Probucol attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits.

1. Probucol was administered to rabbits fed a cholesterol-enriched (2% wt/wt) diet to determine potential anti-atherogenic effects in a preparation in which the disease process is due to elevated plasma concentrations of cholesterol ester-rich very low density lipoproteins (CER-VLDL). 2. Probucol was supplemented to the diet at 1% wt/wt which resulted in plasma concentrations rising steadily to 53 +/- 8 micrograms ml-1 after 14 days, with no significant changes during continued administration. Dietary consumption and body weight gains were comparable in the drug-treated and control groups during the observation period. 3. Probucol treatment did not significantly affect plasma concentrations of total cholesterol, unesterified cholesterol, triglycerides or phospholipids. 4. The concentration of CER-VLDL in plasma and its physicochemical characteristics were not significantly changed during administration of probucol. CER-VLDL from both control and probucol-treated animals was a potent stimulant of the augmentation of the intracellular incorporation of [3H]-oleate into cholesteryl-[3H]-oleate in cultured macrophages. 5. Despite the lack of effect of probucol on concentrations of plasma lipids and the cell interaction characteristics of CER-VLDL, administration of the drug markedly decreased the extent of intimal aortic surface area covered by grossly discernible atherosclerotic lesions from 55.6 +/- 11.8% to 11.6 +/- 1.9% in thoracic sections, and from 49.1 +/- 10.2% to 7.2 +/- 0.4% in abdominal sections. Furthermore, probucol treatment significantly reduced the deposition of total cholesterol in vascular tissue. 6. Probucol reduced the extent of aortic atherosclerosis produced by diet-induced hypercholesterolemia in rabbits. This reduction occurred in the absence of any significant change in the characteristics of plasma lipoproteins that were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of gamma-oryzanol on atheroma formation in hypercholesterolemic rabbits

The anti-oxidant action of ferulic acid in gamma-oryzanol was tested in hypercholesterolemic rabbits to determine its effects on the development of atherosclerosis. Eighteen male New Zealand white rabbits were fed a diet containing 1% cholesterol with or without 1% gamma-oryzanol for 10 weeks. The anti-oxidant effect was assessed by the degree of resistance of low density lipoprotein (LDL) to oxidation by copper sulfate and the effect of the copper oxidized LDL on the incorporation of oleate into cholesteryl ester by mouse peritoneal macrophages. In addition, the levels of plasma cholesterol, triglyceride and lipid peroxide were measured before, during and at the end of cholesterol loading. The amount of free ferulic acid and gamma-oryzanol in plasma and LDL and the area of aortic atherosclerotic plaques were determined. It was found that oleate incorporation into cholesteryl ester by macrophages was significantly reduced in the gamma-oryzanol-treated group compared to the non-treated group, and that the reduction was via a mechanism independent of anti-oxidant action. However, no differences were found in the levels of plasma cholesterol, triglyceride, lipid peroxide or in the areas of atherosclerotic plaques between the two groups. It was concluded that gamma-oryzanol has little or no preventive effects on atherosclerosis in rabbits.     

Prominent lectin-like oxidized low density lipoprotein (LDL) receptor-1 (LOX-1) expression in atherosclerotic lesions is associated with tissue factor expression and apoptosis in hypercholesterolemic rabbits

BACKGROUND: Despite increasing in vitro evidence that lectin-like oxidized low density lipoprotein (LDL) receptor-1 (LOX-1), a cell-surface receptor for oxidized LDL, is implicated in the atherogenesis and thrombus formation, its in vivo participation to the atherosclerotic plaque destabilization, rupture and thrombus formation remains unclear. Here, we compared the in vivo expression of LOX-1, with tissue factor (TF) expression and cell apoptosis, in atherosclerotic lesions of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. METHODS AND RESULTS: We prepared sixty series of cross sections in the aortic arch and the thoracic aorta from four WHHLMI rabbits. LOX-1 and TF expression, as well as apoptotic events were determined by immunohistochemical staining and TUNEL methods, respectively. LOX-1 expression was mainly observed in the macrophage-rich lipid areas of vulnerable plaque-like atheromatous lesions where TF expression and apoptotic events were prominent. LOX-1 expression was positively correlated with TF expression (r=0.53, p<0.0001), apoptotic events (r=0.52, p<0.0001) and morphological vulnerability (r=0.63, p<0.0001). CONCLUSIONS: LOX-1 expression appears to be closely associated with TF expression, apoptotic events and the morphological vulnerability, suggesting the in vivo involvement of LOX-1 in the destabilization and rupture of atherosclerotic lesions and the subsequent thrombus formation. The present findings in hypercholesterolemic rabbits should help advance our understanding of the pathophysiology of atherosclerosis.     

