Zhili Wen

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

Exosomes Secreted from CXCR4 Overexpressing Mesenchymal Stem Cells Promote Cardioprotection via Akt Signaling Pathway following Myocardial Infarction

Background and Objective. Exosomes secreted from mesenchymal stem cells (MSC) have demonstrated cardioprotective effects. This study examined the role of exosomes derived from MSC overexpressing CXCR4 for recovery of cardiac functions after myocardial infarction (MI). Methods. In vitro, exosomes from MSC transduced with lentiviral CXCR4 (ExoCR4) encoding a silencing sequence or null vector were isolated and characterized by transmission electron microscopy and dynamic light scattering. Gene expression was then analyzed by qPCR and Western blotting. Cytoprotective effects on cardiomyocytes were evaluated and effects of exosomes on angiogenesis analyzed. In vivo, an exosome-pretreated MSC-sheet was implanted into a region of scarred myocardium in a rat MI model. Angiogenesis, infarct size, and cardiac functions were then analyzed. Results. In vitro, ExoCR4 significantly upregulated IGF-1α and pAkt levels and downregulated active caspase 3 level in cardiomyocytes. ExoCR4 also enhanced VEGF expression and vessel formation. However, effects of ExoCR4 were abolished by an Akt inhibitor or CXCR4 knockdown. In vivo, ExoCR4 treated MSC-sheet implantation promoted cardiac functional restoration by increasing angiogenesis, reducing infarct size, and improving cardiac remodeling. Conclusions. This study reveals a novel role of exosomes derived from MSCCR4 and highlights a new mechanism of intercellular mediation of stem cells for MI treatment.

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.

Molecular Strategy to Reduce In Vivo Collagen Barrier Promotes Entry of NCX1 Positive Inducible Pluripotent Stem Cells (iPSCNCX1+) into Ischemic (or Injured) Myocardium

Objective The purpose of this study was to assess the effect of collagen composition on engraftment of progenitor cells within infarcted myocardium. Background We previously reported that intramyocardial penetration of stem/progenitor cells in epicardial patches was enhanced when collagen was reduced in hearts overexpressing adenylyl cyclase-6 (AC6). In this study we hypothesized an alternative strategy wherein overexpression of microRNA-29b (miR-29b), inhibiting mRNAs that encode cardiac fibroblast proteins involved in fibrosis, would similarly facilitate progenitor cell migration into infarcted rat myocardium. Methods In vitro: A tri-cell patch (Tri-P) consisting of cardiac sodium-calcium exchanger-1 (NCX1) positive iPSC (iPSCNCX1+), endothelial cells (EC), and mouse embryonic fibroblasts (MEF) was created, co-cultured, and seeded on isolated peritoneum. The expression of fibrosis-related genes was analyzed in cardiac fibroblasts (CFb) by qPCR and Western blot. In vivo: Nude rat hearts were administered mimic miRNA-29b (miR-29b), miRNA-29b inhibitor (Anti-29b), or negative mimic (Ctrl) before creation of an ischemically induced regional myocardial infarction (MI). The Tri-P was placed over the infarcted region 7 days later. Angiomyogenesis was analyzed by micro-CT imaging and immunofluorescent staining. Echocardiography was performed weekly. Results The number of green fluorescent protein positive (GFP+) cells, capillary density, and heart function were significantly increased in hearts overexpressing miR-29b as compared with Ctrl and Anti-29b groups. Conversely, down-regulation of miR-29b with anti-29b in vitro and in vivo induced interstitial fibrosis and cardiac remodeling. Conclusion Overexpression of miR-29b significantly reduced scar formation after MI and facilitated iPSCNCX1+ penetration from the cell patch into the infarcted area, resulting in restoration of heart function after MI.

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.

Ultrastructural Features of Ischemic Tissue following Application of a Bio-Membrane Based Progenitor Cardiomyocyte Patch for Myocardial Infarction Repair

Background and Objective Implantation of cell-sheets into damaged regions of the heart after myocardial infarction (MI) has been shown to improve heart function. However, the tissue morphology following application of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) has not been studied in detail at the level afforded by electron microscopy. We hypothesized that increasing the number of CM derived from iPSC would increase the effectiveness of cell-sheets used to treat ischemic cardiomyopathy. We report here on the ultrastructural features after application of a bio-membrane ‘cell patch’. Methods iPSC-derived progenitor cells were transduced using lentivirus vectors with or without NCX1 promoter. iPSC-CM sheets were transplanted over the transmural MI region in a mouse model of regional ischemic cardiomyopathy. Mice were divided into four groups, 1) Sham; 2) MI; 3) MI + iPSC without NCX1 treated cells (MI + iPSCNull) and 4) MI + iPSC receiving NCX1 promoter treated cells (MI + iPSCNCX1). Echocardiography was performed 4 weeks after cell patch application, followed by histological and transmission electron microscopy (TEM) analysis. Results Large numbers of transplanted CM were observed with significant improvements in left ventricular performance and remodeling in group 4 as compared with group 3. No teratoma formation was detected in any of the treatment groups. Conclusion Manipulation of iPSC yields large numbers of iPSC-CM and favorable morphological and ultrastructural tissue changes. These changes have the potential to enhance current methods used for restoration of cardiac function after MI.

Repair Injured Heart by Regulating Cardiac Regenerative Signals

Cardiac regeneration is a homeostatic cardiogenic process by which the sections of malfunctioning adult cardiovascular tissues are repaired and renewed employing a combination of both cardiomyogenesis and angiogenesis. Unfortunately, while high-quality regeneration can be performed in amphibians and zebrafish hearts, mammalian hearts do not respond in kind. Indeed, a long-term loss of proliferative capacity in mammalian adult cardiomyocytes in combination with dysregulated induction of tissue fibrosis impairs mammalian endogenous heart regenerative capacity, leading to deleterious cardiac remodeling at the end stage of heart failure. Interestingly, several studies have demonstrated that cardiomyocyte proliferation capacity is retained in mammals very soon after birth, and cardiac regeneration potential is correspondingly preserved in some preadolescent vertebrates after myocardial infarction. There is therefore great interest in uncovering the molecular mechanisms that may allow heart regeneration during adult stages. This review will summarize recent findings on cardiac regenerative regulatory mechanisms, especially with respect to extracellular signals and intracellular pathways that may provide novel therapeutics for heart diseases. Particularly, both in vitro and in vivo experimental evidences will be presented to highlight the functional role of these signaling cascades in regulating cardiomyocyte proliferation, cardiomyocyte growth, and maturation, with special emphasis on their responses to heart tissue injury.