PPARdelta-mediated antiinflammatory mechanisms inhibit angiotensin II-accelerated atherosclerosis

Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor delta (PPARdelta) has been shown to improve insulin resistance, adiposity, and plasma HDL levels. However, its antiatherogenic role remains controversial. Here we report atheroprotective effects of PPARdelta activation in a model of angiotensin II (AngII)-accelerated atherosclerosis, characterized by increased vascular inflammation related to repression of an antiinflammatory corepressor, B cell lymphoma-6 (Bcl-6), and the regulators of G protein-coupled signaling (RGS) proteins RGS4 and RGS5. In this model, administration of the PPARdelta agonist GW0742 (1 or 10 mg/kg) substantially attenuated AngII-accelerated atherosclerosis without altering blood pressure and increased vascular expression of Bcl-6, RGS4, and RGS5, which was associated with suppression of inflammatory and atherogenic gene expression in the artery. In vitro studies demonstrated similar changes in AngII-treated macrophages: PPARdelta activation increased both total and free Bcl-6 levels and inhibited AngII activation of MAP kinases, p38, and ERK1/2. These studies uncover crucial proinflammatory mechanisms of AngII and highlight actions of PPARdelta activation to inhibit AngII signaling, which is atheroprotective.     

Bone marrow chimerism prevents atherosclerosis in arterial walls of mice deficient in apolipoprotein E

OBJECTIVE: apolipoprotein E (apoE) is a key regulator in cholesterol-rich lipoprotein metabolism. Inherited deficiency of this protein results in type III hyperlipoproteinemia in humans. ApoE, especially that derived from macrophages, can efficiently protect against development of atherosclerotic lesion. To use stem cell gene therapy or mini-transplant in treating abnormal lipid metabolism and preventing atherosclerosis, a minimal level of bone marrow chimerism must be determined. METHODS: lethally irradiated apoE deficient mice (12-16 weeks of age) fed on normal chow were transplanted with normal bone marrow cells (C57BL/6.Ly5.1) mixed with those of apoE deficient mice (C57BL/6.Ly5.2) at various ratios. Plasma cholesterol levels were determined every 3 weeks for up to 42 weeks. Areas of atherosclerotic lesion in the aortas were quantified 6 months post-transplant. Plasma apoE was measured by Western blot analysis. RESULTS: bone marrow transplantation (BMT) in apoE (-/-) mice resulted in a detectable level of plasma apoE as determined by Western blot analysis. The plasma cholesterol levels in mice with > or = 60% chimerism were normalized by 6 weeks post-transplant. Mice with < or = 40% chimerism showed significant reductions, but not normalization, in the plasma cholesterol levels even at 42 weeks posttransplant. However, atherosclerotic areas observed in 10%-chimeric mice were significantly smaller than those in control mice (P<0.01). Immunohistochemical studies in 10%-chimeric mice revealed foam cells derived from donor marrow (apoE (+/+)) and expressed immunoreactive apoE in the atherosclerotic lesion. The positive signals by Western blot analysis were represented in the plasma of up to 8% of the chimeric mice. CONCLUSION: chimerism of 10%, the minimum level analyzed, was sufficient to reduce the severity of atherosclerosis, although the plasma cholesterol levels were not completely normalized. The results indicate that stem cell gene therapy and mini-transplant may provide possible therapeutic approaches to treat patients with abnormal lipid metabolism and atherosclerosis.     

Reduced atherosclerosis in interleukin-18 deficient apolipoprotein E-knockout mice

OBJECTIVE: Atherosclerosis is an inflammatory disease in which T helper 1 (Th1) immunity has been proposed to play an important role. Nai;ve CD4+ T cells differentiate into interferon-gamma (IFN-gamma) producing Th1 effector cells when stimulated by interleukin-18 (IL-18) and IL-12. We wanted to directly test whether the Th1 pathway is proatherogenic. METHODS: We bred IL-18(-/-) mice with apolipoprotein E(-/-) (apoE(-/-)) mice and assessed atherosclerosis in the aortic root of the offspring. RESULTS: 24-week-old IL-18 deficient apoE(-/-) mice exhibited substantially reduced lesion size (93,866+/-11273 vs. 144,019+/-9667 microm(2) in IL-18(+/+)xapoE(-/-) mice, P=0.005). Lesion cells in compound knockout mice displayed reduced I-A(b) expression, implying reduced local IFN-gamma stimulation. These mice also had an increased proportion of alpha-SM-actin+ smooth muscle cells, compatible with a more stable lesion phenotype. Immunoglobulin G (IgG) subclass analysis of antibodies to malondialdehyde-modified low density lipoprotein indicated increased Th2 and reduced Th1 helper to B cell antibody production. Surprisingly, serum cholesterol and triglyceride levels were significantly higher in IL-18(-/-)xapoE(-/-) mice in spite of their reduced atherosclerosis. However, no changes in lipoprotein cholesterol patterns were registered. CONCLUSION: These data show reduced atherosclerosis and Th1 activity in spite of increased serum cholesterol in IL-18 deficient apoE(-/-) mice. They support a proatherogenic role for IL-18.     