Effect of atorvastatin on tumor necrosis factor alpha serum concentration and mRNA expression of adipose in hypercholesterolemic rabbits

Tumor necrosis factor alpha (TNFalpha) is an inflammatory cytokine involved in atherogenesis. Adipose tissue is an important source of endogenous TNFalpha production. The aim of this study was to evaluate the effect of atorvastatin on TNFalpha serum concentration and mRNA expressions of subcutaneous adipose in hypercholesterolemic rabbits. Sixteen rabbits fed with a high-cholesterol diet for 8 weeks were randomly divided into 2 groups: (1) the high-cholesterol group (n=8) was maintained on a high-cholesterol diet for 6 weeks; (2) the atorvastatin group (n=8) had the same high-cholesterol diet plus atorvastatin (2.5 mg/kg/d) for 6 weeks. A control group (n=5) was fed with a normal diet for 14 weeks. Subcutaneous adipose was collected for mRNA analysis. Additionally, the direct effect of atorvastatin on TNFalpha release and mRNA expression was assayed in primary rabbit adipocytes. TNFalpha levels in serum and adipocyte culture supernatant were measured by ELISA. RT-PCR was used to evaluate TNFalpha mRNA expression in adipose and adipocytes. Serum TNFalpha concentration was significantly associated with serum total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) (both P<0.01). Compared with the control group, rabbits fed with a high-cholesterol diet showed higher levels of TNFalpha serum concentration and mRNA expression of adipose, both of which were significantly reduced by atorvastatin treatment (both P<0.05). TNFalpha mRNA expressions of adipose were significantly correlated with circulating TNFalpha levels among the 3 groups (r=0.51, P<0.05). Atorvastatin dose-dependently inhibited lipopolysaccharide (LPS)-induced TNFalpha secretion and mRNA expression in cultured adipocytes. In conclusion, atorvastatin can directly inhibit TNFalpha expression and secretion in adipocytes. Atorvastatin reduced TNFalpha serum concentration in hypercholesterolemic rabbits, which might be because of its cholesterol-lowering effect and direct inhibition of TNFalpha expression in adipose.     

Antioxidant activity of probucol and its analogues in hypercholesterolemic Watanabe rabbits

Probucol (1) and probucol analogues with the substitutions at the disulfide-linked carbon (2, 3) and an additional substitution at a tert-butyl of each phenolic ring (4) were tested for their ability to lower total serum cholesterol and prevent aortic atherosclerosis in modified Watanabe heritable hyperlipidemic (WHHL) rabbits and to inhibit Cu2(+)-induced lipid peroxidation of isolated plasma low-density lipoproteins (LDL). After 84 days of feeding 1% of each compound in rabbit chow, probucol was effective in lowering serum cholesterol, whereas 2-4 were not. The concentration of drug in serum and LDL was 2 greater than 1 greater than 3 greater than 4. Probucol and analogues prevented Cu2(+)-induced oxidation of LDL in vitro to an extent that directly related to their concentrations in LDL. The decrease in lipid oxidation was directly correlated with the inhibition of both oxidized-LDL-induced cholesteryl ester synthesis in cultured macrophages and to the inhibition of aortic atherosclerosis in vivo. These results show that the antioxidant activity of probucol and analogues is directly related to their concentration in LDL, which may explain their pharmacological activity in reducing atherosclerosis.     

