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A1200 - DNA Methyltransferase 1(DNMT1) Promotes Reactive Oxygen Substances(ROS)-mediated Lung Endothelial Cell Apoptosis in Chronic Obstructive Pulmonary Disease
Author Block: T. Li, X. He, L. Chen, Y. Chen, H. Zeng; Respiratory Department, The Sencond Xiangya Hospital of Central South University, Changsha, China.
RATIONALE: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. Cigarette smoke (CS)-induced mitochondrial damage with increased reactive oxygen species (ROS) production has been implicated in COPD pathogenesis by accelerating apoptosis. DNA methyltransferase 1 (Dnmt1), a member of DNA methyltransferases, is essential for maintain DNA methylation, and participate in a variety of diseases by regulating cell cycle, proliferation and apoptosis. But its role in ROS-derived apoptosis of COPD has not been clarified. Here, we assessed the expression of DNMT1 in vivo of the CS induced COPD mouse model and in vitro of cigarette smoke extract(CSE) induced endothelial cell impairment model, and investigated that whether DNMT1 can promote endothelial cell apoptosis induced by reactive oxygen substances(ROS) in COPD through hypermethylation of apoptosis-suppressor gene. METHODS: Oxidative stress(ROS) impaired endothelial cell model was established by CSE and H2O2 treatment, while, COPD mouse model was established by intermittent cigarette smoke exposure for three months. Antioxidant vitamin E, DNMT inhibitor 5'-aza-2'deoxycytidine(5-AZA) or DNMT1 overexpressed lenti-virus were used to intervene COPD mouse model and oxidative stress impaired endothelial cell model. DNMT1, SOD3 and Bcl-2 expression were determined by quantitative Real-time PCR (qRT-PCR) and western blotting. In situ cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Cell apoptosis in endothelial cells was fully measured with Annexin V (AV) and PropidiumIodide (PI) staining. The oxidative stress was measured by ROS assay. DNA methylation was assessed by bisulfite sequencing PCR(BSP). RESULTS: Lung cell apoptosis in COPD mouse model and oxidative stress impaired endothelial cell model were significantly increased, coinciding with the decreased abundance of Bcl-2 and SOD3, but increased DNMT1 expression. Antioxidant vitamin E intervention decreased DNMT1 expression and oxidative stress, but increased Bcl-2 expression and SOD3 expression in mouse model and endothelial cell. Furthermore, DNMT1 overexpression inhibit Bcl-2 and promote apoptosis both in COPD mouse model and endothelial cell model through hypermethylation of Bcl-2 gene promoter. However, after treated with 5-AZA in mice and cells model, apoptosis was alleviated. These results indicate that the epigenetic regulation of some apoptosis-regulated genes could provide some bases to the identification of the methylation machineries of apoptosis-associated genes for which the DNMT expression acts as a promoting factor. CONCLUSION: Cigarette smoke induced oxidative stress leads to lung endothelial cell apoptosis, while, DNMT1 promotes cell apoptosis through hypermethylation of Bcl-2. Antioxidant and methylation inhibitor may suppress cell apoptosis through DNMT1 in COPD.