Search

Coronary artery disease (CAD) remains a leading cause of death worldwide. To identify genes causally linked to CAD, we previously conducted large-scale genetic analyses, and identified an association between genes in the semaphorin pathway and CAD. Expression profiling in human atherosclerosis-relevant cell types and atherosclerotic plaques identified semaphorin3F (SEMA3F) as a possible candidate causally linked to atherosclerosis. Administration of SEMA3F reduced aortic lesion area in western-type diet-fed Ldlr-/-mice (Control: 12.1±1.4; SEMA3F: 8.1±0.8, % lesion area/total aortic sinus area, p=0.02). In contrast, neointimal formation was increased in Sema3f-/-mice with partial carotid artery ligation (WT: 22.8±2.8; Sema3f-/-: 39.2±4.0; % intimal area/total vessel area, p=0.004). Additionally, SEMA3F administration increased re-endothelialization and reduced restenosis in Ldlr-/-mice that underwent carotid endothelial denudation (Control: 53.3±3.5; SEMA3F: 21.4±3.1; % intimal plaque area/vessel area, p=0.0006). Monocyte adhesion to aortic vascular endothelial cells (VECs) from Sema3f-/-mice was elevated, driven by increased NF-κB activation and increased expression of its target adhesion molecules, VCAM1 and ICAM1, in VECs. Monocyte transmigration through VECs from Sema3f-/-mice was also increased, likely due to increased membrane permeability, assessed by in-vivointravital two-photon microscopy. The increased membrane permeability was modulated in part by decreased mTOR phosphorylation that in turn suppressed vascular endothelial cadherin protein expression. SEMA3F increased thick actomyosin filaments and elicited larger focal adhesions in VECs, suggesting decreased migration. In line with this, SEMA3F inhibited vascular smooth muscle cell (VSMC) migration and maintained the contractile phenotype of VSMCs, which was mediated by the Rho pathway. SEMA3F is causally atheroprotective in models of atherosclerosis and restenosis, mediated, in part, through the modulation of vascular endothelial cell permeability and adhesion molecule expression, and vascular smooth muscle cell migration and phenotype switching.

Objectives:Accumulating evidence suggests a role of semaphorin 3F (Sema3F) in heart development, immune responses and tumor growth inhibition. Genetic analyses suggest that semaphorin signaling associates with coronary artery disease, but the roles of specific semaphorin subtypes remain unexplored. Here, we aim to elucidate a potentially novel role for Sema3F in atherosclerosis.Methods and Results:Atherosclerosis was induced via partial ligation of the common carotid in 12 week old Sema3F-/-and litter-mate control mice fed with the Western type diet (WTD) for 5 weeks. Deletion of Sema3F significantly increased atherosclerotic lesion area in the Sema3F-/-mice (48% increase, p=0.008). Conversely, exogenous Sema3F protein was injected (100ng/kg, ip, 3X weekly) into 12 week old WTD-fed Ldlr-/-mice that had undergone carotid endothelial cell denudation via wire injury for 3 weeks to establish restenosis-driven plaque formation. Sema3F overexpression significantly reduced plaque formation in this model (56% decrease, p=0.0006). Macrophage numbers were significantly lower in plaques from the wire-injured mice overexpressing Sema3F (66% lower, p=0.001), with no changes in smooth muscle cell number. Since macrophage numbers were decreased in the plaques of the mice over-expressing Sema3F, we tested the impact of endothelial Sema3F on monocyte adhesion/migration utilizing a micro-engineered perfusable 3D vasculature model. Aortic endothelial cells from Sema3F-/-and WT mice were seeded onto the flow chamber and allowed to form a confluent monolayer, followed by perfusion with THP-1 monocytes. Significantly greater adhesion/migration of monocytes to endothelial cells from Sema3F-/-were found (50% increase, p=0.01), suggesting that Sema3F mediates its protective effects on atherosclerosis in part through inhibiting monocyte/macrophage infiltration.Conclusions:These data show that Sema3F plays a significant and potentially causal role in atherosclerosis/restenosis, and establish a novel role for semaphorins in large vessel disease. Approaches to increase Sema3F levels may represent a novel therapeutic avenue for the prevention and treatment of atherosclerosis.