Naringin has an antiatherogenic effect with the inhibition of intercellular adhesion molecule-1 in hypercholesterolemic rabbits.

Naringin, a bioflavonoid found in citrus fruit peel, is known to have an antioxidative effect, but its effect on atherosclerosis has not been studied. This study evaluated the effect of naringin on blood lipid levels and aortic fatty streaks, and its action mechanism in hypercholesterolemic rabbits. Male New Zealand white rabbits were fed a 0.25% cholesterol diet and divided into an untreated group (n = 4), a naringin-treated group (n = 5; 500 mg/kg per day), and a lovastatin-treated group (n = 5; 20 mg/kg per day). After 8 weeks, blood was sampled and analyzed biochemically. Aorta and liver were harvested and examined histologically. Cholesterol level in rabbits fed the 0.25% cholesterol diet reached 17 times normal and decreased in the rabbits fed naringin and lovastatin, whose effects were not statistically significant (p > 0.05). However, both naringin and lovastatin effectively decreased the area of fatty streak in thoracic aorta on macroscopic analysis (p < 0.05) and significantly reduced subintimal foam cell infiltration on microscopic morphometry (p < 0.05). These foam cells were macrophages on immunohistochemical analysis. Naringin treatment inhibited hypercholesterolemia-induced intercellular adhesion molecule-1 (ICAM-1) expression on endothelial cells. Hypercholesterolemia caused fatty liver and elevation of liver enzymes, which was prevented by naringin but not by lovastatin. Naringin significantly reduced fatty streak formation and neointimal macrophage infiltration and also inhibited the expression of ICAM-1 in endothelial cells, suggesting that suppression of ICAM-1 contributed to the antiatherogenic effect. Naringin, unlike lovastatin, has a hepatoprotective action.     

Regulation mechanism of ABCA1 expression by statins in hepatocytes

ATP-binding cassette transporter A1 (ABCA1) is predicted to be involved in the control of apolipoprotein AI-mediated cholesterol efflux: biosynthesis of high-density lipoprotein (HDL). However, the effects of HMG-CoA reductase inhibitors (statins) on ABCA1 in the liver and the precise mechanisms of their actions have been obscure. The aims of this study were to determine whether statins (atorvastatin (Ato) and pitavastatin (Pit)) affect hepatic ABCA1 expression and to clarify the mechanisms of their actions using HepG2 cells and the rat liver. We examined alterations in mRNA and protein levels of ABCA1 and peroxisome proliferator-activated receptors (PPARs) by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. In vitro and in vivo studies suggested that Pit increases ABCA1 mRNA level, but not Ato. Pit greatly increased Abca1 mRNA level and also increased the amount of plasma HDL and the mRNA level of PPARα. Clofibrate (PPARα agonist) increased ABCA1 expression in HepG2 cells and rat primary hepatocytes more than did PPAR β/δ and γ agonists. Pit-induced ABCA1 expression alteration was blocked by GW6471 (PPARα antagonist) and by PPARα knockdown. In this study, we demonstrated that Pit affect ABCA1 expression via PPARα in hepatocytes. The strategy to target a PPARα agonist in the liver can lead to increases in ABCA1 expression and HDL level.     

Oren-gedoku-to and Keishi-bukuryo-gan-ryo inhibit the progression of atherosclerosis in diet-induced hypercholesterolemic rabbits

In this study, we examined whether the Kampo formulas Oren-gedoku-to (OGT, Huanglian-jie-du-tang in Chinese) and Keishi-bukuryo-gan-ryo (KBG, Gui-zhi-fu-ling-wan in Chinese) could prevent the progression of atherosclerosis in cholesterol-fed rabbit, an animal model for hypercholesterolemia in vivo. Twenty-four male Japanese white rabbits (2 kg body weight) were divided into four groups. The control group was fed standard rabbit chow containing 1% cholesterol, the OGT group was fed standard rabbit chow containing 1% cholesterol and 1% OGT, the KBG group was fed standard rabbit chow containing 1% cholesterol and 1% KBG, and the vitamin E group was fed standard rabbit chow containing 1% cholesterol and vitamin E (450 mg/1000 g). All four groups were kept on these diets for 8 weeks. At the end of the experiments, the percentage of surface area of the total thoracic aorta with visible plaque was significantly reduced in the OGT and KBG groups. The serum thiobarbituric acid reactive substances of the vitamin E group showed a significantly low value compared with the control group, whereas the serum lipid peroxide levels of the OGT and KBG groups were considerably lower than that of the control groups as well as that of the vitamin E group. Furthermore, the urinary 8-hydroxydeoxyguanosine levels of the OGT and KBG groups were considerably lower than that of the vitamin E group. These results suggest that OGT and KBG prevent the progression of atheromatous plaque by creating a sounder antioxidant defense system than vitamin E.     

