Abstract 112: Cigarette Smoke Directly Produces a Pro-inflammatory/matrix remodeling Phenotype in Cerebral Vascular Smooth Muscle Cells via KLF4: A Potential Mechanism for Cerebral Aneurysm Pathogenesis

Abstract

Cigarette smoke is one of the most significant environmental risk factor for cerebral aneurysm formation. Inflammation, matrix degradation and vascular smooth muscle cell phenotypic modulation are thought to be important in aneurysm pathology yet molecular pathogenic mechanisms are unknown. We investigated the role of cigarette smoke in producing phenotypic modulation and inflammation in cerebral vascular smooth muscle cells (VSMCs) focusing on a potential pivotal role of the transcription factor, KLF4. Methods: Rat cerebral VSMCs were treated with Cigarette Smoke Extract (CSE) at 4 and 40 ug/ml for 4, 24 and 48 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. SMC marker genes (SM-alpha-Actin, 22-alpha and MHC), transcription co-activator, Myocardin, KLF4 and inflammatory/matrix remodeling genes (MMP-2, 3, 9, VCAM-1, MCP-1, and iNOS) were measured using qPCR. siRNA against KLF4 and non-specific siGFP were transfected in cultured VSMCs to examine a potential critical role for KFL4 in this process. As part of in vivo experiments, Pluronic Gel (40% w/v) containing 0.2 - 0.8 mg/ml CSE was applied to the adventitial surface of rat carotid arteries for 24-48 hours. Results: CSE significantly induced KLF4 expression at 4 hours (p<0.05 vs. baseline) preceding phenotypic modulation of VSMCs. CSE directly produced marked phenotypic modulation with decreased expression of SMC marker genes and myocardin while markedly increasing expression of inflammatory/matrix remodeling genes (p<0.05 vs. baseline). Strikingly, this profound phenotypic modulation was reversed with siKLF4. Similarly, decreased expression of marker genes and Myocardin with concomitant upregulation of KLF4 and inflammatory/matrix remodeling genes was noted following in vivo pluronic gel application containing CSE. The most notable increase was noted in MMPs, MCP-1, IL-1 and TNF-alpha expression (all p<0.05 vs. control). Conclusions: Cigarette smoke directly produces marked phenotypic modulation of cerebral vascular smooth muscle cells in vitro and in vivo promoting a pro-inflammatory/matrix remodeling phenotype. These effects appear to be mediated via KLF4. Inflammation, accelerated matrix remodeling and VSMC phenotypic modulation are implicated in human cerebral aneurysms. The molecular mechanisms activated by cigarette smoke outlined here may have critical implications for human cerebral aneurysm pathogenesis and may represent important targets for future therapy. Fig: Gene Expression.