Study on the mechanism of cigarette smoke extract induced actin cytoskeleton reorganization in human umbilical vein endothelial cells

• Main Authors: Bo-Hong, 林柏宏
• Other Authors: 王祖興
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/84923671002397908620

碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 100 === Cigarette smoking is a major cause of cardiovascular disorders. Actin cytoskeleton is a possible key player in responding to inflammatory stimuli and also an early target of cellular oxidative stress. Laboratory previous results also show that cigarette smoke extract (CSE) induces rapid actin cytoskeleton remodeling and up-regulate ICAM-1 (intercellular adhesion molecule-1) expression in human umbilical vein endothelial cells (HUVEC). The purpose of this study is to understand the actin damages and its possible mechanism in human umbilical vein endothelial cells EA.hy926 exposed to cigarette smoke extract (CSE). The results showed that CSE caused dose- and time-dependent reorganization of actin cytoskeleton and cell shrinkage in EA.hy926 cells by staining with rhodamine phalloidin. In addition, CSE increased total protein carbonylation in a dose-dependent manner. Cells co-treated with different chemical compounds, such as reactive oxygen species scavenger (vitamin C, alpha-tocopherol, lipoic acid, glutathione, N-acetylcysteine), reactive carbonyl scavengers (aminoguanidine), calcium chelator (ethylene glycol tetraacetic acid, BAPTA-AM ), transient receptor potential cation channels inhibitor (MRS 1845), and autopgay inhibitor (3-methyladenine) with CSE caused different inhibition of actin reorganization, protein carbonylation, and cell shrinkage in CSE-treated EA.hy926 cells. Among these compounds, lipoic acid, glutathione, EGTA, and MRS 1845 were found to potently inhibit the CSE-induced actin cytoskeleton reorganization and cell shrinkage. Furthermore, immunofluorescence staining analysis showed that the autophagy marker LC3 protein (microtubule-associated protein 1 light chain 3) accumulated at cell cortex in CSE-treated cells. Collectively, these findings suggest that CSE-mediated ROS generation and Ca2+ influx increase through TRPC channels are the key factors that may cause reorganization of actin cytoskeleton and the subsequent cell shrinkage. In addition, CSE-induced inappropriate organization of the actin cytoskeleton may dysregulate autophagy by abnormal accumulation of LC3 proteins at cell cortex.