Short-Chain Acly-CoA Dehydragenase Is Involved In Improving Hypertensive Vascular Remodeling

Objective:Short-chain acyl-CoA dehydrogenase（SCAD）is a member of the acyl-CoA dehydrogenase family.It specifically breaks down short-chain acyl-CoA substrates and is the first rate-limiting enzyme to catalyze mitochondrial fatty acidβoxidation.SCAD is closely related to flavin adenine dinucleotide（FAD）.The SCAD enzyme is a flavin protein,consisting of 4 subunits,each of which contains a molecule of the co-factor FAD.This article aims to explore the relationship between the SCAD expression and hypertension vascular remodeling at the animal level.And we use existing clinical drugs,FAD,to treat hypertensive vascular remodeling model and endothelial cell apoptosis model,and to observe the SCAD expression level,consequently,to explore the role of FAD in improving hypertensive vascular remodeling and maintaining endothelial cell homeostasis.Methods:1.20-month-old model of chronic hypertension vascular remodeling induced by spontaneously hypertensive rats with natural aging were established.Aortic morphological changes were observed by HE staining;Western Blot and RT-PCR were performed to assess the change in SCAD expression.Then,SCAD enzyme activity,aortic FFA content,aortic ATP content,and aortic NO content were detected.the changes of p-eNOS（ser1177）,eNOS,Bcl2,Bax,Cleaved caspase3,NOX-2 and NOX-4 were both detected by Western Blot.Changes of fatty acid oxidation metabolism and aortic apoptosis were analyzed for study the relationship between SCAD and chronic hypertension vascular remodeling.2.SCAD recombinant adenovirus gene therapy was used to treat hypertensive vascular remodeling model.SCAD recombinant adenovirus was injected by the way of tail vein to determine the optimal dose to achieve the best expression of SCAD in rat aorta,which selected from5×108 Viral Particles/mL/d,5×1010 Viral Particles/mL/d and 5×1012 Viral Particles/mL/d.Spontaneously hypertensive rats,aged 12 weeks,were treated with SCAD recombinant adenovirus for 8 weeks.Blood pressure measurement and echocardiography were performed to evaluate the cardiovascular function in rats.Changes of rats aorta in multiple directions were analyzed by HE staining,DHE staining,EVG staining,Sirius red staining,and TUNEL staining.SCAD Immunofluorescence was used to detect SCAD content in rats aorta.Western Blot and RT-PCR were used to detect the expression of SCAD.SCAD enzyme activity,FFA content,NO and ATP contents were detected.The expression of p-eNOS（ser1177）,eNOS,Bcl2,Bax,cleaved caspase3,NOX-2,NOX-4 were also detected by Western Blot.The effects of SCAD recombinant adenovirus on cardiovascular function and aortic remodeling in rats were analyzed and evaluated.3.SCAD knockout mouse model were established.The type of SCAD-/-mice were confirmed by rat tail identification.Western Blot and RT-PCR were used to detect SCAD expression in mice aorta.Blood pressure measurement,echocardiography,heart-to-weight ratio,were both used to evaluate cardiovascular function in SCAD^-/-mice;HE staining,DHE staining,EVG staining,Sirius red staining,and TUNEL staining were performed to analyze the aorta of mice in multiple directions.4.12-week-old spontaneously hypertensive rats were treated with FAD in the tail vein for 8 weeks to observe the relationship between FAD and SCAD,and to investigate the effect of FAD in the treatment of hypertensive vascular remodeling.The blood pressure test was performed to evaluate the anti-hypertensive effect of FAD.SCAD fluorescence was used to detect SCAD content in rats aorta;Western Blot and RT-PCR were performed to detect SCAD expression in rats aorta,SCAD enzyme activity was detected,and the effect of FAD on SCAD expression in rat aorta was analyzed.HE staining,DHE staining,EVG staining,and Sirius Red staining were used to analyze the changes of rats aorta in multiple directions.The FFA content of aorta and serum,ATP content and NO content of aorta were measured.The expression of p-eNOS（ser1177）,eNOS,caveolin-1,PGC-1αwere also detected by Western blot.Then,the effect of FAD on activating SCAD was analyzed and evaluated in blood pressure and aortic remodeling in rats and that effects may be involved in the changes of related-protein.5.Model of endothelial apoptosis was established in human umbilical vein endothelial cells（HUVECs）induced by tert-butyl hydrogen peroxide（t BHP）.Western Blot and RT-PCR were performed to detect the effects of FAD on the expression of SCAD in HUVECs;and SCAD enzyme activity was detected.Content of ROS was detected by DHE staining.Apoptosis of HUVECs was detected by flow cytometry.The FFA content,ATP content