The Role Of Endothelial Cell Function In Arteriovenous Fistula Stenosis Through YAP/TAZ Signaling Pathway

BackgroundThe number of patients with chronic kidney disease（CKD）has been increasing rapidly year by year.According to the annual report of CK-NET,the incidence of uremia receiving renal replacement therapy（RRT）reached 122.19/million population in China,and arteriovenous fistula（AVF）for hemodialysis is the most widely used treatment method at present.Most current researches show that initimal hyperplasia（IH）is the main cause of AVF failure in the middle or late term,and intervention from the formation mechanism of IH is the most ideal treatment.The injury of vascular endothelial cells（EC）,proliferation and migration of vascular smooth muscle cells（VSMCs）,accumulation of extracellular matrix and activation of inflammatory cells will all participate in the neointima formation of fistula,leading to vascular stenosis.EC,as cells directly in contact with blood,can directly feel the hemodynamic and composition changes of blood,and its changes may directly affect nearby cells.The Hippo signaling pathway involved in cell growth and organ size control.The core components of the pathway,Yes-associated protein（YAP）and Transcriptional coactivator with PDZ-binding motif（TAZ）,is a focus of many cell proliferation studies.The role of YAP/TAZ in EC in the intimal hyperplasia of AVF still remain uncertain,which deserves further investigation.Objective1.To investigate the role of YAP/TAZ expression in vascular endothelial cells in the stenosis of arteriovenous fistula in CKD;2.To investigate whether inhibiting the YAP/TAZ signaling pathway in vascular endothelial cells can improve the proliferation of VSMCs in the intimal tissue,and provide a new target for the treatment of fistula stenosis.Methods1.Expression of YAP protein in mouse model of arteriovenous internal fistula with chronic renal disease.Eight-week-old wild-type C57/B6 mice were selected and divided into non-CKD group（8 mice）and CKD group（12 mice）.In order to improve the survival rate of animals,5/6 kidney tissues were resected in the CKD group twice.At the fifth week of the experiment,whether the model of CKD was successful was determined by serum urea nitrogen（BUN）test.Mice in non-CKD group received CKD sham operation.At the fifth week of the experiment,right common carotid artery-external jugular vein anastomosis was performed to establish AVF in both groups of mice,and specimens were collected at the ninth week of the experiment.AVF tissues of mice in the non-CKD group and the CKD group were collected,and tissue proteins and paraffin-embedded sections were extracted.The expression of YAP in the internal fistula tissues was detected by western blot technique,and the hyperplasia of the intima of internal fistula was detected by HE staining.The expression of YAP andα-smooth muscle actin（α-SMA）in internal fistula tissue was detected by immunohistochemistry.The expression of macrophage surface molecule-2（MAC2）and interleukin-1β（IL-1β）were detected by immunofluorescence staining in fistula tissue.The expression of pro-inflammatory factor IL-1β,serum and glucocorticoid-induced kinase 1（SGK1）in fistula tissues were detected by immunohistochemistry.2.Effect of Yap/Taz gene knockout in vascular endothelial cells on endometrial hyperplasia of internal fistula.Cultivation of Yap^f/f/Taz^f/f/VE-cadherin ERCre⁺mice:Yap^f/f/Taz^f/f mice were hybridized and backcrossed with VE-cadherin ERCre⁺mice to obtain offspring mice for DNA extraction and genotype identification.Tamoxifen solution（20 mg/m L）was prepared with corn oil in 6 mice with 3 mutants.Three mice were intraperitoneally injected with 80 mg/kg of tamoxifen solution,and the other three mice were intraperitoneally injected with the same volume of corn oil as control for 5 consecutive days.The mice arteries were collected for immunohistochemical staining to detect the expression of YAP and identify the tamoxifen-induced gene knockout effect.16 mice with 3 mutated genes were selected and underwent 5/6 nephrectomy.The success of CKD model was detected at the 5th week of the experiment.Yap/Taz gene knockout:At the third week of the experiment,8 mice were randomly selected and injected intraperitoneally with tamoxifen corn oil solution for 5 days,and the Yap/Taz gene（Yap/Taz KO）was knocked out in vascular endothelial cells.The other 8 mice were control mice（CTL）.At week 5,16 mice were treated with AVF anastomosis,and at week 9,AVF specimens were collected for paraffin-embedded sections.Relationship between Yap/Taz knockout and intimal hyperplasia of internal fistula:The proliferation of intima of fistula in two groups of mice was detected by HE staining,and the expressions of proliferating label Ki67,proliferating cell nuclear antigen（PCNA）andα-SMA