| Objective:To observe the pulmonary vascular endothelial cell apoptosis, mitochondrial transcription factor A(mtTFA) and COX subunitⅡ(COXⅡ) expression in COPD patients and analysis the relationship among them. To investigate the possible role of them in COPD pathogenesis.Methods:According to the latest COPD diagnostic criteria,21 patients underwent pneumonectomy were divided into 2 groups: non-COPD group and COPD group (stable stage). TUNEL assay was used to assess cell apoptosis of pulmonary vascular endothelial cells. COX activity was measured by spectrophotometry. Semi-quantitative and real-time reverse transcription polymerase chain reaction (RT-PCR and real-time RT-PCR) were used to measure mtTFA mRNA and COXⅡmRNA expression. Moreover, immunohistochemistry and western-blot were used to detect mtTFA protein expression respectively. Western-blot was used to measure COXⅡprotein. Data were presented as Means±SD. Statistical analysis were performed using SPSS version 13.0 and differences were considered significant if P<0.05.Results:The smoking index of COPD group[(33.6±12.0)pack year] was much higher than that in non-COPD group[(12.1±8.9)pack year]. Difference between the two groups was of great significance (P<0.01). The apoptotic index of pulmonary vascular endothelial cells in COPD group[(13.8±1.9)%] was much higher than that in non-COPD group[(5.9±1.0)%]. Difference between the two groups was of great significance (P<0.01). mtTFA mRNA and mtTFA protein were lower in COPD group[(0.59±0.07) and (0.32±0.07)] than those in non-COPD group[(0.78±0.06) and (0.55±0.09)](all P<0.01). COX activity, COXⅡmRNA and COXⅡprotein in COPD group [(4.4±0.9)×10-1U/mg,(0.72±0.12) and (0.62±0.06)]were lower than those in non-COPD group[(7.6±0.4)×10-1U/mg,(0.86±0.16) and (0.89±0.13)] (P<0.01, P <0.05, P<0.01). The correlation analysis showed that the apoptotic index was negatively correlated with FEV1/FVC and FEV1%pre (r=-0.796, r=-0.827, all P<0.01), but positively correlated with smoke index (r=0.703, P<0.01). COX activity was negatively correlated with apoptotic index (r =-0.756, P<0.01). mtTFA potein was positively correlated with FEV1% pre, COXⅡprotein and COX activity(r=0.892, r=0.810, r=0.854, all P<0.01), but negatively correlated with apoptotic index and smoke index (r=-0.749, r=-0.763, all P<0.01).Conclusions:(1) Pulmonary vascular endothelial cell apoptosis is increased in COPD patients. (2) mtTFA expression, COXⅡexpression and COX activity are inhibited in COPD patients. (3)Cigarette smoking may be related with pulmonary vascular endothelial cell apoptosis in COPD through inhibiting mtTFA and COX II expression.Objective:To investigate whether there is aberrant mtTFA methylation in the lung tissue of COPD patients.Methods:Lung tissue of 21 patients underwent pneumonectomy was collected and divided into 2 groups:non-COPD group and COPD group. Genome DNA of lung tissue was extracted and mtTFA methylation satus was detected using Bisulfite DNA sequence (BSP). Statistical analysis were performed using SPSS version 13.0 and differences were considered significant if P<0.05.Results:Several methylated CpGs were found in mtTFA of the lung tissue of COPD group but no methylated CpG was found in non-COPD group. The methylation rate of mtTFA in COPD patients is (27.8±3.6) %. Conclusions:There is aberrant mtTFA methylation in the lung tissue of COPD patients.Objective:To investigate whether mtTFA methylation is involved in CSE-induced HUVEC apoptosis.Methods: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. Then, 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. Real-time RT-PCR was used to detect mtTFA mRNA and COXⅡmRNA. Western-blot and immunocytochemistry were used to detect mtTFA protein. Western-blot was used to detect COXⅡprotein. Moreover, DNA was extracted and mtTFA methylation status was detected by BSP. Data were presented as Means±SD. Statistical analysis were performed using SPSS version 13.0 and differences were considered significant if P<0.05. Results: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% to 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). Compared with the other three groups, apoptotic index increased in CSE-treated group [(44.0±3.6)%](P<0.01). While mtTFA mRNA, mtTFA protein, COX activity, COXⅡmRNA and COXⅡprotein decreased in CSE-treated group(P<0.01, P<0.01, P<0.05, P<0.05, P<0.05). Nevertheless, no significant differences between the control group, AZA group and AZA+CSE group were found (P>0.05). After BSP, several methylated CpGs were found in CSE-treated group but no methylated CpG was found in control group, AZA group and AZA+CSE group.The methylation rate of mtTFA in CSE group is (26.5±2.4)%.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) mtTFA methylation may contribute to CSE inducing HUVEC apoptosis.
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