Endogenous p53 protects vascular smooth muscle cells from apoptosis and reduces atherosclerosis in ApoE knockout mice

Recent studies have indicated that the tumor suppressor gene p53 limits atherosclerosis in animal models; p53 expression is also increased in advanced human plaques compared with normal vessels, where it may induce growth arrest and apoptosis. However, controversy exists as to the role of endogenous levels of p53 in different cell types that comprise plaques. We examined atherosclerotic plaque development and composition in brachiocephalic arteries and aortas of p53-/-/ApoE-/- mice versus wild type p53 controls. p53-/- mice demonstrated increased aortic plaque formation, with increased rates of cell proliferation and reduced rates of apoptosis in brachiocephalic arteries. Although most proliferating cells were monocyte/macrophages, apoptotic cells were both vascular smooth muscle cells (VSMCs) and macrophages. Transplant of p53 bone marrow to p53-/-/ApoE-/- mice reduced aortic plaque formation and cell proliferation in brachiocephalic plaques, but also markedly reduced apoptosis. To examine p53 regulation of these processes, we studied proliferation and apoptosis in macrophages, bone marrow stromal cells and VSMCs cultured from these mice. Although endogenous p53 promoted apoptosis in macrophages, it protected VSMCs and stromal cells from death, a hitherto unknown function in these cells, in part by inhibiting DNA damage response enzymes. p53 also inhibited stromal cell expression of VSMC markers. We conclude that endogenous levels of p53 protect VSMCs and stromal cells against apoptosis, while promoting apoptosis in macrophages, and protect against atherosclerosis development.     

Bone marrow-derived monocyte chemoattractant protein-1 receptor CCR2 is critical in angiotensin II-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice

Angiotensin II (Ang II) is implicated in atherogenesis by activating inflammatory responses in arterial wall cells. Ang II accelerates the atherosclerotic process in hyperlipidemic apoE-/- mice by recruiting and activating monocytes. Monocyte chemoattractant protein-1 (MCP-1) controls monocyte-mediated inflammation through its receptor, CCR2. The roles of leukocyte-derived CCR2 in the Ang II-induced acceleration of the atherosclerotic process, however, are not known. We hypothesized that deficiency of leukocyte-derived CCR2 suppresses Ang II-induced atherosclerosis. METHODS AND RESULTS: A bone marrow transplantation technique (BMT) was used to develop apoE-/- mice with and without deficiency of CCR2 in leukocytes (BMT-apoE-/-CCR2+/+ and BMT-apoE-/-CCR2-/- mice). Compared with BMT-apoE-/-CCR2+/+ mice, Ang II-induced increases in atherosclerosis plaque size and abdominal aortic aneurysm formation were suppressed in BMT-apoE-/-CCR2-/- mice. This suppression was associated with a marked decrease in monocyte-mediated inflammation and inflammatory cytokine expression. CONCLUSIONS: Leukocyte-derived CCR2 is critical in Ang II-induced atherosclerosis and abdominal aneurysm formation. The present data suggest that vascular inflammation mediated by CCR2 in leukocytes is a reasonable target of therapy for treatment of atherosclerosis.     

Rapamycin attenuates atherosclerosis induced by dietary cholesterol in apolipoprotein-deficient mice through a p27 Kip1 -independent pathway

