Objectives Vascular smooth muscle cells (VSMCs) are known to undergo functional changes that can contribute to the pathogenesis of atherosclerosis and restenosis. Wnts are a family of secreted glycoproteins that bind to transmembrane Frizzled receptors and initiate signalling cascades with indispensable roles during cell migration, adhesion, proliferation, and survival. The aim of our study is to investigate the effect and mechanism of Wnt3a on VSMC migration and adhesion by activating the canonical Wnt signalling.
Methods Primary VSMCs were cultured by tissue adherence method and stimulated with recombinant Wnt3a (100 ng/ml). We performed transwell migration and wound healing assays to investigate the effect of Wnt3a treatment on VSMC migration. To observe the influence of Wnt3a on cell-matrix interactions, we performed adhesion assays. The levels of total β-catenin, phosphor-β-catenin (Ser675), total GSK-3β, phosphor-GSK-3β (Ser9), β1-integrin, and ILK protein expression was detected by western blots. Active β1-integrin and total β1-integrin expression on the surface of VSMCs was evaluated by flow cytometry.
Results The migratory ability of VSMCs treated with Wnt3a was significantly increased in the transwell migration assay (P ˂0.05). The number cells that migrated across the polycarbonate membrane was higher in the Wnt3a group (150.9 ± 11.0) than in the control group (77.6 ± 7.4, P ˂ 0.05). Adhesion assay demonstrated that the number of VSMCs that adhered to collagen type I was significantly greater in the Wnt3a-treated group (94.2 ± 7.0) than in the control group (62.2 ± 5.1, P˂0.05). Treatment of VSMCs with Wnt3a upregulated the expression of phosphor-β-catenin (Ser675), phosphor-GSK-3β (Ser9), and ILK, but the expression of total β-catenin, total GSK-3β, and β1-integrin was not significantly different between the two groups. VSMCs that were stimulated with Wnt3a for 3 days bound more active-β1-integrin antibody (5.96 ± 0.82%) than did unstimulated cells (4.76 ± 0.56%, P˂0.05). Flow cytometry results demonstrate that Wnt3a treatment activates β1-integrin without changing its expression levels.
Conclusions VSMCs migration and proliferation contribute to arterial wound repair and thickening of the intimal layer in atherosclerosis, restenosis and transplant vascular disease. These processes are influenced by cell adhesion to molecules present in the ECM, and regulated by the integrin family of cell-surface matrix receptors. An important signalling molecule acting downstream of integrin receptors is ILK. Evidence exists to establish ILK as a molecular adaptor protein linking integrins to the actin cytoskeleton and regulating actin polymerisation. In vitro, ILK can phosphorylate and inactivate GSK-3β to promote nuclear accumulation of β-catenin and then activate the canonical Wnt pathway. But activation of the canonical Wnt pathway how to influence the activity of ILK remains unclear. To test the involvement of canonical Wnt pathway in ILK activation, in this study, we treated cultured rat VSMCs with recombinant Wnt3a and checked ILK protein expression and β1-integrin activity. The results showed that Wnt3a can activate the canonical Wnt pathway and increase ILK protein expression and β1-integrin activity.
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