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Ezetimibe suppresses cholesterol accumulation in lipid-loaded vascular smooth muscle cells in vitro via MAPK signaling.

Qin L, Yang YB, Yang YX, Zhu N, Gong YZ, Zhang CP, Li SX, Liao DF - Acta Pharmacol. Sin. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Stem Cell Regulation and Application, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China [2] Institute of Pharmacy and Pharmacology, South China University, Hengyang 421001, China.

ABSTRACT

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.

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Effects of ezetimibe on nuclear translocation of SREBP-1 in lipid-loaded VSMCs induced by Chol:MβCD. VSMCs were co-incubated with 10 μg/mL Chol:MβCD in the absence or presence of 3 μmol/L ezetimibe for 72 h. SREBP-1 in the cytoplasm (red) or nucleus (blue) was detected by indirect immunofluorescence with double staining. Images were obtained with a fluorescence microscope (×400).
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fig3: Effects of ezetimibe on nuclear translocation of SREBP-1 in lipid-loaded VSMCs induced by Chol:MβCD. VSMCs were co-incubated with 10 μg/mL Chol:MβCD in the absence or presence of 3 μmol/L ezetimibe for 72 h. SREBP-1 in the cytoplasm (red) or nucleus (blue) was detected by indirect immunofluorescence with double staining. Images were obtained with a fluorescence microscope (×400).

Mentions: The SREBP-1 precursor is located on the membrane of the endoplasmic reticulum. When the intracellular cholesterol level changes, active SREBP-1 is transferred into the nucleus to regulate Cav-1 transcription34. To examine the activity of SREBP-1 under ezetimibe treatment, we observed SREBP-1 translocation by double immunofluorescence staining. Compared with the control group, Chol:MβCD stimulated SREBP-1 translocation from the cytoplasm into the nucleus (Figure 3). However, there was a redistribution of SREBP-1 after ezetimibe treatment. A substantial portion of SREBP-1 accumulated in the cytoplasm after ezetimibe treatment, suggesting that ezetimibe reversed the process of SREBP-1 nuclear translocation (Figure 3).


Ezetimibe suppresses cholesterol accumulation in lipid-loaded vascular smooth muscle cells in vitro via MAPK signaling.

Qin L, Yang YB, Yang YX, Zhu N, Gong YZ, Zhang CP, Li SX, Liao DF - Acta Pharmacol. Sin. (2014)

Effects of ezetimibe on nuclear translocation of SREBP-1 in lipid-loaded VSMCs induced by Chol:MβCD. VSMCs were co-incubated with 10 μg/mL Chol:MβCD in the absence or presence of 3 μmol/L ezetimibe for 72 h. SREBP-1 in the cytoplasm (red) or nucleus (blue) was detected by indirect immunofluorescence with double staining. Images were obtained with a fluorescence microscope (×400).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4155523&req=5

fig3: Effects of ezetimibe on nuclear translocation of SREBP-1 in lipid-loaded VSMCs induced by Chol:MβCD. VSMCs were co-incubated with 10 μg/mL Chol:MβCD in the absence or presence of 3 μmol/L ezetimibe for 72 h. SREBP-1 in the cytoplasm (red) or nucleus (blue) was detected by indirect immunofluorescence with double staining. Images were obtained with a fluorescence microscope (×400).
Mentions: The SREBP-1 precursor is located on the membrane of the endoplasmic reticulum. When the intracellular cholesterol level changes, active SREBP-1 is transferred into the nucleus to regulate Cav-1 transcription34. To examine the activity of SREBP-1 under ezetimibe treatment, we observed SREBP-1 translocation by double immunofluorescence staining. Compared with the control group, Chol:MβCD stimulated SREBP-1 translocation from the cytoplasm into the nucleus (Figure 3). However, there was a redistribution of SREBP-1 after ezetimibe treatment. A substantial portion of SREBP-1 accumulated in the cytoplasm after ezetimibe treatment, suggesting that ezetimibe reversed the process of SREBP-1 nuclear translocation (Figure 3).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Stem Cell Regulation and Application, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China [2] Institute of Pharmacy and Pharmacology, South China University, Hengyang 421001, China.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus