Background— Activated macrophages contribute to the pathogenesis of inflammatory diseases such as atherosclerosis. Although Notch signaling participates in various aspects of immunity, its role in macrophage activation remains undetermined. Methods and Results— To explore the role of Notch signaling in inflammation, we examined the expression and activity of Notch pathway components in human primary macrophages in vitro and in atherosclerotic plaques. Macrophages in culture express various Notch pathway components including all 4 receptors (Notch1 to Notch4). Notch3 selectively increased during macrophage differentiation; however, silencing by RNA interference demonstrated that all receptors are functional. The ligand Delta-like 4 (Dll4) increased in macrophages exposed to proinflammatory stimuli such as lipopolysaccharide, interleukin-1β, or minimally-modified low-density lipoprotein in a Toll-like receptor 4– and nuclear factor-κB–dependent fashion. Soluble Dll4 bound to human macrophages. Coincubation of macrophages with cells that expressed Dll4 triggered Notch proteolysis and activation; increased the transcription of proinflammatory genes such as inducible nitric oxide synthase, pentraxin 3 and Id1; resulted in activation of mitogen-activated protein kinase, Akt, and nuclear factor-κB pathways; and increased the expression of Dll4 in macrophages. Notch3 knockdown during macrophage differentiation decreased the transcription of genes that promote inflammation, such as inducible nitric oxide synthase, pentraxin 3, Id1, and scavenger receptor-A. These in vitro findings correlate with results of quantitative immunohistochemistry, which demonstrated the presence of Dll4 and other Notch components within macrophages in atherosclerotic plaques. Conclusion— Dll4-triggered Notch signaling may mediate inflammatory responses in macrophages and promote inflammation.

Abstract-We have previously shown that long-term treatment with an inflammatory cytokine from the adventitia causes the development of coronary vascular lesions, with the accumulation of macrophages. Recent studies in vitro have suggested that small G-protein Rho and its effector, Rho-kinase/ROK/ROCK, may be the key molecules for various cellular functions, including cell adhesion and movement. In this study, we examined whether adventitia-derived macrophages cause the formation of coronary vascular lesions in vivo and, if so, whether Rho-kinase is involved in the process. Porcine coronary segments from the adventitia were chronically treated with monocyte chemoattractant protein-1 alone, oxidized low density lipoprotein alone, or both. Vascular lesion formation (neointimal formation and development of vascular remodeling) was mostly enhanced at the coronary segment cotreated with monocyte chemoattractant protein-1 and oxidized low density lipoprotein, where the phosphorylation of myosin binding subunit of myosin phosphatase was increased, indicating an increased activity of Rho-kinase in vivo. Histological examination demonstrated that macrophages were accumulated at the adventitia and thereafter migrated into the vascular wall. Long-term oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor (hydroxyfasudil) after oral absorption, markedly inhibited the myosin binding subunit phosphorylation, the macrophage accumulation and migration, and the coronary lesion formation in vivo. These results indicate that Rho-kinase is involved in macrophage-mediated formation of coronary vascular lesions in our porcine model in vivo. (Arterioscler Thromb Vasc