Gene expression of LOX-1, VCAM-1, and ICAM-1 in pre-atherosclerotic mice

To identify markers of the earliest stage of atherosclerosis, endothelial dysfunction, we evaluated the gene expression of lectin-like oxidized-low-density-lipoprotein receptor-1 (LOX-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in very young pre-atherosclerotic mice. Furthermore, the plasma levels of the soluble VCAM-1 and ICAM-1 were compared to the gene expression profiles. Gene expressions of LOX-1 and VCAM-1 were up-regulated in young apoE^−/− mice, and thus, it seems probable that these genes play a role in pre-atherosclerosis. Contrarily, the gene expression profile of ICAM-1 did not show any apparent differences between the groups, questioning the involvement of this molecule in the early development of atherosclerosis. Plasma levels of sVCAM-1 and sICAM-1 were similar in all mice and did not correlate with the vascular gene expression of the corresponding genes. It therefore seems likely that these circulating markers are not suited to detect early atherosclerosis.     

Citreoviridin Enhances Atherogenesis in Hypercholesterolemic ApoE-Deficient Mice via Upregulating Inflammation and Endothelial Dysfunction

Vascular endothelial dysfunction and inflammatory response are early events during initiation and progression of atherosclerosis. In vitro studies have described that CIT markedly upregulates expressions of ICAM-1 and VCAM-1 of endothelial cells, which result from NF-κB activation induced by CIT. In order to determine whether it plays a role in atherogenesis in vivo, we conducted the study to investigate the effects of CIT on atherosclerotic plaque development and inflammatory response in apolipoprotein E deficient (apoE^-/-) mice. Five-week-old apoE^-/- mice were fed high-fat diets and treated with CIT for 15 weeks, followed by assay of atherosclerotic lesions. Nitric oxide (NO), vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) were detected in serum. Levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), VEGF, and ET-1 in plaque areas of artery walls were examined. NF-κB p65 expression and NF-κB activation in aorta also were assessed. CIT treatment significantly augmented atherosclerotic plaques and increased expressions of ICAM-1, VCAM-1, VEGF and ET-1 in aorta. Mechanistic studies showed that activation of NF-κB was significantly elevated by CIT treatment, indicating the effect of CIT on atherosclerosis may be regulated by activation of NF-κB.     

Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development

Endothelial cell activation leading to leukocyte recruitment and adhesion plays an essential role in the initiation and progression of atherosclerosis. Vitamin D has cardioprotective actions, while its deficiency is a risk factor for the progression of cardiovascular damage. Our aim was to assess the role of basal levels of vitamin D receptor (VDR) on the early leukocyte recruitment and related endothelial cell-adhesion-molecule expression, as essential prerequisites for the onset of atherosclerosis. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls. Inhibition of NF-κB activation with super-repressor IκBα blunted all signs of endothelial cell activation caused by downregulation of VDR in endothelial cells. In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE^-/- mice, with higher macrophage content. apoE^-/-VDR^-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE^-/-VDR^+/+ mice. Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE^-/-VDR^-/- mice. The results reveal an important role for basal levels of endothelial VDR in limiting endothelial cell inflammation and atherosclerosis.     

Cholesterol increases adhesion of monocytes to endothelium by moving adhesion molecules out of caveolae

Caveolae and its structural protein caveolin-1 (Cav-1) are abundant in vascular endothelial cells (ECs). We examined whether caveolae are involved in monocyte adhesion to ECs responding to a synergy of hypercholesterolemia and inflammation. Treating human umbilical vein ECs with cholesterol enhanced endotoxin lipopolysaccharide (LPS)-induced monocyte adhesion. Use of isolated caveolae-enriched membranes revealed that cell adhesion molecules (CAMs), including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), co-localized with Cav-1 in caveolae. LPS upregulated CAMs expression and increased the co-localization. Cholesterol exposure decreased the level of CAMs in the caveolae. Co-immunoprecipitation and confocal microscopy revealed that ICAM-1 interacted with Cav-1. Electron microscopy showed that ICAM-1 was mainly located in caveolae. Cholesterol exposure decreased this interaction and drove ICAM-1 out of caveolae. Knockdown of Cav-1 reduced the synergistic effects of cholesterol and inflammation. In vivo, ICAM-1 and Cav-1 co-localization was lower in the aortic endothelium of ApoE^−/− mice than in that of wild-type controls. Cav-1 negatively regulates monocyte adhesion by the co-localization of CAMs in caveolae, which is disturbed by cholesterol. Thus, our study suggests a molecular basis underlying the synergistic effects of hypercholesterolemia and inflammation in atherogenesis.     

Docosahexaenoic acid attenuates VCAM-1 expression and NF-κB activation in TNF-α-treated human aortic endothelial cells

This study was conducted to test the hypothesis that n-3 polyunsaturated fatty acids are able to down-regulate expression of adhesion molecules and nuclear factor-κB (NF-κB) activation in vascular endothelial cells, in addition to reducing atherosclerotic lesions in vivo. We report here that docosahexaenoic acid (DHA) reduces atherosclerotic lesions in the aortic arteries of apolipoprotein E knockout (apoE^-/-) mice. Consistent with the observation in animal study, DHA inhibited THP-1 cell adhesion to tumor necrosis factor α (TNF-α)-activated human aortic endothelial cells (HAECs). Expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on the cell surface of HAECs was determined by cell-surface enzyme-linked immunosorbent assay. DHA and eicosapentaenoic acid decreased VCAM-1 expression in a dose-dependent manner in TNF-α treated HAECs, while cis-linoleic acid and arachidonic acid did not have any significant effect on either VCAM-1 or ICAM-1 expression. Moreover, DHA significantly reduced VCAM-1 protein expression in the cell lysates of TNF-α-treated HAECs, as determined by Western blot analysis. In line with NF-κB signaling pathway, DHA suppressed the TNF-α-activated IκBα phosphorylation and degradation as well as IκB kinase-β phosphorylation. Subsequently, translocation of the NF-κB (p50/p65) and AP-1 (c-Fos/c-Jun) subunits was down-regulated by DHA in the nucleus of HAECs. These results suggest that DHA negatively regulates TNF-α-induced VCAM-1 expression through attenuation of NF-κB signaling pathway and AP-1 activation. This study provides evidence that DHA may contribute to the prevention of atherosclerosis and inflammatory diseases in vivo.     

Simvastatin and Benznidazole-Mediated Prevention of Trypanosoma cruzi-Induced Endothelial Activation: Role of 15-epi-lipoxin A4 in the Action of Simvastatin

Trypanosoma cruzi is the causal agent of Chagas Disease that is endemic in Latin American, afflicting more than ten million people approximately. This disease has two phases, acute and chronic. The acute phase is often asymptomatic, but with time it progresses to the chronic phase, affecting the heart and gastrointestinal tract and can be lethal. Chronic Chagas cardiomyopathy involves an inflammatory vasculopathy. Endothelial activation during Chagas disease entails the expression of cell adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) through a mechanism involving NF-κB activation. Currently, specific trypanocidal therapy remains on benznidazole, although new triazole derivatives are promising. A novel strategy is proposed that aims at some pathophysiological processes to facilitate current antiparasitic therapy, decreasing treatment length or doses and slowing disease progress. Simvastatin has anti-inflammatory actions, including improvement of endothelial function, by inducing a novel pro-resolving lipid, the 5-lypoxygenase derivative 15-epi-lipoxin A4 (15-epi-LXA4), which belongs to aspirin-triggered lipoxins. Herein, we propose modifying endothelial activation with simvastatin or benznidazole and evaluate the pathways involved, including induction of 15-epi-LXA4. The effect of 5 μM simvastatin or 20 μM benznidazole upon endothelial activation was assessed in EA.hy926 or HUVEC cells, by E-selectin, ICAM-1 and VCAM-1 expression. 15-epi-LXA4 production and the relationship of both drugs with the NFκB pathway, as measured by IKK-IKB phosphorylation and nuclear migration of p65 protein was also assayed. Both drugs were administered to cell cultures 16 hours before the infection with T. cruzi parasites. Indeed, 5 μM simvastatin as well as 20 μM benznidazole prevented the increase in E-selectin, ICAM-1 and VCAM-1 expression in T. cruzi-infected endothelial cells by decreasing the NF-κB pathway. In conclusion, Simvastatin and benznidazole prevent endothelial activation induced by T. cruzi infection, and the effect of simvastatin is mediated by the inhibition of the NFκB pathway by inducing 15-epi-LXA4 production.     