Activation of immune cells and dysregulated growth and motility of vascular smooth muscle cells contribute to neointimal lesion development during the pathogenesis of vascular obstructive disease. Inhibition of these processes by the immunosuppressant rapamycin is associated with reduced neointimal thickening in the setting of balloon angioplasty and chronic graft vessel disease (CGVD). In this study, we show that rapamycin elicits a marked reduction of aortic atherosclerosis in apolipoprotein E (apoE)-null mice fed a high-fat diet despite sustained hypercholesterolemia. This inhibitory effect of rapamycin coincided with diminished aortic expression of the positive cell cycle regulatory proteins proliferating cell nuclear antigen and cyclin-dependent kinase 2. Moreover, rapamycin prevented the normal upregulation of the proatherogenic monocyte chemoattractant protein-1 (MCP-1, CCL2) seen in the aorta of fat-fed mice. Previous studies have implicated the growth suppressor p27(Kip1) in the antiproliferative and antimigratory activities of rapamycin in vitro. However, our studies with fat-fed mice doubly deficient for p27(Kip1) and apoE disclosed an antiatherogenic effect of rapamycin comparable with that found in apoE-null mice with an intact p27(Kip1) gene. Taken together, these findings extend the therapeutic application of rapamycin from the restenosis and CGVD models to the setting of diet-induced atherosclerosis. Our results suggest that rapamycin-dependent atheroprotection occurs through a p27(Kip1)-independent pathway that involves reduced expression of positive cell cycle regulators and MCP-1 within the arterial wall.     

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.     

Extracellular release of the atheroprotective heat shock protein 27 is mediated by estrogen and competitively inhibits acLDL binding to scavenger receptor-A

We recently identified heat shock protein 27 (HSP27) as an estrogen receptor beta (ERbeta)-associated protein and noted its role as a biomarker for atherosclerosis. The current study tests the hypothesis that HSP27 is protective against the development of atherosclerosis. HSP27 overexpressing (HSP27o/e) mice were crossed to apoE-/- mice that develop atherosclerosis when fed a high-fat diet. Aortic en face analysis demonstrated a 35% reduction (P < or =0.001) in atherosclerotic lesion area in apoE-/-HSP27o/e mice compared to apoE-/- mice, but primarily in females. Serum -HSP27levels were 10-fold higher in female apoE-/-HSP27o/e mice compared to males, and there was a remarkable inverse correlation between circulating HSP27 levels and lesion area in both male and female mice (r(2)=0.78, P < or =0.001). Mechanistic in vitro studies showed upregulated HSP27 expression and secretion in macrophages treated with estrogen or acLDL. Moreover, exogenous HSP27 added to culture media inhibited macrophage acLDL uptake and competed for the scavenger receptor A (SR-A)--an effect that was abolished with the SR-A competitive ligand fucoidan and absent in macrophages from SR-A-/- mice. Furthermore, extracellular HSP27 decreased acLDL-induced release of the proinflammatory cytokine IL-1beta and increased the release of the antiinflammatory cytokine IL-10. HSP27 is atheroprotective, perhaps because of its ability to compete for the uptake of atherogenic lipids or attenuate inflammation.     

Gene expression changes in foam cells and the role of chemokine receptor CCR7 during atherosclerosis regression in ApoE-deficient mice

Atherosclerosis regression is an important clinical goal. In previous studies of regression in mice, the rapid loss of plaque foam cells was explained by emigration to lymph nodes, a process reminiscent of dendritic cells. In the present study, plaque-containing arterial segments from apoE-/- mice were transplanted into WT recipient normolipidemic mice or apoE-/- mice. Three days after transplant, in the WT regression environment, plaque size decreased by approximately 40%, and foam cell content by approximately 75%. In contrast, both parameters increased in apoE-/- recipients. Foam cells were isolated by laser capture microdissection. In WT recipients, there were 3- to 6-fold increases in foam cells of mRNA for liver X receptor alpha and cholesterol efflux factors ABCA1 and SR-BI. Although liver X receptor alpha was induced, there was no detectable expression of its putative activator, peroxisome proliferator-activated receptor gamma. Expression levels of VCAM or MCP-1 were reduced to 25% of levels in pretransplant or apoE-/- recipient samples, but there was induction at the mRNA and protein levels of chemokine receptor CCR7, an essential factor for dendritic cell migration. Remarkably, when CCR7 function was abrogated in vivo by treatment of WT recipients with antibodies to CCR7 ligands CCL19 and CCL21, lesion size and foam cell content were substantially preserved. In summary, in foam cells during atherosclerosis regression, there is induction of CCR7 and a requirement for its function. Taken with the other gene expression data, these results in vivo point to complex relationships among the immune system, nuclear hormone receptors, and inflammation during regression.     

