Atif Ashraf

Genetically manipulated progenitor cell sheet with diprotin A improves myocardial function and repair of infarcted hearts

We postulated that the combination of overexpression of CXCR4 in mesenchymal stem cells (MSC) with diprotin A would enhance MSC recruitment and penetration into ischemic myocardium, leading to an improvement in heart function after myocardial infarction (MI). Male rat MSC were genetically engineered with adenoviral vectors coexpressing CXCR4 and enhanced green fluorescent protein (EGFP) (MSC(CXCR4)), GFP alone (MSC(Null), control), or siRNA-targeted CXCR4 (MSC(siRNA)). Cell sheets were applied over the surface of infarcted left ventricle (LV) in female rats 7 days after ligation of the left anterior descending coronary artery (LAD) pretreated with either vehicle (VEH) or diprotin A (DIP). At 28 days after cell sheet implantation, echocardiography was performed. Hearts were harvested for histological analysis 7 days after LAD ligation or 28 days after cell sheet implantation. DPP-IV and stroma-derived factor-1α (SDF-1α) in the LV were analyzed. Efficacy of engraftment was determined by the presence of Y chromosome in nuclei (Y(ch+)). LV blood vessel density and apoptosis were also analyzed. Myocardial SDF-1α was elevated before placement of the cell sheet in the DIP group compared with vehicle group on day 7 after LAD. On day 28 after cell sheet transplantation, the number of Y(ch+) was increased in the MSC(CXCR4) + VEH group compared with the MSC(Null) + VEH group and further increased in the MSC(CXCR4) + DIP treated group. This enhanced response was associated with increased angiogenesis in both sides of epicardium and improvement of LV function. Combination of gene-manipulated MSC(CXCR4) patch with DIP pretreatment inhibits myocardial ischemia-induced apoptosis, promotes tissue angiogenesis, and enhances cell engraftment, leading to improved LV mechanical function after MI.

Mesenchymal Stem Cells Overexpressing CXCR4 Attenuate Remodeling of Postmyocardial Infarction by Releasing Matrix Metalloproteinase-9

Myocardial infarction (MI) results in loss of myofibers in the ischemic zone of the heart, followed by scar formation. These factors increase barriers to mobilization of mesenchymal stem cells (MSC), thereby impeding their effectiveness in cardiac repair. This study examined MSC overexpressing CXCR4 (MSC(CX4)) to determine penetration into infarcted myocardium by releasing collagen degrading enzyme, matrix metalloproteinase-9 (MMP-9). In vitro, mouse MSC were utilized, including MSC using adenoviral transduction, to express CXCR4/green fluorescent protein (GFP) (MSC(CX4)), Null/GFP (MSC(Null)), MSC treated with siRNA targeting CXCR4 (MSC(siR)), MSC treated with control siRNA(MSC(Con-siR)), MSC(CX4) treated with siRNA targeting MMP-9 (MSC(CX4-siRMP9)) and MMP-14 (MSC(CX4-siRMP14)), MSC derived from MMP-9 knockout mouse with adenoviral transduction for GFP (MSC(MP9-)), or MSC(MP9-) plus overexpressing CXCR4 (MSC(MP9-CX4)). The ability to cross the basement membrane was evaluated in all MSC using a trans-collagen gel invasion assay. The CXCR4 and MMP expression were analyzed by Western blot. In vivo, MSC with various treatments were infused into mice via tail vein injections 7 days after MI. Echocardiography was performed before harvesting hearts for analysis at 4 weeks after MSC injection. Both in vitro and in vivo studies demonstrated upregulation of MMP-9 induced by MSC(CX4), promoting increased GFP(+) cell migration into the infarcted area in comparison to control group. This enhanced response was associated with reduced left ventricular (LV) fibrosis, increased LV free wall thickness, angiogenesis, and improved LV function. Under hypoxic conditions, MMP-9 is upregulated in MSC(CX4), thus facilitating cross of the basement membrane, resulting in an improved remodeling of post-MI tissue.

