The Role And Mechanisms Of AP-2a In Vascular Endothelial Cell Apoptosis Induced By Cigarette Smoke Exposure

Background Chronic Obstructive Pulmonary Disease(COPD) is one of public health problems for its high morbidity and mortality,but its pathogenesis remains enigmatic and there is no good way to treat it. Cigarette smoke is one major etiologic factor of COPD.Besides chronic inflammation,the elastase/antielastase imbalance theory and oxidant/antioxidant imbalances,recently more and more clinical studies have shown apoptosis occurs in alveolar walls induced by cigarette smoking,resulting in progressive cell loss,emphysema and even respiratory failure.Thus,it is the key to interfere with apoptosis in the prevention and treatment of COPD.Transcription factor activator protein-2(AP-2) is a family of cell type-specific developmentally regulated transcription factors.To date,five members of the AP-2 family have been identified and AP-2αis the first one.AP-2αis shown to modulate the expressions of many genes such as VEGF,P⁵³,p^21WAF/CIP,Bcl-2 and Manganese superoxide dismutase(MnSOD),and it has been implicated in vertebrate development,embryogenesis and carcinogenesis. Most of these studies have been focused on the regulation of apoptosis and proliferation.Depending on different cell type,AP-2αhas been shown to promote or inhibit apoptosis.As yet there are no related reports about the expression level of AP-2αin lung tissue from rat COPD models and human vascular endothelial cells ECV304 treated by CSE,and whether AP-2αexerts influence on apoptosis and the activated Caspase-3 induced by CSE,and whether its function is associated with its domain.Objective:To investigate the expression of AP-2αin COPD rat lung tissue and ECV304 cells stimulated by CSE,and furthermore to study the relationship between the expression of AP-2αand apoptosis in ECV304 cells stimulated by CSE and mechanisms.Methods:To replicate rat models of COPD by passive smoking.The apoptosis of alveolar septal cells was detected using Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) methods and active Caspase-3 in the lung tissue was measured by western blot.The expression of AP-2αin rat lung tissue was detected by immunohistochemistry and Western blot.ECV304 cells were treated with different concentrations of CSE for indicated time,then tested for proliferation rate by MTT assay;Apoptosis rate by Hoechst 33258 nucleus staining;and Caspase-3 activation and AP-2αprotein levels by western blot.Mouse AP-2α,transactivation-domain mutated AP-2α(AP-2â–³) and respective control one were transfected into ECV304 cells by adenovirus-induced or plasmid transfection for 24 hours,then cells were treated with 5%CSE for another 24 hours.Cell proliferation rate,apoptosis rate and Caspase-3 activation levels were compared between the two groups. Results:Rat model of COPD were replicated successfully by passive smoking for eighty days.COPD group showed more inflammatory cells infiltration,larger air spaces and significant decrease of FEV_0.3/FVC compared with the normal group;The apoptosis rate of alveolar septal cells and the expression of active Caspase-3 in the lung tissue were increased indeed;the expression of AP-2αwas significantly higher than that in the controls(p＜0.05).In ECV304 cell,5%,10%,15%and 20%CSE treatment for 24h significantly reduced cell proliferation rate(P＜0.005),while 2.5% increased it.5%CSE treatment significantly increased Caspase-3 activation(p＜0.001) and ECV304 cell apoptosis shown by Hoechst 33258 nuclear staining(p＜0.01).Also,5%CSE treatment increased endogenous AP-2αprotein expression in the ECV304 cells.AP-2αOver-expression in ECV304 cells inhibited 5%CSE-induced cell apoptosis and activated Caspase-3 expression(p＜0.05),while the transactivation-domain mutated AP-2α(AP-2â–³) showed no protective effect on CSE-induced cell damage.Conclusions:It is concluded that CSE is one major etiologic factor of COPD;Apoptosis may be one of important mechanisms contributing to COPD;The expression of AP-2αwas significantly higher in lung tissue in COPD group and ECV304 cell stimulated by CSE than that in the controls.AP-2αcan protect ECV304 cell against CSE-induced cell damage and its activation domain is necessary for this function.