Deletion of the phosphoinositide 3-kinase p110gamma gene attenuates murine atherosclerosis

Inflammatory cell activation by chemokines requires intracellular signaling through phosphoinositide 3-kinase (PI3-kinase) and the PI3-kinase-dependent protein serine/threonine kinase Akt. Atherosclerosis is a chronic inflammatory process driven by oxidatively modified (atherogenic) lipoproteins, chemokines, and other agonists that activate PI3-kinase. Here we show that macrophage PI3-kinase/Akt is activated by oxidized low-density lipoprotein, inflammatory chemokines, and angiotensin II. This activation is markedly reduced or absent in macrophages lacking p110gamma, the catalytic subunit of class Ib PI3-kinase. We further demonstrate activation of macrophage/foam cell PI3-kinase/Akt in atherosclerotic plaques from apolipoprotein E (apoE)-null mice, which manifest an aggressive form of atherosclerosis, whereas activation of PI3-kinase/Akt was undetectable in lesions from apoE-null mice lacking p110gamma despite the presence of class Ia PI3-kinase. Moreover, plaques were significantly smaller in apoE-/-p110gamma-/- mice than in apoE-/-p110gamma+/+ or apoE-/-p110gamma+/-mice at all ages studied. In marked contrast to the embryonic lethality seen in mice lacking class Ia PI3-kinase, germ-line deletion of p110gamma results in mice that exhibit normal viability, longevity, and fertility, with relatively well tolerated defects in innate immune and inflammatory responses that may play a role in diseases such as atherosclerosis and multiple sclerosis. Our results not only shed mechanistic light on inflammatory signaling during atherogenesis, but further identify p110gamma as a possible target for pharmacological intervention in the primary and secondary prevention of human atherosclerotic cardiovascular disease.     

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.     

Crucial role of the CCL2/CCR2 axis in neointimal hyperplasia after arterial injury in hyperlipidemic mice involves early monocyte recruitment and CCL2 presentation on platelets

Monocyte chemoattractant protein-1 (also known as CC chemokine ligand 2 [CCL2]) and its receptor CC chemokine receptor 2 (CCR2) play a central role in the inflammatory response and neointimal formation after vascular injury. In the context of hyperlipidemia, this appears to involve neointimal monocyte infiltration. Hence, we investigated the function of the CCL2/CCR2 axis in early monocyte recruitment to injured arteries. Wire-induced injury of the carotid artery in apoE-/- mice caused a rapid increase of JE/CCL2 protein in the vessel wall peaking at 24 hours after injury, whereas serum JE/CCL2 was increased solely at 6 hours and blood cell-associated levels were unaltered, as demonstrated by enzyme-linked immunosorbent assay. Immunohistochemistry revealed intense staining for JE/CCL2 in smooth muscle cells (SMCs) and in association with platelets adherent to the denuded vessel wall 24 hours after injury. In vitro, exogenous or SMC-derived JE/CCL2 binds to the platelet surface and triggers monocyte arrest on adherent platelets but not on SMCs in flow assays. Accordingly, monocyte arrest in ex vivo perfused apoE-/- carotid arteries isolated 24 hours after injury was profoundly inhibited by pretreatment with a JE/CCL2 antibody. In CCR2-/-/apoE-/- mice, neointimal plaque area was reduced by 47% compared with CCR2+/+/apoE-/- mice. Moreover, CCR2 deletion markedly decreased neointimal macrophage content while expanding SMC content. Vascular JE/CCL2 expressed by SMCs and immobilized by adherent platelets after endothelial denudation is crucial for mediating early monocyte recruitment to injured arteries in hyperlipidemic mice. This mechanism may explain reduced neointimal macrophage infiltration and lesion formation in CCR2-deficient apoE-/- mice.     

