The Mechanism Of Up-Regulation Of JAGGED1 In Vascular Endothelial Cells Participating In Intimal Hyperplasia Of Arteriovenous Fistula

BackgroundAccording to China’s chronic kidney disease（CKD）epidemiologic survey estimates,China’s existing patients with CKD about 130 million,about 2%of patients will enter the stage of end-stage renal disease（ESRD）,this part patients need to renal replacement therapy,among them,the hemodialysis（HD）is a common renal replacement therapy.According to the National Blood Purification Case Information Registration Data,by the end of 2014,China had nearly 340,000 hemodialysis patients.For patients with CKD undergoing maintenance hemodialysis,the optimal vascular access has always been arteriovenous fistula（AVF）,but unfortunately,the patent rate after 1 year of AVF is only 60%.Dysfunction of AVF leads to inadequate dialysis,which leads to an increase in central venous catheter use,hospitalization and mortality in dialysis patients.Currently,there are few effective therapies for this clinical problem.Therefore,it is of great significance to further explore the pathogenesis of stenosis and dysfunction of AVF,and to research and develop effective prevention and treatment methods to improve the maturity rate of AVF and prolong the service life of AVF,so as to improve the prognosis and quality of life of hemodialysis patients.The present study showed that is the most common cause of AVF dysfunction is stenosis of the outflow tract caused by intimal hyperplasia（IH）of the vein surrounding the anastomosis.According to the existing research results,intimal hyperplasia of AVF is closely related to the migration and proliferation of vascular smooth muscle cells（VSMC）and fibroblasts,as well as the infiltration of mononuclear macrophages.Our previous research results showed that under chronic kidney disease,the Notch signal of vascular smooth muscle cells and monocyte macrophages was activated,and a series of phenotypic transformations and functional transformations occurred,which eventually led to intimal hyperplasia.However,under the condition of chronic kidney disease,the relationship between vascular endothelial cells（VEC）and IH is not yet clear.Vascular endothelium is a monolayer of cells that separates the blood vessel wall from circulating blood.Endothelial cells are not only a highly selective barrier against the early pathological changes of neointimal hyperplasia,but also synthesize and release molecules to maintain vascular homeostasis.The destruction of endothelial cells and the exposure of related factors can trigger the formation of new intima.Notch signaling pathways exist widely in many organisms and are highly conservative in evolution.Through the combination of Notch ligands and receptors on the surface between adjacent cells,they can realize the regulation of various functions of organisms.Jagged1 is a Notch ligand and plays an important role in vascular remodeling.Studies have shown that the expression of Jagged1 in pulmonary microvascular endothelial cells is closely related to endothelial-mesenchymal transition（Endo MT）during bleomycin-induced pulmonary fibrosis.Objective1.To investigate whether Jagged1 is involved in the process of intimal hyperplasia in arteriovenous fistula of rats with chronic kidney disease;2.To explore the effect of chronic kidney disease on the expression of vascular Endothelial cells Jagged1 and the endothelial-mesenchymal transition.Methods1.Establishment of a rat model of arteriovenous internal fistula with end-to-side anastomosis in chronic kidney disease 18 wild type SD rats aged 8-12 weeks were divided into 2 groups:Sham group,6.Model group（CKD+AVF）,12.a.5/6 major nephrectomy was performed to establish a model of chronic kidney disease:in order to reduce animal mortality,rats in the model group were subjected to secondary nephrectomy,with a total of 5/6 renal tissue removed.4weeks after operation,venous blood of rats was collected by tail vein blood sampling method to detect serum creatinine,so as to determine whether the modeling for chronic kidney disease was successful.b.Common carotid artery-external carotid vein end-to-side anastomosis arteriovenous fistula in rats model was constructed and ultrasonic testing arteriovenous fistula blood flow and cardiac structure:the rat model with chronic kidney disease after the success of the building,implementation of common carotid artery-external carotid arteriovenous end side anastomosis to establish arteriovenous fistula,postoperative use of animal ultrasound instrument fistula blood flow and cardiac structural changes;c.Relationship between Endo MT and intimal hyperplasia of arteriovenous fistula: AVF specimens of rats in the model