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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.