Effects Of Hepatic Overexpression Of MsrA On Lipid Metabolism And Atherosclerosis In Mice

Background Lipid metabolic disorder and inflammation are considered as the risk factors for atherogensis. Liver as a vital organ that plays a major role in regulation of both lipid metabolism and inflammation, and is closely associated with the pathogenesis of athosclerosis in the body. Manipulation of hepatic lipid metabolism and inflammation is a commonly pursued strategy to prevent atherosclerosis progression. Recent findings showed that oxidative modification of methionine residues (methionine sulfoxide) are involved in the occurrence and development of several chronic diseases via influence certain proteins structures and functions. And methionine sulfoxide reductase A (MsrA), as a special protective barrier against protein oxidation in the body that converts methionine-S-sulfoxide (MetSO) to methionine, plays an important role in the regulation of protein function and the maintenance of redox homeostasis in cells. Therefore, MsrA maybe as a target of redox status intervention in the liver, this study was to investigate the effects of hepatic overexpression of hMsrA on inflammation and lipid metabolism and atherosclerotic plaque in atherosclerosis model mice and to explore its related mechanism.Methods In vitro study, lentiviral expression vectors (pWPI-hMsrA-GFP or control pWPI-GFP) were transient transfected using liposome into human hepatocellular carcinoma HepG2 cells, respectively. The reporter gene GFP and MsrA levels in cells were detected by fluorescent microscope or Western blotting. Dihydroethidium bromide (be oxidized to bromide) were used as fluorescent probe to analyze intracellular reactive oxygen species (ROS) levels, cellular red and green fluorescence were detected and ROS level was indicated by the mean fluorescence intensity of cells which calculated by Image J software. The expression levels of inflammation-related and lipoprotein-metabolism-related genes in HepG2 cells were determined by quantitative real-time PCR or Western blotting analysis, to evaluate effects of hMsrA overexpression on inflammation and lipid metabolism in HepG2 cells.In vivo study, lentiviral expression vector (pWPI-GFP or pWPI-hMsrA-GFP) and the packaging plasmid (PCMV and PD2G) were cotransfected into 293T cells by calcium phosphate method, which allows production of lentivirus (Lv-GFP or Lv-MsrA-GFP). The culture medium containing lentivirus particles was harvested and concentrated to obtain high titer of lentivirus concentrated solution. Apolipoprotein E deficient (apoE-/-) mice or scavenger receptor class B type I deficient (SR-BI-/-) mice were used as atherosclerosis model mice, and were randomly divided into two groups of 8 mice per group. Either the recombinant lentivirus containing hMsrA genes (Lv-MsrA-GFP) or lentivirus pWPI (Lv-GFP control group) were injected into mice via the retroorbital venous plexus, respectively. Starting at 2 weeks after lentiviral injection, the mice were fed with AIN76A Western diet for 12 weeks to accelerate the development of atherosclerosis. Beginning at 2 weeks after lentiviral injection, blood samples were collected from mice at 3-4 week intervals and plasma total cholesterol (TC), free cholesterol (FC), triglycerides (TG) and high density lipoprotein cholesterol (HDL-C) were all determined enzymatically, plasma lipoprotein profiles were determined by fast protein liquid chromatography (FPLC), plasma superoxide dismutase (SOD) activity was detected using reagent kit, and plasma paraoxonase-1 (PON1) activity were monitored by continuous dynamic method using paraoxon as a substrate, fecal cholesterol was extracted using isopropanol and determined. Fourteen weeks after lentiviral injection, the extent of atherosclerosis was examined using Oil Red O-stained cross sections of the aortic root and by en face analysis of the aorta. MsrA levels in liver were detected by immnunohistochemical analysis. Liver lipids content were analyzed by enzymatic method. The mRNA levels of inflammatory related and lipid metabolism genes were determined by quantitative real-time PCR (Q-PCR). Western blotting was used to measure protein levels of liver lipid metabolism related genes and plasma apolipoprotein AI (apoAI), PON1 and serum amyloid A (SAA).Results1. Overexpression of hMsrA in HepG2 cells decreased the level of ROS, inhibited the production of inflammatory cytokines TNFa and IL-6, also significantly increased the expression levels of cholesterol-metabolism-related genes Liver X receptor a (LXRa), ATP binding cassette transporter Al (ABCA1) and SR-BI, but not that of low density lipoprotein receptor (LDLR). The results suggested that high level of hMsrA could reduce ROS level and impact on inflammation related and lipoprotein metabolism related genes expression via exert an antioxidant effect.2. GFP fluorescence detection and MsrA immunohistochemistry suggested that intravenous injection of Lv-MsrA-GFP predominantly increased the expression of hMsrA in the liver. Hepatic high-level expression of exogenous gene by lentivirus mediated had no significant adverse effect on the immune and liver function