Abstract 108: Transcriptional and Epigenetic Regulation of MCP1 by Tumor Necrosis Factor-a and Cigarette Smoke: Key Mediators in Aneurysm Formation

Abstract

Background: Although the underlying pathophysiology involved in aneurysm formation and progression is unknown, both cigarette smoke and alterations in the inflammatory cascade play a key role. We investigated the role of TNF-α and cigarette smoke extract (CSE) induction of an inflammatory phenotype in cerebral vascular smooth muscle cells (VSMC) through regulation of Monocyte Chemoattrant Protein-1 (MCP-1) and Kruppel Like Factor-4 (KLF4). Methods: Cultured cerebral VSMC from rat Circle of Willis were treated with increasing doses of TNF-α and CSE. siRNA specific to KLF4 was transfected into cells, followed by TNF-α and CSE treatment. In vivo, experiments were performed by applying TNF-α and CSE infused pluronic gel to rat carotid artery adventitial surface. MCP1 activity was measured with qPCR. Aneurysm induction surgery was performed in rats, and mRNA from the Circle of Willis was measured with qPCR. Chromatin Immunoprecipitation (ChIP) was performed both in vitro and in vivo using anti-KLF4, anti-histone deacetylase 2 (HDAC2), and anti-histone 4 acetylation (H4Ac) antibodies. Results: TNF-α and CSE treatment for 24-hours increases the expression of MCP-1 both in vitro and in vivo. Treatment with siKLF4 abrogates this inflammatory phenotype. Similarly, increased expression of TNF-α, KLF4, and MCP-1 was found after aneurysm induction in vivo. TNF-α and CSE initiated KLF4 binding to the MCP-1 promoter region in ChIP assays. This increases H4 acetylation and decreases HDAC2 binding to the promoter region resulting in MCP-1 stimulation. Conclusion: TNF-αand CSE induce an inflammatory phenotype in cerebral VSMC by regulating the expression of MCP-1. KLF4 plays a critical role in this process through binding to the promoter region and induction of epigenetic changes favoring MCP-1 expression. MCP-1 upregulation and subsequent inflammatory cell recruitment is likely a key mechanism in cerebral aneurysm formation and may be an important target for future therapy.