| Background:Pulmonary hypertension(PH),as one of the major complications of chronic obstructive pulmonary disease(COPD),directly affects the quality of life and prognosis of patients with COPD.Cigarette smoking is the main risk factor for COPD.Much evidence from clinical samples and animal model has shown that pulmonary vascular remodeling was found in mild-moderate COPD patients with cigarette smoking even in smokers with normal lung function without hypoxia.High oxidative stress and chronic inflammation caused by cigarette exposure in COPD patients play an important role in the formation of pulmonary vascular remodeling and PH.In recent years,AMP-activated protein kinase(AMPK),which regulates the balance of cellular energy metabolism,has attracted much attention as an important target for regulating oxidative stress and inflammation.In patients with diabetes and obesity,low AMPK activity in vivo is closely related to the high oxidative stress status,chronic inflammation and the higher incidence of cardiovascular diseases.The activation of AMPK is important for maintaining the normal structure and function of blood vessels.Several studies in systemic vascular remodeling disease such as atherosclerosis and restenosis after balloon injury has shown upregulating the activity of AMPK can protect endothelial function and inhibit the proliferation of arterial smooth muscle cells.The catalytic α subunits of AMPK has two isforms,AMPKα1 and AMPKα2,which seem to play a different role in the maintenance of vascular homeostasis.During the pathological process of pulmonary vascular remodeling,switching of pulmonary arterial smooth muscle cells(PASMCs)from contractile phenotype to proliferative phenotype is a key hallmark,which ultimately lead to pulmonary vascular remodeling and PH.High oxidative stress and chronic inflammation in pulmonarymicroenvironment induced by cigarette smoke is closely related with the pathogenesis of pulmonary arterial remodeling and PH in COPD patients.Thus,we speculated that deregulation of AMPK activity(target for regulation of oxidative stress and chronic inflammation)participated in PASMCs phenotypic switch and excessive proliferation,which contributed toin cigarette smoking-related pulmonary vascular remodeling.Traditional antidiabetic drug metformin is a activator of AMPK.Animal experiments showed that metformin can inhibit the formation of PH induced by monocrotaline.Although these studies did not further discuss the mechanism,these findings showed that metformin may have the protective effect on the pulmonary circulation.Our study aimed to explore the role of AMPK and its different catalytic isformsin the phenotypic switch and excessive proliferation of PASMCs under cigarette smoking exposure,and whether metformin inhibits the phenotypic switch and excessive proliferation of PASMCs by activating AMPK or specific AMPK subtypes.Till now,there is still a lack of effective treatment for PH associated with COPD,once our hypothesis has been verified,it will be helpful not only to elucidate the molecular mechanisms of PH associated with COPD induced by cigarette smoking,but also to provide a new strategy for its early prevention and treatment.Objective:1.To study the relationship between cigarette smoking,systemic inflammation and PH;2.To define the role of AMPKα2 in the phenotypic switch of rat PASMCs induced by cigarette smoke extract(CSE);3.To define the effect of metformin on the phenotypic switch and proliferation of PASMCs induced by CSE and its underlying mechanism.Methods:1.The case-control study was designed to take 111 COPD patients with PH admitted in our department from 2013 April to 2015 March as the case group;after matching with ageandsex,111 COPD patients without PH were enrolled as the control groupin the same period.Collection of patients with age,sex,smoking history,erythrocyte sedimentation rate(ESR),white blood cell(WBC),neutrophil/lymphocyte ratio(NLR),serum C-reactive protein(CRP),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),fibrinogen(FIB),procalcitonin(PCT),arterial blood gas,chest CT and echocardiogram results.SPSS 22 software was used to compare the two groups of data,correlation analysis and Logistic multivariate regression analysis were used to find out the risk factors of PH;2.5%CSE treated rat PASMCs in vitro,CCK-8 method was used to detect cell proliferation.Western blot was used to detect the expression of PASMCs phenotype marker(SMA),AMPK,mTOR protein and their phosphorylation level.The role of different subtypes of AMPKα in the phenotypic switch induced by CSE was explored after AMPKα1 and AMPKα2 gene expression was respectively silenced by lentivirus mediated siRNA;3.The effect of metformin on the phenotypic switch and proliferation of PASMCs induced by CSE and its underlying mechanism were explored by methods as shown in method section 2.Results:1.The smoking index,serum CRP,IL-6 and TNF-α levels of COPD with PH group were significantly higher than that of COPD without PH group(p<0.05);2.The smoking index,serum CRP,IL-6 and TNF-αlevels were positively correlated with pulmonary artery systolic pressure(PASP)(p<0.05),the smoking index was positively correlated with CRP(p<0.05);3.Logistic multivariate regression analysis showed that smoking index,serum CRP,IL-6 and TNF-αare independent risk factors for PH(OR = 6.185,6.740,3.434,2.700,p<0.05);4.CCK-8 results showed that 5%CSE promotes the proliferation of rat PASMCs,western blot showed that 5%CSE can downregulate the expression of PASMCs contractile phenotype marker SMA(p<0.05);5.Using lentivirus mediated siRNA technology successfully silent AMPKα1and AMPKα2 in rat PASMCs,western blot results showed that SMA expression was significantly decreased in AMPKα2 siRNA group(p<0.05);6.CCK-8 results showed that metformin significantly inhibited the proliferation of PASMCs induced by 5%CSE(p<0.05);metformin could not inhibit the proliferation of PASMCs both in AMPKα1 siRNA group and AMPKα2 siRNA group;7.Western blot results showed that metformin could reverse down-regulation of SMA expression induced by 5%CSE(p<0.05);8.Western blot results further confirmed that metformin inhibits PASMCs phenotypic switch induced by 5%CSE in AMPKα2/mTOR dependent pathway,.Conclusions:1.Smoking,systemic inflammatory response(CRP,IL-6,TNF-α)were independent risk factor for PH;2.Smoking and serum CRP,IL-6,TNF-αwas positively correlated with PASP,smoking index was positively correlated with serum CRP;3.5%CSE could downregulate the expression of PASMCs contractile phenotype marker SMA,induce switch of PASMCs from contractile phenotype to proliferative phenotype.4.AMPKα2 but not AMPKα1 played an important role in the phenotypic switch of PASMCs induced by CSE;5.Metformin can reverse the down-regulation of SMA induced by 5%CSE by activating AMPKα2,which maintainthe normal contractile phenotype of PASMCs and inhibit excessive proliferation of PASMCs induced by CSE. |
