Comparison of the effects of alpha-tocopherol,ubiquinone-10 and probucol at therapeutic doses on atherosclerosis in WHHL rabbits

Oxidative modification of lipoproteins may trigger and maintain atherogenesis. We compared the effects of different antioxidants (alpha-tocopherol, probucol, ubiquinone-10) at doses similar to those used in humans in Watanabe Heritable Hyperlipidemic (WHHL) rabbits for 12 months. Aortic lesions were analyzed for their extent and cellular composition of lesions, mean thickness of fibrous caps and density of smooth muscle cells therein, content of antioxidants, non-oxidized and oxidized lipids. Compared to controls, probucol significantly lowered the extent and macrophage content of lesions and increased the existence and smooth muscle cell density of fibrous caps. alpha-Tocopherol supplementation increased the aortic content of vitamin E, but had no decreasing effect on either the accumulation of macrophage-specific antigen in the aorta or lesion size. Nevertheless, both probucol and alpha-tocopherol significantly decreased in vitro LDL oxidizability, measured under typically strong oxidative conditions. Ubiquinone-10 supplement increased lesion size and the fraction of lesions containing fibrous caps; however, LDL oxidizability remained unaffected by ubiquinone-10 treatment. None of the antioxidants tested lowered oxidized lipids within aortic tissue; however, long-term treatment with probucol provided the most effective anti-atherosclerotic effect, while alpha-tocopherol may be pro-atherogenic and ubiquinone-10 exerts ambivalent effects. Our data suggest that (i) widely used oxidation measures, such as ex-vivo LDL oxidizability, do not reflect the degree of atherosclerosis; and (ii) long-term beneficial effects of relatively low doses of antioxidants may be outweighed by high levels of plasma cholesterol in WHHL rabbits.     

In vivo uptake of radiolabeled MDA2, an oxidation-specific monoclonal antibody, provides an accurate measure of atherosclerotic lesions rich in oxidized LDL and is highly sensitive to their regression

To determine whether labeled antibodies against oxidized LDL (OxLDL) offer advantages for quantifying atherosclerosis, we compared in vivo aortic uptake of (125)I-labeled MDA2, a monoclonal antibody against malondialdehyde-lysine epitopes), atherosclerotic surface area, and aortic weight in Watanabe heritable hyperlipidemic and New Zealand White rabbits and in low density lipoprotein receptor-deficient (LDLR(-/-)) and apolipoprotein E-deficient (apoE(-/-)) mice. Absolute and specific uptakes of (125)I-MDA2 were significantly greater in plaque than in normal aortas. Uptake of (125)I-MDA2 significantly correlated with aortic weight and percent atherosclerotic surface area in rabbits and mice. To assess whether (125)I-MDA2 uptake reflects changes in lesion content of OxLDL, in a separate study, extensive atherosclerosis was induced in 4 groups of LDLR(-/-) mice by feeding them a high fat/cholesterol diet for 6 months. A baseline group was euthanized at this time. The remaining groups were fed "regression" diets (chow or chow+1% vitamin E+0.05% vitamin C) or the high fat/cholesterol diet for 6 more months. When atherosclerosis was measured as percent surface area or aortic weight, there was strong progression in the high fat/cholesterol group, moderate progression in the chow group, and no progression in the chow+vitamin E+vitamin C group compared with the baseline group. The (125)I-MDA2 method also yielded a significant increase in atherosclerosis in the high fat/cholesterol group but significant decreases in the chow and chow+vitamin E+vitamin C groups. Immunocytochemistry showed fewer oxidation-specific epitopes in lesions from the chow and chow+vitamin E+vitamin C groups. Thus, the uptake of (125)I-MDA2 correlates well with traditional measures of atherosclerosis but also reflects reduced plaque OxLDL content after hypocholesterolemic intervention.     

