Vascular smooth muscle cells (SMCs) are the major cell type within blood vessels. SMCs exhibit low rates of proliferation, migration, and apoptosis in normal blood vessels. However, increased SMC proliferation, migration, and apoptosis rates radically alter the composition and structure of the blood vessel wall and contribute to cardiovascular diseases, such as atherosclerosis, and restenosis that occur after coronary artery vein grafting and stent implantation. Consequently, therapies that modulate SMC proliferation, migration, and apoptosis may be useful for treating cardiovascular diseases. The family of Wnt proteins, which were first identified in the wingless drosophila, has a well-established role in embryogenesis and development. It is now emerging that Wnt proteins also regulate SMC proliferation, migration, and survival. In this review article, we discuss recently emerging research that has revealed that Wnt proteins are important regulators of SMC behaviour via activation of β-catenin-dependent and β-catenin-independent Wnt signalling pathways.

Objective-Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. Approach and Results-Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs.Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via β-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. was not translated into protein, and recombinant Wnt2 (rWnt2) protein did not increase VSMC proliferation in vitro. 5 The Wnt pathway has also been shown to have a role in cell migration, with Wnt3a involvement in both migration and adhesion of VSMCs through integrin linked kinase regulation of β1-integrin.10 However, on initiation of this study, it was unclear which Wnt proteins modulated VSMC growth factor-induced migration and whether Wntinduced VSMC migration promoted intimal thickening in vivo. Many genes are upregulated by the Wnt pathway, some of which are known to modulate VSMC migration, including Wnt-1-inducible signaling pathway protein-1 (WISP-1/CCN4 Materials and MethodsMaterials and Methods are available in the online-only Data supplement. Nonstandard Abbreviations and Acronyms Results Wnt2 Was Upregulated in Migrating VSMCs In VitroWnt mRNA levels during VSMC migration were assessed using an in vitro scratch wound assay with multiple wounds to stimulate migration of the VSMCs. mRNA was extracted from VSMCs and applied to a focussed Wnt pathway microarray to assess whether the level of expression changed during migration. A significant increase was only observed in Wnt2 mRNA ( Figure 1A), and this change was confirmed using quantitative polymerase chain reaction ( Figure 1A). No significant change was seen in the mRNA levels of any other Wnts (see Table III in the online-only Data Supplement). It was observed by Western blotting ( Figure 1B) and immunocytochemistry ( Figure I in the online-only Data Supplement) that the increase in Wnt2 mRNA was translated into augmented Wnt2 protein levels in migrating VSMCs. Migrating VSMCs on the wound edge could be seen to express higher levels of Wnt2 protein than nonmigratory VSMCs further away from the wound edge ( Figure I in the online-only Data Supplement). Wnt2 Promoted VSMC Migration In VitroAddition of rWnt2 protein significantly increased VSMC migration in vitro, whereas knockdown of Wnt2 using silencing RNA (siRNA) inhibited migration ( Figure 1C). When rWnt2 was ad...

A wealth of evidence has been presented for the involvement of the Wnt pathways in vascular development. Although less evidence exists for the regulation of vascular disease by the Wnt pathways, sufficient evidence exists to propose these pathways act as an important regulator of vascular disease. A greater understanding of Wnt pathways may reveal new therapeutic targets for vascular disease.

Objective-Apoptosis of vascular smooth muscle cells (VSMCs) contributes to thinning and rupture of the atherosclerotic plaque fibrous cap and is thereby associated with myocardial infarction. Wnt protein activation of β-catenin regulates numerous genes that are associated with cell survival. We therefore investigated Wnt/β-catenin survival signaling in VSMCs and assessed the presence of this pathway in human atherosclerotic plaques at various stages of the disease process. Approach and Results-Wnt5a induced β-catenin/T-cell factor signaling and retarded oxidative stress (H 2 O 2 )-induced apoptosis in mouse aortic VSMCs. Quantification of mRNA levels revealed a >4-fold (P<0.05; n=9) increase in the expression of the Wnt/β-catenin responsive gene, Wnt1-inducible secreted protein-1 (WISP-1), which was dependent on cAMP response element-binding protein and sustained in the presence of H 2 O 2 . Exogenous WISP-1 significantly reduced H 2 O 2 -induced apoptosis by 43% (P<0.05; n=3) and was shown using silencing small interfering RNA, to be important for Wnt5a-dependent survival responses to H 2 O 2 (P<0.05; n=3). WISP-1 protein levels were significantly lower (≈50%) in unstable atherosclerosis compared with stable plaques (n=11 and n=14). Conclusions-These results indicate for the first time that Wnt5a induces β-catenin survival signaling in VSMCs viaWISP-1. The deficiency of the novel survival factor, WISP-1 in intimal VSMCs of unstable coronary plaques, suggests that there is altered Wnt/β-catenin/ T-cell factor signaling with progressive atherosclerosis, and restoration of WISP-1 protein might be an effective stabilization factor for vulnerable atherosclerotic plaques. Deregulated Wnt signaling is associated with many diseases including cancer 22 ; however, its involvement in atherosclerosis remains to be fully elucidated.It has been shown previously that Wnt5a is expressed in atherosclerotic plaques, predominantly in macrophages. 25,26 The effect of Wnt5a in atherosclerotic plaques, and its role as a survival factor for VSMCs, is however still to be determined. In this study, we investigated the effect of exogenous Wnt5a on canonical Wnt pathway activation in VSMCs and directly assessed the effect that Wnt5a had on VSMC survival under conditions of oxidative stress. We highlighted an association of Wnt1-inducible secreted protein-1 (WISP-1) expression with atherosclerotic plaque stability and propose that it is a distinct survival factor, which has potential as a plaque stabilizing protein. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results Activation of Wnt/β-Catenin Signaling in VSMCs by Wnt5aWnt5a protein was not detectable in mouse or human VSMC lysates or conditioned media but was readily detectable in macrophage lysates ( Figure IA and IB in the online-only Data Supplement). This was confirmed by quantitative polymerase chain reaction ( Figure IC in the online-only Data Supplement). Moreover, treatment with Wnt5a or H 2 O 2 did not induce expression of Wnt5a prot...