and NO content were detected.The expression of p-eNOS（ser1177）,eNOS,Caveolin-1,PGC-1αwere also detected by Western Blot.And then,the effect of FAD on SCAD was evaluated in the homeostasis of endothelial cells and that effects may be involved in the changes of related-protein.Results:1.From the chronic hypertension vascular remodeling model,it was found that the blood pressure of normal Wistar and SHR rats increased with the age.Compared with old-Wistar group,in old-SHR aorta,the SCAD expression was remarkably reduced,and the fatty acid oxidation metabolism was marked decrease;the FFA was significantly increased in old-SHR aorta,the aortic ATP content was significantly decreased in old-SHR aorta,and the aortic NO content was significantly decreased.Compared with old-Wistar group,in old-SHR group,the aorta had obvious remold,thickened and disordered arrangement of the middle layer.Compared with old-Wistar group,the expressions of the stimulative apoptotic proteins,Cleaved caspase3 and Bax,were significantly up-regulated,and the expression of the inhibitory apoptotic protein,Bcl2,was significantly down-regulated in rats aorta of old-SHR group.Simultaneously,we found that the related-protein expression of oxidative stress,NOX-2,was significantly increased,the eNOS phosphorylation was lower in old-SHR group than old-Wistar group.2.Western blot analysis demonstrated that Ad-SCAD could significant upregulate in SCAD expression at a low dosage（5×10⁸ Viral Particles/mL/d）,therefore,we used a low concentration for subsequent experiment.SCAD expression was over-expressed in the rats aorta by injecting SCAD recombinant adenovirus into the rat tail vein.Compared with Wistar+NS group,the aorta remodeling in SHR+NS group was obvious.Then we found that compared with the SHR+NS group,the upregulation of SCAD expression in the SHRs aorta caused a lower in blood pressure,a remarked improvement in cardiac function,a thinness in vascular wall,a significant reduction in collagen deposition;we also found that the production of ROS was significantly reduced and the apoptosis was relieved in SHRs aorta with Ad-SCAD;The function of Ad-SCAD treatment expression were further revealed by in test of data from FFA assay and ATP production assay,which was cutting the content FFA on serum and aorta,and increasing the systhesis of ATP on aorta.3.Compared with wild-type mice,the cardiovascular function of SCAD knockout mice was significantly reduced.In particular,the systolic and diastolic blood pressure were significantly increased,and ejection fraction and fractional shortening values were significantly reduced.Detection of aortic SCAD protein and mRNA levels from WB and qPCR confirmed that SCAD expression in the vessel wall was significantly knocked down.From the morphological results,compared with wild-type mice,the vascular wall of SCAD knockout mice were significantly changed,the vascular lumen were significantly reduced,the vascular wall thickness/lumen ratio were significantly increased.The content of the collagen and reactive oxygen species had a rise in the vascular wall,and the vessel cells were also significantly given rise to apoptosis.4.From the results of Western blot,qPCR and SCAD immunofluorescence,it was found that FAD can activate the expression of SCAD in SHR rats aorta.Compared with the SHR+NS group,the blood pressure was significantly lower in the SHR+FAD group.Compared with the SHR+NS group,the thickness of the vessel wall was significantly reduced and the smooth muscle arrangement was remarkably improved in the SHR+FAD group.The content of elastic fiber was increased,the content of collagen was significantly reduced,and the production of ROS in the aorta was eliminated.Compared with the SHR+NS group,Caveolin-1 protein expression was significantly down-regulated,PGC-1αprotein expression was significantly increased in the aorta of the SHR+FAD group;and the aorta FFA content was decreased,the ATP content was increased,and eNOS phosphorylation was activated,consequently,make for NO content increasing.5.In tBHP-induced HUVECs apoptosis model,it was found that HUVECs pretreated by FAD can resist the apoptosis for t BHP.The expression of SCAD was significantly increased in the tBHP+FAD group,and the SCAD enzyme activity was significantly increased in comparison with the tBHP group.Compared with the tBHP group,in the tBHP+FAD group,the FFA content was significantly reduced,the ATP content was significantly increased,eNOS phosphorylation was activated,and the NO content was significantly increased;Compared with the tBHP group,the ROS production had a lower,and the apoptosis was significantly relieved in the tBHP+FAD group.