Proatherogenic and antiatherogenic effects of probucol and phytosterols in apolipoprotein E-deficient mice: possible mechanisms of action

BACKGROUND: The effects of probucol and a phytosterol mixture (FCP-3PI) on atherosclerotic lesion formation, plasma lipoproteins, hepatic and lipoprotein lipase activities, antioxidant enzyme activities, and plasma fibrinogen were investigated in apolipoprotein E-knockout (apoE-KO) mice. METHODS AND RESULTS: Three groups of 8 mice were fed a diet containing 9% (wt/wt) fat (controls) or the foregoing diet supplemented with either 1% (wt/wt) probucol (the probucol group) or 2% (wt/wt) FCP-3PI (the FCP-3PI group) for 20 weeks. Compared with controls, atherosclerotic lesion size was 3 times greater in the probucol group, whereas it was decreased by half in the FCP-3PI group. Probucol treatment resulted in high plasma probucol concentrations, which correlated (r=0.69) with the lesion area. HDL cholesterol was reduced (>75%) in the probucol group and slightly increased (14%) in the FCP-3PI-treated group. Postheparin lipoprotein lipase (LPL) activity was significantly reduced in both treatment groups, but only FCP-3PI significantly decreased hepatic lipase activity. Plasma fibrinogen was increased 42% by probucol and decreased 19% by FCP-3PI relative to controls. Probucol significantly increased plasma glutathione reductase, glutathione peroxidase, and superoxide dismutase activities (P<0.05). In contrast to findings in apoE-KO mice, there was no probucol-induced atherosclerosis in their wild-type counterparts fed the same dose for the same period of time. CONCLUSIONS: Antiatherogenic activity of FCP-3PI in apoE-KO mice is associated with an increase in HDL cholesterol concentration along with decreases in hepatic lipase activity and plasma fibrinogen concentrations. Proatherogenic effects of probucol may be related to increased plasma fibrinogen, decreased HDL cholesterol concentrations along with decreased LPL activity, or its direct "toxicity" due to very high plasma concentration. Our studies demonstrate that the antioxidant and cholesterol-lowering properties of probucol do not prevent atherogenesis in this particular animal model.     

The effect of vitamin E, probucol, and lovastatin on oxidative status and aortic fatty lesions in hyperlipidemic-diabetic hamsters

Diabetes mellitus is associated with an increased risk of premature atherosclerosis, which may be due in part to an increased rate of low density lipoprotein (LDL) oxidation. Previous studies have shown that vitamin E, probucol, and lovastatin can reduce the oxidative susceptibility of LDL in normoglycemic animal models; however, few studies have investigated this in conjunction with aortic fatty streak lesion formation in diabetic hyperlipidemic models. Forty-eight Syrian hamsters were made diabetic by intraperitoneal injection of low dose streptozotocin. Diabetic animals (12 animals/groups) received a high saturated fat and cholesterol diet for 12.5 weeks. At 2.5 week of dietary treatments, the diet was supplemented with either: (1) 500 IU/day vitamin E (D+E); (2) 1% probucol w/w of the diet (D+P); (3) 25 mg/kg lovastatin (D+L); or (4) diabetic control (D). An age-matched group of hamsters (n=6) receiving the same diet but not made diabetic (ND) was used as control. At the end of the study, aortic arch foam cell-rich fatty streak lesion, plasma glucose, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), non-HDL-C, triglycerides (TG), phospholipids, alpha-tocopherol, plasma lipid peroxide and the susceptibility of LDL to copper-catalyzed oxidation were determined. Diabetes increased plasma glucose, and when combined with an atherogenic diet resulted in a further increase of plasma lipids. Vitamin E, probucol, and lovastatin significantly reduced plasma TG in the diabetic hamsters fed the atherogenic diet. Vitamin E treatment increased TC, probucol reduced HDL-C without affecting TC; whereas lovastatin reduced TC and selectively decreased non-HDL-C, and significantly reduced fatty streak lesion formation in the aortic arch. While vitamin E and probucol were effective in reducing several indices of oxidative stress including plasma lipid peroxides, cholesterol oxidation products and in vitro LDL oxidation, they had no effect on fatty streak lesion formation. Our results indicate that the LDL in diabetic animals is more susceptible to oxidation than in non-diabetic hamsters and that not only vitamin E and probucol but also lovastatin provide antioxidant protection. It appears that in this combined model of diabetes and hypercholesterolemia, progression of fatty streak lesion formation is mainly associated with changes in TC and non-HDL-C as affected by lovastatin, and is less dependent on the extent of LDL oxidation, changes in plasma TG level and oxidative stress status.     

