Ezetimibe Inhibits Atherosclerosis Via MAPKs Regulating Caveolin1, Cyclin D1 And NFκB Signal Pathways

Atherosclerosis, along with the resultant coronary artery disease, is a leading cause of mortality in industrialized countries. There are many factors taking part in the atherosclerosis progression. Hypercholesterolemia is the major exciting factor for the development of vascular lesions. The increased plasma levels of LDL result in the oxidation or perhaps other modifications of LDL within the vascular wall, which initiating the atherosclerotic response. Three cellular components of the circulation, monocytes, platelets, and T lymphocytes, together with two cell types of the artery wall, endothelial and smooth muscle cells (SMC), interact with ox-LDL and generate atherosclerotic lesions at last. On the other side, atherosclerosis is also a specific example of a chronic inflammatory response. An initiating event is the accumulation of lipids, mainly LDLs in the vessel wall, which subsequently will become modified and trigger an inflammatory process. Ox-LDL can increase the expression of adhesion molecules and the foam cells and activated endothelium may also produce proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, IFN-γand tumor necrosis factor-α(TNF-α), which promote the further development of the inflammatory response. Therefore, hypercholesterolemia and the inflammatory response together result in atherosclerosis. 【The relationship between Ezetimibe and atherosclerosis】Ezetimibe is the first of a new class of selective cholesterol absorption inhibitors. It selectively inhibits the intestinal uptake and absorption of dietary and biliary cholesterol at the brush border of small intestinal enterocytes, confining cholesterol to the intestinal lumen for subsequent excretion. The reductions occurred in LDL-CH, whereas HDL-CH was increased by ezetimibe treatment. However, the mechanisms of ezetimibe to inhibit atherosclerosis are not very clear. We found that ezetimibe can decrease the FC and TC in macrophages treated with Chol:MβCD and inhibited the foam-cell formation by the analysis of HPLC and Oil red O staining. Moreover, ezetimibe decreased the levels of TG, TC, LDL-CH and increased the level of HDL-CH, and further inhibited the formation of vascular lesion in ApoE knockout (ApoE-/-) mice. Therefore, we further investigated the mechanisms of ezetimbe with Oligo Atherosclerosis Microarray. We found that ezetimibe can down-regulate the genes related with: cholesterol/lipid transport and metabolism: SCARB1, OLR1, ADFP, CD36, LPA and LDLR; inflammatory response: CCL2, ILlA, TNF, SELE, ILlR1 and IL7; cell growth and proliferation: HBEGF, PDGFB, PDGFRB and KDR; apoptosis: BCL2, BID and SERPINE1 and up-regulate the genes related with: cholesterol/lipid transport and metabolism: ApoE; inflammatory response: CCLll; cell growth and proliferation: EGR1. Therefore, the possible mechanisms of ezetimibe to inhibit atherosclerosis are regulating cholesterol transport and metabolism, inhibiting inflammatory response and cell proliferation.【Ezetimibe inhibits atherosclerosis via regulating the absorption and transport of cholesterol】It is the most important reason to form atherosclerosis that macrophages and vascular smooth muscle cells absorb ox-LDL and form foam cells. The accumulation of such foam cells constitutes the bulk of the early vascular lesion. Therefore, inhibiting the absorption of cholesterol by macrophages and vascular smooth muscle cells and accelerating the transport of cholesterol are keys to prevent atherosclerosis.In our present study, we found that Chol:MβCD could decrease caveolinl expression, increase the expression of p-ERK1/2 and activate ERK1/2 to translocate into nucleus. However, ezetimibe increased caveolinl expression, inhibited the activating of ERK1/2 and decreased the expression of p-ERK1/2. Moreover, cells pretreated with the ERK1/2 inhibitor PD98059 showed a significant reduction in caveolinl expression and phosphorylated ERK1/2 levels. The analysis of Real-time PCR, immunofluorescent and western blot confirmed that ezetimibe could decrease the expression of SR-BI, CD36 and SREBP-1 and increase ApoE expression. At the same time, we found that caveolinl and SR-BI are interactional proteins and ezetimibe could affect their interaction by immunoprecipitation analysis. Immunohistology also showed that ezetimbe decreased the expression of SR-BI and CD36 and increased caveolinl expression in ApoE-/- atherosclerosis mouse. Therefore, ezetimibe can increase the expression of caveolinl and ApoE to mediate cholesterol transport, which result in the decrease of cholesterol in cells, and decrease the expression of SR-BI and CD36, and further decrease the absorption of cholesterol.