Atorvastatin has hypolipidemic and anti-inflammatory effects in apoE/LDL receptor-double-knockout mice

Statins are first-line pharmacotherapeutic agents for hypercholesterolemia treatment in humans. However the effects of statins in animal models of atherosclerosis are not very consistent. Thus we wanted to evaluate whether atorvastatin possesses hypolipidemic and anti-inflammatory effects in mice lacking apolipoprotein E/low-density lipoprotein receptor (apoE/LDLR-deficient mice). Two-month-old female apoE/LDLR-deficient mice (n=24) were randomly subdivided into 3 groups. The control group of animals (n=8) was fed with the western type diet (atherogenic diet) and in other two groups atorvastatin was added to the atherogenic diet at the dosage of either 10 mg/kg or 100 mg/kg per day for a period of 2 months. Biochemical analysis of lipids, ELISA analysis of monocyte chemotactic protein-1 (MCP-1) in blood, quantification of lesion size and expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) in the atherosclerotic lesion by means of immunohistochemistry and Western blot analysis were performed. The biochemical analysis showed that administration of atorvastatin (100 mg/kg/day) significantly decreased level of total cholesterol, lipoproteins (VLDL and LDL), triacylglycerol, and moreover significantly increased level of HDL. ELISA analysis showed that atorvastatin significantly decreased levels of MCP-1 in blood and immunohistochemical and Western blot analysis showed significant reduction of VCAM-1 and ICAM-1 expression in the vessel wall after atorvastatin treatment (100 mg/kg/day). In conclusion, we demonstrated here for the first time strong hypolipidemic and anti-inflammatory effects of atorvastatin in apoE/LDLR-deficient mice. Thus, we propose that apoE/LDLR-deficient mice might be a good animal model for the study of statin effects on potential novel markers involved in atherogenesis and for the testing of potential combination treatment of new hypolipidemic substances with statins.     

7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species

Abstract

The aim of the present study was to assess the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), monocytic adhesion of human aortic endothelial cells (HAECs), and the production of intracellular reactive oxygen species (ROS), when HAECs were stimulated by 7-ketocholesterol. 7-ketocholesterol enhances surface expression of ICAM-1 and VCAM-1 as determined by EIA, induces their mRNA expression by RT-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells. We confirmed up-regulation of ROS production of HAECs treated with 7-ketocholesterol. Although the surface expression of ICAM-1 and VCAM-1 on HAECs treated with 7-ketocholesterol increased in a time-dependent manner, α-tocopherol inhibited this increase of the surface expression of ICAM-1 and VCAM-1. In the monocytic adhesion assay, adhesion of U937 to HAECs treated with 7-ketocholesterol was enhanced, but monoclonal anti-ICAM-1 and VCAM-1 antibodies reduced the endothelial adhesiveness. In conclusion, this study suggests that the endothelial adhesiveness to monocytic cells that was increased by 7-ketocholesterol was associated with enhanced expression of ICAM-1 and VCAM-1 mediated by ROS production.     

