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.


RESULTS
MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid ((Shh)MSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in (Shh)MSCs which also led to increased expression of angiogenic and pro-survival growth factors in (Shh)MSCs. Significantly improved migration and tube formation was seen in (Shh)MSCs as compared to empty vector transfected MSCs ((Emp)MSCs). Significant upregulation of netrin-1 and iNOS was observed in (Shh)MSCs 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 microl basal DMEM without cells (group-1) or containing 1x10(6) (Emp)MSCs (group-2) and (Shh)MSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of (Shh)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 (Shh)MSCs in group-3 animals.


CONCLUSIONS
Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.
Ahmed
Rafeeq P H
RP
Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America.
Haider
Khawaja Husnain
KH
Shujia
Jiang
J
Afzal
Muhammad Rizwan
MR
Ashraf
Muhammad
M
D000818:Animals; D015153:Blotting, Western; D003331:Coronary Vessels / Q000378:metabolism*; D053823:Hedgehog Proteins / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D059630:Mesenchymal Stem Cells; D018919:Neovascularization, Physiologic; D009414:Nerve Growth Factors / Q000378:metabolism*; D000075388:Netrin-1; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D053823:Hedgehog Proteins; D009414:Nerve Growth Factors; C000617354:Ntn1 protein, rat; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; D052247:Nitric Oxide Synthase Type II; D011493:Protein Kinase C