Emerging evidence showed that resistin induces vascular smooth muscle cell (VSMC) migration, a critical step to initiating vascular restenosis. Mechanistically, adhesion molecule expression and cytoskeletal rearrangement have been observed in this progress. Given that matrix metalloproteinases (MMPs) also regulates cell migration, we hypothesized that MMPs may mediate resistin-induced VSMC migration.
Materials and Methods
Human VSMCs were treated with recombinant human resistin at physiological (10 ng/mL) and pathological (40 ng/mL) concentrations for 24 hours. Cell migration was determinate by Boyden chamber assay. MMP and TIMP mRNA and protein levels were measured with real-time PCR and ELISA. MMP enzymatic activity was measured by zymography on precast gels. In another experiment, neutralizing antibodies against MMP-2 and MMP-9 were co-incubated with resistin in cultured VSMCs. The regulation of MMP by protein kinase C (PKC) was determined by εV1–2, a selective PKCε inhibitor.
Resistin-induced SMC migration was confirmed by Boyden chamber assay. 40ng/mL Resistin increased SMC migration by 3.7 fold. Molecularly, resistin stimulated MMP-2 and - MMP9 mRNA and protein expressions. In contrast, the TIMP-1 and TIMP-2 mRNA levels were inhibited by resistin. Neutralizing antibodies against MMP-2 and MMP-9 effectively reversed VSMC migration. Furthermore, resistin activated PKCε and selective PKCε inhibitor suppressed resistin-induced MMP expression, activity and cell migration.
Our study confirmed that resistin increases vascular smooth muscle cell migration in vitro. Mechanistically, resistin-stimulated cell migration was associated with increased MMP expression and activity, which was dependent on PKCε activation.