Simvastatin reduces atherogenesis and promotes the expression of hepatic genes associated with reverse cholesterol transport in apoE-knockout mice fed high-fat diet
- Guohua Song†1,
- Jia Liu†1, 2,
- Zhenmei Zhao2,
- Yang Yu1,
- Hua Tian1,
- Shutong Yao1,
- Guoli Li2 and
- Shucun Qin1Email author
© Song et al; licensee BioMed Central Ltd. 2011
Received: 22 December 2010
Accepted: 18 January 2011
Published: 18 January 2011
Statins are first-line pharmacotherapeutic agents for hypercholesterolemia treatment in humans. However the effects of statins on atherosclerosis in mouse models are very paradoxical. In this work, we wanted to evaluate the effects of simvastatin on serum cholesterol, atherogenesis, and the expression of several factors playing important roles in reverse cholesterol transport (RCT) in apoE-/- mice fed a high-fat diet.
The atherosclerotic lesion formation displayed by oil red O staining positive area was reduced significantly by 35% or 47% in either aortic root section or aortic arch en face in simvastatin administrated apoE-/- mice compared to the control. Plasma analysis by enzymatic method or ELISA showed that high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-I (apoA-I) contents were remarkably increased by treatment with simvastatin. And plasma lecithin-cholesterol acyltransferase (LCAT) activity was markedly increased by simvastatin treatment. Real-time PCR detection disclosed that the expression of several transporters involved in reverse cholesterol transport, including macrophage scavenger receptor class B type I, hepatic ATP-binding cassette (ABC) transporters ABCG5, and ABCB4 were induced by simvastatin treatment, the expression of hepatic ABCA1 and apoA-I, which play roles in the maturation of HDL-C, were also elevated in simvastatin treated groups.
We demonstrated the anti-atherogenesis effects of simvastatin in apoE-/- mice fed a high-fat diet. We confirmed here for the first time simvastatin increased the expression of hepatic ABCB4 and ABCG5, which involved in secretion of cholesterol and bile acids into the bile, besides upregulated ABCA1 and apoA-I. The elevated HDL-C level, increased LCAT activity and the stimulation of several transporters involved in RCT may all contribute to the anti-atherosclerotic effect of simvastatin.
Statins are class of drug used to lower cholesterol levels by inhibiting the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), which plays a central role in the production of cholesterol in the liver. Increased cholesterol levels have been associated with cardiovascular diseases (CVD), and statins are therefore first-line pharmacotherapeutic agents for the treatment of these diseases in humans. Recently a great line of animal species have been used to study the pathogenesis and potential treatment of the lesions of atherosclerosis. However until now the effects of statins in animal models of atherosclerosis are not very consistent. The apoE-knockout (apoE-/-) mouse is a well-established genetic mouse model of atherogenic hypercholesterolemia, in which mice spontaneously develop atherosclerosis with similar features to those observed in human type III familial hyperlipoproteinemia. Florian Bea et al. has reported simvastatin increased serum cholesterol and atherosclerotic lesion size in apoE-/- mice fed a chow diet although the plaque stability was increased . In this work, we wanted to evaluate the effects of simvastatin on serum cholesterol and atherogenesis in apoE-/- mice fed a high-fat diet.
Reverse cholesterol transport (RCT) is believed to be crucial for preventing atherogenesis and hence the development of most cardiovascular diseases. This anti-atherogenic mechanism involves export of cholesterol from lipid-laden macrophages in the artery wall back to the liver for eventual excretion. The process of RCT can be divided into three stages: 1) the efflux of cellular cholesterol to high-density lipoprotein (HDL) from peripheral cells. Macrophage ATP-binding cassette transporter (ABC) A1, ABCG1 and scavenger receptor class B type I (SR-BI) all play important roles in the first stage. 2) the transport of HDL-cholesterol (HDL-C) in blood to the liver. The cholesterol in HDL is converted to cholesteryl esters (CE) by the enzyme lecithin-cholesterol acyltransferase (LCAT) and carried as CE in the core of the HDL particle to the liver. 3) the delivery of cholesterol esters to hepatocytes from HDL . After uptaking of HDL-accociated cholesterol into hepatocytes by heptic SR-BI, secretion of cholesterol, bile acids, and phospholipid into the bile is regulated by the respective transporters ABCG5, ABCG8, ABCB4 and ABCB11. Simvastatin has been reported to improve RCT in type 2 diabetic patients with hyperlipidemia . However, the effects of simvastatin on the important factors playing roles in RCT has not been elucidated. In the present study, we hypothesized that simvastatin might promote the expression of several factors involved in RCT. Therefore, we investigated the effects of simvastatin on the expression of several transporters, including macrophage ABCA1, ABCG1 and SR-BI, hepatic SR-BI, ABCG5, ABCG8, ABCB4 and ABCB11. Besides transporters, we also detected the effects of simvastatin on plasma LCAT activities and apoA-I concentrations, which also play roles in RCT in apoE-/- mice.
