Abstract 1412: Sonic Hedgehog Gene Transfer Promotes Angiogenesis and Myocardial Regional Blood Flow via PKC Dependent Netrin Expression

Abstract

Background: Hedgehog signaling effects endothelial and fibroblast cell migration and vascularization of various tissues during embryonic development. We propose here that stem cell based Shh gene delivery to the heart promotes neovascularization and enhances regional blood flow (RBF) in the infartced heart. Methods and Results: The pCMVShh plasmid was constructed and used for non-viral transfection of primary mesenchymal stem cell (MSC) culture. Successful transfection and expression of Shh in transfected MSCs ( Shh MSCs) was confirmed by immunoflourescence, real-time PCR and Western blotting. Real-time PCR based array showed upregulation of angiogenic factors in Shh MSCs including netrin (>62-fold) and iNOS (>100-fold). The expression of netrin and iNOS was PKC dependent and was abolished in the presence of 2.5 μ M chelerythrin. Transduction of iNOS gene into MSCs increased netrin expression implying a role for iNOS downstream of PKC. Western blot showed that 45-KDa PKM proteolytic subunit of PKC was involved in this activity. Shh MSCs conditioned medium was biologically active and caused greater migration and tube formation of human vascular endothelial in vitro in comparison with untransfected MSCs ( non-Shh MSCs) conditioned medium. For in vivo studies, 70 μ l DMEM without cells (group-1) or containing 1x10 6 male non-Shh MSC (group-2), or Shh MSCs (group-3) were transplanted into female rat model of acute coronary artery ligation. S ry -gene studies on day-4 after cell engraftment showed higher survival of Shh MSCs ( p < 0.05 vs non-Shh MSC). Immunostaining for vWillebrand Factor-VIII and smooth muscle actin showed robust angiogenesis with distinctly large vessels in group-3 ( p < 0.001 vs group-2). RBF measured by fluorescent microspheres was increased ( p < 0.01 ) and infarction size was attenuated ( p < 0.05 ) in group-3 as compared with control groups. Left ventricle ejection fraction (52.3±2.6%) and fractional shortening (21.8±1.2%) were more preserved in group-3 as compared with control groups. Conclusions: Ex-vivo Shh gene delivery improves angiogenesis and RBF in the infarcted heart to preserve global heart function through the upregulation of multiple angiogenic cytokines including iNOS and netrin.