Smooth Muscle–Selective Inhibition of Nuclear Factor‐κB Attenuates Smooth Muscle Phenotypic Switching and Neointima Formation Following Vascular Injury

Abstract

BackgroundVascular proliferative diseases such as atherosclerosis are inflammatory disorders involving multiple cell types including macrophages, lymphocytes, endothelial cells, and smooth muscle cells (SMCs). Although activation of the nuclear factor‐κB (NF‐κB) pathway in vessels has been shown to be critical for the progression of vascular diseases, the cell‐autonomous role of NF‐κB within SMCs has not been fully understood.Methods and ResultsWe generated SMC‐selective truncated IκB expressing (SM22α‐Cre/IκBΔN) mice, in which NF‐κB was inhibited selectively in SMCs, and analyzed their phenotype following carotid injury. Results showed that neointima formation was markedly reduced in SM22α‐Cre/IκBΔN mice after injury. Although vascular injury induced downregulation of expression of SMC differentiation markers and myocardin, a potent activator of SMC differentiation markers, repression of these markers and myocardin was attenuated in SM22α‐Cre/IκBΔN mice. Consistent with these findings, NF‐κB activation by interleukin‐1β (IL‐1β) decreased expression of SMC differentiation markers as well as myocardin in cultured SMCs. Inhibition of NF‐κB signaling by BAY 11‐7082 attenuated repressive effects of IL‐1β. Of interest, Krüppel‐like factor 4 (Klf4), a transcription factor critical for regulating SMC differentiation and proliferation, was also involved in IL‐1β‐mediated myocardin repression. Promoter analyses and chromatin immunoprecipitation assays revealed that NF‐κB repressed myocardin by binding to the myocardin promoter region in concert with Klf4.ConclusionsThese results provide novel evidence that activation of the NF‐κB pathway cell‐autonomously mediates SMC phenotypic switching and contributes to neointima formation following vascular injury.

Role of Krüppel-Like Factor 4 and its Binding Proteins in Vascular Disease

“…The mutation of each of these elements decreases the transcriptional activity of the Cdkn1a gene, thus suggesting that these cis-elements are functional. In addition, the overexpression of KLF4 increases the binding 1β) is able to repress the Myocd expression and concomitantly decrease the expression of SMC differentiation markers in cultured SMCs 48) . The effects of IL-1β on the Myocd expression have been shown to be mediated in part by KLF4, because: (1) IL-1β induces the KLF4 expression; and (2) the repressive effects of IL-1β on SMC differentiation marker genes as well as Myocd are attenuated in Klf4-deficient cultured SMCs 48) .…”

“…In addition, the overexpression of KLF4 increases the binding 1β) is able to repress the Myocd expression and concomitantly decrease the expression of SMC differentiation markers in cultured SMCs 48) . The effects of IL-1β on the Myocd expression have been shown to be mediated in part by KLF4, because: (1) IL-1β induces the KLF4 expression; and (2) the repressive effects of IL-1β on SMC differentiation marker genes as well as Myocd are attenuated in Klf4-deficient cultured SMCs 48) . The Myocd promoter contains a consensus KLF4 binding site, 5'-(G/A)(G/A)GG(C/T)G(C/T)-3' 49) , and a consensus nuclear factor (NF)-κB binding site 48,50) .…”

“…The effects of IL-1β on the Myocd expression have been shown to be mediated in part by KLF4, because: (1) IL-1β induces the KLF4 expression; and (2) the repressive effects of IL-1β on SMC differentiation marker genes as well as Myocd are attenuated in Klf4-deficient cultured SMCs 48) . The Myocd promoter contains a consensus KLF4 binding site, 5'-(G/A)(G/A)GG(C/T)G(C/T)-3' 49) , and a consensus nuclear factor (NF)-κB binding site 48,50) . The mutation of each of these elements abolishes the IL-1β-induced repression of Myocd transcription in cultured SMCs 48) .…”

“…The Myocd promoter contains a consensus KLF4 binding site, 5'-(G/A)(G/A)GG(C/T)G(C/T)-3' 49) , and a consensus nuclear factor (NF)-κB binding site 48,50) . The mutation of each of these elements abolishes the IL-1β-induced repression of Myocd transcription in cultured SMCs 48) . Moreover, KLF4 and p65 are enriched within the Myocd promoter in injured carotid arteries, as determined using chromatin immunoprecipitation assays 48) .…”

“…The mutation of each of these elements abolishes the IL-1β-induced repression of Myocd transcription in cultured SMCs 48) . Moreover, KLF4 and p65 are enriched within the Myocd promoter in injured carotid arteries, as determined using chromatin immunoprecipitation assays 48) . Of interest, the results of coimmunoprecipitation assays have shown that KLF4 interacts with p65, although these assays were performed by inducing the overexpression of proteins in non-SMCs 51) .…”