Abstract 1846: Coordinated Interaction between Survival Signaling and Angio-Competent Molecules Enhances Donor Cell Survival and Angiogenesis in the Infarcted Heart
We have previously shown that transplantation of mesenchymal stem cells (MSCs) co-overexpressing angiopoietin-1 (Ang) and Akt (Akt/AngMSCs) showed improved survival and enhanced angiogenesis in the infarcted myocardium. Here we studied the molecular mechanism for enhanced angiogenesis after Akt/AngMSCs engraftment. MSCs were transduced with genes encoding for GFP (GFPMSCs) as a control, Akt (AktMSCs), Ang (AngMSCs), and Akt/AngMSCs. Subsequent to 8h anoxia, LDH assay showed that Akt/AngMSCs had the highest survival among all cell groups (p=0.01). Cell lysate and conditioned medium (CM) were collected from each cell group after 3 days of gene transduction. Real-time PCR based array showed more than 2-fold increase in expression of HIF-1α, HIF-3α, HO-1, EPAS1, PECAM, CD54, CD53, and Flt-1 in Akt/AngMSCs. Immunoblotting on the nuclear fraction for HIF-1α and the cytosolic fraction for HO-1 showed higher levels (p<0.05) of these proteins in Akt/AngMSCs than other groups. CM from Akt/AngMSCs induced significantly higher formation of tubular structures and mobilization of human umbilical vein endothelial cells (HUVEC) during in vitro Matrigel and invasion assays respectively. Analysis of CM for the secreted prosurvival paracrine factors showed higher levels of SFRP-2 (4 fold) in Akt/AngMSCs. Moreover, adult rat cardiomyocytes and HUVEC treated with CM from Akt/AngMSCs for 30 min showed increased resistance to oxidant stress (100μM H2O2 for 4 h) (p<0.01 vs controls). For in vivo studies, 70μl DMEM without cells (group-1) or containing 3x106 non-transduced MSC (group-2) or Akt/AngMSCs (group-3) were injected intramyocardially after coronary artery ligation. Immunohistology for Von Willebrand factor and smooth muscle actin after 6 weeks of cell engraftment showed highest blood vessel density (per 0.155mm2 ) in the infacrt (42±9) and peri-infarct (55±11) regions in group-3 (p<0.05 vs all groups). Echocardiography at 6 weeks showed higher ejection fraction in group-3 (p<0.05). Coordinated ex-vivo overexpression of Ang and Akt potentiates release of HIF-1α dependent angiogenic mediators to enhance endothelial differentiation and survival of transplanted MSCs.