Yi Fu

Genetic Ace2 deficiency accentuates vascular inflammation and atherosclerosis in the ApoE knockout mouse.

Rationale: Angiotensin-converting enzyme (ACE)2 opposes the actions of angiotensin (Ang) II by degrading it to Ang 1-7. Objective: Given the important role of Ang II/Ang 1-7 in atherogenesis, we investigated the impact of ACE2 deficiency on the development of atherosclerosis. Methods and Results: C57Bl6, Ace2 knockout (KO), apolipoprotein E (ApoE) KO and ApoE/Ace2 double KO mice were followed until 30 weeks of age. Plaque accumulation was increased in ApoE/Ace2 double KO mice when compared to ApoE KO mice. This was associated with increased expression of adhesion molecules and inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, and an early increase in white cell adhesion across the whole aortae on dynamic flow assay. In the absence of a proatherosclerotic (ApoE KO) genotype, ACE2 deficiency was also associated with increased expression of these markers, suggesting that these differences were not an epiphenomenon. ACE inhibition prevented increases of these markers and atherogenesis in ApoE/ACE2 double KO mice. Bone marrow macrophages isolated from Ace2 KO mice showed increased proinflammatory responsiveness to lipopolysaccharide and Ang II when compared to macrophages isolated from C57Bl6 mice. Endothelial cells isolated from Ace2 KO mice also showed increased basal activation and elevated inflammatory responsiveness to TNF-&agr;. Similarly, selective inhibition of ACE2 with MLN-4760 also resulted in a proinflammatory phenotype with a physiological response similar to that observed with exogenous Ang II (10−7 mol/L). Conclusions: Genetic Ace2 deficiency is associated with upregulation of putative mediators of atherogenesis and enhances responsiveness to proinflammatory stimuli. In atherosclerosis-prone ApoE KO mice, these changes potentially contribute to increased plaque accumulation. These findings emphasize the potential utility of ACE2 repletion as a strategy to reduce atherosclerosis.

Caveolin-1 Plays a Critical Role in the Differentiation of Monocytes into Macrophages

Objective—Monocyte to macrophage differentiation is an essential step in atherogenesis. The structure protein of caveolae, caveolin-1, is increased in primary monocytes after its adhesion to endothelium. We explore the hypothesis that caveolin-1 plays a role in monocyte differentiation to macrophages. Methods and Results—Both phorbol myristate acetate–induced THP-1 and colony-stimulating factor–induced primary monocyte differentiation was associated with an increase in cellular caveolin-1 expression. Overexpression of caveolin-1 by transfection increased macrophage surface markers and inflammatory genes, whereas caveolin-1 knockdown by small interfering RNA or knockout reduced these. Also, caveolin-1 knockdown inhibited the differentiation–induced nuclear translocation of early growth response 1 (EGR-1) through extracellular signal-regulated kinase phosphorylation, further decreased the binding of EGR-1 to CD115 promoter, thus decreasing EGR-1 transcriptional activity. In functional assays, caveolin-1 inhibited transmigration but promoted phagocytosis in the monocyte–macrophage lineage. Decreasing caveolin-1 inhibited the uptake of modified low-density lipoprotein and reduced cellular lipid content. Finally, we showed that caveolin-1 knockout mice displayed less monocyte differentiation than wild-type mice and that EGR-1 transcription activity was also decreased in these mice because of the inhibition of extracellular signal-regulated kinase phosphorylation. Conclusion—Caveolin-1 promotes monocyte to macrophage differentiation through the regulation of EGR-1 transcriptional activity, suggesting that phagocytic caveolin-1 may be critical for atherogenesis.

ABCA12 regulates ABCA1-dependent cholesterol efflux from macrophages and the development of atherosclerosis

ABCA12 is involved in the transport of ceramides in skin, but it may play a wider role in lipid metabolism. We show that, in Abca12-deficient macrophages, cholesterol efflux failed to respond to activation with LXR agonists. Abca12 deficiency caused a reduction in the abundance of Abca1, Abcg1, and Lxrβ. Overexpression of Lxrβ reversed the effects. Mechanistically, Abca12 deficiency did not affect expression of genes involved in cholesterol metabolism. Instead, a physical association between Abca1, Abca12, and Lxrβ proteins was established. Abca12 deficiency enhanced interaction between Abca1 and Lxrβ and the degradation of Abca1. Overexpression of ABCA12 in HeLa-ABCA1 cells increased the abundance and stability of ABCA1. Abca12 deficiency caused an accumulation of cholesterol in macrophages and the formation of foam cells, impaired reverse cholesterol transport in vivo, and increased the development of atherosclerosis in irradiated Apoe(-/-) mice reconstituted with Apoe(-/-)Abca12(-/-) bone marrow. Thus, ABCA12 regulates the cellular cholesterol metabolism via an LXRβ-dependent posttranscriptional mechanism.

Selective Endothelial Overexpression of Arginase II Induces Endothelial Dysfunction and Hypertension and Enhances Atherosclerosis in Mice

Background Cardiovascular disorders associated with endothelial dysfunction, such as atherosclerosis, have decreased nitric oxide (NO) bioavailability. Arginase in the vasculature can compete with eNOS for L-arginine and has been implicated in atherosclerosis. The aim of this study was to evaluate the effect of endothelial-specific elevation of arginase II expression on endothelial function and the development of atherosclerosis. Methodology/Principal Findings Transgenic mice on a C57BL/6 background with endothelial-specific overexpression of human arginase II (hArgII) gene under the control of the Tie2 promoter were produced. The hArgII mice had elevated tissue arginase activity except in liver and in resident peritoneal macrophages, confirming endothelial specificity of the transgene. Using small-vessel myography, aorta from these mice exhibited endothelial dysfunction when compared to their non-transgenic littermate controls. The blood pressure of the hArgII mice was 17% higher than their littermate controls and, when crossed with apoE −/− mice, hArgII mice had increased aortic atherosclerotic lesions. Conclusion We conclude that overexpression of arginase II in the endothelium is detrimental to the cardiovascular system.

