Disruption Of SM22α Enhances Inflammatory Response Induced By TNF-α In Vascular Smooth Muscle Cells

Objective:Endothelial injury-induced inflammatory response in vascular smooth muscle cells (VSMC) is the main pathological basis of intimal hyperplasia. Tumor necrosis factor-α(TNF-α) plays an important role in the inflammatory response, proliferation and apoptosis of VSMCs. TNF-αinduces the expression of various inflammatory factors through activation of intracellular signaling pathways related to inflammation, especially NF-κB pathway. Smooth muscle 22 alpha (SM22α) is a 22 kDa protein highly expressed in differentiated VSMCs. Sm22-/- mice showed enhanced in?ammatory response and oxidative stress upon carotid denudation,with NF-κB activation and nuclear translocation. The aim of this study was to explore the role of SM22αin TNF-α-induced inflammatory response of VSMCs in vitro,MethodsVSMC was isolated from the thoracic aorta of Sprague-Dawley rats and were cultured. Western blot analysis and immunostaining analysis were performed to detect the expression and cellular localization of SM22α, NF-κB and phosphor-Akt, respectively. Dihydroethidium (DHE) staining was used to detect the products of ROS in VSMCs.Results1 TNF-αinduces nuclear translocation and activation of NF-κB in VSMCsWestern blot showed that the nuclear translocation of NF-κB reached the peak (p<0.05) following TNF-α(10 ng/ml) stimulation for 30 min,with unchanged total NF-κB protein. The results suggest that TNF-αinduces imflammation response in VSMCs. 2 Knockdown of SM22αenhances TNF-α-induced nuclear translocation of NF-κBsiSM22αwas transfected into VSMCs to silence SM22αexpression. Western blot showed that the expression of SM22αwas significantly reduced in siSM22α-transfected VSMCs, compared with control (p<0.05). Immunostaining analysis showed the level of nuclear NF-κB p65 was increased in VSMCs following knockdown of SM22αor induction with TNF-α. Moreover, combination of SM22αknockdown with TNF-αinduction resulted in the increased nuclear translocation of of NF-κB p65, compared with control (p<0.05).3 Knockdown SM22αenhances TNF-α-induced IKK phosphorylationWestern blot showed the lower level of IKK phosphorylation in quiescent VSMCs. Given TNF-αor knockdown of SM22α, the levels of IKK phosphorylation were increased (P <0.05). Moreover, in VSMCs stimulated by TNF-αwith knockdown of SM22α, IKK phosphorylation level was higher than that of TNF-αalone (P <0.05). Similarly, knockdown SM22αenhances TNF-α-induced Akt phosphorylation and oxidative stress in VSMCs, respectively.4 Overexpression SM22αinhibits TNF-α-induced nuclear translocation of NF-κB p65VSMCs were treated with ATRA to upregulate the expression of SM22α, and then stimulated with TNF-α. Western blot analysis showed that high expression of SM22αinhibited NF-κB p65 nuclear translocation stimulated with TNF-α. Similarly, overexpression of exogenous SM22αin pAd-SM22α-infected VSMCs inhibited TNF-α-induced NF-κB p65 nuclear translocation. These results suggest that SM22αsuppress TNF-α-induced inflammatory response in VSMCs.Conclusions1 TNF-αinduces nuclear translation and activation of NF-κB.2 Knockdown SM22αenhances TNF-α-induced NF-κB nuclear translation, activation of Akt pathway and oxidative stress in VSMCs.3 Overexpression of SM22αinhibits TNF-α-induced nuclear translocation and activation of NF-κB p65.