is associated with downregulation of the functional receptor for severe acute respiratory syndrome coronavirus 2 ACE2 in individuals with obesity.

Obesity is a risk factor for coronavirus disease 2019 (COVID-19) infection, with studies demonstrating the prevalence of individuals with obesity admitted with COVID-19 ranging between 30 and 60%. We determined whether early changes in microRNAs (miRNAs) are associated with dysregulation of angiotensin-converting enzyme 2 (ACE2), the specific functional receptor for severe acute respiratory syndrome coronavirus 2. ACE2 is a membrane-bound enzyme that catalyzes the conversion of angiotensin II to angiotensin 1-7 the latter having cardioprotective and vasorelaxation effects. Quantitative real-time PCR analysis of plasma samples for circulating miRNAs showed upregulation of miR-200c and miR-let-7b in otherwise healthy individuals with obesity. This was associated with significant downregulation of ACE2, a direct target for both miRNAs, in individuals with obesity. Correlation analysis confirmed a significant negative correlation between ACE2 and both the miRNAs. Studies showed that despite being the functional receptor, inhibition/downregulation of ACE2 did not reduce the severity of COVID-19 infection. In contrast, increased angiotensin II following inhibition of ACE2 may increase the severity of the disease. Taken together, our novel results identify that upregulation of miR-200c may increase the susceptibility of individuals with obesity to COVID-19. Considering miRNA are the earliest molecular regulators, the level of circulating miR-200c could be a potential biomarker in the early identification of those at the risk of severe COVID-19.

Cardiac Progenitor Cells and Adipocyte Stem Cells from Same Patients Exhibit In Vitro Functional Differences.

Cardiac progenitor cells (CPCs) and adipocyte stem cells (ASCs) are widely tested for their efficacy in repairing the diseased heart with varying results. However, no study has directly compared the functional efficacy of CPCs and ASCs collected from the same patient. CPCs and ASCs were isolated from the right atrial appendage and epicardial adipose tissue of the same patients, using explant culture. The flow cytometry analysis confirmed that both the cell types express common mesenchymal stem cells markers CD90 and CD105. ASCs, in addition, expressed CD29 and CD73. The wound-healing assay demonstrated that CPCs migrate faster to cover the wound area. Both cell types were resistant to hypoxia-induced cell death when exposed to hypoxia and serum deprivation; however, the ASCs showed increased proliferation. Conditioned medium (CM) collected after culturing serum-deprived CPCs and ASCs showed differential secretion patterns, with ASC CM showing an increased IGF-1 level, while CPC CM showed an increased FGF level. Only CPC CM reduced hypoxia-induced apoptosis in AC-16 human ventricular cardiomyocytes, while vascular network formation by endothelial cells was comparable between CPC and ASC CM. In conclusion, ASCs and CPCs exhibit differential characteristics within the same patient, and in vitro studies showed that CPCs have marginally superior functional efficacy.

Diabetes induces dysregulation of microRNAs associated with survival, proliferation and self-renewal in cardiac progenitor cells.

AIMS/HYPOTHESIS: Diabetes mellitus causes a progressive loss of functional efficacy in stem cells, including cardiac progenitor cells (CPCs). The underlying molecular mechanism is still not known. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate genes at the post-transcriptional level. We aimed to determine if diabetes mellitus induces dysregulation of miRNAs in CPCs and to test if in vitro therapeutic modulation of miRNAs would improve the functions of diabetic CPCs. METHODS: CPCs were isolated from a mouse model of type 2 diabetes (db/db), non-diabetic mice and human right atrial appendage heart tissue. Total RNA isolated from mouse CPCs was miRNA profiled using Nanostring analysis. Bioinformatic analysis was employed to predict the functional effects of altered miRNAs. MS analysis was applied to determine the targets, which were confirmed by western blot analysis. Finally, to assess the beneficial effects of therapeutic modulation of miRNAs in vitro and in vivo, prosurvival miR-30c-5p was overexpressed in mouse and human diabetic CPCs, and the functional consequences were determined by measuring the level of apoptotic cell death, cardiac function and mitochondrial membrane potential (MMP). RESULTS: Among 599 miRNAs analysed in mouse CPCs via Nanostring analysis, 16 miRNAs showed significant dysregulation in the diabetic CPCs. Using bioinformatics tools and quantitative real-time PCR (qPCR) validation, four altered miRNAs (miR-30c-5p, miR-329-3p, miR-376c-3p and miR-495-3p) were identified to play an important role in cell proliferation and survival. Diabetes mellitus significantly downregulated miR-30c-5p, while it upregulated miR-329-3p, miR-376c-3p and miR-495-3p. MS analysis revealed proapoptotic voltage-dependent anion-selective channel 1 (VDAC1) as a direct target for miR-30c-5p, and cell cycle regulator, cyclin-dependent protein kinase 6 (CDK6), as the direct target for miR-329-3p, miR-376c-3p and miR-495-3p. Western blot analyses showed a marked increase in VDAC1 expression, while CDK6 expression was downregulated in diabetic CPCs. Finally, in vitro and in vivo overexpression of miR-30c-5p markedly reduced the apoptotic cell death and preserved MMP in diabetic CPCs via inhibition of VDAC1. CONCLUSIONS/INTERPRETATION: Our results demonstrate that diabetes mellitus induces a marked dysregulation of miRNAs associated with stem cell survival, proliferation and differentiation, and that therapeutic overexpression of prosurvival miR-30c-5p reduced diabetes-induced cell death and loss of MMP in CPCs via the newly identified target for miR-30c-5p, VDAC1.