Swimming reduces the severity of atherosclerosis in apolipoprotein E deficient mice by antioxidant effects

OBJECTIVE: It was shown that aerobic exercise training may protect against the development of atherosclerosis. However, the precise mechanisms are still unknown. We assessed the hypothesis that exercise training reduced the severity of experimental atherosclerosis in apolipoprotein (apo) E-deficient mice by antioxidant effects. METHODS: Exercise training (45 min swimming, 3 times/week) was conducted on apo E-deficient mice fed a high fat diet. Over 8 and 16 weeks on alternate days, mice were treated with and without exercise, and additional exercise-treated mice were orally given 25 mg/kg/day of NG-nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase (NOS). In addition, the effect of L-arginine against L-NAME was also tested. RESULTS: Fatty streak formation at 8 weeks and fibrofatty plaques at 16 weeks developed in apo E-deficient mice fed a high fat diet, and were suppressed in mice treated with swimming for 8 and 16 weeks. In contrast, atherosclerotic lesions were not ameliorated in mice treated with exercise training associated with oral L-NAME. However, in mice treated with swimming associated with L-NAME and L-arginine, the atherosclerotic lesions were reduced. Immunohistochemical analysis revealed that macrophage and CD4+ cell accumulation in the fatty streak lesions was suppressed in mice treated with exercise, but not in those treated with exercise associated with L-NAME administration. The severity of atherosclerotic lesions was inversely correlated with the endothelial NOS expression and the expression of an endogenous antioxidant protein, thioredoxin. Namely, the expression of thioredoxin in mice treated with exercise was suppressed compared with mice without exercise. Plasma thiobarbituric acid-reactive substance levels were significantly lower in groups with exercise than in those without exercise or with exercise associated with L-NAME administration, suggesting exercise-induced less lipid peroxidation. Differences in lesion area did not correlate with any significant alterations in serum lipid levels. The exercise load used in the current study did not affect energy metabolism efficacies in the hearts. CONCLUSION: Exercise training, in which the load did not affect energy metabolism efficacy of the heart, suppressed atherosclerosis by antioxidant effects via the vascular NO system.     

Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E.

To develop a murine model system to test the role of monocyte-derived macrophage in atherosclerosis, the osteopetrotic (op) mutation in the macrophage colony-stimulating factor gene was bred onto the apolipoprotein E (apoE)-deficient background. The doubly mutant (op/apoE-deficient) mice fed a low-fat chow diet had significantly smaller proximal aortic lesions at an earlier stage of progression than their apoE-deficient control littermates. These lesions in the doubly mutant mice were composed of macrophage foam cells. The op/apoE-deficient mice also had decreased body weights, decreased blood monocyte differentials, and increased mean cholesterol levels of approximately 1300 mg/dl. Statistical analysis determined that atherosclerosis lesion area was significantly affected by the op genotype and gender. The confounding variables of body weight, plasma cholesterol, and monocyte differential, which were all affected by op genotype, had no significant additional effect on lesion area once they were adjusted for the effects of op genotype and gender. Unexpectedly, there was a significant inverse correlation between plasma cholesterol and lesion area, implying that each may be the result of a common effect of macrophage colony-stimulating factor levels. The data support the hypothesis that macrophage colony-stimulating factor and its effects on macrophage development and function play a key role in atherogenesis.     

瑞舒伐他汀对载脂蛋白E基因敲除小鼠血管内皮黏附性的影响

目的 观察瑞舒伐他汀抗血管壁炎症的作用.方法 载脂蛋白E基因敲除小鼠80只和C57BL/6小鼠20只,均为8～9周龄.分为2周药物处理组和6周药物处理组,每组各40只载脂蛋白E基因敲除小鼠和10只C57BL/6小鼠.载脂蛋白E基因敲除小鼠每日1次皮下注射不同浓度的瑞舒伐他汀,剂量分别为0、1、5和20 mg/kg.药物处理满2周或6周时,心内穿刺取血,并收获小鼠主动脉.结果 经瑞舒伐他汀处理2周或6周后,载脂蛋白E基因敲除小鼠血浆总胆固醇和低密度脂蛋白胆固醇明显下降,但甘油三酯和高密度脂蛋白胆固醇均无明显变化.经瑞舒伐他汀处理2周后,20 mg/kg组载脂蛋白E基因敲除小鼠主动脉内皮一单核细胞黏附率有明显下降;经瑞舒伐他汀处理6周后,5、20 mg/kg组载脂蛋白E基因敲除小鼠主动脉内皮一单核细胞黏附率均有明显下降.经定量RT-PCR分析,瑞舒伐他汀20 mg/kg能明显抑制载脂蛋白E基因敲除小鼠主动脉VCAM-1、MCP-1 mRNA表达.结论 瑞舒伐他汀具有抑制动脉粥样硬化早期炎症反应的作用.     

