Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Caffeine inhibits the proliferation of vascular smooth muscle cells (VSMCs); however, little is known about the mechanism(s). Here, we demonstrated that caffeine decreased VSMC proliferation and induced autophagy in an in vivo vascular injury model of restenosis. Further, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of mTOR signaling in these cells. Genetic deletion of the key autophagic gene, ATG5, and its adaptor protein, SQSTM1/p62, showed the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased Wntsignaling and the expression of two Wnt target genes, AXIN2 and Cyclin D1. This effect was mediated by autophagic degradation of a key member of the Wnt signaling cascade, DVL2, by caffeine to decrease Wnt signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and Dvl2were also shown to interact with each other, and the overexpression of Dvl2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings have demonstrated that induction of autophagy by caffeine significantly reduced vascular restenosis.Caffeine reduced VSMC proliferation by inhibiting Wnt signaling via stimulation of autophagy.Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.

We previously conducted genetic analyses, and identified semaphorin signaling as associating with coronary artery disease. Of the semaphorins, human vascular expression profiling suggested SEMA3F as potentially linked to atherogenesis. In hyperlipidemic mice, SEMA3F reduced aortic lesion area, and increased fibrous cap endothelial content, leading to plaque stability. In a disturbed-flow-mediated endothelial dysfunction-driven lesion model, the absence of Sema3f increased plaques, further implicating SEMA3F in endothelial function. Monocyte adhesion to Sema3f-/- vascular endothelial cells (VECs) was elevated, driven by increased PI3K activity, leading to increased NF-κB-mediated elevation in VCAM1 and ICAM1 expression, suggesting that SEMA3F reduces VEC PI3K activity. Increased permeability led to increased monocyte transmigration through Sema3f-/- VECs, and mTOR phosphorylation was decreased, suppressing VE-cadherin expression and cell-cell adherens junction stability. Actomyosin fiber formation was decreased in Sema3f-/- VECs, which was reversed by PI3K inhibition, further implicating SEMA3F in adherens junction stability. In Sema3f-/- vascular smooth muscle cells (VSMCs), active PI3K was also increased. PI3K facilitates VSMC proliferation, migration, and pro-atherogenic phenotype switching, which were reduced by SEMA3F. In agreement, in a model of VSMC proliferation and migration-induced neointima formation, SEMA3F reduced plaques. Semaphorin3F is causally atheroprotective. SEMA3F’s suppression of VEC and VSMC PI3K activation may contribute to its atheroprotection.