Interleukin 10 antioxidant effect decreases leukocytes/endothelial interaction induced by tumor necrosis factor α.

ABSTRACT Little is known about the endothelial mechanisms involved in the anti-inflammatory effects of interleukin 10 (IL-10). The goal of this study was to evaluate the effects of IL-10 on endothelial oxidative stress and endothelial inflammation induced by tumor necrosis factor &agr; (TNF-&agr;). Production of reactive oxygen species (ROS) in perfused human umbilical vein endothelial cells (HUVECs) was studied by fluorescent microscopy using dichlorodihydrofluorescein diacetate. Tumor necrosis factor &agr; (1 ng/mL) was added to the perfusion medium in the absence and presence of IL-10 (1 ng/mL). The role of phosphatidylinositol 3-kinase (PI3-kinase) was assessed using wortmannin and LY 2940002 (inhibitors of PI3-kinase). Specific inhibition of p110 &agr; and p110 &ggr;/&dgr; PI3-kinase subunits was studied using A66 and TG100–115. As well, levels of ceramide and intercellular adhesion molecule 1 (ICAM-1) expression were measured. Finally, the effect of IL-10 on TNF-&agr;–induced leukocyte/endothelium interaction was examined using an ex vivo perfused vessel model. Interleukin 10 significantly reduced dichlorodihydrofluorescein diacetate fluorescence induced by TNF-&agr; in HUVECs (12.5% ± 3.2% vs. 111.7% ± 21.6% at 60 min). Pretreatment by LY2940002 or wortmannin restored ROS production induced by TNF-&agr; in the presence of IL-10. In HUVECs treated by TNF-&agr; + IL-10, inhibition of p110 &agr; PI3-kinase subunit significantly increased ROS production, whereas p110 &ggr;/&dgr; inhibition did not have a significant effect. Pretreatment with IL-10 significantly decreased TNF-&agr;–induced increased levels of ceramide (TNF-&agr; vs. TNF-&agr; + IL-10: 6,278 ± 1,013 vs. 1,440 ± 130 pmol/mg prot), as well as ICAM-1 expression and leukocyte adhesion (TNF-&agr; vs. TNF-&agr; + IL-10: 26.8 ± 2.6 vs. 6.7 ± 0.4 adherent leukocytes/field at 15 min). Interleukin 10 decreases the level of inflammation induced by TNF-&agr; in endothelial cells by reducing the TNF-&agr;–induced ROS production, ICAM-1 expression, and leukocyte adhesion to the endothelium. The antioxidant effect of IL-10 is mediated through PI3-kinase and is paralleled by a decrease in ceramide synthesis induced by TNF-&agr;.

High-density lipoprotein inhibits human M1 macrophage polarization through redistribution of caveolin-1.

Monocyte‐derived macrophages are critical in the development of atherosclerosis and can adopt a wide range of functional phenotypes depending on their surrounding milieu. High‐density lipoproteins (HDLs) have many cardio‐protective properties including potent anti‐inflammatory effects. We investigated the effects of HDL on human macrophage phenotype and the mechanisms by which these occur.

701 HIGH INTRALUMINAL PRESSURE INDUCES ENDOTHELIAL INFLAMMATION

Background and Aims: Hypertension is a potent risk factor for coronary artery disease. The contributing mechanism however, remains elusive. We hypothesise that high intraluminal pressure per se induces vascular inflammation and the adhesion cascade. Methods and Results: Using a specialised pressure vessel chamber we show that elevations in intraluminal pressure increases gene expression of adhesion molecules and functional leukocyte adhesion to the endothelium in harvested vessels (P < 0.001). We also show that endothelial microparticles are shed from pressurised HUVECs (P < 0.05). Low shear stress (LSS: 1.6 dynes/cm2) coupled with pressure (120 mmHg) increased leukocyte adhesion compared to HSS (16 dyne/cm2; 90 ± 23 vs 24 ± 9 leukocytes/field respectively; n = 5-6; P < 0.001). However, LSS on its own (with no pressure) had no influence (4 ± 3 leukocytes/field; n = 4) suggesting that circumferential stretch plays a critical role. Electron microscopy images of pressurised vessels demonstrate caveolae disassembly, resulting in reduced caveolae number. To investigate the signalling pathways involved, we demonstrate that this acute inflammatory response was reduced with the use of inhibitors of NADPH oxidase (P < 0.001) and mitochondrial permeability (P < 0.05). Increased reactive oxygen species production was also seen at 120 mmHg in HUVECs perfused with the global ROS dye DCFH (60 mmHg: 3 ± 6 vs 120 mmHg: 149 ± 65 intensity units; n = 5–7; P < 0.05). Conclusion: We conclude that high intraluminal pressure induces vascular inflammation via circumferential stretch acting on mechano-sensors on the endothelium triggering the release of ROS and consequential adhesion molecule expression. Inhibiting these pathways may prove to be therapeutically beneficial for hypertensive patients to help reduce the risk of atherosclerosis.