Stage-specific regulation of signalling pathways to differentiate pluripotent stem cells to cardiomyocytes with ventricular lineage.

BACKGROUND: Pluripotent stem cells (PSCs) can be an ideal source of differentiation of cardiomyocytes in vitro and during transplantation to induce cardiac regeneration. However, differentiation of PSCs into a heterogeneous population is associated with an increased incidence of arrhythmia following transplantation. We aimed to design a protocol to drive PSCs to a ventricular lineage by regulating Wnt and retinoic acid (RA) signalling pathways. METHODS: Mouse embryonic stem cells were cultured either in monolayers or three-dimensional hanging drop method to form embryonic bodies (EBs) and exposed to different treatments acting on Wnt and retinoic acid signalling. Samples were collected at different time points to analyse cardiomyocyte-specific markers by RT-PCR, flow cytometry and immunofluorescence. RESULTS: Treatment of monolayer and EBs with Wnt and RA signalling pathways and ascorbic acid, as a cardiac programming enhancer, resulted in the formation of an immature non-contractile cardiac population that expressed many of the putative markers of cardiac differentiation. The population exhibited upregulation of ventricular specific markers while suppressing the expression of pro-atrial and pro-sinoatrial markers. Differentiation of EBs resulted in early foetal like non-contractile ventricular cardiomyocytes with an inherent propensity to contract when stimulated. CONCLUSION: Our results provide the first evidence of in vitro differentiation that mimics the embryonic morphogenesis towards ventricular specific cardiomyocytes through regulation of Wnt and RA signalling pathways.

Acute Weight Loss Restores Dysregulated Circulating MicroRNAs in Individuals Who Are Obese.

CONTEXT: Obesity is a global epidemic and an independent risk factor for several diseases. miRNAs are gaining interest as early molecular regulators of various pathological processes. OBJECTIVE: To examine the miRNA signatures in women who are obese and determine the response of miRNAs to acute weight loss. METHODS: Plasma samples were collected from women who are obese (n = 80) before and after acute weight loss (mean, 7.2%). Plasma samples from age-matched lean volunteers (n = 80) were used as controls. Total RNA was extracted from the plasma samples and subjected to NanoString analysis of 822 miRNAs. The expression level of candidate miRNAs was validated in all participants using quantitative real-time PCR analysis. RESULTS: NanoString analysis identified substantial dysregulation of 21 miRNAs in women who are obese that were associated with impaired glucose tolerance, senescence, cardiac hypertrophy, angiogenesis, inflammation, and cell death. Acute weight loss reversed the expression pattern of 18 of these miRNAs toward those seen in the lean control group. Furthermore, real-time PCR validation of all the samples for 13 miRNAs with at least twofold upregulation or downregulation confirmed substantial dysregulation of all the chosen miRNAs in women who are obese at baseline. After acute weight loss, the levels of seven miRNAs in women who are obese and who are lean were comparable, with no statistically significant evidence for differences between the two groups. CONCLUSIONS: Our study has provided evidence that the circulating miRNAs associated with various disorders are dysregulated in women who are obese. We also found that seven of these miRNAs showed levels comparable to those in lean controls after acute weight loss in women who are obese.