Ezetimibe suppresses cholesterol accumulation in lipid-loaded vascular smooth muscle cells in vitro via MAPK signaling
Abstract
Li QIN1, 2, #, Yun-bo YANG1, 3, 4, #, Yi-xin YANG3, Neng ZHU4, 5, Yong-zhen GONG1, 6, Cai-ping ZHANG2, Shun-xiang LI1, Duan-fang LIAO1, *
1Division of Stem Cell Regulation and Application, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China; 2Institute of Pharmacy and Pharmacology, South China University, Hengyang 421001, China; 3Matthew Mailing Centre for Translational Transplantation Studies, London Health Sciences Centre, Western University, London N6A5A5, Canada; 4The Second Xiang-Ya Hospital, Central South University, Changsha 410011, China; 5The Second Affiliated Hospital, South China University, Hengyang 421001, China; 6Department of Pharmacology, Central South University, Changsha 410011, China
Aim: To investigate the mechanisms of anti-atherosclerotic action of ezetimibe in rat vascular smooth muscle cells (VSMCs) in vitro.
Methods: VSMCs of SD rats were cultured in the presence of Chol:MβCD (10 μg/mL) for 72 h, and intracellular lipid droplets and cholesterol levels were evaluated using Oil Red O staining, HPLC and Enzymatic Fluorescence Assay, respectively. The expression of caveolin-1, sterol response element-binding protein-1 (SREBP-1) and ERK1/2 were analyzed using Western blot assays. Translocation of SREBP-1 and ERK1/2 was detected with immunofluorescence.
Results: Treatment with Chol:MβCD dramatically increased the cellular levels of total cholesterol (TC), cholesterol ester (CE) and free cholesterol (FC) in VSMCs, which led to the formation of foam cells. Furthermore, Chol:MβCD treatment significantly decreased the expression of caveolin-1, and stimulated the expression and nuclear translocation of SREBP-1 in VSMCs. Co-treatment with ezetimibe (3 μmol/L) significantly decreased the cellular levels of TC, CE and FC, which was accompanied by elevation of caveolin-1 expression, and by a reduction of SREBP-1 expression and nuclear translocation. Co-treatment with ezetimibe dose-dependently decreased the expression of phosphor-ERK1/2 (p-ERK1/2) in VSMCs. The ERK1/2 inhibitor PD98059 (50 μmol/L) altered the cholesterol level and the expression of p-ERK1/2, SREBP-1 and caveolin-1 in the same manner as ezetimibe did.
Conclusion: Ezetimibe suppresses cholesterol accumulation in rat VSMCs in vitro by regulating SREBP-1 and caveolin-1 expression, possibly via the MAPK signaling pathway.
Keywords: atherosclerosis; vascular smooth muscle cells; cholesterol; ezetimibe; caveolin-1; SREBP-1; MAPK
This work was supported by grants from the National Natural Science Foundation of China (No 81000946, No 81270359, No 81173047, and No 12JJ5068) and the Construct Program of the Pharmaceutical Science Key Discipline in Hunan province.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail dfliao66@aliyun.com
Received 2013-11-20 Accepted 2014-07-14
Keywords:
1Division of Stem Cell Regulation and Application, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China; 2Institute of Pharmacy and Pharmacology, South China University, Hengyang 421001, China; 3Matthew Mailing Centre for Translational Transplantation Studies, London Health Sciences Centre, Western University, London N6A5A5, Canada; 4The Second Xiang-Ya Hospital, Central South University, Changsha 410011, China; 5The Second Affiliated Hospital, South China University, Hengyang 421001, China; 6Department of Pharmacology, Central South University, Changsha 410011, China
Aim: To investigate the mechanisms of anti-atherosclerotic action of ezetimibe in rat vascular smooth muscle cells (VSMCs) in vitro.
Methods: VSMCs of SD rats were cultured in the presence of Chol:MβCD (10 μg/mL) for 72 h, and intracellular lipid droplets and cholesterol levels were evaluated using Oil Red O staining, HPLC and Enzymatic Fluorescence Assay, respectively. The expression of caveolin-1, sterol response element-binding protein-1 (SREBP-1) and ERK1/2 were analyzed using Western blot assays. Translocation of SREBP-1 and ERK1/2 was detected with immunofluorescence.
Results: Treatment with Chol:MβCD dramatically increased the cellular levels of total cholesterol (TC), cholesterol ester (CE) and free cholesterol (FC) in VSMCs, which led to the formation of foam cells. Furthermore, Chol:MβCD treatment significantly decreased the expression of caveolin-1, and stimulated the expression and nuclear translocation of SREBP-1 in VSMCs. Co-treatment with ezetimibe (3 μmol/L) significantly decreased the cellular levels of TC, CE and FC, which was accompanied by elevation of caveolin-1 expression, and by a reduction of SREBP-1 expression and nuclear translocation. Co-treatment with ezetimibe dose-dependently decreased the expression of phosphor-ERK1/2 (p-ERK1/2) in VSMCs. The ERK1/2 inhibitor PD98059 (50 μmol/L) altered the cholesterol level and the expression of p-ERK1/2, SREBP-1 and caveolin-1 in the same manner as ezetimibe did.
Conclusion: Ezetimibe suppresses cholesterol accumulation in rat VSMCs in vitro by regulating SREBP-1 and caveolin-1 expression, possibly via the MAPK signaling pathway.
Keywords: atherosclerosis; vascular smooth muscle cells; cholesterol; ezetimibe; caveolin-1; SREBP-1; MAPK
This work was supported by grants from the National Natural Science Foundation of China (No 81000946, No 81270359, No 81173047, and No 12JJ5068) and the Construct Program of the Pharmaceutical Science Key Discipline in Hunan province.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail dfliao66@aliyun.com
Received 2013-11-20 Accepted 2014-07-14