Abstract TMP26: Binding of KLF4 and HDAC2 to The Promoter Region of SM22alpha Mediates Phenotypic Modulation In vitro and In vivo: Implications in Cerebral Aneurysm Formation

Abstract

Cerebral Vascular Smooth Muscle Cell (VSMC) phenotypic modulation appears to play an important role in cerebral aneurysm formation and progression, yet the molecular mechanisms involved remain unknown. We investigated the role of inflammatory cytokine, Tumor Necrosis Factor-alpha (TNF-α) and cigarette smoke in directly mediating phenotypic modulation in VSMC. We hypothesize that this may, in part, occur through the binding of the transcription factor, KLF4, to the promoter region of SM22alpha, thereby downregulating VSMC differentiation and contractile genes. This may be critical in the pathogenesis of cerebral aneurysm formation. Methods: Cultured cerebral VSMC from rat Circle of Willis were treated with increasing doses of TNF-α (10ng/ml & 50ng/ml) and cigarette smoke extract (CSE; 10ug/ml & 40ug/ml) from 2 to 24hrs. VSMC were transfected with siRNA specific to KLF4. In vivo experiments included application of pluronic gel (containing TNF-α & CSE) to rat carotid artery adventitia. Expression of SM22alpha and KLF4 was measured with qPCR. Chromatin Immunoprecipitation (ChIP) was performed both in vitro and in vivo after treatment (TNF-α and CSE) and incubation with anti-KLF4, anti-HDAC2, anti-H4Ac and anti-H3K9Ac. Results: Both TNF-α and CSE independently suppress SM22alpha and increase expression of KLF4 in vitro and in vivo. siKLF4 inhibits the effects of TNF-α and CSE on KLF4 and SM22alpha. ChIP assays demonstrate that KLF4 binds directly to the promoter region of SM22alpha and further recruits HDAC2 to the promoter region. This complex leads to histone modifications leading to decreased acetylation of H4 and H3K9, resulting in decreased expression of SM22alpha. Conclusion: KLF4 is a key mediator of TNF-α and CSE induced phenotypic modulation of cerebral VSMC. Phenotypic modulation has been implicated in cerebral aneurysm formation in humans. KLF4 dependent regulation of cerebral VSMC phenotypic modulation may be an underlying molecular mechanism involved in the pathogenesis of cerebral aneurysm formation and a target for future therapies.