The Effect Of Cytochrome C Oxidase Subunit â…¡ Methylation In Pulmonary Vascular Endothelial Cell Apoptosis Of Chronic Obstructive Pulmonary Disease

Chapter 1.Pulmonary vascular endothelial apoptosis and COX expression in COPDObjective:To observe the pulmonary vascular endothelial cell apoptosis,COX activity and COXâ…¡expression in COPD patients,and to investigate the possible mechanism of COPD pathogenesis.Methods:According to the latest COPD diagnostic criteria and WHO standards for smoking-related investigation,20 patients underwent pneumonectomy were divided into 3 groups:group A,non-smokers without COPD;group B,smokers without COPD;and group C,smokers with COPD. TUNEL assay was used to assess cell apoptosis of pulmonary vascular endothelial cells.COX activity was measured by spectrophotometry. Semi-quantitative and fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR and RQ-PCR) were used to measure COX subunitâ…¡(COXâ…¡) mRNA expression.Moreover, immunohistochemistry and Western Blot were used to detect COXâ…¡protein expression respectively.Data were presented as Means±SD.Statistical analyses were performed using SPSS version 13.0 and differences were considered significant if P＜0.05.Results:The apoptotic index of pulmonary vascular endothelial cells in group C[(13.8±1.8)%]was much higher than that in group B[(9.6±0.8)%] and group A[(5.9±1.0)%],differences between every two groups were of great significance(all P＜0.05).COX activity,COXâ…¡mRNAΔΔCt value and COXâ…¡protein OD value in group A were(7.6±0.4)×10^-1U/mg, (0.86±0.19) and(0.99±0.14) respectively.And those in group B were (6.0±0.6)×10^-1U/mg,(0.81±0.15) and(0.81±0.09);in group C were (4.4±0.7)×10^-1U/mg,(0.76±0.17) and(0.60±0.07).Each variant decreased in turn and differences between every two groups were siganificant(all P＜0.05).The correlation analyses showed that the apoptotic index was negatively correlated with FEV₁/Pre(r=-0.846,P＜0.01),but positively correlated with smoke index(r=0.794,P＜0.01),while COX activity was positively correlated with FEV₁/Pre(r=0.812,P＜0.01),but negatively correlated with smoke index(r=-0.783,P＜0.01).Conclusions:(1) Pulmonary vascular endothelial cell apoptosis is increased in COPD patients.(2) COXâ…¡expression and COX activity are inhibited in COPD patients.(3) Cigarette smoking may be related with pulmonary vascular endothelial cell apoptosis and inhibition COXâ…¡expression and COX activity.(4) COX mediates pulmonary vascular endothelial cell apoptosis in COPD. Chapter 2.The methylation status of COXâ…¡in COPD and its association with COPDObjective:To investigate whether there is COXâ…¡methylation in COPD and the relationship between COXâ…¡methylation and COPD.Methods:Lung tissues of 20 patients underwent pneumonectomy were collected and devided into 3 groups:group A,non-smokers without COPD; group B,smokers without COPD;and group C,smokers with COPD.Blood samples of another 10 in patients with COPD and 20 healthy controls were collected and divided into 3 other groups:group D,non-smokers without COPD;group E,smokers without COPD;and group F smokers with COPD. Extracted genome DNA in either lung tissues or peripheral bloods,and detected the COXâ…¡methylation in them using methylation-specific PCR. Quantitative data were presented as Means±SD,and analyzed using One-Way ANOVA.While methylated frequencies of COXâ…¡were presented as percentage and analyzed using Fisher's exact test.Statistical analyses were performed using SPSS version 13.0 and differences were considered significant if P＜0.05.Results:The methylation frequencies of COXâ…¡in lung tissues were 71.4%(5/7) in group A,71.4%(5/7) in group B and 66.7%(4/6) in group C. Through Fisher exact test,no difference among them was found(P＞0.05). while in blood samples,methylated frequencies of COXâ…¡were 0%(0/10) in group D,50%(5/10) in group E and 60%(6/10) in group F.Differences between group D and group E or group F were statistic significant(all P＜0.05),but no difference was found between group E and group F(P＞0.50).Conclusions:There is COXâ…¡methylation in COPD patients,and this aberrant methylation may be associated with cigarette smoke.Chapter 3.Methylation status of COXâ…¡in CSE-induced HUVEC apoptosis and the effect of 5-aza-2'-deoxycytidine interventionObjective:To investigate whether COX activity and COXâ…¡methylation are involved in CSE-induced HUVEC apoptosis.Methods:In part one,cultured HUVECs were exposed to CSE at various concentrations and for different durations.TUNEL assay was used to detect apoptosis.COX activity and caspase-3 activity were measured by spectrophotometry.In part two,HUVECs were divided into 4 groups: control group;AZA group;CSE group and AZA+CSE group.Apoptotic index and COX activity were assessed in each group.Fluorescence RQ-PCR and Western Blot were used to detect COXâ…¡mRNA and COXâ…¡protein, respectively.Moreover,DNA was extracted and COXâ…¡methylation was detected by MSP.Data were presented as Means±SD.Statistical analyses were performed using SPSS version 13.0 and differences were considered significant if P＜0.05.Results:Part one,CSE-induced HUVEC apoptosis increased in a dose-and time-dependent manner with obvious cell death in 5%and 10% CSE-treated groups.COX activity decreased paralleled to CSE concentration and duration.Caspase-3 activity increased with CSE at a lower concentration of 0.5%-2.5%,but suddenly decreased after 5%and 10%CSE-treatment.This CSE-induced apoptosis in HUVEC was found negatively correlated with COX activity(r=-0.884,P＜0.01),but independent of caspase-3 activity(r=-0.236,P＞0.05).Part two,compared with control group,apoptotic index increased in CSE-treated group [(44.0±3.6)%]and AZA+CSE-treated group[(21.7±2.3)%].While COX activity,COXâ…¡mRNA and COXâ…¡protein expression decreased in these two groups[(5.94±0.36)×10^-1U/mg,(0.82±0.06) and(0.73±0.05) in CSE-treated group;(6.83±0.26)×10^-1U/mg,(1.18±0.08) and(1.10±0.12) in AZA+CSE-treated group].Differences of variants between every two of these three groups were significant(all P＜0.05).Nevertheless,no significant difference between control group and AZA group was found(P＞0.05).After MSP,positive COXâ…¡methylation(semi-methylation) in CSE group and AZA+CSE group was found.Conclusions:(1) CSE induces HUVEC apoptosis in a dose- and time-dependent manner.This apoptosis is independent of caspase-3 activation and is mediated by COX.(2) CSE inhibits COXâ…¡transcription via inducting methylation of COXâ…¡.(3) AZA demethylation may partially reverse CSE -induced COXâ…¡methylation,and serve as a protective factor of COX and HUVEC.