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Background-Remodeling of the arterial extracellular matrix participates importantly in atherogenesis and plaque complication. Increased expression of the elastinolytic and collagenolytic enzyme cathepsin L (Cat L) in human atherosclerotic lesions suggests its participation in these processes, a hypothesis tested here in mice. Methods and Results-We generated Cat L and low-density lipoprotein receptor (LDLr) ϩ T cells, macrophages, and smooth muscle cells. Mechanistic studies showed that Cat L contributes to the degradation of extracellular matrix elastin and collagen by aortic smooth muscle cells. Smooth muscle cells from LDLr Ϫ/Ϫ Cat L Ϫ/Ϫ mice or those treated with a Cat L-selective inhibitor demonstrated significantly less degradation of elastin and collagen and delayed transmigration through elastin in vitro. Cat L deficiency also significantly impaired monocyte and T-lymphocyte transmigration through a collagen matrix in vitro, suggesting that blood-borne leukocyte penetration through the arterial basement membrane requires Cat L. Cysteine protease active site labeling demonstrated that Cat L deficiency did not affect the activity of other atherosclerosis-associated cathepsins in aortic smooth muscle cells and monocytes. Conclusions-Cat L directly participates in atherosclerosis by degrading elastin and collagen and regulates blood-borne leukocyte transmigration and lesion progression.

Rationale A high-fat diet accompanied by hypertriglyceridemia increases an individual’s risk for developing atherosclerosis. An early event in this process is monocyte recruitment through binding to VCAM-1 upregulated on inflamed arterial endothelium. Diets high in polyunsaturated fatty acids (PUFAs) may provide athero-protection by ameliorating this effect. Objective We investigated the acute regulation of VCAM-1 expression in human aortic endothelial cells (HAEC) in response to triglyceride-rich lipoproteins (TGRL) isolated from subjects following consumption of a high-fat meal. Methods and Results Postprandial TGRL isolated from 38 subjects were categorized as pro- or anti-atherogenic according to their capacity to alter the inflammatory response of HAEC. Pro-atherogenic TGRL increased expression of VCAM-1, ICAM-1, and E-selectin by ~20% compared to stimulation with TNFα alone, while anti-atherogenic TGRL decreased VCAM-1 expression by ~20% while still upregulating ICAM-1. The relative atherogenicity of TGRL positively correlated with particle density of TG, ApoCIII, ApoE, and cholesterol. Ω3-PUFA mimicked the effect of anti-atherogenic TGRL by down-regulating VCAM-1 expression. TGRL exerted this differential regulation of VCAM-1 by reciprocally modulating expression and activity of the transcription factor IRF-1 and expression of microRNA 126 (miR-126). Overexpression or silencing of IRF-1 or miR-126 expression recapitulated the pro- or anti-atherogenic regulation of VCAM-1. Conclusions In response to a high-fat meal, TGRL bias the inflammatory response of endothelium via transcriptional and post-transcriptional editing of VCAM-1. Subjects with an anti-inflammatory response to a high-fat meal produced TGRL that was enriched in non-esterified fatty acids, decreased IRF-1 expression, increased miR-126 activity, and diminished monocyte arrest.

Background Endothelial dysfunction and monocyte migration are key events in the pathogenesis of atherosclerosis. Non-muscle myosin light-chain kinase (nmMLCK), the predominant MLCK isoform in endothelial cells, has been shown to contribute to vascular inflammation by altering endothelial barrier function. However, its impact on atherogenesis remains unknown. Methods and Results We investigated the role of nmMLCK in the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE−/−) mice fed an atherogenic diet for 12 weeks. Histopathological examination demonstrated that nmMLCK deficiency (apoE−/− nmmlck−/−) reduced the size of aortic lesions by 53%, lipid contents by 44% and macrophage deposition by 40%. Western blotting and reverse-transcription polymerase chain reaction revealed the expression of nmMLCK in aortic endothelial cells and peripheral blood monocytes. Measurements of transendothelial electric resistance indicated that nmMLCK deficiency attenuated endothelial barrier dysfunction caused by thrombin, oxLDL and TNF-α. In monocytes, nmMLCK deficiency reduced their migration in response to the chemokine MCP-1. Further mechanistic studies showed that nmMLCK acted through both myosin light chain (MLC) phosphorylation-coupled and -uncoupled pathways; the latter involved Src signaling. Moreover, depletion of Src via gene silencing, site-specific mutagenesis or pharmacological inhibition of Src greatly attenuated nmMLCK-dependent endothelial barrier dysfunction and monocyte migration. Conclusions nmMLCK contributes to atherosclerosis by regulating endothelial barrier function and monocyte migration via mechanisms involving not only kinase-mediated MLC phosphorylation but also Src activation.