were detected by immunohistochemistry.Inflammatory cells and cytokines in internal fistula tissue:leukocyte common antigen（LCA,also known as CD45）,MAC2,and intercellular cell adhesion molecule-1 were detected by immunohistochemistry.Vascular cell adhesion molecule-1（VCAM-1）,IL-1βand connective tissue growth factor（CTGF）in fistula tissue.3.Yap/Taz regulates the function of EC which promotes the proliferation of VSMCs.Separation of primary EC:Yap^f/f/Taz^f/f/VE-cadherin ERCre⁺mouse lung tissues were isolated by magnetic bead method and cultured in vitro.After 24 h treatment with 4 u M tamoxifen,EC were set as the specific Yap/Taz knockout EC group（Yap/Taz KO EC）.Untreated EC were set as control group（CTL EC）.Yap/Taz and EC functions:Lipid metabolite 1 u M lysophosphatidic acid（LPA）and 2 u M Sphingosine-1-phosphate（S1P）were stimuli of EC.After 24 hours,the expressions of YAP,CTGF,PCNA and SGK1were detected by western blot,and the changes of cell migration ability were detected by scratch test.CTL EC/VSMCs and Yap/Taz KO EC/VSMCs were co-cultured in transwell plate,respectively,and the PCNA expression of VSMCs was detected by Western blot.Si RNA interference technique was used to silence the expression of CTGF in EC（si-CTGF EC）.EC CTL/VSMCs and si-CTGF EC/VSMCs were mixed and co-cultured in 6-well plates,respectively.The changes of Ki67 expression in VSMCs were observed by immunofluorescence double staining.The effect of CTGF on the proliferation and migration ability of VSMCs:VSMCs were stimulated with 100ng/ml and 200 ng/ml CTGF for 24h,respectively,and PCNA expression was detected by western blot.The change of migration ability of VSMCs under 100 ng/ml CTGF was detected by scratch test.Results1.The expression of YAP protein was increased in internal fistula tissues of CKD miceAVF modeling was successful in 7 mice（7/8）in the non-CKD group,and in 8 mice（8/12）in the CKD group.The protein expression of YAP in the fistula tissue was significantly increased in western blot,the lumen area of AVF in the CKD group was decreased in HE staining,and the expression of YAP andα-SMA were increased in immunohistochemical staining,which was statistically significant compared with that in the non-CKD group（P<0.05）.Immunohistochemical staining showed that the positive staining rates of MAC2,IL-1βand SGK1 in the internal fistula tissue were increased,and the difference was statistically significant compared with that in the non-CKD group（P<0.05）.2.Yap/Taz gene knockout in vascular endothelial cells alleviates endometrial hyperplasia of internal fistulaImmunohistochemistry showed that YAP staining of the arterial endoderm of mice treated with tamoxifen was negative,and that of the endoderm of control mice was positive,indicating that conditional knockout of the Yap/Taz gene in the vascular endothelial cells of mice was successful.CKD and AVF were successfully constructed in seven（7/8）mice in the Yap/Taz KO group and eight（8/8）mice in the CTL group.HE staining showed an increase in AVF lumen area in Yap/Taz KO group,while immunohistochemical staining showed a decrease inα-SMA,PCNA and Ki67 positive staining rates in fistula tissue,and the difference was statistically significant compared with that in CTL group（P<0.05）.Immunohistochemical staining showed that the positive staining rates of inflammatory cell markers MAC2,CD45,inflammatory cytokines ICAM-1,VCAM-1,pro-inflammatory cytokine IL-1βand growth factor CTGF were decreased in the Yap/Taz KO group,and the difference was statistically significant compared with the CTL group（P<0.05）.3.Yap/Taz regulates the function of vascular endothelial cells and promotes the proliferation of vascular smooth muscle cellsAfter S1P and LPA stimulation,the expression of PCNA,SGK1 and CTGF in Yap/Taz KO EC was decreased in western blot,and the difference was statistically significant compared with that in Yap/Taz KO EC（P<0.05）.The mobility of Yap/Taz KO EC was lower than that of CTL EC in scratch test（P<0.05）.Western blot results showed that the expression of PCNA in VSMCs co-cultured with Yap/Taz KO EC was lower than that co-cultured with CTL EC（P<0.05）.Immunofluorescence dual staining showed that the proliferation of VSMCs co-cultured with si-CTGF EC was decreased compared with that co-cultured with CTL EC（P<0.05）.Western blot results showed that PCNA expression of VSMCs treated with CTGF was higher than that of control VSMCs（P<0.05）.In the scratch experiment,the mobility of VSMCs under CTGF stimulation was higher than that of the control group（P<0.05）.ConclusionCKD activates the Yap/Taz in EC,promotes the infiltration of inflammatory cells in the neointima of AVF,induces the expression of CTGF to promote the proliferation of VSMCs,which participates in the occurrence and development of AVF neointima formation.