丙丁酚对动脉粥样硬化小鼠抗氧化作用的比较

目的观察丙丁酚对高脂高胆固醇饲养的C57BL/6J小鼠和载脂蛋白E基因缺陷(载脂蛋白E-/-)小鼠抗氧化作用的影响,比较丙丁酚对两种小鼠的作用差异。方法以高脂高胆固醇饲料饲喂小鼠,制作高脂血症模型。采用酶比色法及相关检测试剂盒分别测定小鼠血脂水平及与氧化应激相关的指标;采用双抗夹心ELISA法测定氧化型低密度脂蛋白的水平;采用改良的Eckerson方法测定对氧磷酶1的活性,采用逆转录聚合酶链式反应检测对氧磷酶1mRNA的表达。结果与两种小鼠各自的基础饲料组比较,高脂高胆固醇组血脂水平均有升高;与各自的高脂高胆固醇组比较,丙丁酚处理后,C57BL/6J小鼠血清的甘油三酯、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇水平分别降低了41.0%、43.6%、44.8%和41.3%(P<0.05),载脂蛋白E-/-小鼠血清的甘油三酯、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇水平,分别降低了36.2%、28.2%、31.3%、33.9%(P<0.05)。小鼠主动脉内膜的动脉粥样硬化病变与各自高脂高胆固醇组比较,C57BL/6J小鼠经丙丁酚处理后病变面积减少了61.1%,载脂蛋白E-/-小鼠经丙丁酚处理后病变面积减少了27.2%(P<0.05)。与各自基础饲料组比较,高脂血症小鼠丙二醛及氧化型低密度脂蛋白的水平增高,而总抗氧化能力和超氧化物歧化酶水平降低(P<0.05);与各自的高脂高胆固醇组比较,丙丁酚处理组小鼠的总抗氧化能力和超氧化物歧化酶水平上升,丙二醛及氧化型低密度脂蛋白的水平则降低(P<0.05)。两组小鼠高脂高胆固醇组血清对氧磷酶1的活性较基础饲料组均下降,经高脂饮食加丙丁酚处理后对氧磷酶1活性比单纯高脂饮食组有所增加;肝脏对氧磷酶1mRNA的表达在高脂高胆固醇组下调,而丙丁酚处理组则上调(P<0.05)。结论丙丁酚可以减轻高脂血症小鼠动脉粥样硬化病变,减轻高脂高胆固醇饮食诱导的氧化应激,而载脂蛋白E基因缺陷则一定程度减弱丙丁酚的上述作用。     

The effect of pharmacological doses of different antioxidants on oxidation parameters and atherogenesis in hyperlipidaemic rabbits

The oxidation hypothesis of atherosclerosis implies that antioxidants are able to inhibit lipoprotein oxidation in the arterial wall and thereby retard atherogenesis. Since most of the animal studies performed have used very high doses of antioxidants, it is to date unknown whether antioxidants are effective antiatherosclerotic agents when given in pharmacological doses. Here we addressed this question using homozygous Watanabe heritable hyperlipidaemic (WHHL) rabbits as an animal model of atherosclerosis. The rabbits were divided into four groups, each consisting of ten animals. They received either a standard diet or a diet containing 4.3 mg ubiquinone-10, or 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. After 12 months, the extent of aortic atherosclerosis was assessed as the intima thickness, media thickness and intima-to-media ratio in 14 cross sections equally distributed over the whole aorta. To evaluate the antioxidant effects of the diet, lipophilic and hydrophilic antioxidants, lipids, fatty acids and plasma oxidizability were measured after 0, 3 and 6 months of feeding. We found that supplementation with probucol significantly decreased aortic intima-to-media ratio compared to controls. The antiatherosclerotic action of probucol was accompanied by its beneficial action on plasma oxidizability and some plasma antioxidants. No decrease in aortic atherosclerosis was measured in ubiquinone-10- and vitamin E-supplemented rabbits, despite the fact that both antioxidants decreased plasma oxidizability and ubiquinone-10 increased the plasma levels of antioxidants. Taken together, these data suggest that pharmacological doses of probucol retard atherogenesis in WHHL rabbits by an antioxidant mechanism, while ubiquinone-10 and vitamin E at these dosages are ineffective in this highly hyperlipidaemic model. The measurement of some oxidation-related parameters in plasma, such as lipophilic antioxidants, polyunsaturated fatty acids and lipoprotein oxidizability, may be useful in assessing the risk of atherogenesis in humans.     

