Matrix metalloproteinases modulated by protein kinase Cε mediate resistin-induced migration of human coronary artery smooth muscle cells

Abstract

Background: Emerging evidence showed that resistin induces vascular smooth muscle cell (VSMC) migration, a critical step in initiating vascular restenosis. Adhesion molecule expression and cytoskeletal rearrangement have been observed in this progress. Given that matrix metalloproteinases (MMPs) also regulate cell migration, we hypothesized that MMPs may mediate resistin-induced VSMC migration.

Methods: Human VSMCs were treated with recombinant human resistin at physiologic (10 ng/mL) and pathologic (40 ng/mL) concentrations for 24 hours. Cell migration was determined by the Boyden chamber assay. MMP and tissue inhibitor metalloproteinase (TIMP) mRNA and protein levels were measured with real-time PCR and ELISA. MMP enzymatic activity was measured by zymography. In another experiment, neutralizing antibodies against MMP-2 and MMP-9 were coincubated with resistin in cultured VSMCs. The regulation of MMP by protein kinase C (PKC) was determined by εV1-2, a selective PKCε inhibitor.

Results: Resistin-induced smooth muscle cell (SMC) migration was confirmed by the Boyden chamber assay. Forty nanograms/milliliter resistin increased SMC migration by 3.7 fold. Additionally, 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, but selective PKCε inhibitor suppressed resistin-induced MMP expression, activity, and cell migration.

Conclusions: Our study confirmed that resistin increased vascular smooth muscle cell migration in vitro. In terms of mechanism, resistin-stimulated cell migration was associated with increased MMP expression, which was dependent on PKCε activation.

Figure 1

In vitro effect of resistin on human coronary artery smooth muscle cell migration in Boyden chamber assay. Two concentrations of resistin at physiological (10ng/mL) and pathological (40 ng/mL) concentrations were tested. TNF-alpha was used as a positive control. Values are expressed as the number of migrated cells, and each bar represents the mean±SEM of quadruplicate determinations.

Figure 2

Effect of resistin on MMP-2 and MMP-9 expression in human coronary artery smooth muscle cells. Resistin was tested at physiological (10ng/mL) and pathological (40 ng/mL) concentrations. Left panels, MMP-2 mRNA and protein data; right panels, MMP-9 mRNA and protein data. Values are expressed as percentage of the control, and each bar represents the mean±SEM of triplicate determinations in 2–3 independent experiments.

Figure 3

Effect of resistin on TIMP mRNA expression in human coronary artery smooth muscle cells. Resistin was tested at physiological (10ng/mL) and pathological (40 ng/mL) concentrations. Left panel, TIMP-1 mRNA data; right panel, TIMP-2 mRNA data. Values are expressed as percentage of the control, and each bar represents the mean±SEM of triplicate determinations in 2–3 independent experiments.

Figure 4

Effect of MMP-2 and MMP-9 antibodies on resistin-induced cell migration of human coronary artery smooth muscle cells (Boyden chamber assay). The migratory effect of resistin was exerted at the concentration of 40 ng/mL. Anti MMP-2 and MMP-9 as well as nonspecific IgG was tested at the same concentration of 10µg/mL. Values are expressed as the number of migrated cells, and each bar represents the mean±SEM of quadruplicate determinations.

Figure 5

Effects of resistin on PKCε activation and PKCε inhibitor (εV1–2) on resistin-induced PKCε activation. Values are expressed as percentage of the control, and each bar represents the mean±SEM of duplicate determinations in 2 independent experiments.

Figure 6

Effect of PKCε inhibitor on MMP and TIMP mRNA expressions in human coronary artery smooth muscle cells. Resistin was tested at the concentration of 40 ng/mL. Top panels, MMP mRNA data; bottom panels, TIMP mRNA data. Values are expressed as percentage of the control, and each bar represents the mean±SEM of triplicate determinations in 2 independent experiments.

Figure 7

Effect of PKCε inhibitor on MMP activity in human coronary artery smooth muscle cells. Resistin was tested at the concentration of 40 ng/mL. Left panel, MMP-2 activity; right panel, MMP-9 activity. Values are expressed as percentage of the control, and each bar represents the mean±SEM of duplicate determinations in 2 independent experiments.

Figure 8

Effect of PKCε inhibitor on resistin-induced cell migration of human coronary artery smooth muscle cells (Boyden chamber assay). The migratory effect of resistin was exerted at the concentration of 40 ng/mL. Values are expressed as the number of migrated cells, and each bar represents the mean±SEM of quadruplicate determinations.