Sonic Hedgehog gene delivery to the rodent heart promotes angiogenesis via iNOS/netrin-1/PKC pathway

• Published in: PloS one 2010-01, Vol.5 (1), p.e8576-e8576
• Main Authors: Ahmed, Rafeeq P H, Haider, Khawaja Husnain, Shujia, Jiang, Afzal, Muhammad Rizwan, Ashraf, Muhammad
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; Angiogenesis; Animals; Biochemistry/Cell Signaling and Trafficking Structures; Blood vessels; Blotting, Western; Bone marrow; Cell Biology/Gene Expression; Cell survival; Comparative analysis; Coronary Vessels - metabolism; Developmental Biology/Stem Cells; Echocardiography; Embryogenesis; Embryonic development; Embryonic growth stage; Fluorescence; Gene transfer; Genetic modification; Genetically modified organisms; Growth factors; Heart; Heart attack; Heart function; Heart transplantation; Hedgehog protein; Hedgehog Proteins - genetics; Immunofluorescence; In Situ Hybridization, Fluorescence; In vivo methods and tests; Ischemia; Kinases; Mesenchymal Stem Cells; Mesenchyme; Microspheres; Myocardial infarction; Neovascularization, Physiologic; Nerve Growth Factors - metabolism; Netrin-1; Nitric oxide; Nitric Oxide Synthase Type II - metabolism; Nitric-oxide synthase; Polymerase chain reaction; Protein kinase C; Protein Kinase C - metabolism; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; Signaling; Stem cell transplantation; Stem cells; Studies; Survival; Tumor Suppressor Proteins - metabolism; Western blotting
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0008576

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. 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. Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.