Animals and Experimental Design
21 male apoE-/- mice were purchased from laboratory animal center of Academy of Military Medical Sciences (Beijing). All experiments were approved by the laboratory animals' ethical committee of Taishan Medical University and followed national guidelines for the care and use of animals. At 5 weeks of age, the 21 apoE-/- mice were randomly divided into 2 groups, the model group (n = 11, vehicle treated group) and the simvastatin (5 mg/kg/d) treated group (n = 10). All groups were fed an atherogenic high-fat diet (15.8% fat and 1.25% cholesterol) supplemented with vehicle or simvastatin for 6 weeks. Vehicle or simvastatin were administered intragastrically once daily.
After 6 weeks of treatment, blood was collected from the retro-orbital sinus of the mice without dietary exposure for 12 h. Concentrations of plasma total cholesterol (TC), HDL-C, and triglycerides (TG) were determined by enzymatic methods (BioSino). Non-HDL-C was calculated as TC minus HDL-C. Plasma concentrations of apolipoprotein A-I (apoA-I) was determined by ELISA kit (BlueGene). Endogenous lecithin-cholesterol acyltransferase (LCAT) activity was measured as the utilization rate of free cholesterol (FC) in native plasma according to the method by Ly et al. . The FC content was measured colorimetrically by using a kit (Biovision) in pentuplicate by a microplate reader (Tecan) at zero time and after 1 hr at 37°C. LCAT activity was expressed as nanomoles FC consumed per hr per ml plasma.
The proximal aorta attached to the heart was used to prepare cross-sections. Cryosections (8 μm) were cut from the site where the aorta valve cups appear at the aorta root, collected and stained with oil red O and brilliant green. The volume of stained lipids (μm2) was calculated from five sections for an animal. For determination of atherosclerotic plaque area in aortic arch, the aortic arch were dissected and stained en face with oil red O. Quantitative analysis of plaque area was performed by 2 blinded observers using Image-Pro Plus software 4.5 (Media Cybernetics).
Tissues or cells were harvested and protein extracts prepared as previously described . They were then subjected to western blot analyses using anti-ABCA1, ABCG1, SR-BI, apoA-I, and β-actin antibodies (Santa Cruz). The proteins were visualized and quantified using a chemiluminescence method (Pierce) and Quantity One (Bio-Rad) software program.
Isolation of Peritoneal Macrophages
Three days after intraperitoneal thioglycollate injection, peritoneal macrophages were harvested from six weeks simvastatin or vehicle treated apoE-/- mice. Following collection, peritoneal macrophages were immediately frozen at -80°C until RNA or protein extraction.
Quantitative Real-Time PCR
Primers used for real-time PCR analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) test with the GraphPad Prism programme ver.4.0. Results are expressed as means ± SD. P values less than 0.05 were considered significant.
Simvastatin decreases atherosclerotic lesion formation in apoE-/- mice fed a high-fat diet
Effect of simvastatin on plasma lipoprotein profiles and plasma LCAT activities
Effect of Simvastatin on plasma lipoprotein lipids and apoA-I concentrations in apoE-/- mice
Simvastatin (5 mg/kg)
Animal number (n)
24.69 ± 5.34
22.07 ± 4.12
1.13 ± 0.37
1.24 ± 0.47
22.06 ± 4.87
18.93 ± 3.89
2.63 ± 0.35
3.14 ± 0.34**
4.24 ± 1.01
11.58 ± 2.03**
Effect of simvastatin on the expression of hepatic genes involved in cholesterol metabolism
Effect of simvastatin on the expression of transporter genes involved in cholesterol efflux in peritoneal macrophages
In the present study, we confirmed simvastatin-treatment not only promoted plasma HDL-C, apoA-I levels, LCAT activities and macrophage SR-BI expression, but also increased the expression of hepatic ABCB4 and ABCG5 besides upregulated hepatic ABCA1 and apoA-I, and reduce atherosclerotic lesion size in apoE-/- mice fed a high-fat diet. It is well-known that statins are first-line pharmacotherapeutic agents for hypercholesterolemia treatment in humans. However the effects of statins on atherosclerosis and lipid levels in mouse models are very paradoxical. Nachtigal P et al. have shown atorvastatin could increase plasma HDL-C and decrease TC levels in apoE/LDL receptor-double-knockout mice fed an atherosclerotic diet . Previous studies [1, 7, 8] in which the simvastatin administered to the apoE-deficient mice showed that the plasma cholesterol levels of the treated animals fed a chow diet were significantly elevated compared to those of the untreated animals. In our study, we confirmed simvastatin-treatment did not affect plasma TC, TG and non-HDL-C levels, and significantly decreased the atherosclerotic lesion size in apoE-/- mice fed a high-fat diet. Our data was in agreement with Sparrow et al. reported  that the administration of simvastatin to apoE-deficient mice fed a high cholesterol diet did not alter their plasma TC levels, Sparrow et al. also found simvastatin decreases aortic cholesterol accumulation in apoE-/- mice besides increased anti-inflammotory effects, but the underlying mechanisms was not yet established. In the present study we found that the simvastatin administrated animals displayed atheroprotection may involved the promoted reverse cholesterol transport by up-regulated HDL-C and related genes expression, including genes important for the formation of HDL-C and genes important for secretion of cholesterol, bile acids, and phospholipid into the bile. In addition, the discrepancy of the effects of statin-treatment on lipid levels in apoE-/- mice might be attributable in part to the possibility that the dyslipidemic profile of apoE-/- mice fed a high-fat diet whose lipid profile is VLDL dominant, is different from and more severe than that fed a chow diet.