Gene manipulated peritoneal cell patch repairs infarcted myocardium

A gene manipulated cell patch using a homologous peritoneum substrate was developed and applied after myocardial infarction to repair scarred myocardium. We genetically engineered male rat mesenchymal stem cells (MSC) using adenoviral transduction to over-express CXCR4/green fluorescent protein (GFP) (MSC(CXCR4)) or MSC(Null) or siRNA targeting CXCR4 (MSC(siRNA)). Gene expression was studied by real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). Cells were cultured on excised peritoneum for 9 days. Two weeks after left anterior descending (LAD) coronary artery ligation in female hearts, the peritoneum patch was applied over the scarred myocardium, cell side down. Efficacy of engraftment was determined by presence of GFP positive cells. One month after cell implantation, echocardiography was performed and hearts were harvested for histological analysis. Left ventricle (LV) fibrosis, LV anterior wall thickness (AWT) and blood vessel density at the margins of the graft were measured. There was significant up-regulation of the chemokines in the MSC(CXCR4) group cultured under normoxic conditions when compared to the MSC(Null) group and a further increase was observed after exposure to hypoxia. One month after cell transplantation with the peritoneum patch, substantial numbers of GFP-positive cells were observed in and around the infarcted myocardium in MSC(CXCR4) group. LV AWT, LV fibrosis and LV function were significantly improved in the MSC(CXCR4) group as compared to these same variables in the MSC(Null) control. These salutary effects were absent in MSC(siRNA) group. The gene manipulated MSC-seeded peritoneum patch promotes tissue nutrition (angiogenesis), reduces myocardial remodeling, and enhances heart function after myocardial infarction.

Suicide Gene Reveals the Myocardial Neovascularization Role of Mesenchymal Stem Cells Overexpressing CXCR4 (MSCCXCR4)

Background Our previous studies indicated that MSCCXCR4 improved cardiac function after myocardial infarction (MI). This study was aimed to investigate the specific role of MSCCXCR4 in neovascularization of infarcted myocardium using a suicide gene approach. Methods MSCs were transduced with either lentivirus-null vector/GFP (MSCNull as control) or vector encoding for overexpressing CXCR4/GFP. The MSC derived-endothelial cell (EC) differentiation was assessed by a tube formation assay, Dil-ac-LDL uptake, EC marker expression, and VE-cadherin promoter activity assay. Gene expression was analyzed by quantitative RT-PCR or Western blot. The suicide gene approach was under the control of VE-cadherin promoter. In vivo studies: Cell patches containing MSCNull or MSCCXCR4 were transduced with suicide gene and implanted into the myocardium of MI rat. Rats received either ganciclovir (GCV) or vehicle after cell implantation. After one month, the cardiac functional changes and neovascularization were assessed by echocardiography, histological analysis, and micro-CT imaging. Results The expression of VEGF-A and HIF-1α was significantly higher in MSCCXCR4 as compared to MSCNull under hypoxia. Additionally, MSCCXCR4 enhanced new vessel formation and EC differentiation, as well as STAT3 phosphorylation under hypoxia. STAT3 participated in the transcription of VE-cadherin in MSCCXCR4 under hypoxia, which was inhibited by WP1066 (a STAT3 inhibitor). In addition, GCV specifically induced death of ECs with suicide gene activation. In vivo studies: MSCCXCR4 implantation promoted cardiac functional restoration, reduced infarct size, improved cardiac remodeling, and enhanced neovascularization in ischemic heart tissue. New vessels derived from MSCCXCR4 were observed at the injured heart margins and communicated with native coronary arteries. However, the derived vessel networks were reduced by GCV, reversing improvement of cardiac function. Conclusion The transplanted MSCCXCR4 enhanced neovascularization after MI by boosting release of angiogenic factors and increasing the potential of endothelial differentiation.