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 (ShhMSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in ShhMSCs which also led to increased expression of angiogenic and pro-survival growth factors in ShhMSCs. Significantly improved migration and tube formation was seen in ShhMSCs as compared to empty vector transfected MSCs (EmpMSCs). Significant upregulation of netrin-1 and iNOS was observed in ShhMSCs in PI3K 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 µl basal DMEM without cells (group-1) or containing 1×106EmpMSCs (group-2) and ShhMSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of ShhMSCs 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 ShhMSCs 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.