NOX4Regulates The Phenotypic Transformation Of Adventitial Fibroblasts

Background:The treatments of coronary artery disease (CHD), either percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), are limited by restenosis. The restenosis is a very complex process that so many cells and factors take part in it. Recently, many researchers have found that adventitial fibroblasts (AF) play an important role in the process of restenosis. The transformation of AF induced by various factors is the crux in the process of restenosis. Although TGF?1is considered as the most common factor of the transformation, the previous underlying mechanism is unclear. Some studies have found that NDAPH oxidase4(NOX4) has significant expression in the cardiovascular system and involves in many physiological, pathological process by way of reactive oxygen species (ROS). However, NOX4take part in the process of AF transformation or not is unclear. Here, we explored the role of NOX4in TGF?1-induced AF transformation and its molecular mechanism.Part I Culture of rat thoracic aorta adventitia fibroblastsObject: Inorder to get the adventitia fibroblasts for further researchMethods: The rats from animal center of Wuhan University were decapitated. The thoracic aorta was cut. The adventitia was separated, cut into1mm x1mm small pieces, and used tissue explant method to culture. The cell was determinate by immunohistochemical method.Results: The AF cells cultured by tissue explant method were identified with immunohistochemical method. The expression of a-SMA was negative. The expression of vimentin was positive. After3passages culture, cells can be used for further research.Conclusion:The AF cells cultured by tissue explant method can be used for further research. Part ? The role of NOX4in adventitia fibroblast transformationObject: NOX4plays an important role in the cardiovascular system. So in this part, we exptored the role of NOX4in adventitia fibroblast transformation.Methods: The AF cells were cultured in vitro. The cells were serum-free for12hours prior to treatment. The blank group, TGF ?1group, and TGF ?1+siRNA group were established as follow:The blank group: Cells were cultured in2%FBS medium for24h; TGF ?1group:Cells were cultured in2%FBS medium+TGF?1(lOng/ml) for24h; experimental group: after transfection of the NOX4siRNA, the cells were cultured in2%FBS medium+TGF?1(10ng/ml) for24h. The variation of NOX4was roughly tested by immunofluorescence. As with a-SMA, the expression of NOX4was assessed by PCR and Western blotting. The cell migration was measured by Transwell assay for12h and24h.Results:(1) Comparing with blank group, the expression of NOX4in the TGF ?1group increased greatly (Western blotting:0.796�0.0616VS0.531�0.0444, P<0.05; PCR:0.7293�0.05280VS0.2470�0.01659, P<0.05). NOX4siRNA can obviously reduce the expression of NOX4(Western blotting:0.420�0.0279VS0.796�0.0616, P<0.05; PCR:0.2081�0.02166VS0.7293�0.05280, P<0.05).(2) Knockdown of NOX4with siRNA obviously prevent the expression of a-SMA (Western blotting:0.3647�0.06642VS0.6352�0.09138, P<0.05; PCR:0.3375�0.04136VS0.5400�0.03158, P<0.05).(3) TGF?1can promote cell migration (12h:20.600�1.5166VS3.800�0.8367, P<0.05;24h:50.800�2.5884VS11.400�1.3416, P<0.05). Knockdown of NOX4with siRNA obviously prevent cell migration (12h:7.000�0.7071VS20.600�1.5166, P<0.05;24h:20.400�1.8166VS50.800�2.5884, P<0.05).Conclusion:(1) In the process of AF transformation, the expression of NOX4increased significantly. The cell migration was enhanced.(2) Knock down the expression of NOX4by NOX4siRNA can reduce the AF phenotypic transformation and cell migration. Part ? mechanism of NOX4regulating adventitia fibroblast transformationObject: We have demonstrated that NOX4played an important role in the process of AF transformation. In this part, we want to explore the mechanism of NOX4regulating adventitia fibroblast transformation.Methods: The AF cells were cultured in vitro. The cells were serum-free for12hours prior to treatment. NAC (a ROS inhibitor) and U0126(a specific inhibitor of MEK) pre-intervention was used to investigate the effects of signaling pathway. The expression of a-SMA was assessed by PCR and Western blotting. The phosphorylation levels of MEKl/2and ERKl/2were assessed by Western blotting.Resplt:(1) NAC pre-intervention prevented the expression of a-SMA (Western blotting:0.2332�0.01294VS0.4700�0.06351, P<0.05; PCR:0.1959�0.01940VS0.4642�0.03505, P<0.05), and reduced the phosphorylation levels of MEK1/2and ERKl/2(p-MEK1/2:0.4342�0.02450VS0.6748�0.03225, P<0.05)(p-ERK1/2:2.2359�0.1069VS4.9034�0.35521, P<0.05).(2) U0126pre-intervention prevented the expression of a-SMA(Western blotting:0.2337�0.01166VS0.4334�0.05675, P<0.05; PCR:0.1468�0.02986VS0.3968�0.06183, P<0.05), and reduced the phosphorylation level of ERKl/2(2.9315�0.41269VS7.0608�0.45744, P<0.05).Conclusion:(1) When function of ROS was inhibited by NAC, AF transformation was reduced. The phosphorylation levels of MEKl/2and ERK1/2were decreased.(2) When the MEK-ERK signal pathway was blocked, AF transformation was reduced.(3) NOX4regulates AF transformation though the NOX4-ROS-MEK-ERK signal pathway.