The Effect Of Ezetimibe On Atherosclerosis And Its Potential Mechanisms

Background:Atherosclerosis is the chronic non-infective inflammation in the arterial walls. Atherosclerosis-associated cardiovascular and cerebral disease is one of the chief causes of death in the developed countries. Endothelial injury, lipid disorder and chronic inflammation are the critical issues in the pathological process of atherosclerosis. Since lowering cholesterol concentration is the basis of anti-atherosclerotic therapy, statins have been widely adopted in preventing and treating atherosclerosis-associated cardiovascular disease (ASCVD). However, plasma concentrations of lower density lipoprotein cholesterol (LDL-C) in some patients were still far from the target under the application of high dose of statins.Ezetimibe, as a cholesterol absorption inhibitor widely used in treating the patients with hypercholesterolemia, targets at a polytopic transmembrane protein localized at the apical membrane of enterocytes and the canalicular membrane of hepatocytes, called Niemann-Pick Cl-Like1(NPC1L1) protein. Since NPC1L1protein functions as a sterol transporter to mediate intestinal cholesterol absorption and counter-balances hepatobiliary cholesterol excretion, ezetimibe exerts the dual effects on lipid metabolism, inhibiting exogenous cholesterol absorption from diet and promoting a compensatory increase of endogenous cholesterol synthesis, and ultimately leads to reductions of LDL-C and total cholesterol (TC) concentrations in humans. However, the effects of ezetimibe on atherosclerosis and its potential mechanisms are still controversial so far.Some clinical trials had been employed to evaluate the effects of ezetimibe on lipid profiles, cardiovascular outcomes or the surrogate endpoints, most of them were combined to statin therapy. Most recently, the results from IMPROVE-IT trial were so encouraging that the addition of ezetimibe versus placebo to simvastatin therapy had a6.4%lower risk of major cardiovascular events in patients with acute coronary syndromes, including subsequent heart attack, stroke, cardiovascular death, rehospitalization for unstable angina and procedures to restore blood flow to the heart. However, reports of ezetimibe monotherapy on ASCVD are exceedingly rare.Therefore, in the present study, we determined the effects of ezetimibe on the development of atherosclerosis in apoE-/-mice on high-fat diet, with or without atorvastatin administration. As a result, ezetimibe not only reduced serum TC and LDL-C concentrations, but suppressed the formation of atherosclerotic lesions by decreasing serum inflammation factors and macrophage accumulation in the lesions. Our findings provide with basic research data and clinical therapeutic strategy for ASCVD patients. Objectives:The present studies aimed to investigate the effect of ezetimibe with or without on atherosclerosis in apoE knockout male mice on high-fat diet and the potential mechanisms.Methods and Results:Materials and MethodsSixty male apoE-/-mice on C57BL/6background (8-12weeks old) were originally purchased from the Beijing Vital River Laboratory Animal Technology Corporation and bred in-house. Mice were housed under specific pathogen-free (SPF) conditions, and fed normal laboratory diet. One week prior to drug administration, all mice were placed on a diet containing0.15%(wt/wt) cholesterol and21%(wt/wt) milk fat. Mice were randomly divided into four groups, administered with ezetimibe, atorvastatin, ezetimibe plus atorvastatin or vehicle7days after high-fat diet and throughout the whole study. Mice were inspected daily and weighed weekly. Ezetimibe and atorvastatin were dissolved in drinking water at concentrations that gave approximate doses of10mg/kg/day and20mg/kg/day when delivered by daily gavage, respectively. Systolic blood pressures were measured on conscious mice using noninvasive tail-cuff systems (CODA6; Kent Scientific Corp, Torrington, CT, U.S.A.) as described previously. Systolic blood pressures were measured one week before drug administration to record baseline blood pressures, and repeated on weeks4during the whole study.Mice were terminated after28days of drug administration, with blood harvested from the left ventricle and aortic arch immersion fixed with10%neutral buffer formalin. Serum cholesterol concentrations were determined using an enzymatic assay kit. Serum concentrations of alanine aminotransferase were measured by a lactic acid dehydrogenase enzymatic method (ALT). Serum concentrations of aspartate transaminase were measured by malate dehydrogenase enzymatic method (AST). Serum concentrations of monocyte chemoattractant protein-1(MCP-1), tumor necrosis factor (TNF-α) and transforming growth factor (TGF-β1) were measured with ELISA kits according to manufacturer’s recommendation.Aortic root was embedded with OCT and frozen at-20℃. Atherosclerosis was assessed on the intimas of both aortic arches by en face technique and also using cross-sections (10μm thick) of aortic roots as described previously. Oil Red O staining was used to assist in visualization of lesions. Quantitative analysis of atherosclerosis was performed using Image-Pro software as described previously. Aortic roots containing atherosclerosis were serially cross-sectioned (10μm