We have shown that in vitro reprogramming of MSCs maximizes their survival and angiogenic potential in the infarcted heart. The salient findings of the study are: 1- Genetic modification of MSCs with Shh transgene results in multifold increased iNOS upregulation and NO production, an effect which was associated with higher level of angiogenic growth factor expression including VEGF, Ang-1 and netrin-1. 2- The induction of iNOS in ShhMSCs occurred in PKC dependent manner. 3- ShhCM mediated increased angiogenic response was abrogated in the presence of Shh and netrin-1 specific antibodies. 4- Intramyocardial engraftment of ShhMSC induced significant angiogenesis, improved regional blood flow and significantly preserved global heart function. We propose that the increased angiogenesis is due to the production of NO with simultaneous upregulation of multiple angio-competent factors including netrin-1. The results from the present study also indicated that genetically engineered ShhMSCs promoted migration of endothelial progenitor cells which may be attributed to the dramatic upregulation of MMP-9 (4-fold) in ShhMSCs.
Sonic hedgehog (Shh) together with Desert hedgehog (Dhh) and Indian hedgehog (Ihh) are three members of the hedgehog gene family identified in the mammals 
. Hedgehog signaling involves binding of hedgehog with its ptch-1 receptor which in turn releases its inhibitory effect on Smoothened (smo
). Activated smo
then initiates signaling events which lead to regulation of transcriptional factors belonging to the Gli
family and its relevant downstream genes 
. Shh is a secretary protein and therefore Shh gene delivery has been assessed in experimental animal models of the infarcted heart 
. Results from these studies indicate that postnatal reconstitution of Shh signaling resulted in tissue preservation and repair, and that gene therapy with Shh not only prevented fibrosis, it also promoted angiogenesis and regional blood flow. Enhanced angiogenesis may be attributed to the multiple effects of localized Shh transgene expression. Activation of Shh pathway upregulates the expression of multiple angiogenic cytokines, including VEGF, angiopoietins, SDF-1α and IGF-1 and development of capillary network 
. Our results were in harmony with these data and further showed uniquely that Shh gene overexpression up-regulated iNOS, netrin-1 and HGF in addition to the already reported cytokines. HGF is a mitogen of mesenchymal origin, and is also reported to stimulate NO production and endothelial cell motility through upregulation of iNOS 
. Put together, these molecular changes lead to a significant increase in biologically active NO production in Shh
iNOS is expressed following inflammatory or growth factor stimulation of cells unlike its counterpart eNOS (endothelial nitric oxide synthase) and nNOS (neuronal nitric oxide synthase) which are constitutively expressed. Activation of iNOS can produce copious amounts of NO for longer duration 
. Generated from the NOS enzyme activity, NO is recognized as an important regulator of cardiovascular system functionality. Whereas NO inhibits proliferation of smooth muscle cells, gene delivery of iNOS to endothelial cells is protective for the cells via NO release without an influence on their proliferation 
. Secondly, endothelial cell migration is an essential component of several vascular processes including the maintenance of endothelial integrity and angiogenesis. It is suggested that endothelial cells must exhibit a phenotype of non-directional motility as a prerequisite for responding to stimuli for migration. The same study proposes that NO plays an obligatory role in eliciting this phenotype. More recent studies have shown that NO generated from iNOS activity modulates the expression of matrix metalloproteinase-9 (MMP-9), an enzyme responsible for degradation of extracellular matrix, which significantly influenced cell migration 
. A few previous studies have reported increased MMP-9 the Shh-expressing cells, which was attenuated by the inhibition of EGF receptor activation or blocking the EGF receptor and ligand interaction 
. In doing so, Shh has been shown to directly stimulate EGFR signaling. In the present study, prior treatment of cells with cyclopamine and chel abrogated the expression of iNOS and angiogenic growth factors including VEGF, Ang-1 and netrin-1 thus suggesting that these pro-angiogenesis relevant molecular events were PKC dependent.
Netrins are important in axonal guidance, regulation and maintenance of central nervous system and are also involved in the development of mammary gland, lung, pancreas, and blood vessel 
. Even though there is some controversy, most recent studies suggested that netrin-1 functions as a pro-angiogenic factor. In vivo studies also indicated that netrins promoted neovascularization and reperfusion in a murine model of peripheral vascular disease and also induced migration, proliferation and tube formation during in vitro studies involving multiple endothelial cell lines 
. Characterizing a mechanism for netrin-1 induced angiogenesis, a critical role for NO has been elucidated subsequent to feed-forward ERK1/2 and eNOS activation in endothelial cells treated with netrin-1 
. Our results showed that netrin-1 expression increased significantly in Shh
MSCs both at the protein and RNA levels in PKC (PKM) dependent fashion.
Another interesting finding in the present study was the presence of PKM fragment of PKC in Shh
MSCs at 72-h after transfection with Shh plasmid. PKM is a constitutively active 40-kDa catalytic fragment of PKC 
. The regulatory role of PKC in Shh signaling has been reported previously 
. In a study involving NIH 3T3 cells, PKC-δ was integral to hedgehog signaling to promote proliferative activity of the cells 
. In our present study, treatment of Shh
MSCs with both the PKC inhibitor chel and Shh inhibitor cyclopamine abolished PKM fragment which indicated that PKM was downstream of Shh and that PKC was essential for its upregulation. White et al. (2007) while experimenting on intact sea urchin spermatozoa showed PKC, most likely through its cleavage into active catalytic product PKM, was the central signaling mediator associated with maintenance of sperm mobility 
. PKC inhibitors such as chel and calphostin-C, as well as staurosporine, were found to rapidly arrest the motility of sea urchin spermatozoa freshly released into seawater. At the same time, these inhibitors prevented the motility-associated increase in phosphorylation of several PKC substrates. Pretreatment of Shh
MSCs with these inhibitors abrogated PKM with concurrent abrogation of iNOS and netrin-1.
Another novel finding of our study supported by molecular and histological data was the improved survival of Shh
MSCs post engraftment. In view of the reported data that massive loss of the transplanted cells occurs after transplantation remains a major determinant of the effectiveness of heart cell therapy, Shh
MSCs are at an advantage in terms of their survival after engraftment. Histological studies at eight weeks after cell transplantation showed a marked reduction in infarct size and preservation of host myocardium in Shh
MSCs transplanted group as compared with Emp
MSC and DMEM groups. The cytoprotective effects were accompanied by a significant increase in capillary density and a higher number of mature blood vessels (smooth muscle actin positive cells) in Shh
MSCs group. These results are consistent with earlier studies using direct injection of Shh plasmid into the infarcted heart 
In conclusion ShhMSCs showed upregulation of several angiogenic cytokines and signaling molecules including Ang-1, VEGF, IGF, HGF, iNOS and netrin-1. Additionally, ShhMSCs also generated NO at significantly higher levels. These molecular changes were mediated by iNOS/netrin/PKC signaling pathway downstream of Shh gene overexpression which combined with stem cell transplantation could be a promising strategy for the treatment of an infarcted heart.