Angiotensin II infusion induces site-specific intra-laminar hemorrhage in macrophage colony-stimulating factor-deficient mice

Angiotensin II (AngII) infusion promotes macrophage infiltration into the aortic wall resulting in several forms of vascular pathology. To determine the causal role of macrophages in these vascular diseases, we used osteopetrotic (op) male mice in which a natural mutation ablates production of M-CSF and results in severe depletion of monocytes. AngII infusion into apoE-/- mice resulted in increased atherosclerosis that was attenuated in op mice. AngII infusion in op mice unexpectedly produced grossly discernable thickening of the proximal thoracic aorta characterized by intra-mural hematoma. This pathology was also observed in apoE+/+ x op male mice, and therefore, independent of hyper-lipidemia. No perceptible structural properties of aortas from op mice could be discerned prior to AngII infusion. Regional effects in the contractile response to phenylephrine were noted in aortic rings with enhanced responsivity in the upper thoracic aortas of op mice compared to those from +/+ mice. No differences in contractile response were noted in aortic rings from the lower thorax. In conclusion, deficiency of M-CSF attenuated AngII-induced atherosclerosis but led to an unanticipated pathology of intra-laminar hemorrhage in the upper aortic regions.     

遗传性混合型高脂血症小鼠的自发性动脉粥样硬化

目的为探讨高甘油三酯血症对动脉粥样硬化的影响,建立了脂蛋白脂肪酶和载脂蛋白E双基因缺陷的混合型高甘油三酯和高胆固醇血症动物模型。方法比较了双基因以及载脂蛋白E基因缺陷小鼠的血浆甘油三酯,总胆固醇和高密度脂蛋白胆固醇的水平,并对整条主动脉和主动脉流出道进行了动脉粥样硬化病变定量检测。结果发现双基因缺陷小鼠血浆甘油三酯是载脂蛋白E基因缺陷小鼠的3.8倍(5.904±0.505比1.536±0.860g/L),血浆总胆固醇也略有增加,但高密度脂蛋白胆固醇则无明显差异。两种小鼠的主动脉全长及流出道均有明显的粥样硬化,斑块面积占全部动脉的37.2%±10.7%比44.6%±18.1%,但之间无显著差异。结论载脂蛋白E基因缺陷合并脂蛋白脂肪酶缺陷虽然可以出现血浆甘油三酯增高,但动脉粥样硬化病变并没有加重。这可能与血管壁局部脂蛋白脂肪酶的活性相应降低而对动脉粥样硬化抑制作用有关。     

Accumulation of biglycan and perlecan, but not versican, in lesions of murine models of atherosclerosis

Proteoglycan accumulation within the arterial intima has been implicated in lipoprotein retention and in atherosclerosis progression in humans. Two commonly studied murine models of atherosclerosis, the apolipoprotein E (apoE)-deficient (apoE-/-) mouse and the low density lipoprotein receptor-deficient (LDLR-/-) mouse, develop arterial lesions similar to those of human atherosclerosis. However, specific proteoglycan classes that accumulate in lesions of these mice and their relation to the retention of specific apolipoproteins have not been previously determined. In this report, we characterized the distribution of proteoglycans (versican, biglycan, and perlecan) and apolipoproteins (apoB, apoA-I, and apoE) in proximal aortic lesions of chow-fed apoE-/- and LDLR-/- mice at 10, 52, and 73 weeks of age. We observed that similar to the apoE-/- mice, the LDLR-/- mice develop intermediate and advanced plaques within 52 weeks of age. Perlecan and biglycan (both are proteoglycans) appeared early in lesion development with distinct expression patterns as the plaques advanced. Versican, a major proteoglycan detected in human plaques, was mostly absent in both strains. ApoA-I and apoB were detected in early through advanced lesions in regions of proteoglycan accumulation in both strains. Our results indicate that proteoglycans may contribute to the retention of lipoproteins at the earliest stage of atherosclerosis in murine models of atherosclerosis.     

普伐他汀预防载脂蛋白E小鼠动脉粥样硬化的抗氧化机制与3-硝基酪氨酸的关系

目的探讨普伐他汀预防载脂蛋白E-/-小鼠动脉粥样硬化的抗氧化机制与3-硝基酪氨酸(3-nitrotyrosine,3-NT)的关系。方法将8周龄小鼠36只分为3组,空白对照组、模型组、普伐他汀组,每组12只。通过病理检测主动脉根动脉粥样硬化病变,Western blot法检测主动脉内3-NT和诱导型一氧化氮合酶的表达。结果普伐他汀组动脉粥样硬化病变的相对面积、3-NT和诱导型一氧化氮合酶的表达量显著低于模型组;模型组病变的相对面积、3-NT和诱导型一氧化氮合酶的表达量显著高于空白对照组。结论普伐他汀预防动脉粥样硬化的抗氧化机制之一可能是通过减少诱导型一氧化氮合酶致3-NT减少来实现的。     

