TNF-α Induces Phenotypic Modulation in Cerebral Vascular Smooth Muscle Cells: Implications for Cerebral Aneurysm Pathology

Abstract

Little is known about vascular smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. Tumor necrosis factor-alpha (TNF-a) has been associated with aneurysms, but potential mechanisms are unclear. Cultured rat cerebral SMCs overexpressing myocardin induced expression of key SMC contractile genes (SM-a-actin, SM-22a, smooth muscle myosin heavy chain), while dominant-negative cells suppressed expression. Tumor necrosis factor-alpha treatment inhibited this contractile phenotype and induced pro-inflammatory/matrix-remodeling genes (monocyte chemoattractant protein-1, matrix metalloproteinase-3, matrix metalloproteinase-9, vascular cell adhesion molecule-1, interleukin-1 beta). Tumor necrosis factor-alpha increased expression of KLF4, a known regulator of SMC differentiation. Kruppel-like transcription factor 4 (KLF4) small interfering RNA abrogated TNF-a activation of inflammatory genes and suppression of contractile genes. These mechanisms were confirmed in vivo after exposure of rat carotid arteries to TNF-a and early on in a model of cerebral aneurysm formation. Treatment with the synthesized TNF-a inhibitor 3,6-dithiothalidomide reversed pathologic vessel wall alterations after induced hypertension and hemodynamic stress. Chromatin immunoprecipitation assays in vivo and in vitro demonstrated that TNFa promotes epigenetic changes through KLF4-dependent alterations in promoter regions of myocardin, SMCs, and inflammatory genes. In conclusion, TNF-a induces phenotypic modulation of cerebral SMCs through myocardin and KLF4-regulated pathways. These results demonstrate a novel role for TNF-a in promoting a pro-inflammatory/matrix-remodeling phenotype, which has important implications for the mechanisms behind intracranial aneurysm formation.

Down-regulation of Insulin Receptor Substrate 1 during Hyperglycemia Induces Vascular Smooth Muscle Cell Dedifferentiation

“…Myocardin suppression may also regulate the proliferative response to injury because myocardin expression was robust in wild-type animals but nearly absent in IRS-1 Ϫ/Ϫ animals that had a hyperproliferative response to injury, and myocardin is known to modulate this response (31). Other variables, such as TNF␣, PDGF, and oxidized LDL, that regulate VSMC differentiation stimulate KLF-4 expression, thereby resulting in enhancement of VSMC dedifferentiation (30,(32)(33). During vascular injury, KLF-4 is up-regulated, and this is accompanied by dedifferentiation (34 -35).…”

“…This finding was replicated in our study, and we noted an enhanced dedifferentiation response in the absence of IRS-1, suggesting that IRS-1 is also functioning during injury to attenuate smooth muscle cell proliferation by this mechanism. KLF-4 can also be induced by other atherogenic stimuli, such oxidized phospholipids and advanced glycation end products (32)(33)(34)(35)(36).…”

Review of Cerebral Aneurysm Formation, Growth, and Rupture

“…A series of in vitro and in vivo studies by the Jefferson group found that the phenotypic modulation of SMCs in CAs was induced by tumor necrosis factor-α (TNF-α). 17,18 Specifically, TNF-α inhibited the contractile phenotype of SMCs and induced proinflammatory/matrixremodeling genes, namely MCP-1, MMPs, vascular cell adhesion molecule 1, and interleukin 1β (IL-1β). 17 This process was mediated by Kruppel-like transcription factor 4, a known regulator of SMC differentiation.…”

“…17,18 Specifically, TNF-α inhibited the contractile phenotype of SMCs and induced proinflammatory/matrixremodeling genes, namely MCP-1, MMPs, vascular cell adhesion molecule 1, and interleukin 1β (IL-1β). 17 This process was mediated by Kruppel-like transcription factor 4, a known regulator of SMC differentiation. In a follow-up study, the same group found that phenotypic modulation of SMCs was reversed with a TNF-α inhibitor after aneurysm induction.…”

Inflammation in human cerebral aneurysms: pathogenesis, diagnostic imaging, genetics, and therapeutics

“…[25] TNF-α has been previously identified as a contributor to the phenotypic modulation of VSMCs in vivo following hemodynamic stress. [26] The transcription factor, NF-κB, has also been found to be essential in the activation and recruitment of macrophages as it influences the expression of a number of pro-inflammatory genes. [5,6] Aoki et al [27] demonstrated the activation of NF-κB and expression of downstream genes; these changes seen in the vessel wall of the early stages of aneurysm development in a rat model.…”