Abstract 88: Blockade of ETS-1 Attenuates EGF-Induced Collagen Loss in Carotid Plaques of Patients With Carotid Stenosis

Abstract

Introduction: Coronary thrombosis and rupture of plaques leading to stroke are major causes of morbidity and mortality. Here, we examined the role of v-ets erythroblastosis virus E26 oncogene homologue-1 (ETS-1) on the epidermal growth factor (EGF)-induced destabilization of collagen type I (Col-Iα1) and collagen type III (Col-IIIα1) involving matrix metalloproteinase-1 (MMP-1) and MMP-9 in isolated vascular smooth muscle cells (VSMCs) from the plaques of symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. To understand the molecular mechanisms underlying EGFR-regulated ETS-1, the signaling pathways directing ETS-1 production was investigated and examined if EGF-induced loss of fibrillar collagens in patients with carotid stenosis is regulated by ETS-1. Methods: VSMCs isolated from carotid plaques of both AS and S patients were treated with or without EGF. The mRNA transcripts and protein for ETS-1, MMP-9 and -1, EGFR, Col-I(α1) and Col-III(α1) were analyzed by qPCR and immunofluorescence, respectively. Results: Treatment of VSMCs with EGF significantly increased ETS-1 expression and immunoreactivity in S compared to AS. EGFR inhibitor (AG1478) decreased ETS-1 expression and upregulated Col-I(α1) and Col-III(α1) in EGF-treated VSMCs of both groups. In the transfection experiments, overexpression of ETS-1 in VSMCs increased mRNA transcripts of MMP-9 and MMP-1 and decreased the collagen mRNA transcripts. Knock-down of ETS-1 gene with either siRNA or transfection with dominant-negative form (ETS-DN) blocked EGF-induced MMP-1 and MMP-9 expression and increased mRNA transcripts for Col-I(α1) and Col-III(α1) in S compared to AS group. Inhibitors of p38MAPK (SB202190) and JNK-II (SP600125) decreased ETS-1, MMP-1 and MMP-9 transcripts and increased collagen transcripts in EGF-treated VSMCs, suggesting that p38MAPK and JNK pathways are involved in EGFR-induced collagen loss. Conclusion: These findings revealed a novel mechanism of ETS-1-regulated EGF-induced collagen loss in human carotid plaques and this could be a leading cause of plaque instability in patients with carotid stenosis. Selective blockade of ETS-1 and EGFR may be novel strategy and promising target for treating unstable and vulnerable plaques.