Dietary cosupplementation with vitamin E and coenzyme Q(10) inhibits atherosclerosis in apolipoprotein E gene knockout mice

Intimal oxidation of LDL is considered an important early event in atherogenesis, and certain antioxidants are antiatherogenic. Dietary coenrichment with vitamin E (VitE) plus ubiquinone-10 (CoQ(10), which is reduced during intestinal uptake to the antioxidant ubiquinol-10, CoQ(10)H(2)) protects, whereas enrichment with VitE alone can increase oxidizability of LDL lipid against ex vivo oxidation. In the present study, we tested whether VitE plus CoQ(10) cosupplementation is more antiatherogenic than either antioxidant alone, by use of apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet without (control) or with 0.2% (wt/wt) VitE, 0.5% CoQ(10), or 0.2% VitE plus 0.5% CoQ(10) (VitE+CoQ(10)) for 24 weeks. None of the supplements affected plasma cholesterol concentrations, whereas in the VitE and CoQ(10) groups, plasma level of the respective supplement increased. Compared with control, plasma from CoQ(10) or VitE+CoQ(10) but not VitE-supplemented animals was more resistant to ex vivo lipid peroxidation induced by peroxyl radicals. VitE supplementation increased VitE levels in aorta, heart, brain, and skeletal muscle, whereas CoQ(10) supplementation increased CoQ(10) only in plasma and aorta and lowered tissue VITE: All treatments significantly lowered aortic cholesterol compared with control, but only VitE+CoQ(10) supplementation significantly decreased tissue lipid hydroperoxides when expressed per parent lipid. In contrast, none of the treatments affected aortic ratios of 7-ketocholesterol to cholesterol. Compared with controls, VitE+CoQ(10) supplementation decreased atherosclerosis at the aortic root and arch and descending thoracic aorta to an extent that increased with increasing distance from the aortic root. CoQ(10) significantly inhibited atherosclerosis at aortic root and arch, whereas VitE decreased disease at aortic root only. Thus, in apoE-/- mice, VitE+CoQ(10) supplements are more antiatherogenic than CoQ(10) or VitE supplements alone and disease inhibition is associated with a decrease in aortic lipid hydroperoxides but not 7-ketocholesterol.     

Plasma insulin levels predict atherosclerotic lesion burden in obese hyperlipidemic mice

Despite a clear association between obesity, insulin resistance and atherosclerosis in humans, to date, no animal models have been described in which insulin resistance is associated with atherosclerotic lesion burden. Using two mouse models of obesity-induced hyperlipidemia:leptin deficient (ob/ob) mice on an apolipoprotein E deficient (apoE-/-) or low density lipoprotein receptor deficient (LDLR-/-) background, we sought to determine metabolic parameters most closely associated with atherosclerotic lesion burden. Total plasma cholesterol (TC) levels in ob/ob;apoE-/- mice and ob/ob;LDLR-/- mice were indistinguishable (682+/-48 versus 663+/-16, respectively). Analysis of lipoprotein profiles showed that cholesterol was carried primarily on VLDL in the ob/ob;apoE-/- mice and on LDL in the ob/ob;LDLR-/- mice. Plasma triglycerides (TG) were 55% lower (P<0.001), non-esterified fatty acids (NEFA) were 1.5-fold higher (P<0.01), and insulin levels were 1.7-fold higher (NS) in ob/ob;apoE-/- mice compared to ob/ob;LDLR-/- mice. Other parameters such as body weight, fat pad weight, and glucose levels were not different between the groups. Aortic sinus lesion area of ob/ob;apoE-/- mice was increased 3.2-fold above ob/ob;LDLR-/- mice (102,455+/-8565 microm2/section versus 31,750+/-4478 microm2/section, P<0.001). Lesions in ob/ob;apoE-/- mice were also more complex as evidenced by a 7.7-fold increase in collagen content (P<0.001). Atherosclerotic lesion area was positively correlated with body weight (P<0.005), NEFA (P=0.007), and insulin (P=0.002) levels in the ob/ob;LDLR-/- mice and with insulin (P=0.014) in the ob/ob;apoE-/- mice. In contrast, lesion burden was neither associated with TC and TG, nor with individual lipoprotein pools, in either animal model. These data provide a direct demonstration of the pathophysiologic relevance of hyperinsulinemia, NEFA, and increased body weight to atherosclerotic lesion formation.     