Critical role of endothelial cell activation in hypoxia-induced vasoocclusion in transgenic sickle mice

Activation of vascular endothelium plays an essential role in vasoocclusion in sickle cell disease. The anti-inflammatory agents dexamethasone and adhesion molecule-blocking antibodies were used to inhibit endothelial cell activation and hypoxia-induced vasoocclusion. Transgenic sickle mice, expressing human alpha-, beta(S)-, and beta(S-Antilles)-globins, had an activated vascular endothelium in their liver, lungs, and skin, as exhibited by increased activation of NF-kappaB compared with normal mice. NF-kappaB activation increased further in the liver and skin after sickle mice were exposed to hypoxia. Sickle mice had decreases in red blood cell (RBC) velocities and developed vasoocclusions in subcutaneous venules in response to hypoxia. Dexamethasone pretreatment prevented decreases in RBC velocities and inhibited vasoocclusions and leukocyte-endothelium interactions in venules after hypoxia. Dexamethasone treatment inhibited NF-kappaB, VCAM-1, and ICAM-1 expression in the liver, lungs, and skin of sickle mice after hypoxia-reoxygenation. VCAM-1 or ICAM-1 blockade with monoclonal antibodies mimicked dexamethasone by inhibiting vasoocclusion and leukocyte adhesion in sickle mice, demonstrating that endothelial cell activation and VCAM-1 and ICAM-1 expression are necessary for hypoxia-induced vasoocclusion in sickle mice. VCAM-1, ICAM-1, and vasoocclusion increased significantly 3 days after dexamethasone discontinuation, possibly explaining rebounds in vasoocclusive crises observed after withdrawal of glucocorticosteroids in sickle patients. We conclude that anti-inflammatory treatments that inhibit endothelial cell activation and adhesion molecule expression can inhibit vasoocclusion in sickle cell disease. Rebounds in vasoocclusive crises after dexamethasone withdrawal are caused by rebounds in endothelial cell activation.     

Simvastatin modulates TNFalpha-induced adhesion molecules expression in human endothelial cells

Adhesion and transendothelial migration of leukocytes into the vascular wall is a crucial step in atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. We investigated the effect of simvastatin, an inhibitor of HMG-CoA reductase administered to reduce plasma levels of LDL-cholesterol, on the expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) by human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor alpha (TNFalpha). We found the expression to be significantly inhibited by the drug in a time and concentration-dependent manner and to a greater extent in the case of VCAM-1 as compared with ICAM-1. In TNFalpha-stimulated HUVEC, simvastatin decreased VCAM-1 and ICAM-1 mRNA levels, inhibited TNFalpha-induced activation of nuclear factor kappaB (NF-kappaB) and enhanced expression of peroxisome proliferator-activated receptor alpha (PPARalpha). These effects were associated with reduction of adherence of monocytes and lymphocytes to HUVEC. The present findings suggest that the benefits of statins in vascular disease may include the inhibition of expression of VCAM-1 and ICAM-1 through effects on NF-kappaB.     

Lack of apoE causes alteration of cytokines expression in young mice liver

To investigate the effect of apolipoprotein E (apoE) on cytokine expression profile of the liver of young mice, quantitative RT-PCR (qRT-PCR) assay and cytokine antibody array for multiplex analysis of 62 cytokines have been used to analyze characteristics of expression of cytokines in the liver of 6-week-old apoE-null (apoE^−/−) mice. The levels of plasma cytokines were also analyzed. The mRNA level of IL-1β, IL-2, IL-6, ICAM-1, VCAM-1, MCP-1, NF-κB (p65), IFN-γ and IκB-α were increased significantly in apoE^−/− mice comparative to wild-type (WT) mice. IL-4, IL-10 and GM-CSF, however, were slightly decreased. Compared with WT, levels of 21 cytokines altered twofold or more in apoE^−/− mice, including 10 cytokines increased and 11 decreased. Expression patterns of IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and VCAM-1 showed identical trend between cytokine antibody array and qRT-PCR analysis. Moreover, levels of IL-1β, IFN-γ and IL-6 in the plasma were elevated, while IL-4 was lightly decreased in apoE^−/− mice compared to those in WT mice. These results implied that promotion of type I immune response in the liver of young apoE^−/− mice due to alteration of these cytokines, and the phenotypes may be caused by the regulation of NF-κB. The inflammation and lipid metabolism dysfunction in the liver cooperated in dysfunction of the liver in young apoE^−/− mice.     