【Ezetimibe inhibits the proliferation and migration of vascular smooth muscle cells via MAPKs regulating CyclinD1 signal pathways】In response to vascular injury, medial smooth muscle cells proliferate and migrate into the intima, which contributes to the intimal proliferate and the formation of atherosclerosis lesion. The proloferation of VSMCs is controlled by the cell cycle. The mammalian cell cycle is subject to be controlled by numberous cyclin-CDK complexes. The cyclinD1 and Rb/E2F play key role in the cell cycle progression to regulate cells from G1 to S phase.Flow cytometry analysis indicated that ezetimibe delayed the VSMC cell cycle progression from G1 to S phase when induced by Chol:MBCD. Scratch-wound assay also showed that ezetimibe inhibited VSMC migration. To further investigate the molecular mechanism of ezetimibe suppressing VSMC proliferation and migration, we performed western blot, immunofluorescent, transfection and luciferase analysis. We found that ezetimibe decreased the expression of CyclinD1, CDK4, pRb and decreased the promoter activities of CyclinD1, E2F and AP1 in VSMCs treated with Chol:MβCD.MAPKs play important role in cell cycle regulation. We found that ezetimibe decreased the expression of p-MEK1, p-ERK1/2, JNK1, c-JUN and inhibited the activation ERK1/2 and JNK1 to translocate into nucleus in VSMCs treated with Chol:MβCD. Moreover, cells pretreated with the ERK1/2 inhibitor PD98059 showed a significant reduction in the expression of p-MEK1, p-ERK1/2 and CyclinD1.Thus, ezetimibe inhibits the proliferation and migration of vascular smooth muscle cells via suppressing ERK1/2 activation to decrease the expression of Cyclin D1 and CDK4, and decreases the phosphorylated Rb levels, and further inhibits E2F activity.【Ezetimibe inhibits inflammatory response of atherosclerosis via MAPKs regulating NF k B signal pathways】Atherosclerosis is regarded as a chronic inflammatory disease of the vessel wall. The transcription factor NFkB is one of the key regulators of inflammation responses. Upon activation, NFkB can mediate the induction of genes, many of which take important role in atherosclerotic development. Activated NFkB has been detected in endothelial cells, smooth muscle cells, and macrophages in atherosclerotic plaques.In our present study, ELISA analysis showed that ezetimibe could decrease the level of C-reactive protein in ApoE-/- atherosclerosis mouse. Therefore, we do the further research to study the mechanisms of ezetimibe suppressing inflammatory response of atherosclerosis. Western blot showed that ezetimibe decreased NFkB p⁶⁵ expression and increased IKB expression in THP-1 cells treated with PMA and Chol:MβCD. Immunofluorescent analysis also indicated that ezetimibe inhibited NFkB p⁶⁵ activation to translocate into nucleus. However, cells pretreated with the ERK1/2 inhibitor PD98059 showed a significant reduction in NFkB p⁶⁵ expression. Moreover, we found ezetimibe decreased the full-long promoter activity of TNF-αin THP-1 cells treated with Phorbol 12-myristate 13-acetate (PMA) and Chol:MβCD by transfection and luciferase analysis. But ezetimibe has not significant effect on the promoter activity of TNF-αthat lacks the NFkB blinding region. At the same time, we found that ezetimibe decreased TNF-αcontent by ELISA analysis. Therefore, Ezetimibe inhibits inflammatory response of atherosclerosis via suppressing ERK1/2 activation to decrease the expression of NFkB p⁶⁵ and increases I K B expression, and further decreases TNF-αexpression. In conclusion, ezetimibe mediates the absorption and transport of cholesterol via MAPKs regulating caveolinl/SR-BI; inhibits the proliferation and migration of VSMCs via MAPKs regulating Cyclin D1 signal pathways; inhibits inflammatory response via MAPKs regulating NF K B signal pathways, and further prevents the progression of atherosclerosis.