The Role And Mechanism Of TRPC6 Channal In Atherosclerosis

Background:Atherosclerosis?AS?is a chronic inflammatory disease of the blood vessels that is characterized by subendothelial lipid deposition and plaque formation.Coronary heart disease,myocardial infarction,stroke and other cardiovascular diseases with AS as the main pathological basis have always been the primary threat to human health.It is still an important task of medical researchers to thoroughly study the pathological mechanism of AS and find potential prevention and control targets for AS.TRPC6 is one of the most important members of the transient receptor potential canonical family.Studies have shown that TRPC6 is involved in the pathogenesis of various cardiovascular diseases such as cardiac hypertrophy and heart failure.However,the role of TRPC6 in AS has not been reported.Objective:This research intends to identify the role of TRPC6 in AS from whole animal,tissue,cellular and molecular levels by studying the expression of TRPC6 in plaques of AS patients,then establishing a high-fat diet-induced ApoE^-/-mice AS model,and adopting genetic intervention strategies by breeding Trpc6 and ApoE double gene knockout mice.Moreover,the role of TRPC6 in the regulation of macrophage foaming,inflammation,polarization,proliferation and apoptosis during AS are further elucidated.The current study will provide an experimental basis for understanding the pathological mechanism of AS and finding new prevention and treatment targets for AS.Methods:1)Human carotid AS tissues were obtained during autopsy and the expression of TRPC6 in human AS plaques was detected by immunohistochemistry.The 8-week-old male ApoE^-/-mice were fed with high-fat diet for 12 weeks to establish a mouse AS model.H&E and Oil Red O staining were used to evaluate the occurrence of aortic AS plaques.Immunohistochemistry method was used to detect the expression of TRPC6 protein in mouse aortic AS plaques.Immunofluorescence staining was used to recognize the main cell type involved in the regulation of TRPC6 in AS.2)Trpc6^-/-ApoE^-/-?DKO?mice and their littermate Trpc6^+/+ApoE^-/-mice were obtained by crossbreeding ApoE^-/-mice and Trpc6^-/-mice.After genotyping,mice were fed with high-fat diet for 12 weeks.Ultrasonography of carotid artery was used to detect vessel diameter,vessel wall thickness,and blood flow resistance index.H&E staining and Oil Red O staining were used to evaluate aortic AS plaques.Serum lipids?T-CHO,TG,LDL-C,and HDL-C?levels were measured by enzymatic method.Serum levels of inflammatory cytokines?TNF-?,IL-1?,IL-6?were measured by ELISA.Immunohistochemistry and Sirius Red staining were used to detect the content of macrophages,smooth muscle cells and collagen fiber in plaque,assessing plaque stability.3)Transcriptome sequencing technology was used to detect differentially expressed genes and their enrichment in aorta of DKO and ApoE^-/-mice.Bone marrow-derived macrophages were extracted and cultured successfully and used to establish an ox-LDL induced foam cell model.Oil Red O staining was used to evaluate the foam cell formation level.The expression of TRPC6 protein in foamed macrophages was determined by Western Blot.The qPCR analysis was used to detect lipoprotein uptake,cholesterol excretion,cholesterol ester hydrolysis and free cholesterol esterification related genes expression.The secretion of pro-inflammatory cytokines in foamed macrophages was detected by ELISA.NF?B activity was detected by Western Blot.IFN-?+LPS or IL-4were used to induce macrophage M1 or M2 polarization and polarization related gene expression levels were detected by qPCR analysis.TUNEL method was used to detect apoptotic cells in AS plaques.Apoptosis related protein cleavage or expression levels were detected by Western Blot.BrdU method was used to detect cell proliferation in AS plaques.Results:1)Mouse AS model was successfully established.The expression of TRPC6 in human and mouse AS plaques were significantly increased and the average optical density of immunohistochemistry staining increased by about 6.5-fold?P<0.05?and 14.2-fold?P<0.01?,respectively.The up-regulation of TRPC6 expression was most notable in macrophages compared with smooth muscle and endothelial cells.2)DKO mice were successfully obtained.After Trpc6 gene knockout,AS induced vessel narrowing,vessel wall thickening and increased blood flow resistance were all significantly alleviated.The aortic sinus AS plaque area decreased from 0.510±0.020 mm² to 0.279±0.075 mm²?P<0.01?and Oil Red O positive staining area decreased from 0.405±0.028 mm² to 0.240±0.069 mm²?P<0.05?.In the intima of en face aorta,the proportion of AS plaque area decreased from?15.86±2.80?%to?9.08±3.02?%?P<0.05?.There was no significant change in the content of serum lipids,but inflammatory factors including TNF-?,IL-1?and IL-6 were significantly reduced.The content of macrophages in the plaques was significantly reduced,while the smooth muscle content increased,suggesting an increase in plaque stability.3)After Trpc6 gene knockout,there were 1595 differential expressiongenes in the aorta of AS mice,of which about 3/4 were down-regulated,and down-regulated differentially expressed genes were enriched in immune and inflammatory gene sets.Ox-LDL could successfully induce the macrophage foaming model and TRPC6was up-regulated by about 1.5-fold?P<0.05?in foamed macrophages.After Trpc6knockout:the lipid droplet content in foamed cells was reduced by 24%?P<0.05?,and the expression of Marco gene mediating lipoprotein uptake was downregulated by 86%?P<0.01?.The secretion of inflammatory cytokines?TNF-?,IL-1?and IL-6?in foamed marcopahges was markedly reduced;The activity of NF?B was markedly decreased;The macrophage M1 polarization was weakened and the polarization to M2 was enhanced;The level of macrophage apoptosis in AS plaques was significantly decreased by 44%?P<0.05?,and the activation or expression level of apoptosis-related proteins in foamed marcophages were significantly decreased.Conclusion:1)TRPC6 is highly expressed in human and mouse AS plaques,especially in macrophages,suggesting that it may be involved in the formation of AS plaques.2)After Trpc6 knockout,the occurrence of AS plaques significantly reduced with the plaque stability enhanced.3)TRPC6 participates in the pathological process of AS by regulating macrophage foaming,inflammation,polarization and apoptosis.In conclusion,TRPC6 is involved in the occurrence and development of AS and is a potential new target for AS drug intervention.