Sonic Hedgehog Gene Delivery to the Rodent Heart Promotes Angiogenesis via iNOS/Netrin-1/PKC Pathway

Background: We hypothesized that genetic modification of mesenchymal stem cells (MSCs) with Sonic Hedgehog (Shh) transgene, a morphogen during embryonic development and embryonic and adult stem cell growth, improved their survival and angiogenic potential in the ischemic heart via iNOS/netrin/PKC pathway. Methods/Principal Findings: MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid ((MSCs)-M-Shh). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in (MSCs)-M-Shh which also led to increased expression of angiogenic and pro-survival growth factors in (MSCs)-M-Shh. Significantly improved migration and tube formation was seen in Shh MSCs as compared to empty vector transfected MSCs ((MSCs)-M-Emp). Significant upregulation of netrin-1 and iNOS was observed in (MSCs)-M-Shh in P13K independent but PKC dependent manner. For in vivo studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 mu l basal DMEM without cells (group-1) or containing 1x10(6) (MSCs)-M-Emp (group-2) and (MSCs)-M-Shh (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of (MSCs)-M-Shh post engraftment. Histological studies combined with fluorescent microspheres showed increased density of functionally competent blood vessels in group-3 and group-4. Echocardiography showed significantly preserved heart function indices post engraftment with (MSCs)-M-Shh in group-3 animals. Conclusions/Significance: Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.