Abstract 261: EBP50/NHERF-1 Regulates Basal and TNFα-induced Mitochondrial Dynamics in Vascular Smooth Muscle Cells

Abstract

Mitochondrial dysfunction has been associated with the phenotypic switch of VSMC and vascular disease. Changes in mitochondrial dynamics (fission and fusion) are linked to VSMC proliferation and metabolism during vascular remodeling. Mitochondrial fission and fusion are regulated by several key molecules, including dynamin-related protein 1 (Drp1) and mitofusin2 (Mfn2). We previously showed that scaffolding Ezrin-radixin-moesin binding phosphoprotein of 50 kDa (EBP50/NHERF1) increases inflammatory responses and proliferation of VSMC. These actions are mediated by the activation of PKCζ under inflammatory stimuli and the stabilization of the S-phase kinase associated protein 2 (Skp2), a component of an E3 ligase that promotes proliferation. Thus, EBP50 knockout (EBP50 -/- ) mice are protected neointimal hyperplasia following arterial injury. Here we test the hypothesis that EBP50 regulates mitochondrial dynamics and responses to inflammatory stimuli in vascular smooth muscle cells (VSMC). We found that EBP50 knockdown, by decreasing Skp2 levels, increased FoxO1 stability and nuclear localization leading to higher mitofusin-2 (Mfn-2) expression. In contrast, inhibition of FoxO1 reduced Mfn2 levels. High resolution morphological analysis with both TEM and confocal microscopy revealed that mitochondria were more elongated in EBP50 -/- than WT VSMC. In WT VSMC, TNFα induced PKCζ-mediated phosphorylation of Drp1. In contrast, EBP50 -/- VSMC exhibited significantly reduced Drp1 phosphorylation following TNFα treatment. Live-cell 3D imaging followed by morphological analysis showed that TNFα elicited rapid and significantly greater mitochondria fragmentation in WT compared to EBP50 -/- VSMC. Finally, EBP50 -/- VSMC exhibited lower extracellular acidification rate (ECAR) than WT VSMC consistent with their lower proliferation. Collectively, these findings delineate a new mechanism of regulation of mitochondrial dynamics by the scaffolding protein EBP50 in response to inflammatory stimuli. Therefore EBP50 can be viewed as a potential therapeutic target for vascular proliferative diseases.