普罗布考对高脂喂养LDLR-/-小鼠动脉粥样硬化的影响及机制研究

目的探讨普罗布考对高脂喂养LDLR-/-小鼠动脉粥样硬化的影响并初步阐明其作用机制。方法14只8周龄LDLR-/-雄性小鼠,予以高脂喂养4周后随机分为高脂组（继续高脂饮食,n=7）和普罗布考组（高脂加0．5％普罗布考,n=7）,喂养14周后处死,检测血浆中甘油三酯（TG）、总胆固醇（TG）和高密度脂蛋白胆固醇（HDL—C）水平;取主动脉进行油红0染色观察斑块生长情况;制作主动脉根部冰冻切片,分别进行油红0、Masson染色,检测主动脉根部斑块面积和胶原含量;ELISA检测血浆中炎症因子IL-12p70水平。结果普罗布考组小鼠血浆TG、HDL—C显著低于高脂组（P均〈0．01）,TG无显著改变（P〉0．05）;油红O染色结果显示,两组间主动脉斑块占主动脉比例无显著差异（P〉0．05）,但普罗布考组小鼠主动脉根部斑块面积显著高于高脂组（P〈0．01）;Masson染色结果显示,普罗布考组小鼠主动脉根部斑块中胶原比例显著高于高脂组（P〈0．01）;ELISA结果显示,普罗布考组小鼠血浆IL-12p70水平显著低于高脂组（P〈0．05）。结论普罗布考可降低高脂喂养的LDLR-/-小鼠血浆TG、HDL-C和IL-12p70水平,促进主动脉根部斑块形成,但增加主动脉根部斑块中胶原含量。     

Paraoxonase 1 (PON1) attenuates macrophage oxidative status: studies in PON1 transfected cells and in PON1 transgenic mice

OBJECTIVE: High density lipoprotein (HDL)-associated paraoxonase 1 (PON1), hydrolyzes oxidized lipids in oxidized low density lipoprotein (LDL) and thus protects against atherosclerosis development. Increased susceptibility to atherosclerosis observed in PON1 knockout (PON1(0)) mice was associated with increased LDL lipid peroxidation as well as increased macrophage oxidative stress. Thus, the aim of the present study is to characterize the direct effect of PON1 on oxidative status processes in macrophages. METHODS AND RESULTS: We used in vitro and in vivo models of PON1 expression in macrophages, as PON1 is not synthesized by these cells. Peritoneal macrophages (MPM) harvested from PON1(0) mice were transfected with human (hPON1). These cells exhibited reduced total peroxide levels by 47% and decreased capacity to release superoxide anions by 69%, associated with a small but significant increment of the reduced form of glutathione (GSH), a major cellular anti-oxidant, compared to control cells. MPM were also harvested from PON1 transgenic (PON1Tg) mice. Unexpectedly, these cells expressed hPON1 (mRNA and activity). Compared to MPM derived from control C57BL/6J mice, PON1Tg mouse MPM exhibited 35% decreased cellular total peroxide levels, decreased capacity to produce superoxide anions and 47% decreased capacity to oxidize LDL. PON1Tg mouse MPM were also characterized by 51% increased levels of GSH, compared to control MPM. Similarly, MPM harvested from PON1Tg on the genetic background of the atherosclerotic apolipoprotein E knockout (PON1Tg/E(0)) mice also exhibited decreased oxidative stress, compared to E(0) mouse MPM. Aortas obtained from these mice were characterized by decreased lipid peroxide levels, decreased capacity to oxidize LDL, and also increased GSH levels, compared to aortas obtained from E(0) mice. The decreased macrophage and aortic oxidative stress in PON1Tg/E(0) mice was associated with 2.7-fold decreased atherosclerotic lesion size in comparison to E(0) mice. CONCLUSIONS: PON1 directly reduced macrophage and aortic oxidative status, which was associated with decreased superoxide anion production and increased glutathione content. These phenomena could be responsible for the observed attenuated atherosclerosis development in PON1Tg mice in comparison to control mice.     

