| Background & Objective: Tetraspanins have been shown to associate with each other and with other integral membrane proteins, including integrins,the major receptors for extracellular matrix (ECM) adhesive proteins. Although the physiological function of tetraspanin CD151 is largely unknown,in vitro functional studies showed that CD151 is involved in cell adhesion,motility,and polarity. High CD151 expression was found to be associated with a poor prognosis in lung,colon,and prostate cancer. Monoclonal antibodies to CD151 inhibited in vivo metastasis of human cancer cells and transfection of CD151 cDNA into different tumor cell lines resulted in enhanced cell motility and metastasis. This implies that CD151 does not only play an important role in normal physiological processes, but also in pathological events,such as tumor cell invasion and metastasis. Recent research has identified CD151 as a potential target for therapeutic angiogenesis. However, little is known about mechanism(s) of CD151-induced cell proliferation,migration and angiogene- sis.Therapeutic angiogenesis that may be beneficial in the treatment of ischemia has recently been substantiated by a large amount of experimental data. Ischemic heart diseases develop as a consequence of coronary atherosclerotic lesion formation. Coronary collateral vessels and microvascular angiogenesis develop as an adaptive response to myocardial ischemia,which ameliorate the function of the damaged heart. Endothelial cell proliferation and migration play critical roles in angiogenesis. Angiogenesis is the formation of new blood vessels from pre-existing vessels,a physiological or pathological neovascularization process in response to tissue ischemia and tumor growth or metastasis,which is complex and involves several discrete steps such as extracellular matrix degradation,proliferation and migration of endothelial cells,and morphological differentiation of endothelial cells to form tubes.Many growth factors and hormones have been shown to regulate cell proliferation, migration and angiogenesis,including the activation of eNOS activity,via the PI3K/Akt signaling pathway.We hypothesized that CD151 may activate PI3K/Akt pathway and exert affects angiogenesis. To test whether CD151 promotes neovascularization and angiogenesis after myocardial infarction,we used rAAV for direct delivery of the human CD151 gene into the rat myocardium,evaluating the effect of CD151 on PI3K pathway and neovascularization. Therefore,the purpose of this study was to determine the mechanisms by which CD151 induces angiogenesis and promotes neovascularization after myocardial infarction.Methods and results: pAAV-CD151 and pAAV-anti-CD151 were constructed, and used to transiently transfect to ECV304 mediated with LipofectamineTM 2000. After transfection,the expression of CD151 was measured by western blot. Cell migration assay was performed using boyden transwell,proliferation assay was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) method and tube formation test was examined on Matrigel. eNOS activity was assayed by L-[3H] citrulline production from L-[3H] arginine. The involvement of eNOS was explored using eNOS inhibitor (L-NAME) and observed the effects in the process. We found that CD151 promotes cell migration,proliferation and tube formation. In addition, CD151 increases eNOS activity. Moreover,cell migration,proliferation and tube formation induced by CD151 are inhibited when L-NAME is used,which indicated that an involvement of eNOS in CD151-induced cell migration,cell proliferation,tube formation.Then,we produced the rAAV particles by transfections of 293 cells,and the titer of virus was determined by quantitative real-time PCR. Here we showed that CD151 promotes human umbilical vein endothelial cell (HUVEC) proliferation,migration and tube formation in vitro, accompanied by increased phosphorylation of Akt and eNOS, leading to increased eNOS activity and nitric oxide (NO) levels after rAAV-CD151 infection,whereas infection with rAAV-anti-CD151 attenuated the effects of CD151,which suggested that CD151 can activate PI3K/Akt pathway. Moreover, inhibitors of PI3K (LY294002) and eNOS (L-NAME) can attenuate CD151-induced cell proliferation and cell migration,which suggests that PI3K/Akt pathway mediates the effects of CD151.In order to know whether rAAV-CD151 promotes neovascularization and the mechanism involved,rats were subjected to sham surgery or coronary artery ligation. We used rAAV for direct delivery of the human CD151 gene into the rat myocardium. At 4 weeks after coronary artery ligation,human CD151 mRNA was detected using RT-PCR. Measurement of capillary density was evaluated using immunostaining for von Willebrand factor. Hemodynamic variables and physiological parameters were monitored,too. Western blot analysis for CD151,PI3K,phosphor-Akt,total Akt, phosphor-eNOS and total eNOS was performed. In addition,we also observed the effect of CD151 on the expression of VEGF using western blot analysis. We found that CD151 gene delivery could increase the expression of CD151 at gene level and protein level. Overexpression of CD151 could increase the number of microvessels in the ischemic myocardium and significantly improved the hemodynamic variables after myocardial infarction. In addition, CD151 could activate PI3K pathway, including activation of Akt and eNOS, but not affect the expression of VEGF.Conclusions: 1.CD151 promotes ECV 304 migration,proliferation and tube formation. The mechanism is that overexpression of CD151 increases eNOS activity. This result also suggests that eNOS is involved in the angiogenic effecs of CD151. 2.Activation of PI3K/Akt signaling pathway mediates CD151-induced HUVECs proliferation and migration. 3.CD151 could promote neovascularization and improve ventricular function after myocardial infarction in rats. The mechanism may be that CD151 can activate PI3K pathway and promote neovascularization via PI3K pathway,without affecting ischemia- induced VEGF expression. |
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