CircMAPK1 promotes the proliferation and migration of vascular smooth muscle cells through miR-22-3p/ methyl-CpG binding protein 2 axis

Background and aimsAtherosclerosis is a chronic inflammatory disease. The proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to intimal hyperplasia. CircRNAs are class of endogenous RNA and implicated in the various biological processes. However, the role of circRNAs in atherosclerosis remains largely unknown.Methods and resultsMice models of atherosclerosis were established using APOE^?/- mice fed with high-fat diet. High-throughput sequencing was performed to profile the expression of circRNAs in atherosclerosis. A total of 1289 circRNAs were identified. Six circRNAs were up-regulated and 12 circRNAs were down-regulated in the atherosclerotic plaque tissues. Then we focused on circMAPK1, which showed a high level in atherosclerosis. Silencing circMAPK1 suppressed the proliferation and migration of VSMCs. Further study showed that circMAPK1 bound with miR-22-3p. CircMAPK1 silencing increased the level of miR-22-3p and suppressed the level of MECP2, a known target of miR-22-3p. Interestingly, suppression of miR-22-3p rescued the effect of circMAPK1 silencing on the proliferation and migration of VSMCs.ConclusionCircMPAK1 promoted the proliferation and migration of VSMCs through miR-22-3p/MECP2 axis. Our study revealed the role of circMAPK1 in atherosclerosis and shed lights on the treatment of 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.     

Lesion progression and plaque composition are not altered in older apoE-/- mice lacking tumor necrosis factor-alpha receptor p55

BACKGROUND: Inflammatory processes are an integral component of the initiation, progression, and destabilization of atherosclerotic lesions. Tumor necrosis factor-alpha (TNF-alpha) is considered a primary mediator of inflammatory processes. METHODS AND RESULTS: The role of TNF-alpha in plaque progression and plaque destabilization was investigated in the innominate arteries of older TNF-alpha receptor p55 deficient mice that were generated on a hyperlipidemic apolipoprotein E deficient background (p55-/- apoE-/-). There were no significant differences in levels of circulating cytokines, plaque progression, plaque composition or features of plaque destabilization in p55-/- apoE-/- compared to wild type (p55+/+ apoE-/-) mice. CONCLUSIONS: Progression and destabilization of advanced atherosclerotic lesions does not seem to be mediated via the TNF-alpha receptor p55.     

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.     

Macrophage p53 deficiency leads to enhanced atherosclerosis in APOE*3-Leiden transgenic mice

Cell proliferation and cell death (either necrosis or apoptosis) are key processes in the progression of atherosclerosis. The tumor suppressor gene p53 is an essential gene in cell proliferation and cell death and is upregulated in human atherosclerotic plaques, both in smooth muscle cells and in macrophages. In the present study, we investigated the importance of macrophage p53 in the progression of atherosclerosis using bone marrow transplantation in APOE*3-Leiden transgenic mice, an animal model for human-like atherosclerosis. APOE*3-Leiden mice were lethally irradiated and reconstituted with bone marrow derived from either p53-deficient (p53(-/-)) or control (p53(+/+)) donor mice. Reconstitution of mice with p53(-/-) bone marrow did not result in any hemopoietic abnormalities as compared with p53(+/+) transplanted mice. After 12 weeks on an atherogenic diet, APOE*3-Leiden mice reconstituted with p53(-/-) bone marrow showed a significant (P=0.006) 2.3-fold increase in total atherosclerotic lesion area as compared with mice reconstituted with p53(+/+) bone marrow. Although likely a secondary effect of the increased lesion area, p53(-/-) transplanted mice also showed significantly more lesion necrosis (necrotic index, 1.1+/-1.3 versus 0.2+/-0.7; P=0.04) and lesion macrophages (macrophage area, 79.9+/-40.0 versus 39.7+/-27.3x10(3) micrometer(2) per section; P=0.02). These observations coincided with a tendency toward decreased apoptosis (terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei going from 0.42+/-0.39 to 0.14+/-0.15%, P=0.071), whereas the number of proliferating cells (5'-bromo-2'-deoxyuridine-positive nuclei) was not affected (3.75+/-0.98 versus 4.77+/-2.30%; P=0.59). These studies indicate that macrophage p53 is important in suppressing the progression of atherosclerosis and identify a novel therapeutic target for regulating plaque stability.     