Urokinase-type plasminogen activator plays a critical role in angiotensin II-induced abdominal aortic aneurysm

We have previously demonstrated that urokinase-type plasminogen activator (uPA) is highly expressed in the aneurysmal segment of the abdominal aorta (AAA) in apolipoprotein E-deficient (apoE-/-) mice treated with angiotensin II (Ang II). In the present study, we tested the hypothesis that uPA is essential for AAA formation in this model. An osmotic minipump containing Ang II (1.44 mg/kg per day) was implanted subcutaneously into 7- to 11-month-old male mice for 1 month. Ang II induced AAA in 9 (90%) of 10 hyperlipidemic mice deficient in apoE (apoE-/-/uPA+/+ mice) but in only 2 (22%) of 9 mice deficient in both apoE and uPA (apoE-/-/uPA-/- mice) (P<0.05). Although the expansion of the suprarenal aorta was significantly less in apoE-/-/uPA-/- mice than in apoE-/-/uPA+/+ mice, the aortic diameters of the aorta immediately above or below the suprarenal aorta were similar between the 2 groups. Ang II induced AAA in 7 (39%) of 18 strain-matched wild-type C57 black/6J control mice. The incidence was significantly higher in atherosclerotic apoE-deficient (apoE-/-) mice, in which 8 (100%) of 8 mice developed AAA. Only 1 (4%) of 27 uPA-/- mice developed AAA after Ang II treatment. We conclude the following: (1) uPA plays an essential role in Ang II-induced AAA in mice with or without preexisting hyperlipidemia and atherosclerosis; (2) uPA deficiency does not affect the diameter of the nonaneurysmal portion of the aorta; and (3) atherosclerosis and/or hyperlipidemia promotes but is not essential for Ang II-induced AAA formation in this model.     

Vitamins C and E downregulate vascular VEGF and VEGFR-2 expression in apolipoprotein-E-deficient mice

Anti-angiogenic therapy reduces both plaque growth and intimal neovascularization in apolipoprotein-E-deficient mice (apoE-/-). Vascular endothelial growth factor (VEGF) has been suggested as playing a role in the development of atherosclerosis. We examined the hypothesis that VEGF and VEGF receptor-2 (VEGFR-2) expression is upregulated in apoE-/- and, since it could be driven by oxidative stress, tested whether dietary supplementation with vitamins C and E could downregulate it.Two-month-old apoE-/- received vitamin C combined with alpha- or beta-tocopherol for 4 weeks. Aortic VEGF and VEGFR-2 expression were measured by RT-qPCR and western blot.ApoE-/- showed significantly higher expression of aortic VEGF and VEGFR-2 mRNA (P<0.001) and protein (P<0.001) than wild-type mice, as well as increased plasma VEGF (P<0.001). Vitamin C and alpha-tocopherol significantly reduced aortic VEGF and VEGFR-2 expression in apoE-/- (P<0.001), circulating VEGF (P<0.01) and plasma lipid peroxidation (P<0.01). apoE-/- receiving vitamin C and beta-tocopherol showed diminished lipid peroxidation and VEGFR-2, but only partial reduction of VEGF expression.These data demonstrate that augmented VEGF and VEGFR-2 expression in apoE-/- vasculature can be downregulated by vitamins C and E, at least partially through oxidative stress reduction. This novel mechanism could contribute to explaining the beneficial effects of antioxidant vitamins in experimental atherosclerosis.     

Atherosclerosis progression in LDL receptor-deficient and apolipoprotein E-deficient mice is independent of genetic alterations in plasminogen activator inhibitor-1

Impaired fibrinolysis has been linked to atherosclerosis in a number of experimental and clinical studies. Plasminogen activator inhibitor type 1 (PAI-1) is the primary inhibitor of plasminogen activation and has been proposed to promote atherosclerosis by facilitating fibrin deposition within developing lesions. We examined the contribution of PAI-1 to disease progression in 2 established mouse models of atherosclerosis. Mice lacking apolipoprotein E (apoE-/-) and mice lacking the low density lipoprotein receptor (LDLR-/-) were crossbred with transgenic mice overexpressing PAI-1 (resulting in PAI-1 Tg(+)/apoE-/- and PAI-1 Tg(+)/LDLR-/-, respectively) or were crossbred with mice completely deficient in PAI-1 gene expression (resulting in PAI-1-/-/apoE-/- and PAI-1-/-/LDLR-/-, respectively). All animals were placed on a western diet (21% fat and 0.15% cholesterol) at 4 weeks of age and analyzed for the extent of atherosclerosis after an additional 6, 15, or 30 weeks. Intimal and medial areas were determined by computer-assisted morphometric analysis of standardized microscopic sections from the base of the aorta. Atherosclerotic lesions were also characterized by histochemical analyses with the use of markers for smooth muscle cells, macrophages, and fibrin deposition. Typical atherosclerotic lesions were observed in all experimental animals, with greater severity at the later time points and generally more extensive lesions in apoE-/- than in comparable LDLR-/- mice. No significant differences in lesion size or histological appearance were observed among PAI-1-/-, PAI-1 Tg(+), or PAI-1 wild-type mice at any of the time points on either the apoE-/- or LDLR-/- genetic background. We conclude that genetic modification of PAI-1 expression does not significantly alter the progression of atherosclerosis in either of these well-established mouse models. These results suggest that fibrinolytic balance (as well as the potential contribution of PAI-1 to the regulation of cell migration) plays only a limited role in the pathogenesis of the simple atherosclerotic lesions observed in the mouse.     

