Publication Date:
2010-11-19
Description:
Abstract 563 Sonic hedgehog (Shh) is a morphogen regulating epithelial-mesenchymal interactions during embryogenesis. In post-natal life, Shh is a potent indirect angiogenic agent, able to upregulate different families of angiogenic growth factors. Furthermore, Shh gene transfer enhances the contribution of bone marrow (BM)-derived endothelial progenitor cells to myocardial neovascularization. In this study, we tested the beneficial potentials of Shh gene therapy in an experimental model of peripheral limb ischemia, a disease characterized by occlusion of vessels of the arterial circulation of the legs, often secondary to atherosclerosis, thrombosis, and/or inflammatory processes. Ischemia of the left hindlimb was induced by excising the femoral artery, from its proximal origin as a branch of the external iliac artery till the bifurcation into saphenous and popliteal arteries. Blood flow was measured in ischemic and contralateral hindlimbs by laser Doppler perfusion imaging at days 0, 7, 14, 21, and 28 after ischemia. At day 28, mice were sacrificed and adductor muscles were analyzed for capillary and arteriole density. An additional set of 1-year-old C57BL/6J mice and 8-weeks-old C57BL/6J mice were used to study the effects of Shh gene therapy on mobilization of BM-derived endothelial progenitors in the course of ischemia. Mice were treated with 200 μ g phShh or empty plasmid and hindlimb ischemia was induced 7 days after treatment. Peripheral blood samples were obtained at days 2, 4, and 7 after induction of ischemia. FACS analyses were used to detect cells Lin- and Sca-1+CD34+, Sca-1+CD133+, Sca-1+ Flk-1+, CD14+ Flk-1+, CD14+ CD34+. We also used a BM transplantation model to test the effects of Shh gene therapy on BM-derived cells in the setting of hindlimb ischemia. Wild-type recipient mice received 2×106 BM cells from nls-Ptc1-LacZ mice. Six weeks laster, mice received treatment with 200 μ g phShh or empty plasmid in the hindlimb muscles. Seven days after treatment, ischemia of the left hindlimb was induced. X-gal positive cells were counted on hindlimb muscles 10 days after ischemia. We also measured local expression levels of VEGF165, Ang-1, and SDF-1α proteins in mice treated with 200 μ g phShh or empty plasmid 7 days after ischemia. At day 28 after ischemia, blood perfusion ratio between the ischemic and the contralateral leg was 0.97 ± 0.01 in Shh-treated mice and 0.68 ± 0.03 in control animals (p=0.008) (Figure 1a, b). Capillary and arteriole density were significantly higher in the phShh–treated muscles compared to controls (p=0.02; p=0.03, respectively). In both young and middle-aged animals, phShh treatment resulted in significant increase of the number of circulating CD45-/Sca-1+/Flk-1+ and CD45-/Sca-1+/CD133+ cells (p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
Permalink