thick/section) from the ascending aortas to the aortic sinus as described previously. Immunostaining was performed to identify macrophages as described previously.Data are presented as means±standard error of means (SEM). SigmaPlot version12.5was used for statistical analyses. Four-group comparisons were performed using one-way ANOVA for normally and equally distributed data and Kruskal-Wallis one-way ANOVA on Rank analysis for data having failed either normality or equal variance test. Weekly body weight and systolic blood pressure were analyzed using two way repeated measures ANOVA. A P<0.05was considered to be statistically significant.ResultsCharacteristics of Study MiceSixty male apoE-/-mice were fed on high-fat diet and administrated with ezetimibe (lOmg/kg/day), atorvastatin (20mg/kg/day), ezetimibe(10mg/kg/day) plus atorvastatin (20mg/kg/day), or the vehicle, respectively. During the28-day duration,21mice (35%) died of the delivery by daily gavage. Mice on high-fat diet increased the average body weight of4.68gram on28days. Ezetimibe with or without atorvastatin given in drinking water were well tolerated as determined by daily observation and body weight measurements on a weekly base. Administration of atorvastatin seemed to abate the increase of mice average body weight during the whole study, but had no significance as compared to the mice administered with vehicle (P>0.05). In mice on high-fat diet, administration of ezetimibe with or without atorvastatin had no effects on body weight and systolic blood pressure.Both Ezetimibe and Atorvastatin Reduced Serum Cholesterol ConcentrationsOur studies demonstrated that either ezetimibe or atorvastatin administration reduced serum concentrations of total cholesterol (TC) and LDL cholesterol (LDL-C, P<0.001respectively). Consistently, ezetimibe monotherapy or combination with atorvastatin significantly decreased serum concentrations of high density lipoprotein cholesterol (HDL-C, P<0.05). Neither ezetimibe nor atorvastatin administration altered serum concentrations of triglyceride in apoE-/-mice. Compared to mice administrated with vehicle, neither ezetimibe nor atorvastatin administration led to the elevation of serum hepatic enzymes, such as alanine aminotransferase and aspartate transaminase.Ezetimibe Decreased Atherosclerotic Lesions As Well As AtorvastatinWe determined the effects of ezetimibe with or without atorvastatin in apoE-/-mice with high-fat diet on atherosclerotic lesion areas in aortic roots (from aortic sinuses to ascending aorta) and percent of lesion areas on intimas of aortic arches. Administration of either ezetimibe or atorvastatin decreased lesion areas from aortic sinuses to ascending aortas compared to mice administered with vehicle (P<0.001). Consistently, either ezetimibe or atorvastatin administration also profoundly decreased percent of lesion areas on intimas of aortic arches compared to mice administered with vehicle (P<0.001). Although combined administration of ezetimibe and atorvastatin decreased both lesion areas from aortic sinuses to ascending aortas and percent of lesion areas on intimas of aortic arches compared to mice administered with vehicle (P<0.001), the combination effects on atherosclerotic lesions did not exceed those of the monotherapy of either ezetimibe or atorvastatin (P>0.05).Ezetimibe Reduced Serum Concentrations of Inflammatory CytokinesTo investigate the potential mechanisms of those drugs on attenuating atherosclerosis, serum concentrations of MCP-1, TNF-α and TGF-β1were measured as an index of circulating inflammatory markers. Consistently, serum concentrations of MCP-1and TNF-a were reduced by administration of either ezetimibe or atorvastatin, compared to the vehicle (P<0.01). Neither ezetimibe nor atorvastatin influence serum concentrations of TGF-β1(P>0.05). Furthermore, the combined administration of ezetimibe and atorvastatin administration profoundly reduced serum concentrations of MCP-1and TNF-a compared to mice administered with vehicle (P<0.01). However, the combination effects of ezetimibe and atorvastatin administration on serum MCP-1and TNF-a concentrations did not exceed those of the monotherapy of either ezetimibe or atorvastatin (P>0.05).Ezetimibe Decreased Accumulation of Macrophages in Atherosclerotic LesionsConsistent to the previous reports, atherosclerotic lesions in apoE-/-mice on high-fat diet have a characteristic of pronounced macrophages presented in the lesions and adventitia. Administration of ezetimibe with or without atorvastatin significantly decreased the accumulation of macrophages in the lesions, compared to mice administered with vehicle (P<0.05).Conclusions:In the present studies, we found that administration of ezetimibe with or without atorvastatin prevented the development of atherosclerosis in apoE-/-male mice on high-fat diet. Ezetimibe administration with or without atorvastatin significantly reduced serum concentrations of total cholesterol and lower density lipoprotein, but did not change serum triglyceride concentrations. The anti-atherosclerotic effect of ezetimibe depended not only on its lowering lipid profiles, but on its anti-inflammation, including the decrease of serum MCP-1, TNF-α concentrations, and accumulation of macrophages in the atherosclerotic lesions.