The Study Of Rosiglitazone Improving HDL Function In Atherosclerotic Model

Study BackgroundCoronary artery disease (CAD) is one of severe diseases threatening human health. In China, with the improvement of living standards, the incidence of CAD has increased year by year. In the last decade, several studies have indicated the importance of the quantity of high-density lipoprotein cholesterol (HDL-C) is an independent CAD risk assessment. Clinical and epidemiological studies have demonstrated a negative correlation between HDL-C level and the incidence as well as mortality of CAD. However, recent studies have indicated that the level of HDL-C cannot react the whole function of HDL well.The bulk of HDL exerts a broad scope of antiatherogenic effects. And the HDL which has normal function, is called functional HDL. The antiatherogenic functions of HDL include reverse cholesterol transport (RCT), anti-inflammatory, anti-oxidative, anti-aggregatory, anti-coagulant, and pro-fibrinolytic effects. The major cardiovascular protective effects of HDL may be attributed to its role in RCT. The process of RCT involves the transfer of excess cholesterol from membranes of peripheral cells and lipid loaded macrophages (foam cells) to the liver, with final excretion of catabolized cholesterol into bile. Several transmembrane transpoters, such as adenosine triphosphate binding cassette transporters A1 (ABCA1), scavenger receptor class B type I (SR-BI) and apolipoprotein A-1 (apo-A1), play important roles in RCT. The anti-oxidative function of HDL is its another important antiatherogenic function. HDL prevents LDL oxidizing phospholipids, and it hydrolyzes oxidized phospholipids once they are formed. Now it is thought that the antioxidant properties of HDL are determined mainly by its associated antioxidant enzymes, such as paraoxonase 1 (PON1), apo-A1, lecithin-cholesterol acyltransferase (LCAT) and so on. Some studies have demostrated that the quantity and activity of PON 1, which is associated with antioxidative function and CAD, was not also significantly higher in CAD groups than that in normal control groups, but also had a negtive correlation with the severity of coronary artery disease.However, HDL particles as a result of multifactorial actions of chronic inflammation and acute phase responses would lost its normal biological function, even exhibit pro-inflammatory, pro-oxidative and inhibiting RCT function, which is now called dysfunctional HDL. HDL forms a structurally and functionally heterogeneous class of lipoproteins. The modification of HDL associated protein interacts directly with the HDL particles whole function. Several studies have demostrated that myeloperoxidase(MPO) involves in the process of arterial wall oxidative modification. The activated MPO isolated from human atherosclerotic lesions, has been shown to interact with HDL. MPO impairs the ability of apo-A1 to inhibit RCT by oxidating apo-Al selectively and then by nitrating and chlorinating tyrosine residues in apo-Al. Therefore, it is thought that MPO may be correlate with the mechanism of dyfunction HDL.Thiazolidinediones (i.e., rosiglitazone), the agonists of the peroxisome proliferator activated receptor gamma (PPARy), are extensively used in the treatment of type 2 diabetes. Recent studies found that TZDs have the better effects of lowering blood sugar than other sulfonylurea hypoglycemic agents and it can directly affect vascular cells. TZDs also can reduce inflammation and inhibit cell proliferation and migration of smooth muscle cells, endothelial cells and monocytes/macrophages, which are by the way of inhibiting a variety of inflammatory factors (such as C-reactive protein, interleukin 2, tumor necrosis factor-a, IFN-γ, etc.), regulating cell cycle, inducing apoptosis and so on. And it can promote macrophage-foam cell RCT by inducing genes associated with HDL metabolism in SR-B1, ABCA1, ATP binding cassette transporter G1 (ABCG1) expression. And it maintains the balance of intracellular lipid for inhibiting AS formed. But there are less reports that the effects of PON1 activity, MPO activity and ABCA1/SR-B1 expression in aortic plaque on the rosiglitazone in the AS model at home and abroad. Therefore, we have built AS rabbit model, and study the effects of HDL function on rosiglitazone and its mechanism.ObjectivesWe set out to investigate the change of the expression of ABC A1 and SR-B1 and the anti-inflammative and anti-oxidative function of HDL in AS rabbits models. And we study on the the effects of rosiglitazone treatment on the above indexs and its potential anti-atherosclerotic mechanisms.Methods1. Experimental animals18 New Zealand white male rabbits (weighing [2.0±0.1]kg) were randomly divided into three groups:Control group, the animals were fed with regular diet; Atherosclerosis group, the animals were fed with a high fat diet supplemented with 1%(w/w) cholesterol,8% lard (w/w) and 0.05% cholate (w/w); and Rosiglitazone group, the animals were fed with the same high fat diet plus Rosiglitazone (0.5mg/kg body weight/day), n=6 in each group.2. Serum lipid analysisBlood lipid analysis was performed at 0 week and 12 weeks end of the experiment. Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (HDL-C) concentrations were measured by using a automated biochemical analyzer.3. Assessment of the anti-oxidantive function of HDLSerum PON1 activity was assayed using synthetic substrates. MPO activity was determined by a MPO activity kit according to the manufacturer's instructions. 4. Detection of ABCA1 and SR-B1 mRNA in liver tissues(1) The total RNA was isolated from rabbit liver by a Trizol reagent. The quantitative analyses of ABCA1 and SR-B1 mRNA level on rabbit liver tissues were performed by real-time quantitative polymerase chain reaction (RTQ-PCR).(2) For microscopic evaluation of ABC A1 and SR-B1 protein expression in the lesions of aorta, serial paraffin sections of the aortic arch were immunohistochemically stained with the following antibodies against ABCA1 and SR-B1. The expression of ABC A1 and SR-B1 (including percentage of ABCA1 or SR-B1 protein positive area and staining mean density in the lesions by immunohistochemical staining) were measured with Image pro plus 6.0 special image analysis software.5. Aortic atherosclerosis for histological examination and quantificationAt the end of 12 weeks, after sacrifice of rabbits, the entire aorta was moved, fixed in 10% neutral buffered formaldehyde solution for 48 hours. All segments were embeded in paraffin stained with hematoxylin and eosin (HE) for histological examination. The percentage plaque area, which was defined as surface area of plaque/surface area of whole intima, and the aortic intima-media thickness were calculated for evaluating the degree of aortic atherosclerosis.6. Statistical analysisDatas are presented as mean±S.E.M. SPSS 13.0 software was used for statistical analysis. ANCOVA was used for analyzing differences in variables of lipids between groups at 12 weeks. Post-hoc comparisons were made among the various groups using least significant difference (LSD) method. One-way ANOVA was used for analyzing differences in variables between groups at the same time point. Post-hoc comparisons were made among the various groups using least significant difference (LSD) method. When variances are not homogeneity, Welch method was used for analyzing differences in variables between groups at the same time point, and Dunnett's T3 method was used for Post-hoc comparisons. Independent sample t-test was used for analyzing the differences in variables between two groups at the same time point. Paired-Samples t-test was used for analyzing the differences in variables between paired groups at the beginning and at the end of experiments. Coefficients of correlation (r) were calculated by Pearson correlation analysis. P＜0.05 indicated a statistically significant difference.Results1. There were no significant differences in TC (P=0.525, F=0.672), TG(P=0.665, F=0.420), VLDL-C (P=0.505, F=0.614), LDL-C (P=0.978, F=0.023), HDL-C (P=0.982, F=0.018), NHDL-C (P=0.473, F=0.788) among the three groups at the baseline. After 12 weeks of experiment, compared with control group, the levels of serum TC (23.26±3.30 vs 1.79±0.21, P=0.000), TG (1.58±0.25 vs 1.02±0.15, P=0.000), LDL-C (18.09±4.04 vs1.09±0.23,P=0.000), VLDL-C(4.06±1.42 vs 0.25±0.14, P=0.000), HDL-C (1.11±0.09 vs 0.45±0.12,P=0.000), NHDL-C (22.15±3.22 vs 1.34±0.10,P=0.000) in AS group were significantly increased; compared with AS group, the levels of serum HDL-C (1.94±0.30 vs 1.11±0.09, P=0.000) were significantly increased and TC (19.78±1.68 vs 23.26±3.30, P=0.010), VLDL-C(2.28±1.03 vs 4.06±1.42, P=0.008), NHDL-C(17.51±1.43 vs 22.15±3.22, P=0.002) were significantly reduced in rosigitazone group.Each group were compared within groups:after 12 weeks experiment, in control group, there were no significant differences in the levels of serum TC (1.85±0.12 vs 1.79±0.21, t=0.538, P=0.614),TG (1.04±0.19 vs 1.02±0.15, t=0.162,P=0.878),LDL-C (1.00±0.20 vs 1.09±0.23, t=-0.566, P=0.596),VLDL-C (0.38±0.10 vs 0.25±0.14, t=1.485, P=0.198),HDL-C(0.47±0.12 vs 0.45±0.12, t=0.369, P=0.727),NHDL-C (1.38±0.14 vs 1.34±0.10, t=0.475, P=0.655); In AS group, the levels of serum TC (1.77±0.15 