Trimethylamine N-oxide Promotes Phenotypic Switching Of Vascular Smooth Muscle Cells

Objective: Phenotypic switching of vascular smooth muscle(VSMCs)cells plays an important role in pathophysiological processes such as atherosclerosis(AS)and restenosis after angioplasty.The purpose of this study is to investigate the role and mechanism of Trimethylamine N-oxide(TMAO)in the phenotypic switching of VSMCs.Methods: 1.Eighteen Sprague Dawley(SD)male rats were randomly divided into three groups: the control group(n=6),the normal feed operation group(n=6)and the high TMAO group(n=6).The high TMAO group was given 1 % Choline diet,the control group and the normal feed operation group were given basic maintenance feed.After 12 weeks of feeding,rats in the normal diet operation group and high TMAO group underwent balloon injury to the left common carotid artery.The control group had only surgical operations and no balloon implantation.After operation,each group was anesthetized after feeding with the original diet for 2 weeks,and the blood was collected from the retroorbital venous plexus.The content of plasma TMAO was detected by high performance liquid chromatography tandem mass spectrometry,and the intima neovascularization area and media thickness were determined by H-E staining.2.(1)Primary vascular smooth muscle cells of rat thoracic aorta were cultured and stimulated by TMAO.Western Blot was used to detect α-smooth muscle actin(α-SMA),smooth muscle myosin heavy chain(SM-MHC),osteopontin(OPN)and collagen I(COL-1),intercellular adhesion molecule 1(ICAM-1)to determine whether TMAO can induce phenotypic switch of VSMCs.(2)The proliferation of VSMCs was measured by CCK-8 cell proliferation kit.(3)To determine the migration of VSMCs by scratch test and Transwell test to determine whether TMAO can enhance the migration ability of VSMCs.(4)In order to further explore the mechanism of phenotypic switching of VSMCs induced by TMAO,the expression of Myocardin(MYOCD)and kruppel-4-like transcription factor(KLF4),which is the key factor of phenotypic switching of VSMCs,was detected by Western Blot.Results: 1.Compared with normal diet group,the content of plasma TMAO in high TMAO group increased(P < 0.01).The results showed that 1% choline diet could significantly increase the content of plasma TMAO in rats.Neointima appeared in both operation groups compared with the control group,and the neointimal area in the high TMAO operation group was larger than that in the normal feed operation group(the neointimal area in the control group,normal feed operation and high TMAO group: 0 vs 0.032 ±0.014 vs 0.361 ±0.008,P <0.01,statistically significant).The media thickness was measured in the control group,normal diet operation group and high TMAO group: 0.078 ±0.013vs0.086 ±0.013vs0.096 ±0.003,P=0.213,the thickness increased slightly,but there was no statistical significance.The above results indicate that the model is successful and suggest that TMAO can aggravate the degree of neointima.2.(1)Western Blot showed that the expression of α-SMA and SM-MHC decreased and the expression of OPN increased,indicating that TMAO can induce phenotypic switching of VSMCs.(2)Western Blot showed that the expression of COL-1 was increased,which suggested that TMAO could enhance the ability of VSMCs to secrete extracellular matrix(ECM).At the same time,the expression of ICAM-1 increased after VSMCs was treated with TMAO.(3)The results of CCK8 experiment showed that compared with the control group,TMAO could enhance the proliferation ability of VSMCs,and scratch test and Transwell test showed that TMAO could enhance the migration ability of VSMCs,and the difference was statistically significant(P<0.01).(4)Further studies on the mechanism showed that TMAO could reduce the expression of Myocardin and increase the expression of KLF4,while Myocardin and KLF4 were the core factors of phenotypic switching of VSMCs.Therefore,we believe that TMAO is a new factor that induces phenotypic switching in VSMCs.Conclusion:1.TMAO can aggravate the formation of neointima in injured vessels.2.TMAO can promote the phenotypic switching of vascular smooth muscle cells and obtain the ability of high migration,high proliferation and high secretion of ECM.3.The mechanism of phenotypic switching induced by TMAO in vascular smooth muscle cells is achieved by reducing the expression of Myocardin and increasing the expression of KLF4.