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

<div><h3>Background</h3><p>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.</p><h3>Methods/Principal Findings</h3><p>MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid (<sup>Shh</sup>MSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in <sup>Shh</sup>MSCs which also led to increased expression of angiogenic and pro-survival growth factors in <sup>Shh</sup>MSCs. Significantly improved migration and tube formation was seen in <sup>Shh</sup>MSCs as compared to empty vector transfected MSCs (<sup>Emp</sup>MSCs). Significant upregulation of netrin-1 and iNOS was observed in <sup>Shh</sup>MSCs in PI3K independent but PKC dependent manner. For <em>in vivo</em> studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 µl basal DMEM without cells (group-1) or containing 1×10<sup>6</sup><sup>Emp</sup>MSCs (group-2) and <sup>Shh</sup>MSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and <em>sry-</em>quantification revealed improved survival of <sup>Shh</sup>MSCs 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 <sup>Shh</sup>MSCs in group-3 animals.</p><h3>Conclusions/Significance</h3><p>Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.</p></div>