| BackgroundArteriovenous fistula(AVF)is the preferred vascular access in maintenance hemodialysis patients and is known as the "lifeline" of patients with uremia.However,the survey showed that the primary AVF had a patency rate of 60% at one year,a patency rate of only 51% at two years;the secondary patency rate was 71% at one-year,and 64% at two years.It is an important reason that results in insufficient dialysis in the population,increased central venous catheter use,mortality,and high hospitalization.At present,there are no effective clinical measures to prevent AVF from losing power.Therefore,it would have important implications for improving the long-term survival rate of MHD patients and improving the quality of life,with better understanding of the pathogenesis of AVF stenosis and dysfunction,exploring effective prevention and treatment strategies,and prolonging the service life of autogenous AVF.The mechanisms of AVF failure include: 1.Vascular injury: After autologous AVF construction,intimal injury is caused by shear stress changes,chronic inflammatory reactions,hypoxia,uremic toxins,and repeated dialysis punctures.2.The hemodynamic changes: after the establishment of AVF,the blood flow to the reflux vein increases,the distal endothelium of the anastomosis faces with non-physiological high shear stress,the blood flow gradient is unstable,and the shear stress on the vein side increases accordingly.Although the level of shear stress gradually decreases with the expansion of blood vessels and the reestablishment of laminar flow in the process of AVF maturation,the EC of the anastomosis venous end may be subjected to a sustained wall shear stress.3.Uremia: the uremic toxin was accumulated in patients with ESRD,high levels of phosphorus and calcium,inflammation,oxidative stress and inflammatory factors,can itself lead to changes in the body's multiple organ systems and blood vessels.Studies found that the occurrence of vascular endothelial dysfunction and neointimal hyperplasia was earlier than that of arteriovenous fistula aceess in hemodialysis patients.4.Inflammation: chronic persistent metabolic inflammation is a major feature of CKD.Under this micro-inflammatory environment,the secretion of many inflammatory factors increases.In animal models and clinical researches,inflammatory cell infiltration was found in AVF local.These inflammatory cells can promote the release of inflammatory mediators,ECM deposition and thrombosis,and then develop into AVF dysfunction.Vascular smooth muscle cells are an important component of the arterial media,with phenotypic plasticity.The phenotypic transition and proliferation of smooth muscle cells are regulated by various signaling pathways.When the environment changes,the differentiated and mature contractile phenotype can be re-differentiated to a synthetic phenotype,involving in the repair of vascular injury,the formation of new blood vessels,the reconstruction of blood vessels and so on.Our published studies have confirmed that the phenotypic conversion,abnormal proliferation,and migration of vascular smooth muscle cells caused neointimal hyperplasia,which is the pathological basis of arteriovenous fistula stenosis and failure.Notch signaling pathway is highly conserved in evolution,which has homologous structure in many species,and is a regulator of cell development,differentiation,and apoptosis.It is one of the major mechanisms mediating cell-to-cell contact with signaling.In mammals,there are four Notch receptors(Notch1,Notch2,Notch3,Notch4)and five ligands(Jagged-1,Jagged-2,Delta-like 1,Delta-like 3,Delta-like 4).The activation of Notch signaling is achieved through the intercellular contact between the ligand and the Notch receptor in verse interaction.The activation of Notch signaling requires a three-step protease cleavage reaction and is finally released in the activated form,the Notch intracellular domain(NICD).In the previous studies,researchers mostly studied the activation of Notch pathway by using endothelial cells and smooth muscle cells to co-culture in vitro or adding soluble Jagged-1 to cell culture medium.Our previous researches and foreign reports have shown that Notch signaling pathway plays an important role in the phenotypic transition and proliferation of smooth muscle cells,but the specific mechanism of Notch signaling in the intimal hyperplasia of AVF remains unclear.In this study,firstly,the phenotypic transformation of smooth muscle cells and activation of Notch signal in AVF were detected by immunohistochemistry and immunofluorescence.The serum from uremic patients was used for the first time to culture smooth muscle cells and endothelial cells in vitro.ELISA,Western blot and QPCR technology were used to revealed that uremic serum can upregulate the expression of Notch1 and vascular endothelial cell ligand Jagged 1 in vascular smooth muscle cells.Therefore,Notch signaling pathway in vascular smooth muscle cells was activated,and it would promote the phenotype transition from contractile type to synthetic type,and the migration,abnormal proliferation of vascular smooth muscle cells.We also preliminary explored the mechanism of intimal hyperplasia in arteriovenous fistula by the promotion of uremic serum.Objective:1.To study the effect of uremic serum on phenotye transformation and function of vascular smooth muscle cells.2.To investigate the mechanism of uremic serum on phenotype switch of vascular smooth muscle cells,arteriovenous fistula stenosis and dysfunction.Methodology:1.