Quantitative trait locus analysis of circulating adhesion molecules in hyperlipidemic apolipoprotein E-deficient mice

Circulating soluble adhesion molecules have been suggested as useful markers to predict several clinical conditions such as atherosclerosis, type 2 diabetes, obesity, and hypertension. To determine genetic factors influencing plasma levels of soluble vascular cell adhesion molecule-1 (VCAM-1) and P-selectin, quantitative trait locus (QTL) analysis was performed on an intercross between C57BL/6J (B6) and C3H/HeJ (C3H) mouse strains deficient in apolipoprotein E-deficient (apoE^−/−). Female F₂ mice were fed a western diet for 12 weeks. One significant QTL, named sVcam1 (71 cM, LOD 3.9), on chromosome 9 and three suggestive QTLs on chromosomes 5, 13 and 15 were identified to affect soluble VCAM-1 levels. Soluble P-selectin levels were controlled by one significant QTL, named sSelp1 (8.5 cM, LOD 3.4), on chromosome 16 and two suggestive QTLs on chromosomes 10 and 13. Both adhesion molecules showed significant or an apparent trend of correlations with body weight, total cholesterol, and LDL/VLDL cholesterol levels in the F₂ population. These results indicate that plasma VCAM-1 and P-selectin levels are complex traits regulated by multiple genes, and this regulation is conferred, at least partially, by acting on body weight and lipid metabolism in hyperlipidemic apoE^−/− mice. Zuobiao Yuan and Zhiguang Su contributed equally.     

Butyrate inhibits cytokine-induced VCAM-1 and ICAM-1 expression in cultured endothelial cells: the role of NF-kappaB and PPARalpha

Adhesion and migration of leukocytes into the surrounding tissues is a crucial step in inflammation, immunity, and atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. Butyrate, a natural short-chain fatty acid produced by bacterial fermentation of dietary fiber, has been attributed with anti-inflammatory activity in inflammatory bowel disease. Butyrate in vitro is active in colonocytes and several other cell types. We have studied the effect of butyrate on expression of endothelial leukocyte adhesion molecules by cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with butyrate-inhibited tumor necrosis factor-alpha (TNFalpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) in a time and concentration-dependent manner. Butyrate at 10 mM/L inhibited interleukin-1 (IL-1)-stimulated VCAM-1 and ICAM-1 expression. The effect of butyrate on cytokine-stimulated VCAM-1 expression was more pronounced than in the case of ICAM-1. Butyrate decreased TNFalpha-induced expression of mRNA for VCAM-1 and ICAM-1. Suppressed expression of VCAM-1 and ICAM-1 was associated with reduced adherence of monocytes and lymphocytes to cytokine-stimulated HUVEC. Butyrate inhibited TNFalpha-induced activation of nuclear factor-kappaB (NF-kappaB) in HUVEC. Finally, butyrate enhanced peroxisome proliferator-activated receptor-alpha (PPARalpha) expression in HUVEC. These results demonstrate that butyrate may have anti-inflammatory properties not only in colonocytes but also in endothelial cells. The anti-inflammatory and (perhaps) antiatherogenic properties of butyrate may partly be attributed to an effect on activation of NF-kappaB and PPARalpha and to the associated expression of VCAM-1 and ICAM-1. The present findings support further investigations on the therapeutic benefits of butyrate in several pathological events involving leukocyte recruitment.     

7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species

The aim of the present study was to assess the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), monocytic adhesion of human aortic endothelial cells (HAECs), and the production of intracellular reactive oxygen species (ROS), when HAECs were stimulated by 7-ketocholesterol. 7-ketocholesterol enhances surface expression of ICAM-1 and VCAM-1 as determined by EIA, induces their mRNA expression by RT-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells. We confirmed up-regulation of ROS production of HAECs treated with 7-ketocholesterol. Although the surface expression of ICAM-1 and VCAM-1 on HAECs treated with 7-ketocholesterol increased in a time-dependent manner, alpha-tocopherol inhibited this increase of the surface expression of ICAM-1 and VCAM-1. In the monocytic adhesion assay, adhesion of U937 to HAECs treated with 7-ketocholesterol was enhanced, but monoclonal anti-ICAM-1 and VCAM-1 antibodies reduced the endothelial adhesiveness. In conclusion, this study suggests that the endothelial adhesiveness to monocytic cells that was increased by 7-ketocholesterol was associated with enhanced expression of ICAM-1 and VCAM-1 mediated by ROS production.     