Aortic atherosclerotic plaque injury in apolipoprotein E deficient mice

The acute platelet response and chronic smooth muscle cell (SMC) proliferation following aortic injury in apolipoprotein E-deficient mice was investigated. The purpose of this study was to evaluate whether thrombus formation would occur following plaque injury, to determine the type of thrombus that developed, and to evaluate SMC proliferation. Aortic injury was performed by squeezing the aorta between forceps. The response to injury reflects the findings primarily associated with plaque disruption. An attempt was made to exclude the use of injured vascular segments that showed marked injury to the media to minimize the effects that medial SMCs may have in thrombus formation. Acute and chronic experiments following injury were terminated at 30 min and at 2 weeks, respectively. Injury in normal and heterozygous mice and nonplaque injury in apolipoprotein E-deficient mice were accompanied by endothelial denudation. In apolipoprotein E-deficient mice, plaque injury, which released plaque contents, foam cells and fragments of foam cells, was followed by thrombus formation that contained degranulating platelets mixed with fibrin. Large platelet-fibrin aggregates were in close contact with disrupted plaques and were mixed with foam cell debris. In addition, small thrombi were in nonplaque areas following plaque disruptions. These thrombi were not associated with injury to the media and most likely represent a heightened thrombogenicity associated with plaque disruption. At 2 weeks following injury, a thickened neointima was present in both wild type and mutant mice. Lipid filled cells were seen only in the media but not in the intima of apo E−/−vessels at 2 weeks. The results suggest that plaque injury in homozygous apolipoprotein E-deficient mice promotes platelet-fibrin thrombus formation and that these thrombi are primarily associated with disrupted plaque contents. The results also suggest that the platelet response and SMC proliferation induced by aortic injury are not altered by hyperlipidemia caused by apolipoprotein E deficiency.     

P-selectin glycoprotein ligand-1 deficiency leads to cytokine resistance and protection against atherosclerosis in apolipoprotein E deficient mice

Adhesive interactions between endothelial cells and leukocytes contribute to atherosclerotic plaque growth. However, mechanism(s) responsible for endothelial priming and deactivation in inflammatory diseases such as atherosclerosis are not clear. Apolipoprotein E deficient mice were generated with deficiency of P-selectin glycoprotein ligand-1 (Psgl-1(-/-), ApoE(-/-)). On both standard chow and Western diet, Psgl-1(-/-), ApoE(-/-) mice were protected against atherosclerosis compared to Psgl-1(+/+), ApoE(-/-) controls. Psgl-1(-/-), ApoE(-/-) mice also showed reduced leukocyte rolling and firm attachment on endothelial cells, however, adoptively transferred Psgl-1(+/+), ApoE(-/-) leukocytes into Psgl-1(-/-), ApoE(-/-) hosts displayed similar reduced rolling as Psgl-1(-/-), ApoE(-/-) leukocytes. Hematopoietic deficiency of Psgl-1 conferred resistance to the effects of interleukin-1β (IL-1β) on leukocyte rolling along with reduced circulating levels of sP-sel and sE-sel. Antibody blockade of Psgl-1 also reduced endothelial activation in response to IL-1β, eliminated leukocyte rolling, and was protective against atherosclerosis in ApoE(-/-) mice. Monocyte depletion with clodronate restored the endothelial response to IL-1β in Psgl-1(-/-) mice. This study suggests that Psgl-1 deficiency leads to reduced atherosclerosis and adhesive interactions between endothelial cells and leukocytes by indirectly regulating endothelial responses to cytokine stimulation.     

Effect of macrophage-derived apolipoprotein E on established atherosclerosis in apolipoprotein E-deficient mice

Apolipoprotein E-deficient (apoE(-/-)) mice have hyperlipidemia and develop spontaneous atherosclerosis in a time-dependent manner. Although macrophage-derived apoE has been shown to prevent the development of atherosclerosis in apoE(-/-) mice, whether it would induce regression of established atherosclerosis is unknown. To determine this, 8-week-old apoE(-/-) mice were transplanted with apoE(+/+) bone marrow. Four weeks after transplantation, when plasma cholesterol levels had reached normal levels, a group of mice (n=12) were killed and their aortic lesions were measured and used as a baseline to judge regression. Twelve and 20 weeks after transplantation, aortic lesion areas of the mice were 9340+/-2184 micrometer(2) (mean+/-SEM, n=8) and 12 211+/-1433 micrometer(2) (n=9), respectively, values not significantly different from the lesion areas of the baseline mice (12 347+/-2487 micrometer(2); n=12, P>0.05). In contrast, apoE(-/-) mice reconstituted with apoE(-/-) bone marrow developed severe atherosclerotic lesions (453 036+/-29 767 micrometer(2), n=7) 20 weeks after transplantation. These data suggest that macrophage-derived apoE was insufficient to induce significant regression of established atherosclerotic lesions in apoE(-/-) mice, although it was sufficient to eliminate hypercholesterolemia and prevent progression of aortic lesions.