(1)The intimal thickness of fistula was compared with normal vessles by elastic-Van Gieson staining and the expression of fibroblast-specific protein-1(FSP-1),proliferatin cell nuclear antigen(PCNA)was observed by immunohistochemical staining.In addition,the changes in the expression of ?-SMA and SMMHC were compared between before and after the operation of uremia patients.(2)The primary human aortic smooth muscle cells were cultured in vitro and divided into the following groups according to the serum and its proportion in medium: serum-free group,normal serum group(10%),and 5%,10%,15%,20% uremia serum group,treated for 24 h.The expression of ?-SMA,smooth muscle 22?(SM22?),FSP-1,PCNA protein was assessed by Western blot.The mRNA expression of smooth muscle myosin heavy chain(SMMHC),SM22?,FSP-1,PCNA in serum-free group,normal serum group,and 10% uremia serum group was detected by QPCR.2.(1)To compare Notch intracellular domain(NICD)expression between normal blood vessels and internal fistula by immunohistochemical staining and immunofluorescence.(2)Western blot was used to investigated the expression of NICD protein in serum-free group,normal serum group(10%),5%,10%,15% and 20% uremia serum group.(3)The mRNA expression of Hes1,Hes5,Notch1,Notch2,Notch3 in serum-free group,normal serum group,and 10% uremia serum group was detected by QPCR.(4)Smooth muscle cells in serum-free group,normal serum group,and 10% uremia serum group were pre-treated with DAPT,Notch signal inhibitor,then SM22?,FSP-1 protein and mRNA expression was analyzed by Western blot and QPCR.Also the mRNA expression of Hes1 was detected.We compared the results with smooth muscle cells treated without DAPT.(5)The migration function of vascular smooth muscle cells in serum-free group,normal serum group,and 10% uremia serum group was detected by scratch test.3.(1)The Jagged 1 expression which is a ligand of Notch would be detected by immunohistochemical staining between normal vessel and AVF.(2)Western blot was used to investigated the expression of Jagged 1 protein of HASMCs in serum-free group,normal serum group(10%)and 10% uremia serum group.(3)To compare Jagged 1 protein expression of vascular endothelial cell within serum-free group,normal serum group(10%)and 10% uremia serum group by ELISA.Results1.The effect of uremic serum on phenotye transformation and function of vascular smooth muscle cells.(1)Elastic-Van Gieson staining(EVG)showed that the intimal thickness of arteriovenous fistula was obviously increased,comparing with normal vessles;and immunohistochemical staining displayed the expression of ?-SMA,SMMHC was decreased in AVF 1 week after surgery.In the neointima,the positive rate of FSP-1 and PCNA was increased than normal vascular tissue,the difference was statistically significant(P<0.05).These results reveal that phenotype of VSMCs in the hyperplasia of AVF has changed.(2)Compared with the normal serum group,the expression of ?-SMA,SM22? in the uremia serum groupswas decreased significantly(P<0.05),and the expression of FSP-1,PCNA was increased significantly(P<0.05),showing a concentration dependence on uremia serum all.The mRNA expression of SMMHC,SM22? in 10% uremia serum group was decreased(P<0.05),comparing with the normal serum group,thus the mRNA expression of FSP-1 and PCNA was increased(P<0.05).These results demonstrated the uremic serum could accelerate the de-differentiation of vascular smooth muscle cells,and the vascular smooth muscle cell phenotypic transformation by deflating to synthetic.2.The Notch signal pathway was activated by uremia serum.(1)NICD was mainly expressed in the nucleus of neointima in AVF,and FSP-1 was also positive expression in these cells.It told that the activation of Notch signaling pathway existed in AVF.(2)Compared with the normal serum group,the expression of NICD protein in smooth muscle cells treated with 10% uremia serum was increased(P<0.05),and the result showed a serum concentration dependence.(3)Comparing with the normal serum group,the mRNA expression of Hes1,Hes5,Notch1,Notch3 in 10% uremia serum group was increased(P<0.05),but Notch2 mRNA expression was no significant different from cells in the normal serum group.This indicated uremic serum could activate the Notch signal pathway,contributing to the proliferation of vascular smooth muscle cell.(4)The protein and mRNA expression of SM22? was up-regulated in cells pre-treated with DAPT(P<0.05),rather,the expression of FSP-1 and Hes1 was down-regulated in cells pre-treated with DAPT(P<0.05).(5)Comparing with the normal serum group,the migration of vascular smooth muscle cells in 10% uremia serum group was significantly declined(P<0.05).3.The expression of Jagged 1 in vascular endothelial cells were up-regulated,and cytokines in uremic serum(1)The expression of Jagged 1 in AVF was increased(P<0.05),also mainly in the neointima.It mean that uremia could up-regulate Jagged 1 expression.(2)Compared with the normal serum group,the expression of Jagged 1 protein of HASMCs in the 10% uremic serum group was increased(P<0.05).(3)The expression of Jagged 1 protein in HUEVCs was increased with 10% uremic serum group(P<0.05).ConclusionThe experimental results hinted that uremic serum through increase the exprssion of Jagged 1,which is a ligand of Notch signal,activate Notch signaling pathway,and promote vascular smooth muscle cell phenotypic transformation by deflating to synthetic,that could be one of mechanisms contributing to a fistula neointimal hyperplasia in uremia patients.Uremic serum has inhibitory effect on the migration of vascular smooth muscle cells. |
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