ESM-1 promotes adhesion between monocytes and endothelial cells under intermittent hypoxia

Intermittent hypoxia (IH), the key property of obstructive sleep apnea (OSA), is closely associated with endothelial dysfunction. Endothelial-cell-specific molecule-1 (ESM-1, Endocan) is a novel, reported molecule linked to endothelial dysfunction. The aim of this study is to evaluate the effect of IH on ESM-1 expression and the role of ESM-1 in endothelial dysfunction. We found that serum concentration of ESM-1, inter-cellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) is significantly higher in patients with OSA than healthy volunteers (p < 0.01). The expression of ESM-1, hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF) was significantly increased in human umbilical vein endothelial cells (HUVECs) by treated IH in a time-dependent manner. HIF-1α short hairpin RNA and vascular endothelial growth factor receptor (VEGFR) inhibitor inhibited the expression of ESM-1 in HUVECs. ICAM-1 and VCAM-1 expressions were significantly enhanced under IH status, accompanied by increased monocyte–endothelial cell adhesion rate ( p < 0.001). Accordingly, ESM-1 silencing decreased the expression of ICAM-1 and VCAM-1 in HUVECs, whereas ESM-1 treatment significantly enhanced ICAM-1 expression accompanied by increasing adhesion ability. ESM-1 is significantly upregulated by the HIF-1α/VEGF pathway under IH in endothelial cells, playing a critical role in enhancing adhesion between monocytes and endothelial cells, which might be a potential target for IH-induced endothelial dysfunction.     

Long-term effects of resveratrol supplementation on suppression of atherogenic lesion formation and cholesterol synthesis in apo E-deficient mice

Atherosclerosis is a chronic inflammatory disease of the arteries resulting from interactions between lipids, monocytes, and arterial wall cells. The effects of resveratrol supplements (RV, 0.02% and 0.06% each, w/w) with regard to the modulation of lipid profiles, cholesterol synthesis, and anti-atherogenesis were examined in apo E-deficient (apo E^−/−) mice fed a normal diet. The concentration of total-cholesterol (total-C) and LDL-cholesterol (LDL-C) in plasma was significantly lower in the resveratrol-supplemented groups compare to the control group over the entire experimental period. The plasma HDL-C concentration was significantly elevated, and the ratio of HDL-C/total-C was significantly higher in the CF and RV groups than in the control group. Plasma paraoxonase (PON) activity was significantly higher in the 0.06% resveratrol group. The hepatic HMG-CoA reductase (HMGR) activity was significantly lower in the clofibrate and resveratrol groups than in the control group. Resveratrol supplements attenuated the presence of atherosclerotic lesions and periarterial fat deposition in the apo E^−/− mice. The presence of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in atherosclerotic vessels was diminished in the resveratrol-supplemented apo E^−/− mice. These results provide new insight into the anti-atherogenic and hypocholesterolemic properties of resveratrol in apo E^−/− mice that were fed a normal diet.     

Vascular Cellular Adhesion Molecule-1 (VCAM-1) Expression in Mice Retinal Vessels Is Affected by Both Hyperglycemia and Hyperlipidemia

Background

Inflammation has been proposed to be important in the pathogenesis of diabetic retinopathy. An early feature of inflammation is the release of cytokines leading to increased expression of endothelial activation markers such as vascular cellular adhesion molecule-1 (VCAM-1). Here we investigated the impact of diabetes and dyslipidemia on VCAM-1 expression in mouse retinal vessels, as well as the potential role of tumor necrosis factor-α (TNFα).

