Yuliang Feng

Ischemic Preconditioning Potentiates the Protective Effect of Stem Cells through Secretion of Exosomes by Targeting Mecp2 via miR-22

Mesenchymal stem cells (MSCs) have potential application for the treatment of ischemic heart diseases. Besides differentiation properties, MSCs protect ischemic cardiomyocytes by secretion of paracrine factors. In this study, we found exosomes enriched with miR-22 were secreted by MSCs following ischemic preconditioning (ExoIPC) and mobilized to cardiomyocytes where they reduced their apoptosis due to ischemia. Interestingly, by time-lapse imaging, we for the first time captured the dynamic shedding of miR-22 loaded exosomes from cytosol to extracellular space. Furthermore, the anti-apoptotic effect of miR-22 was mediated by direct targeting of methyl CpG binding protein 2 (Mecp2). In vivo data showed that delivery of ExoIPC significantly reduced cardiac fibrosis. Our data identified a significant benefit of ExoIPC for the treatment of cardiac diseases by targeting Mecp2 via miR-22.

Heat Shock Improves Sca‐1+ Stem Cell Survival and Directs Ischemic Cardiomyocytes Toward a Prosurvival Phenotype Via Exosomal Transfer: A Critical Role for HSF1/miR‐34a/HSP70 Pathway

Stem cell‐based therapy is a promising intervention for ischemic heart diseases. However, the functional integrity of stem cells is impaired in an ischemic environment. Here, we report a novel finding that heat shock significantly improves Sca‐1+stem cell survival in an ischemic environment by the regulation of the triangle: heat shock factor 1 (HSF1), HSF1/miR‐34a, and heat shock protein 70 (HSP70). Initially we prove that HSP70 is the key chaperone‐mediating cytoprotective effect of heat shock in Sca‐1+cells and then we establish miR‐34a as a direct repressor of HSP70. We found that HSP70 was downregulated in heat shocked Sca‐1+ stem cells (HSSca‐1+ cells). Intriguingly, we demonstrate that the downregulation of miR‐34a is attributed to HSF1‐mediated epigenetic repression through histone H3 Lys27 trimethylation (H3K27me3) on miR‐34a promoter. Moreover, we show that heat shock induces exosomal transfer of HSF1 from Sca‐1+ cells, which directs ischemic cardiomyocytes toward a prosurvival phenotype by epigenetic repression of miR‐34a. In addition, our in vivo study demonstrates that transplantation of HSSca‐1+ cells significantly reduces apoptosis, attenuates fibrosis, and improves global heart functions in ischemic myocardium. Hence, our study provides not only novel insights into the effects of heat shock on stem cell survival and paracrine behavior but also may have therapeutic values for stem cell therapy in ischemic heart diseases. Stem Cells 2014;32:462–472

MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF

MicroRNAs have been appreciated in various cellular functions, including the regulation of angiogenesis. Mesenchymal-stem-cells (MSCs) transplanted to the MI heart improve cardiac function through paracrine-mediated angiogenesis. However, whether microRNAs regulate MSC induced angiogenesis remains to be clarified. Using microRNA microarray analysis, we identified a microRNA expression profile in hypoxia-treated MSCs and observed that among all dysregulated microRNAs, microRNA-377 was decreased the most significantly. We also validated that vascular endothelial growth factor (VEGF) is a target of microRNA-377 using dual-luciferase reporter assay and Western-blotting. Knockdown of endogenous microRNA-377 promoted tube formation in human umbilical vein endothelial cells. We then engineered rat MSCs with lentiviral vectors to either overexpress microRNA-377 (MSCmiR-377) or knockdown microRNA-377 (MSCAnti-377) to investigate whether microRNA-377 regulated MSC-induced myocardial angiogenesis, using MSCs infected with lentiviral empty vector to serve as controls (MSCNull). Four weeks after implantation of the microRNA-engineered MSCs into the infarcted rat hearts, the vessel density was significantly increased in MSCAnti-377-hearts, and this was accompanied by reduced fibrosis and improved myocardial function as compared to controls. Adverse effects were observed in MSCmiR-377-treated hearts, including reduced vessel density, impaired myocardial function, and increased fibrosis in comparison with MSCNull-group. These findings indicate that hypoxia-responsive microRNA-377 directly targets VEGF in MSCs, and knockdown of endogenous microRNA-377 promotes MSC-induced angiogenesis in the infarcted myocardium. Thus, microRNA-377 may serve as a novel therapeutic target for stem cell-based treatment of ischemic heart disease.

