Abstract 374: Epigenetic Mechanisms Regulate TNF-α-Induced Phenotypic Modulation in Cerebral Vascular Smooth Muscle Cells: Implications for Cerebral Aneurysm Biology

Abstract

Objectives: Inflammation and cerebral Vascular Smooth Muscle Cell (VSMC) phenotypic modulation appear to be involved in cerebral aneurysm formation. Tumor Necrosis Factor-alpha (TNF-α) is found in increased quantity in ruptured and un-ruptured cerebral aneurysms. Our previous work demonstrates that TNF-α potently induces cerebral VSMC phenotypic modulation . We explored the underlying mechanisms involved in these changes. Methods: Rat cerebral VSMCs were treated with TNF-alpha at 50 ng/ml (based on preliminary data) for 2, 6 and 24 hours. Cells at 80-90% confluence were fixed with formaldehyde. DNA was sheared using sonication. Chromatin immune-precipitation was performed using the following antibodies: 1) anti-KLF4 (Krupple Like Factor 4), 2) anti-HDAC2 (Histone Deacetylase 2), 3) anti-H3K9Ac (Histone 3 Lysine 9 Acetylation), 4) anti-H3K27triMe (Histone 3 Lysine 27 Tri-Methylation) and 5) anti-H4Ac (Histone 4 Acetylation). qPCR was performed with primers specific to CArG containing promoter regions of differentiation marker genes {(alpha-Actin and Myosin Heavy Chain (MHC)} and Myocardin. Results were normalized against input DNA. As part of in vivo experiments Pluronic Gel (40% w/v) containing TNF-alpha at 30-60 ug/ml was applied to rat carotid vessels for 6-8 hours. Vessels were harvested and frozen in liquid nitrogen and chromatin immune-precipitation was performed. Results: TNF-alpha stimulation promoted profound phenotypic modulation of cerebral VSMCs. We demonstrated an increased percent enrichment of alpha-actin, MHC and Myocardin promoters with KLF4, HDAC2, and H3K27triMe antibodies and decreased enrichment with H3K9Ac antibody. H4Ac antibody showed decreased enrichment of alpha-Actin and MHC promoter in vivo. Hence, there is evidence of direct binding of the transcription factor, KLF4, to marker gene promoter regions. Along with direct effects, KLF4 exerts indirect effects via binding with HDAC2 which deactylates H3 and H4 histones. Although, KLF4 demonstrated increased binding at 24 hours but HDAC2 did not. HDAC2 binding was more significant at early time points. These effects promote heterochromatin formation and decrease SRF binding to VSMC gene CArG box chromatin which in turn results in decreased transcription of marker genes and phenotypic modulation. Conclusions: We have demonstrated underlying molecular mechanisms involved in repression of cerebral VSMCs marker genes following exposure to TNF-alpha. TNF-alpha has a role in inflammation, matrix degradation and phenotypic modulation. This suggests a novel mechanism whereby epigenetic control of chromatin structure may play an important role in regulating VSMC gene expression in response to inflammatory cytokines like TNF-alpha which has implications for the pathogenesis of cerebral aneurysms.