vs 23.26±3.30, t=-16.603, P=0.000),TG (1.10±0.14 vs 1.58±0.25, t=-4.498, P-0.006),LDL-C (1.00±0.20 vs 18.09±4.04, t=-10.251, P=0.000). VLDL-C (0.30±0.11 vs 4.06±1.42, t=-6.061, P=0.002),HDL-C (0.48±0.12 vs 1.11±0.09, t=-14.615,P=0.000),NHDL-C (1.29±0.14 vs 22.15±3.22, t=-16.045,P=0.000) were significantly increased; In rosigitazone group, the levels of serum TC (1.78±0.12 vs 19.78±1.68, t=-25.288, P=0.000),TG (1.02±0.14vs 1.54±0.15, t=-5.184, P=0.004),LDL-C (0.98±0.14 vs 15.57±1.81, t=-20.228, P=0.000),VLDL-C (0.34±0.20 vs 2.28±1.03, t=-4.247, P=0.008),HDL-C (0.47±0.07 vs 1.94±0.30, t=-10.561, P=0.000),NHDL-C (1.32±0.11 vs 17.51±1.43, t=-26.067, P=0.000) were significantly increased.2. Serum PON1 activity toward phenyl acetate was significantly inhibited in atherosclerosis group compared to control [(72.26±12.03) U/ml vs. (96.77±5.58) U/ml, P=0.000]. As expected, serum PON1 activity was markedly elavated in rosiglitazone group in comparison with atherosclerosis group [(105.18±8.49)U/ml vs. (72.26±12.03) U/ml, P=0.000].3. Serum MPO activity in atherosclerosis group was substantially higher than that of the control group (85.67±17.92 vs 14.94±6.36 U/L,P=0.000). But the MPO activity was significantly inhibited in rosiglitazone group compared to atherosclerosis group (54.45±10.99 vs 85.67±17.92 U/L, P=0.018).4. At the 12th treatment week end, expression of ABCA1mRNA (0.61±0.11 vs. 1.01±0.03, P=0.001) and SR-B1 mRNA (0.63±0.11 vs.1.00±0.02,P=0.001) in hepatocytes were both significantly lower in atherosclerosis group rabbits than in control groups rabbits. Compared with the atherosclerosis group, the rosiglitazone group showed a significantly higher level of expression of ABCA1 (1.29±0.27 vs 0.61±0.11,P=0.003)and SR-B1 (0.63±0.11 vs 1.39±0.23,P=0.000) in hepatocytes at the mRNA levels.5. The histomorphometric analysis of atherosclerotic lesion was performed by means of HE at the 12th experimental week end:the mild to moderate atherosclerosis characterized by local thickening of the intima-media was observed in AS group. Compared with control group, the aortic intima-media thickness (IMT) (527.42±85.16μm vs.203.21±30.61μm, P=0.000) is significantly increased in AS group. Compared with AS group, rosigitazone reduced aortic IMT (291.46±50.18μm vs.527.42±85.16μm, P=0.000) and the percentage plaque area (5.88±3.31% vs. 27.78±12.00%, P=0.002) significantly.6. Immunohistochemical staining revealed substantial ABCA1 and SR-B1 protein infiltration in the aortic plaques in both atherosclerosis group and rosiglitazone group rabbits. However, in control group, there were no lesion, plaque and immunohistochemical staining in aortic wall. In further, we respectively quantitated the two protein expression by (percentage of ABC A1 or SR-B1 protein positive area and staining intensity in the lesions by immunohistochemical staining) using Image pro plus 6.0 special image analysis software. Compared to atherosclerosis group, we found ABCA1 protein expression was significantly increased at both staining mean density (0.22±0.03 vs.0.17±0.03, t=-2.661,P=0.024) and the percentage of ABCA1 protein positive area (47.06±4.93 vs.24.13±9.85, t=-5.100, P=0.000) levels in the rosiglitazone group. However, there was no significant difference in SR-B1 protein expression of aortic plaques between two groups (staining mean density:0.20±0.03 vs.0.18±0.03, t=-0.756, P=0.467; the percentage of SR-B1 protein positive area:24.74±13.04 vs.18.81±7.58, t=-0.964, P=0.358).7. IMT had a negtive correlation with serum PON1 activity (r=-0.675, P=0.002), whereas it had a positive correlation with serum MPO activity (r=0.774, P=0.000).Conclusions1. Rosigitazone increased serum HDL-C concerntration and reduced serum TC, VLDL-C and NHDL-C levels. 2. Rosigitazone promoted the function of HDL-included reverse cholesterol transport (RCT) by up-regulating the protein expression of ABCA1 and SR-B1 in the aortic plaques and their mRNA expression in hepatic cells.3. Rosigitazone promoted the role of HDL in anti-inflammation and anti-oxidation by decreasing serum myeloperoxidase and increasing paraoxonase 1 activity in atherosclerotic rabbits.4. Rosigitazone reduced intima-media thickness (IMT) and the percentage plaque area in atherosclerotic models and had the function of anti-atherosclerosis.5. Serum HDL-C level is the level of cholesterol in HDL particles, but cannot represent HDL function accurately. Rosiglitazone not only increases HDL-C level, but also reduces AS plaque formation and improves HDL functon by increasing the expression of ABC A1 and SR-B1 in macrophages and hepatic cells,elevating PON1 activity and reducing MPO activity.