Accelerated atherosclerosis in C57Bl/6 mice transplanted with ApoE-deficient bone marrow

Apolipoprotein E (apoE), a high affinity ligand for lipoprotein receptors, is synthesized by the liver and extrahepatic tissues, including cells of the monocyte/macrophage cell lineage. The role of monocyte/macrophage-derived apoE in atherogenesis was assessed by transplantation of apoE-deficient (apoE-/-) bone marrow into normolipidemic C57Bl/6 mice. No significant effect could be demonstrated on serum apoE levels in C57Bl/6 mice, transplanted with apoE-deficient bone marrow compared with control transplanted mice. Furthermore, no consistent effect on serum cholesteryl esters and triglyceride concentrations could be demonstrated on either a standard chow diet or a high cholesterol diet. Quantitative analysis of atherosclerosis in mice transplanted with apoE-deficient bone marrow, after two months on a high cholesterol diet, revealed a 4-fold increase in the atherosclerotic lesion area as compared to animals transplanted with apoE+/+ bone marrow. Analysis of the ability of apoE-deficient macrophages to release cholesterol after loading with acetylated LDL revealed that the release of cholesterol from apoE-deficient macrophages was impaired as compared to wild-type macrophages in the absence and the presence of specific cholesterol acceptors. In conclusion, apoE production by macrophages retards the formation of atherosclerotic plaques, possibly by mediating cholesterol efflux. We anticipate that pharmacological approaches to increase apoE synthesis and/or secretion by macrophages might be beneficial for the treatment of atherosclerosis.     

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.     

Disruption of tumor necrosis factor-alpha gene diminishes the development of atherosclerosis in ApoE-deficient mice

Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) have been implicated in atherogenesis. However, the precise role of TNF-alpha in atherogenesis is still unclear. To examine the effect of TNF-alpha on atherogenesis, we generated compound-deficient mice in apolipoprotein E (apoE) and TNF-alpha (apoE-/-/TNF-alpha-/-) and compared them with apoE-/- mice. Although serum total cholesterol levels were markedly elevated in both apoE-/-/TNF-alpha-/- and apoE-/- mice compared to wild-type mice, no differences were observed between apoE-/-/TNF-alpha-/- and apoE-/- mice. The atherosclerotic plaque area in the aortic luminal surface of apoE-/-/TNF-alpha-/- mice (n=8, 3.1+/-0.4%) was significantly smaller than that of apoE-/- mice (n=7, 4.7+/-0.4%, p<0.001) despite the lack of difference in serum cholesterol levels. The atherosclerotic lesion size in the aortic sinus of apoE-/-/TNF-alpha-/- mice (n=10, 5.1+/-0.3 x 10(5)microm(2)) was also significantly smaller than that of apoE-/- mice (n=11, 7.0+/-0.3 x 10(5)microm(2), p<0.0001). RT-PCR analysis indicated that the expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in apoE-/- than apoE-/-/TNF-alpha-/- mice. Macrophages from apoE(-/-) mice showed higher uptake level of oxidized LDL and increased expression level of scavenger receptor class A (SRA) compared to those from apoE-/-/TNF-alpha-/- mice. These results indicate that TNF-alpha plays an atherogenic role by upregulating the expressions of ICAM-1, VCAM-1 and MCP-1 in the vascular wall, and by inducing SRA expression and oxidized LDL uptake in macrophages.