RAAV-mediated CD151 Gene Delivery Promoted Functional Neovascularization And The Mechanism Involved

Background & ObjectiveTherapeutic angiogenesis has been identified as a good potential way in the studies of coronary heart disease. The benefit of therapeutic angiogenesis, which makes the formation of compensatory circulation and restoration of myocardial blood flow become possible after myocardial ischemia at right time, lies in retrieving more myocardial cell and reducing myocardial ischemia, so as to protect the heart function and improve the clinical symptom and prognosis of patients.CD151, a tetraspanin superfamily protein, is reported to form a structural and functional complex with various integrins. As "transmembrane linker", CD151 regulates the signal transduction through integrins. While more researches concernes about the relationship of integrins and angiogenesis, more and more studies become focus on CD151. Although the physiological function of CD151 is largely unknown, in vitro functional studies show that CD151 involves in the cell morphology, adhesion, migration, spreading, hemidesmosome structure formation, and the angiogenesis related to integrins.We have reported that in vivo CD151 gene delivery increases the number of microvessels and improved exercise tolerance in a rat hind-limb ischemia model, and increases myocardial capillaries densities of a rat myocardial ischemia model. These researches have identified CD151 as a potential target for therapeutic angiogenesis. However, increasing evidence indicates that angiogenesis response unlikely contributes to improve the regional blood flow, which tends to depend more on arterioles than on capillaries. Whether CD151-induced neovascularization can effectively promote the blood flow is still unclear. Further, the explicit signaling mechanism by which CD151 regulates blood vessel formation and maintenance has not been well elucidated. The purpose of this study was to evaluate whether CD151 induces functional neovascularization and coronary collateralization, and to determine the signaling pathways involved.Methods and Results1. pAAV-CD151 and pAAV-antiCD151 were constructed. Then, we produced the rAAV particles by transfections of 293 cells, and the titer of virus was determined by Northern blot. Twenty-two pigs, 1 month of age, were randomized into four groups. Four normal pigs underwent no operation were served as the control group. The remaining eighteen pigs, served as three viral administered groups, underwent coronary artery ligation and received intramyocardial viral injection. rAAV- GFP group (n=6), rAAV-CD151 group (n=6) and rAAV-antiCD151 group (n=6) animals respectively received direct intra- myocardial injection of rAAV-GFP, rAAV-CD151 and rAAV-antiCD151 (1×10¹² viron particles per pig, at 10 sites) correspondingly. Eight weeks after viral administration, the expression of CD151 protein was measured by western blot and immunohistochemistry, and CD151 mRNA was detected by RT-PCR. The densities of capillaries and arterioles were determined using immunohistochemistry. Coronary angiography was done to evaluate collateral circulation of the occluded artery. ¹³N-NH₃ PET and echocardiography were applied to evaluate the regional myocardial perfusion and other myocardial functions. Western blot was performed for assessing the signaling mechanisms. In addition, myocardium NO concentration was assayed. We have established the acute myocardial infarction model in pigs successfully. rAAV-mediated CD151 gene delivery promotes the CD151 protein expression in myocardial tissue. Overexpression of CD151 markedly increased the densities of capillaries and arterioles, showed better collateral circulation, significantly enhanced the regional myocardial perfusion, reduced myocardial ischemia and improved the myocardial contraction, wall motion and wall thickness. Conversely, antiCD151 gene delivery reversed these changes above. In addition, CD151 activated FAK, ERK, PI3K, Akt and eNOS, and increased NO level. 2. rAAV-CD151 mediated CD151 gene delivery into ECV304. After transfection, the expression of CD151 was measured by western blot. Proliferation assay was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5, diphenyltetrazolium bromide (MTT) method; Cell migration assay was performed using Boyden transwell; and tubule formation was determined by matrigel tests. In addtion, the potential involvement of MAPKs signaling pathways was explored. Here, we showed that CD151 promoted ECV304 proliferation, migration and tubule fomation in vitro, accompanied by phosphorylation of ERK, leading the activation of ERK. By contrast, overexpression of CD151 did not affect the activity of p38MAPK protein. Moreover, inhibitors of ERK (PD098059) can attenuate CD151-induced cell proliferation, cell migration and tubule formation in vitro, which suggests that ERK mediate the effects of CD151. However, inhibitors of p38MAPK (SB203580) had no effect on the CD151 -induced cell proliferation, cell migration and tubule formation.Conclusions1 . Intramuscular transduction of rAAV-CD151 actually promotes functional neovascularization responses in a pig myocardial infarction model, leading to a greater enhancement of blood flow restoration. The mechanism may be that CD151 can activate FAK, PI3K, MAPKs pathways and promote neovascularization via the MAPKs and PI3K pathways.2. Overexpression of CD151 promotes ECV304 proliferation, migration and tubule formation. The mechanism is that ERK signaling pathway is involved in the angiogenic effects of CD151.