Plasma concentrations of HDL-C have strong inverse correlations with risk of atherosclerotic cardiovascular disease, independently of LDL-C levels . ApoA-I is the major protein component of HDL-C in plasma. HDL-C and apoA-I exert anti-inflammatory, anti-oxidant, anti-thrombotic, promoting RCT and vasodilating properties that could all potentially be involved in their anti-atherogenic effects . Here, we showed that simvastatin-treatment increased plasma levels of HDL-C and apoA-I. Our finding was in agreement with another report, which has shown that 8 weeks of simvastatin-treatment has a tendency to increase plasma HDL-C levels in apoE-/- mice fed a high cholesterol diet . Furthermore, apoA-I is mainly synthesized by liver and hepatic ABCA1 exert anti-atherogenic effect via its contribution to HDL-C formation by promoting lipid efflux to apoA-I from hepatocytes . Our previous study have reported that rosuvastatin selectively stimulates apolipoprotein A-I but not apolipoprotein A-II synthesis in Hep G2 cells . And in this study, our results showed that the expressions of hepatic apoA-I and ABCA1 were increased by simvastatin treatment in vivo, these data gave us a clue that the elevated plasma HDL-C level by simvastatin in this study might be closely associated with the enhanced hepatic apoA-I and ABCA1 expression stimulated by simvastatin.
One of the important anti-atherogenic effects of HDL-C is to facilitate the efflux of cholesterol from peripheral tissues and transport it back to the liver in a process called RCT . Recently, several key molecules have been identified to play pivotal roles on RCT [16–18]. Macrophage ABCA1 facilitates cholesterol efflux from cells to lipid-poor apoA-I, but not HDL [16, 17], whereas another two macrophage transporters, ABCG1, and SR-BI are involved in the cholesterol efflux from macrophages mediated by HDL, but not apoA-I [18, 19]. In our study, we showed that simvastatin induced macrophage SR-BI expression, which might lead to an increased HDL-mediated cholesterol efflux from macrophages. Furthermore, LCAT which bound to HDLs in plasma and play roles in RCT is an enzyme that converts free cholesterol into cholesteryl ester and is critical for the maturation and maintenance of normal HDL metabolism in humans  and mice . At present, the molecular mechanism(s) that regulates plasma LCAT concentrations is not well understood. LCAT activity depends in part on the amount of the enzyme in plasma, and in part on the substrate and cofactors available to the enzyme , such as apoA-I, an activator of LCAT, and apoA-II, an inhibitor of LCAT activity. Our results showed that simvastatin-treatment did not influence the mRNA expression of hepatic LCAT and apoA-II, but significantly increased plasma LCAT activities, hepatic apoA-I and plasma apoA-I concentrations. The discrepancy in hepatic LCAT expression and plasma LCAT activity might attributable to a post-transcriptional regulation of LCAT or the upregulation of apoA-I, the activator of LCAT, by simvastatin.
Besides macrophage transporters and plasma LCAT, several hepatic transporters are also involved in the process of RCT. The direct selective uptake of HDL-C by the liver is mediated by SR-BI. However, in our study, the expression of hepatic SR-BI was not altered in simvastatin-treated mice. After uptaking of HDL-associated cholesterol by SR-BI, secretion of cholesterol, bile acids, and phospholipid into the bile is regulated by the respective transporters ABCG5, ABCG8, ABCB4 and ABCB11. Here, we showed for the first time that the mRNA expression of ABCG5 and ABCB4 was upregulated by simvastatin-treatment. These finding suggest that simvastatin may exert anti-atherosclerotic effects by stimulating the expression of transporters involved in the process of RCT, including macrophage SR-BI, hepatic ABCG5 and ABCB4. The mechanisms in which simvastatin stimulated higher expressions of RCT related genes in apoE-/- mice deserve further investigation.
In conclusion, we confirmed here for the first time simvastatin increased the expression of hepatic ABCB4 and ABCG5 besides upregulated ABCA1 and apoA-I in apoE-/- mice. Our data show that in vivo administration of simvastatin effectively attenuates atherosclerotic lesion formation in apoE-/- mice fed a high-fat diet by targeting several pathways involved in atherogenesis. Elevated plasma apoA-I and HDL-C levels, increased plasma LCAT activities, and the stimulation of reverse cholesterol transporter gene expression may all contribute to the beneficial effect of simvastatin. Our findings provide a novel insight into the anti-atherosclerotic effects of simvastatin besides its lipid-lowering effect.
This research was supported by the Taishan Scholars Foundation of Shandong Province (zd056, zd057) and Special Research Funding of Taishan Medical University (2008). We also thank the colleagues in our laboratory, for their discussion and support.
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