M. C. Zuniga2, G. Raghuraman2, W. Zhou1,2 1Stanford University,Vascular Surgery,Palo Alto, CA, USA 2VA Palo Alto Healthcare Systems,Surgical Services,Palo Alto, CA, USA
Introduction: Dysfunction of vascular smooth muscle cells (VSMCs) is a key process in vascular restenosis and atherosclerotic plaque formation. VSMCs are known to transform from a differentiated contractile state to a dedifferentiated synthetic state, characterized by increased proliferative and migratory capacity. Adipokine resistin, found elevated in obese and diabetic patients, has been suggested as a risk factor for cardiovascular complications, and we have shown that PKC-epsilon (PKC-ε) is involved in resistin-induced VSMC migration. In this study we examined the role of PKC-ε and its time-dependent effects on resistin-induced VSMC dysfunction.
Methods: Primary human coronary artery smooth muscle cells were treated with resistin at pathological plasma concentration, 40 ng/mL, for 0–7 days, and selective PKC-ε inhibitor (εV1-2) and activator (ψεRACK) were used at a 1 µM concentration. Migration was quantified with the Boyden chamber assay. Dedifferentiation was evaluated by examining protein expression of the VMSC lineage markers, smoothelin and SM22α, and the structural markers, α-actin and calponin. Cell proliferation was determined by computing VSMCs growth over a period of 7 days. Data was analyzed with one-way ANOVA, and a p-value of <0.05 was considered significant.
Results: Resistin induced more than a 2-fold increase in VSMC migration as compared to the untreated control (Figure 1 c). Significant increase in VSMC proliferation and significant decreased expression of all VSMC markers was observed after 3 days of treatment (Figure 1 a-b). Timelines for ψεRACK showed that short pre-treatment (30 min) had a protective effect in resistin-induced VSMC migration whereas prolonged pre-treatment (≥ 6 h) augmented resistin-mediated effects. On the other hand, εV1-2 required longer treatment times (≥ 12 h) to mitigate resistin-induced VSMC migration. Combined exposures (ψεRACK for 30 min; then εV1-2 during assay time) significantly decreased resistin-induced migration and proliferative growth to similar levels of untreated controls (Figure 1 b-c).
Conclusion: Resistin promoted VSMC migration, proliferation, and loss of lineage and structural markers. We demonstrated that acute PKC-ε activation followed by prolonged PKC-ε inhibition may provide an optimal therapeutic strategy for resistin-induced VSMC restenosis.
