| BACKGROUND & OBJECTIVENovel angiogenesis-inducing therapies have been developed recently to stimulate collateral vessel formation and to improve muscles function in the treatment of occlusive vascular diseases. Angiogenesis requires signals that are transduced from growth factor receptors and integrins. Therapeutic angiogenesis has focus on the growth factors, such as vessel endothelial growth factors, hepatic growth factors, for a long time, but the clinical trials have showed limited clinical efficacy. In response to an obstruction or occlusion of a major artery, compensation can only come from low-resistance connections between a donor artery in a non-ischemic regions and the postocclusion arterial system of the recipient ischemic regions (arteriogenesis). For getting the satisfactory result, it is necessary to explore more powerful pro-neovascularizing factors, and use them in combination rationally.The Tetraspanin membrane protein CD151 is expressed, especially high in endothelial cells, epithelial cells, and platelets. In vitro functional studies have demonstrated an important role of CD151 in cell migration and integrin-initiated morphogenesis. CD151 is a cell surface protein that belongs to the tetrapanin superfamily. It forms complexes with many laminin-binding integrins a3β1, a6β1, a6β4 et al. and is codistributed with these integrins in many tissues at sites of cell-matrix interactions. CD151-inegrin complex may contribute to the cell biological processes, including cell adhesion, cell motility, metastasis and formation and stability of hemidesmosomes. It is not known that promoting CD151 expression in ischemic tissues could stimulate neovascularization, improve recovery of blood flow and function of the ischemic organ.We thus hypothesize that an increased expression of CD151 may stimulate neovascularization in vivo. Our study is aimed to evaluate the efficiency of pAAV-mediated CD151 gene delivery in promoting neovascularization and improving heart function after myocardial infarction in rats, and explore a new target of the neovascularization after myocardial ischemia.METHODS AND RESULTSFirstly, Functional fragment of CD151 gene with HAtag was amplified by RT-PCR, and inserted into the vector pAAV. So, the recombined plasmid pAAV-CD151 was packed successfully. The pAAV-GFP plasmid was selected as control.The rat Acute myocardial infarction (AMI) model was established by ligation of the left anterior descending coronary artery in male Spague-Dawley(SD) rats. The surviving rats were grouped randomly as MI control, pAAV-CD151 group and pAAV-GFP group(n=12/group). pAAV-CD151 and pAAV-GFP plasmid were delivered by direct injection in myocardium. Sham-operated group (n=12) was taken randomly as non-infarction control. Four weeks after AMI, the following endpoints were measured : (1)Rats heart function and left ventricular remodeling were assessed by echocardiographic evaluation and LV catherization. (2) The expression of CD151 and MMP-9 (matrix metalloproteinase-9) were detected by Western blot. (3)Micro vessels density (MVD) counting of infarct myocardium in rats observed by factor VIII related antigen immunochemical staining was conducted to value the pro-angiogenesis effect of CD151 delivered by gene transfection in vivo.Our immunoblot analysis showed that transfection with pAAV-CD151 could apparently lead to CD151 overexpression in rat myocardium. In comparison with the MI control (OD value 1.07±0.13), the expression of CD151(OD value 1.98±0.23) in pAAV-CD151 group was significantly increased (P<0.01). The micro vessels density (MV) counting in pAAV-CD151-AMI group(385.4±79.9n/mm2) was significantly higher than the MI control(240.8±40.3n/mm2) (P<0.01). All indexes reflecting left ventricular contraction function such as left ventricular ejection fraction, LVPSP and ±dp/dtmax in CD151 group were statistically increased than MI control group(P<0.01).CONCLUSIONpAAV-CD151 mediated CD151 gene delivery into rat myocardium induced an efficient and stable expression. pAAV-mediated CD151 stable expression stimulates neovascularization, and thereby improves blood perfusion in a rat myocardial infarction modeLAs a result, the rat heart function after ischemic injury was reformed. All these findings suggest that CD151 could be a new insight for the neovascularization therapy in the ischemic disease, and pAAV-mediated CD151 gene transfer may be useful for treatment of ischemic disease. CD151 has an effective role in pro-angiogenesis in infarct myocardium in vivo, thereby improves left ventricular function through valid revascularization.This study demonstrates that1) pAAV-CD151 promotes the CD151 protein expression in rat myocardium by direct injection.2) CD151 stable expression stimulates neovascularization in rat ischemic myocardium, and thereby improves heart function in a rat myocardial infarction model.
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