Protein kinase G1 α overexpression increases stem cell survival and cardiac function after myocardial infarction.

Background We hypothesized that overexpression of cGMP-dependent protein kinase type 1α (PKG1α) could mimic the effect of tadalafil on the survival of bone marrow derived mesenchymal stem cells (MSCs) contributing to regeneration of the ischemic heart. Methods and Results MSCs from male rats were transduced with adenoviral vector encoding for PKG1α (PKG1αMSCs).Controls included native MSCs (NatMSCs) and MSCs transduced with an empty vector (NullMSCs). PKG1α activity was increased approximately 20, 5 and 16 fold respectively in PKG1αMSCs. PKG1αMSCs showed improved survival under oxygen and glucose deprivation (OGD) which was evidenced by lower LDH release, caspase-3/7 activity and number of positive TUNEL cells. Anti-apoptotic proteins pAkt, pGSK3β, and Bcl-2 were significantly increased in PKG1αMSCs compared to NatMSCs and NullMSCs. Higher release of multiple prosurvival and angiogenic factors such as HGF, bFGF, SDF-1 and Ang-1 was observed in PKG1αMSCs before and after OGD. In a female rat model of acute myocardial infarction, PKG1αMSCs group showed higher survival compared with NullMSCs group at 3 and 7 days after transplantation as determined by TUNEL staining and sry-gene quantitation by real-time PCR. Increased anti-apoptotic proteins and paracrine factors in vitro were also identified. Immunostaining for cardiac troponin I combined with GFP showed increased myogenic differentiation of PKG1αMSCs. At 4 weeks after transplantation, compared to DMEM group and NullMSCs group, PKG1αMSCs group showed increased blood vessel density in infarct and peri-infarct areas (62.5±7.7; 68.8±7.3 per microscopic view, p<0.05) and attenuated infarct size (27.2±2.5%, p<0.01). Heart function indices including ejection fraction (52.1±2.2%, p<0.01) and fractional shortening (24.8%±1.3%, p<0.01) were improved significantly in PKG1αMSCs group. Conclusion Overexpression of PKG1α transgene could be a powerful approach to improve MSCs survival and their angiomyogenic potential in the infarcted heart.

CXCR4 attenuates cardiomyocytes mitochondrial dysfunction to resist ischaemia-reperfusion injury

The chemokine (C‐X‐C motif) receptor 4 (CXCR4) is expressed on native cardiomyocytes and can modulate isolated cardiomyocyte contractility. This study examines the role of CXCR4 in cardiomyocyte response to ischaemia‐reperfusion (I/R) injury. Isolated adult rat ventricular cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury. In response to H/R injury, the decrease in CXCR4 expression was associated with dysfunctional energy metabolism indicated by an increased adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio. CXCR4‐overexpressing cardiomyocytes were used to determine whether such overexpression (OE) can prevent bio‐energetic disruption‐associated cell death. CXCR4 OE was performed with adenoviral infection with CXCR4 encoding‐gene or non‐translated nucleotide sequence (Control). The increased CXCR4 expression was observed in cardiomyocytes post CXCR4‐adenovirus transduction and this OE significantly reduced the cardiomyocyte contractility under basal conditions. Although the same extent of H/R‐provoked cytosolic calcium overload was measured, the hydrogen peroxide‐induced decay of mitochondrial membrane potential was suppressed in CXCR4 OE group compared with control group, and the mitochondrial swelling was significantly attenuated in CXCR4 group, implicating that CXCR4 OE prevents permeability transition pore opening exposure to overload calcium. Interestingly, this CXCR4‐induced mitochondrial protective effect is associated with the enhanced signal transducer and activator of transcription 3 (expression in mitochondria. Consequently, in the presence of H/R, mitochondrial dysfunction was mitigated and cardiomyocyte death was decreased to 65% in the CXCR4 OE group as compared with the control group. I/R injury leads to the reduction in CXCR4 in cardiomyocytes associated with the dysfunctional energy metabolism, and CXCR4 OE can alleviate mitochondrial dysfunction to improve cardiomyocyte survival.