The Effect And Mechanism Of TGFβ On Arteriovenous Fistula Maturation Under CKD Environment

Objectives:Hemodialysis is currently the most commonly used and effective renal replacement therapy for patients with end-stage renal disease.How to maintain the normal use of hemodialysis access has become the focus of both doctors and patients.Superficial arterialization—Autologous arteriovenous fistulas(AVF)and artificial vascular fistulas(grafts)are the most widely used and effective vascular access for dialysis in clinical practice since they have a higher blood flow and lower risk of infection.Compared with other dialysis access,AVF has a lower infection and complication rate,and is the preferred vascular access for dialysis patients.However,due to a series of disease factors,AVF has a low patency rate and is prone to dysfunction.Therefore,how to maintain the basic function of AVF and improve the long-term patency rate is the focus of clinical attention and an urgent problem to be solved.Therefore,it is particularly important to explore the mechanisms of AVF maturation and dysfunction to maintain the normal function of AVF.Previous studies have shown that the main reason for the dysfunction of arteriovenous fistula is the obstruction of the venous outflow tract,and the formation of a large amount of neointimal hyperplasia on the inner wall of the venous segment at the arteriovenous junction causes lumen stenosis.Other studies have shown that endothelial cells(EC)play an important role in neointimal hyperplasia,and endothelial-mesenchymal transition(EndMT)is an important way for EC to participate in angiogenesis while TGFβ can regulate EndMT.In addition,studies have shown that the development of AVF is differential in animals with and without chronic kidney disease(CKD).Therefore,we explored the mechanism of AVF maturation and the influence and mechanism of CKD on it,and provided a new solution for clinical improvement of AVF maturation and long-term patency rate.MethodsPart I: Detect the histological and protein molecular changes in clinical AVF specimens,determine the difference between AVF and normal veins and the corresponding molecular level changes;Part II: By constructing mouse models of CKD and AVF,set up five groups,including(sham+0/6Nx),(sham+5/6Nx),(AVF+3/6Nx),(AVF+5/6Nx),to observe the characteristics of AVF remodeling under normal and CKD conditions,and explore its related molecular mechanisms;Part III: According to the results of the previous part of the study,it was found that the content of TGFβ1 in AVF was increased and the TGFβsignaling pathway was activated under CKD conditions,so we designed and used specific endothelial cell TGFβ I and II receptor gene knockout mice to verify our hypothesis;Part IV: Use in vitro cell experiments to further explore and validate the relevant mechanisms.ResultsPart I: Compared with the control vein,the thickness of vessel wall(intima-media),lumen area,total collagen 1 and total fibronectin content of AVF were significantly increased.The expression of TAK1 in EC and smooth muscle cells(SMC),the proportion of EC co-expressing CD31 and Notch-3 and Vimentin were significantly increased;the contents of mesenchymal cell markers Notch-3 and Vimentin were also increased;and phosphorylated TAK-1(p TAK-1)and total TAK1 and its downstream signaling effectors JNK and p38 increased immunoreactivity,while the rate of phosphorylated p TAK-1and total TAK1 was unchanged.Part II: At postoperative day21,the AVF diameter of CKD(AVF+5/6Nx)mice was smaller than that of mice without CKD(AVF+0/6Nx and AVF+3/6Nx);sham-operated groups(sham+0/6Nx and sham+5/6Nx)had no significant changes in the vessel wall,and the AVF venous outflow tract vessel wall thickness was significantly thicker in other groups,and the group with CKD was larger than the group without CKD.Western bolt analysis shows that endothelial marker protein(VE-cadherin)in the sham-operated group(sham+0/6Nx and sham+5/6Nx)did not change significantly,but the other three groups had different degrees of reduction,and the changes of CKD groups were greater than those in the non-CKD group,and the expression levels of mesenchymal cell protein markers like α-actin,Notch 3,Vimentin,and FSP-1 showed an opposite trend to endothelial markers.Therefore,we concluded that EndMT exists in AVF,and CKD can significantly increase the expression of EndMTrelated proteins;TGFβ1 and TAK1 were significantly higher in the CKD group than in the non-CKD group,as well as p-Smad2.Part III: The diameter of AVF in EC knockout TGFβ receptor(TGFβR-EC-KO)mice was significantly higher than that in the control group,and there was a significant difference on the 14 th day after surgery.The AVF vessel wall thickness,EndMT-related protein expression,TGFβ signaling pathway and its downstream molecules in TGFβR-EC-KO)mice were lower than those in the control group.Part IV: TGFβ1 can stimulate HUVEC to produce EndMT;the expression levels of EndMT-related proteins Notch3,Vimentin,FSP-1 and related inflammatory molecules ICAM-1 and Fibronectin in TGFβR-ECKO cells were significantly decreased compared with the control group(P <0.0001),while the endothelial cell marker molecule VE-Cadherin was significantly increased,proving that endothelial cells in the experimental group maintained their endothelial properties,while a large number of endothelial cells in the control group developed EndMT.ConclusionTGFβ regulates AVF vascular remodeling through canonical and noncanonical TGFβ signaling pathways in the chronic kidney disease environment;specific inhibition of the TGFβ signaling pathway in endothelial cells can effectively alleviate EndMT to promote AVF maturation.