Modulation of matrix metalloproteinase-1, its tissue inhibitor, and nuclear factor-kappa B by losartan in hypercholesterolemic rabbits

Upregulation of angiotensin II receptor, may be involved in the initiation and progression of atherosclerosis. To examine the contribution of AT1 receptor in the expression of matrix metalloproteinase-1 (MMP-1) and its tissue inhibitor (TIMP-2) in lipid-deposited arterial tissues, New Zealand white rabbits were given high-cholesterol chow (with losartan 25 mg/d or vehicle) for 10 weeks. Losartan reduced the areas of sudanophilia in the aorta of rabbits fed high-cholesterol diet (p < 0.01 vs. control). Losartan also significantly decreased the enhanced mRNA expression of MMP-1 and TIMP-2 in aortas of rabbits with high-cholesterol diet. Losartan-treated rabbits revealed a reduction in immunohistochemical expression of MMP-1, whereas TIMP-2 expression became localized to the intima. In addition, losartan treatment reduced the activation of NF-kappa B by inhibiting the degradation of its inhibitor I kappa-B alpha. These observations demonstrate that AT1 receptor blockade with losartan reduces lipid deposition and exerts potent inhibitory effects on NF-kappa B activation and modulates the expression of MMP-1 and TIMP-2 in hypercholesterolemic rabbits.     

Anti-hyperlipidemic and anti-atherosclerotic effects of Pinus eldarica Medw. nut in hypercholesterolemic rabbits

Background

Previous studies suggest that chemical constituents present in Pinus eldarica Medw (P. eldarica) nut possess antioxidant properties that may positively influence lipid profile.The present study was conducted to evaluate the efficacy of P. eldarica nut on the experimental atherosclerosis development in hypercholesterolemic rabbits.

Methods

Forty male 6 months old white New Zealand rabbits (1.8–2 kg) were randomly assigned into five equal groups. One group was kept as control (normal) group, fed on standard rabbit diet and other 4 groups were fed on high cholesterol diet (HCD). Out of four HCD groups one group was kept as control (HCD) and other three groups were treated with different doses (50, 100 and 200 mg/kg/day) of P. eldarica nut for 8 weeks. Percentage of aortic wall area changes as indication of atherosclerosis development and fasting blood cholesterol, LDL, HDL and triglyceride levels were determined in all groups.

Results

The results indicate that fasting blood cholesterol and aortic atherosclerotic involvements in 200 mg/kg/day and 100 mg/kg/day P. eldarica nut extract treated groups significantly decreased as compared to the high cholesterol-diet control group.

Conclusion

P. eldarica nut lowers blood cholesterol level and aortic atherosclerotic involvement in hypercholesterolemic rabbits.     

Effect of antioxidants on atherosclerotic plaque formation in balloon-denuded and irradiated hypercholesterolemic rabbits