in mice, and no alterations were abeserved in body weight, spleen, or plasma Alanine transaminase (ALT) activity. Compared with Lv-GFP apoE-/- mice, the hepatic MsrA level in Lv-MsrA-GFP mice was increased 3.4 fold, and the hepatic MsrA level in Lv-MsrA-GFP SR-BI-/- mice was increased 2.38 fold when compared with that of SR-BI-/- mice in control group. The experimental animal models that hepatic high expression of hMsrA were obtained successfully.3. Effects of hepatic high-level expression of hMsrA on Western-diet-fed apoE-/-mice:1) Hepatic inflammatory cytokines TNFa and IL-6 mRNA levels in Lv-MsrA-GFP mice were obviously down-regulated, the activities of SOD and PON1 in plasma were significantly increased, and the plasma protein levels of PON1 and apoAI were also significantly increased, wheres plasma SAA level was obviously reduced, compared with those in Lv-GFP mice; 2) Hepatic overexpression of hMsrA significantly reduced plasma TC and TG levels. The FPLC profile indicated that the reduction in plasma TC and TG was due to decreases in VLDL and LDL fractions, but not in HDL fraction; 3) Hepatic overexpression of hMsrA significantly enhanced cholesterol selective uptake, conversion and excertion in liver, and increased fecal cholesterol content (increased by 24.6%), reduced hepatic steatosis in apoE-/- mice fed a Westen-diet, by upregulated the expression of liver cholesterol-metabolism-related genes SR-BI, apoAI, LXRa, ABCA1/G8 and Cholesterol 7-alpha-hydroxylase (CYP7A1)/Cholesterol 27-alpha-hydroxylase(CYP27A1), and coordinately regulated the mRNA or protein levels of cholesteryl ester hydrolase (CEH)/acetyl-CoA acetyltransferase (ACAT) that are closely associated with cholesterol conversion in liver; 4) Hepatic overexpression of hMsrA significantly down-regulated the mRNA levels of lipogenic genes acetyl CoA carboxylase-a (ACCa)/fatty acid synthase (FASN), and abviously reduced the amount of TG in the liver and plasma TG levels; 5) Hepatic high-level expression of hMsrA significantly reduced the development of atherosclerosis in apoE-/- mice. Quantitative analysis of the aortic root cross sections demonstrated a significant decrease in mean lesion area in Lv-MsrA-GFP mice compared with that of Lv-GFP mice (0.50±0.06 vs 0.66±0.06 mm2; P<0.01). En face analysis of aortas revealed that the percentage of lesion area in Lv-MsrA-GFP mice was also significantly reduced in the aortic arch and the total aorta (P<0.01 and P<0.05, respectively). These results showed that hepatic high level of hMsrA in apoE-/- mice improved liver and plasma redox status, inhibited inflammation, regulated lipid metabolism by promotion of cholesterol selective uptake, transformation and bile excretion, inhibited liver TG synthesis, lead to diminish hepatic steatosis, and also attenuate atherosclerosis in mice.4. Effects of hepatic high-level expression of hMsrA on SR-BI-/- mice fed a Western diet:1) Hepatic inflammatory cytokines TNFa and IL-6 mRNA levels in Lv-MsrA-GFP mice were obviously down-regulated, plasma SAA level was obviously reduced, wheres the activities of PON1 in plasma were significantly increased, compared with those in Lv-GFP mice.2) Hepatic high-expression of hMsrA significantly decreased plasma TG and free cholesterol (FC) levels, increased plasma HDL-C content, but no obvious alteration of plasma TC levels.3) Hepatic overexpression of hMsrA in SR-BI-/- mice also significantly down-regulated the mRNA levels of ACCa and FASN, inhibited triglyceride synthesis.4) In SR-BF-/-mice, hepatic high-level expression of hMsrA has no longer upregulated the expression of liver cholesterol-metabolism-related genes SR-BI, apoAI, LXRa, ABCA1/G8 and CYP27A1/7A1, and also no alteration of TC levels in feces, liver. But hepatic high-level expression of hMsrA significantly decreased FC levels in liver and plasma in SR-BI-/- mice fed a Western diet, by upregulated the liver protein levels of AC AT, LXRa and ABCA1, and plasma apoAI protein level, respectively. Above results confirmed that hepatic high level of hMsrA improved SR-BI-/- mice redox status, inhibited inflammation and liver TG synthesis, reduced the ratio of FC in liver or plasma, but not altered TC levels in plasma, liver and feces. The reason may be related to SR-BI gene deficiency, resulting in unable to regulate cholesterol selective uptake by liver in SR-BF-/- mice.Conclusions MsrA overexpression in liver can significantly diminish hepatic steatosis and attenuate atherosclerosis in apoE-/- and SRBI-/- mice, the mechanism is involved in reducing the oxidative status in local liver tissue and circulatory system, improvement in anti-inflammation and regulation of lipid metabolism. Effects of hepatic MsrA on lipids regulation mainly include decrease of liver and plasma cholesterol levels via promotion of the process of liver SR-BI-mediated cholesterol uptake, improvement cholesterol esterification and biliary excretion and inhibition of TG synthesis. In conclusion, hepatic MsrA, play an effective action in regulation lipid metabolism and inflammation, may be an important target protein for the prevention and treatment of atherosclerosis-related cardiovascular diseases.