Dietary homocysteine promotes atherosclerosis in apoE-deficient mice by inducing scavenger receptors expression

Elevated plasma homocysteine (Hcy) levels have been recognized as an independent risk factor for atherosclerosis leading to cardiovascular diseases. However, the mechanisms contributing to atherosclerosis have not been delineated. Since, scavenger receptors mediated uptake of oxidized-LDL (oxLDL) by macrophages resulting in foam cell formation is an early event in atherosclerosis, we hypothesized that atherogenic effects of Hcy may be mediated via regulating expression of scavenger receptor(s). We have tested this hypothesis using apoE-/- female mice fed normal rodent chow (NC) diet or NC supplemented with Hcy in drinking water (9 g/L). Hcy-fed mice showed increased fatty streak lesions in aortic sinus/root compared to NC group without alterations in plasma lipid profiles. Similar findings were observed in the enface analysis of the descending aorta. To determine the molecular mechanisms underlying Hcy-mediated progression of fatty streak lesions, expression of scavenger receptors such as CD36 and lectin-like oxidized LDL binding protein-1 (LOX-1) in the aortic lesions were analyzed. Interestingly, Hcy-fed mice had increased immuno-positive staining for CD36 and LOX-1 in the atherosclerotic lesions compared to NC-fed mice. In vitro analyses showed neither Hcy nor HcyLDL directly affect the expression of CD36 and LOX-1 on mouse macrophages. However, Hcy supplementation in apoE-/- mice resulted in elevated oxLDL levels in plasma. Since oxLDL has been shown to upregulate the expression of CD36 and LOX-1, these findings suggest that Hcy may exert its atherogenic effect in part by elevating the levels of oxLDL. Interestingly, interaction of monocytes with Hcy-activated endothelial cells resulted in upregulation of CD36 expression on monocytes, suggesting a possible mechanism by which Hcy may upregulate CD36 expression at the lesion site. Further, these findings suggest a novel mechanism by which Hcy may promote atherogenesis.     

Retardation of atherosclerosis by overexpression of catalase or both Cu/Zn-superoxide dismutase and catalase in mice lacking apolipoprotein E

Oxidative stress has been suggested to potentiate atherogenesis. However, studies that have investigated the effect of antioxidants on atherosclerosis showed inconsistent results, ie, atherosclerosis was either retarded or not changed by dietary antioxidants. This report directly examined the effect of overexpressing Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and/or catalase on atherosclerosis and lipid peroxidation in mice lacking apolipoprotein E (ApoE-/-). Based on lipid staining of the en face of the aorta tree and the serial sections of the proximal aorta, ApoE-/- mice overexpressing catalase or both Cu/Zn-SOD and catalase had smaller and relatively early stages of atherosclerotic lesions (eg, foam cells and free lipids) when compared with ApoE-/- mice, who developed more advanced lesions (eg, fibrous caps and acellular areas). In addition, the retarded development of atherosclerosis was correlated with a reduced F2-isoprostanes in the plasma and aortas in ApoE-/- mice overexpressing catalase or both Cu/Zn-SOD and catalase. In contrast, the levels of F2-isoprostanes and atherosclerosis in the ApoE-/- mice overexpressing Cu/Zn-SOD alone were comparable to ApoE-/- control mice. These observations implied that endogenously produced hydrogen peroxide, but not superoxide anions, contributed to the formation of oxidized lipids and the development of atherosclerosis in ApoE-/- mice.