Methodology/Principal Findings

Expression of VCAM-1 was examined by confocal immunofluorescence microscopy in vessels of wild type (wt), hyperlipidemic (ApoE^−/−) and TNFα deficient (TNFα^−/−, ApoE^−/−/TNFα^−/−) mice. Eight weeks of streptozotocin-induced diabetes resulted in increased VCAM-1 in wt mice, predominantly in small vessels (<10 µm). Diabetic wt mice had higher total retinal TNFα, IL-6 and IL-1β mRNA than controls; as well as higher soluble VCAM-1 (sVCAM-1) in plasma. Lack of TNFα increased higher basal VCAM-1 protein and sVCAM-1, but failed to up-regulate IL-6 and IL-1β mRNA and VCAM-1 protein in response to diabetes. Basal VCAM-1 expression was higher in ApoE^−/− than in wt mice and both VCAM-1 mRNA and protein levels were further increased by high fat diet. These changes correlated to plasma cholesterol, LDL- and HDL-cholesterol, but not to triglycerides levels. Diabetes, despite further increasing plasma cholesterol in ApoE^−/− mice, had no effects on VCAM-1 protein expression or on sVCAM-1. However, it increased ICAM-1 mRNA expression in retinal vessels, which correlated to plasma triglycerides.

Conclusions/Significance

Hyperglycemia triggers an inflammatory response in the retina of normolipidemic mice and up-regulation of VCAM-1 in retinal vessels. Hypercholesterolemia effectively promotes VCAM-1 expression without evident stimulation of inflammation. Diabetes-induced endothelial activation in ApoE^−/− mice seems driven by elevated plasma triglycerides but not by cholesterol. Results also suggest a complex role for TNFα in the regulation of VCAM-1 expression, being protective under basal conditions but pro-inflammatory in response to diabetes.     

Angiotensin II modulates interleukin-1β-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-κB crosstalk

Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1β-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-κB, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1β-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1β, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE^−/−) mice. VCAM-1 and iNOS expression were higher in ApoE^−/− than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE^−/− mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin II can differentially modulate inflammatory gene expression in aortic smooth muscle cells through influencing ERK-NF-κB crosstalk, which may contribute to angiotensin II-induced inflammatory disorders related to cardiovascular diseases.     

Lack of modulatory effect of simvastatin on indoxyl sulfate-induced activation of cultured endothelial cells

AimsEndothelial dysfunction is a common manifestation of chronic kidney disease (CKD). The protein-bound uremic toxins have emerged as important factors associated with cardiovascular disease and the outcome of CKD. The effect of indoxyl sulfate (IS) on endothelial cells remains unclear.Main methodsHuman umbilical endothelial cells (HUVEC) were incubated using IS at two concentrations: 100 μM and 1000 μM over two periods of time: 16 and 48 h. HUVEC were also pre-treated with simvastatin to examine its effect. RT-PCR was used to assess changes in the gene expression of intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), Monocyte chemotactic protein-1 (MCP-1), E-selectin, and angiotensin receptor type 1 (AT1R). Protein abundance of the investigated molecules was assessed by immunoblotting.Key findingsTreatment with 100 μM IS for 16 h induced a 2-fold increase in the expression of ICAM-1, VCAM-1, and MCP-1. At a concentration of 1000 μM, there was a 2–3-fold increase. An extended treatment period at low concentrations was associated with a 2–3 fold increase and the increase of ICAM-1 and VCAM-1 was more prominent under high concentration. Results of immunoblotting confirmed an increase in the abundance of ICAM-1, VCAM-1 and MCP-1. No significant change was noted in E-selectin and AT1R according to concentration or treatment duration. Pre-treatment with simvastatin did not alter IS-induced changes.SignificanceIS increased the expression of adhesion molecules of endothelial cells exhibiting a concentration and duration dependent pattern. Simvastatin did not demonstrate any effect on IS-associated endothelial activation.     

Inhibition of cholesteryl ester transfer protein by anacetrapib does not impair the anti-inflammatory properties of high density lipoprotein

Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2 weeks, or from normal human subjects treated either placebo, 20 mg, or 150 mg anacetrapib daily for 2 weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.