Abstract 372: Regulation of Nox1 and Nox2 by Cigarette Smoke in Cerebral Vascular Smooth Muscle Cells: A Role of Oxidative Stress in Cerebral Aneurysm Formation

Abstract

Objectives: Cigarette smoke is one of the most significant environmental risk factor for cerebral aneurysm formation and progression. Factors including inflammation and matrix degradation are considered to be important in aneurysm formation. Reactive Oxygen Species (ROS) play a pathophysiological role in vascular inflammation, proliferation, migration and angiogenesis. Among the major sources of ROS in vascular smooth muscle cells (VSMCs) are NAD(P)H oxidase (Nox). We investigated the role of Cigarette Smoke Extract (CSE) in regulation of Nox1 and Nox2 isoforms in cerebral VSMCs both in vitro and in vivo. Methods: Rat cerebral VSMCs were treated with CSE at 10 and 40 ug/ml for 4 and 24 hours. The level of water soluble components of cigarette smoke and nicotine levels present in the CSE overlap with plasma levels of these constituents in human smokers. RNA was extracted and qPCR was performed. Cerebral VSMCs were transfected with siNox1 and siNox2 for 24 and 72 hours and further incubated with CSE for 24 hours. As part of in vivo studies, Pluronic Gel (40% w/v) containing 0.2-0.8 mg/ml of CSE was applied to the adventitial surface of rat carotid arteries for 24 hours. Vessels were harvested for mRNA quantification. Results: CSE decreased the expression of VSMC marker genes and myocardin, increased expression of KLF4 (a transcription factor) and proinflammatory/matrix remodeling marker genes including MMPs, VCAM1 and MCP1. In addition CSE increased the expression of both Nox1 and Nox2 at 4 and 24 hours but was more significant at 4h. Preliminary studies with siNox1 and siNox2 decreased the expression of KLF4, and reversed inflammatory markers gene expression caused by CSE stimulation. siNox1 increased the expression of marker genes including alpha-Actin and 22-alpha. Expression of Nox1 and Nox2 was markedly increased following in vivo application of pluronic gel. Conclusions: These findings indicate that cerebral VSMCs contain Nox1 and Nox2, are potently regulated by CSE and play an important role in NAD(P)H oxidase driven ROS production. CSE induces marked phenotypic modulation of vertebral VSMC while concomitantly increasing expression of Nox1 and Nox2. Furthermore, alteration of Nox expression abrogates aspects of CSE induced VSMC phenotypic modulation. These molecular changes implicate oxidative stress in vascular pathology underlying cerebral aneurysms and may provide a potential target for future therapeutic strategies.