Homocysteine Promotes Migration And Invasion Of Adventitial Fibroblasts Via AT1R To Accelerate Atherosclerosis

Background The adventitia has been an active participant in the process of homocysteine（Hcy）promoting atherosclerosis,however as an important part of adventitia,the specific mechanism of adventitial fibroblasts（AFs）in this process is unclear.Objective The present study aimed to clarify the effect of homocysteine on adventitial fibroblasts and its relationship with angiotensin II type 1 receptor（AT1R）.Methods Animal experiment:Apolipoprotein E-deficient mice were used to establish the murine model of atherosclerosis.Hyperhomocysteinemia was induced and treated by feeding them 1.5%methionine.A total of21 Apo E-/-mice（male,8 weeks）were randomly divided into control group（Control）,hyperhomocysteinemia group（HHcy）and HHcy+telmisartan（10 mg/kg/d）gavage group（HHcy+Telmi）.After 12 weeks of feeding,the mice were killed.The lipid content and plaque area of atherosclerotic plaque in aortic root were observed by Oil Red O staining,and the expression of ER-TR7,a specific marker of AFs in tunica adventitia,was detected by immunofluorescence.The expression of interleukin-6（IL-6）,monocyte chemoattractant protein-1（MCP-1）and macrophages（Mac3）in atherosclerotic plaques of mouse aorta and aortic root was detected by immunohistochemistry,and the stability of plaques was detected by Hematoxylin Eosin（HE）and Masson staining.Cytological experiment:culture of AFs by aortic tissue attachment in SD male rats.Then proceed as follows:（1）Hcy（0-400μmol/L）was added to the culture medium of AFs for 48h,and Cell Counting Kit-8 was used to detect the cell proliferation;（2）Wound-healing assay and Transwell matrigel invasion assays were used to detect the effect of Hcy on the migration and invasion of AFs;（3）Hcy（200μmol/L）was added to the culture medium of AFs for（0-72）h,and the content of hydrogen peroxide（H₂O₂）was detected by Hydrogen Peroxide assay kit and Reactive Oxygen Species（ROS）was detected by Reactive Oxygen Species Assay Kit;（4）The expression of AT1R,IL-6 and matrix metalloproteinase9（MMP-9）were determined by Western blot after Hcy（0-72h）and（0-400μmol/L）was added to the culture medium of AFs in a time and concentration gradient manner;（5）Hcy（200μmol/L）stimulated AFs and HEK293A cells transfected with AT1R plasmid（0-60）min with or without telmisartan（1mmol/L）,pretreated cells for 1h,extracted protein,and detected PKC and ERK1/2 signal pathways.Results Increased plaque area of the aortic root,and increased the expression of IL-6,MCP-1,and Mac-3 were found in the HHcy group compared with the control group,while the expression in the HHcy+Telmi group decreased.Compared with the control group,the HHcy group had adventitial fibroblasts marker protein ER-TR7 expression in the plaque and the entire layer of the blood vessel wall,while this phenomenon was reduced in the HHcy+Telmi group,indicating that HHcy induced adventitial fibroblast migration and AT1R mediated this process.Hcy could increase the production of adventitial fibroblast H₂O₂,ROS,and IL-6,indicating that Hcy could activate oxidative stress and inflammatory response.Subsequent scratch and transwell experiments confirmed that Hcy could induce adventitial fibroblast migration and invasion,increase adventitial fibroblast AT1R expression,and increase the phosphorylation levels of adventitial fibroblasts PKC and ERK1/2,whereas telmisartan could inhibit these effects.Moreover,in AT1R plasmid transfected HEK293A cells,Hcy could increase the phosphorylation levels of PKC and ERK1/2,and telmisartan could also inhibit it,indicating that Hcy could activate the downstream PKC and ERK1/2 signaling pathway of AT1R.Conclusions Hcy promotes adventitial inflammation,induces adventitial fibroblast migration,and aggravates atherosclerosis via AT1R activation.