The data provide evidence for a novel function of ADAM15 in regulating endothelial barrier properties. The mechanisms of ADAM15-induced hyperpermeability involve Src/ERK1/2 signalling independent of junction molecule shedding.

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Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms1,2. Interruption of IL18 action reduces atherosclerosis in mice3,4. This study shows that the absence of IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E-deficient (Apoe−/−) mice, nor does it affect IL18 cell surface binding or signaling. IL18 antibody-mediated immunoprecipitation identified an interaction between IL18 and Na-Cl co-transporter (NCC), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney5. Yet, we find NCC expression and colocalization with IL18r in atherosclerotic lesions and both molecules form a complex. IL18 also binds to the cell surface and induces cell signaling and down-stream cytokine expression in NCC-transfected COS-7 cells that do not express IL18r. In Apoe−/− mice, combined deficiency of IL18r and NCC, but not single deficiency, protects mice from atherosclerosis. Peritoneal macrophages from Apoe−/− mice or those lacking IL18r or NCC respond to IL18 binding or IL18 induction of cell signaling and cytokine and chemokine production, but those with combined deficiency of IL18r and NCC do not. This study identifies NCC as an IL18-binding protein that coordinates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

DeVerse JS, Sandhu AS, Mendoza N, Edwards CM, Sun C, Simon SI, Passerini AG. Shear stress modulates VCAM-1 expression in response to TNF-␣ and dietary lipids via interferon regulatory factor-1 in cultured endothelium. Am J Physiol Heart Circ Physiol 305: H1149-H1157, 2013. First published August 9, 2013; doi:10.1152/ajpheart.00311.2013.-Dyslipidemia is a primary risk factor for cardiovascular disease, but the specific mechanisms that determine the localization of atherosclerotic plaques in arteries are not well defined. Triglyceride-rich lipoproteins (TGRL) isolated from human plasma after a high-fat meal modulate TNF-␣-induced VCAM-1 expression in cultured human aortic endothelial cells (HAECs) via an interferon regulatory factor (IRF)-1-dependent transcriptional mechanism. We examined whether fluid shear stress acts as a mediator of IRF-1-dependent VCAM-1 expression in response to cytokine and dietary lipids. IRF-1 and VCAM-1 were examined by immunofluorescence in TNF-␣-stimulated HAEC monolayers exposed to TGRL and a linear gradient of shear stress ranging from 0 to 16 dyn/cm 2 in a microfluidic device. Shear stress alone modulated TNF-␣-induced VCAM-1 expression, eliciting a 150% increase at low shear stress (2 dyn/cm 2 ) and a 70% decrease at high shear stress (12 dyn/cm 2 ) relative to static. These differences correlated with a 60% increase in IRF-1 expression under low shear stress and a 40% decrease under high shear stress. The addition of TGRL along with cytokine activated a fourfold increase in VCAM-1 expression and a twofold increase in IRF-1 expression. The combined effect of shear stress and TGRL on the upregulation of membrane VCAM-1 was abolished by transfection of HAECs with IRF-1-specific small interfering RNA. In a healthy swine model, elevated levels of endothelial IRF-1 were also observed within atherosusceptible regions of the aorta by Western blot analysis and immunohistochemistry, implicating arterial hemodynamics in the regulation of IRF-1 expression. These data demonstrate direct roles for fluid shear stress and postprandial TGRL from human serum in the regulation of IRF-1 expression and downstream inflammatory responses in HAECs.atherosclerosis; hemodynamics; hypertriglyceridemia; endothelial dysfunction; inflammation; vascular cell adhesion molecule 1; tumor necrosis factor-␣ ATHEROSCLEROSIS is a multifactorial disease characterized by the focal accumulation of lipid-rich plaques at arterial sites that correlate strongly with regions of disturbed blood flow. Diets high in saturated and nonesterified fats contribute to a state of metabolic dysregulation that is associated with chronic lowgrade inflammation and activation of stress responses that accelerate the progression of atherosclerosis (18,21). Elevated levels of circulating lipoproteins and cytokines, such as TNF-␣, contribute to inflammatory responses that culminate in the preferential recruitment of leukocytes to the endothelium via the upregulation of cell adhesion molecules, including VCAM-1 (24). Despite the recognition that...