The oxidative modification of low-density lipoprotein (LDL) hypothesis implies that antioxidants should be effective in suppressing atherosclerosis. This study is designed to test the potential of antioxidants to inhibit atherosclerotic plaque progression in balloon-denuded and irradiated hypercholesterolemic rabbits. Rabbits were fed with a 1% cholesterol diet supplemented with or without a mixture of antioxidants (vitamin E, vitamin C, selenium, zinc, copper, manganese, N-acetylcysteine, glutamine). At 7 days both iliac arteries were balloon denuded, and 4 weeks later, 1 iliac artery underwent endovascular irradiation (n=12), while the contralateral was sham treated (n=12). Four weeks after irradiation, animals were euthanized, and arteries were fixed and processed for histo- or immunohistochemistry for determining the plaque area, macrophage count, and oxidized LDL-positive areas. Plasma antioxidant levels were significantly higher in the animals fed with antioxidant diet. Plasma (thiobarbituric acid-reactive substances) and arterial tissue oxidized LDL (immunoreactive to specific oxidized LDL antibody) levels were significantly higher in the irradiated as compared with nonirradiated animals (0.69+/-0.09 and 31.05+/-4.21 versus 0.24+/-0.04 and 18.42+/-4.62, P<0.001 and 0.05), and antioxidants partially lowered the oxidized LDL levels (0.35+/-0.14 and 25.41+/-4.82, P<0.001 and 0.01). Plaque area in the irradiated animals was 175% greater than in nonirradiated animals (P<0.05). Antioxidant supplementation resulted in a 50% decrease in plaque area of both control and irradiated animals. Antioxidants reduced both the cholesterol-induced and radiation-enhanced circulating and tissue oxidized LDL levels, resulting in reduced plaque.     

普罗布考抑制再狭窄与其调节功能性血管重构的关系

目的　研究普罗布考抗动脉成形术后再狭窄与其调节功能性血管重构的关系。方法　用 3 5F球囊导管构建兔动脉粥样硬化动脉成形术后再狭窄模型。动脉成形术 2wk后取其胸主动脉 ,固定染色并进行计算机图像分析 ;观察血管环对乙酰胆碱诱导的舒张百分率 ,然后再观察其对内源性收缩物质去甲肾上腺素 (NA)、KCl以及 5 羟色胺 (5 HT)的收缩反应性。同时 ,在动脉成形术 2wk后留其血清 ,观察普罗布考对动脉成形术后血清一氧化氮 (NO)含量的影响。结果　普罗布考明显增加血管管腔面积 ,减少新生内膜面积 ;可保护动脉成形术后血管的舒张功能 ,保护内皮细胞合成与释放内皮源性舒张因子NO ,降低血管对内源性收缩物质的收缩反应性。结论　普罗布考能提高扩张性血管重塑 ,抑制收缩性血管重塑 ,从而防止动脉成形术后管腔缩窄防止再狭窄的发生     

Chronic ethanol consumption up-regulates protein-tyrosine phosphatase-1B (PTP1B) expression in rat skeletal muscle

Aim:

To investigate the potential effects of chronic ethanol intake on protein-tyrosine phosphatase-1B (PTP1B) and the insulin receptor signaling pathway in rat skeletal muscle.

Methods:

Rats received ethanol treatment at a daily dose of 0 (control), 0.5 (group L), 2.5 (group M) or 5 g·kg⁻¹ (group H) via gastric gavage for 22 weeks. In vivo insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp. Expression of PTP1B in skeletal muscles was examined at both the mRNA (real-time PCR) and protein (Western blot) levels. PTP1B activity was assayed with a p-nitrophenol phosphate (PNPP) hydrolysis method. Changes of insulin signaling in skeletal muscle were analyzed with Western blotting.

Results:

The activity and expression of PTP1B were dose-dependently elevated 1.6 and 2.0 fold in the skeletal muscle by ethanol, resepctively, at the doses of 2.5 and 5 g·kg⁻¹·d⁻¹. Total IRβ and IRS-1, as well as their phosphorylated forms, were decreased by ethanol at the two higher doses. Moreover, chronic ethanol consumption resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase, inhibition of Akt phosphorylation and reduced levels of mitogen-activated protein kinase phosphorylation.

Conclusion:

Chronic ethanol intake at 2.5 and 5 g·kg⁻¹·d⁻¹ sufficient doses can down-regulate the expression of IRβ, P-IRβ, and IRS-1, as well as the phosphorylated forms of IRS-1 and Akt, in rat skeletal muscle, possibly through increased PTP1B activity.