group and carotid artery,jugular vein sham group were collected and paraffin-embedded and sectioning were performed.The degree of intimal hyperplasia of arteriovenous fistula was detected by HE,EVG and Masson staining.The expression ofα-SMA in the proliferative intima of AVF was detected by immunohistochemistry.Expression ofα-SMA and v WF in proliferative intima was detected by immunofluorescence double staining.d.Expression of Jagged1 in the intima of arteriovenous fistula:the expression of Jagged1 in the intima of AVF hyperplasia was detected by immunohistochemistry.2.Effects of chronic kidney disease on the expression of Jagged1 and Endo MT in vascular endothelial cellsHuman umbilical vein endothelial cells（HUVEC）were cultured in vitro with ECM medium containing 10%fetal bovine serum+1%endothelial growth factor+1%penicillin&streptomycin antibiotic.a.Effect of uremia serum on HUVEC Jagged1 protein expression and Endo MT: Using 10%fetal calf serum（FBS）,10%of normal serum（NS）and 10%uremic serum（US）to stimulate HUVEC 24 hours,respectively applied immune protein imprinting technology to detect HUVEC Jagged1,Endo MT-related protein(endothelial markers between VE-cadherin and mesenchymal markers ofα-SMA expression;Application of scratch test HUVEC migration ability of change.b.Effect of silence of Jagged1 gene expression on Endo MT induced by uremia serum:The expression of Endo MT-related proteins（endothelial marker ve-cadherin and mesenchymal markerα-SMA）was detected by immunoblot after stimulation with 10%uremia serum for 24 hours by si RNA transfection.The change of migration ability of HUVEC was detected by scratch test.c.Jagged1 regulates the effect of HUVEC NF-κB expression on Endo MT:Immunoblotting was used to detect the effects of uremia serum stimulation on the expression of HUVEC NF-κB and the silencing of Jagged1 gene expression on the expression of HUVEC NF-κB,VE-cadherin,andα-SMA.Results1.Endo MT was observed in AVF of CKD ratstsa.Compared with the sham operation group,the serum creatinine of the model group was significantly increased at the 4th week after the 5/6 nephrectomy（P<0.05）,suggesting that the CKD model was successfully constructed.The success rate of CKD modeling was 83.33%.b.The rats in the model group underwent ultrasound examination of internal fistula at 4 and 8 weeks after AVF operation,and the results showed that the AVF average blood flow velocity and average blood flow at 8^th week after AVF operation were significantly lower than that at 4^th week,with statistically significant differences（P<0.05）,suggesting that the AVF of CKD rats was successfully constructed.Five AVF rats were successfully modeled,and the success rate of modeling was 55.56%.At the 8^th week after AVF operation,all the rats in the model group had stenosis of AVF.c.Cardiac ultrasound imaging in rats showed that,compared with the sham group, the anterior and posterior walls of the left ventricle were significantly thickened and the left ventricular mass was significantly increased in the model group at 8 weeks after AVF surgery,and the difference was statistically significant（P<0.05）.d.AVF vascular histopathology:HE,Masson and EVG staining showed that compared with normal arterial and venous vascular tissues of rats in the sham operation group,the AVF vascular wall of rats in the model group showed thickened intima,lumen stenosis,a large amount of collagen deposition in the proliferated intima,and elastic fiber disorder.e.AVF vascular tissue immunohistochemical staining,the result shows:the model group rats AVF vascular tissues,hyperplasia of endometrium tissue in a large number ofα-SMA staining positive cells,and within the lumen side cortex appearsα-SMA positive staining,while in the control group rats normal arteries and veins in the organization,α-SMA express positive only in the blood vessels of membrane muscular layer.The results of immunofluorescence double staining showed that the lumen medial cortex of the intima hyperplasia of AVF showed cells co-expressed byα-sma/v WF.The above results suggested the presence of Endo MT in the intimal tissue with AVF hyperplasia.f.Jagged1 immunohistochemical staining results of AVF vascular tissue showed that:in the AVF vascular tissue of rats in the model group,Jagged1 staining positive cells were found in the proliferative intimal tissue,and Jagged1 discontinuous staining positive cells were found in the endovascular cortex of the lumen.However,in normal arterial and venous tissues of rats in the sham operation group,Jagged1 was only expressed in vascular endothelial cells,and the expression was weak.It is suggested that the expression of Jagged 1 in intimal vascular endothelial cells of AVF hyperplasia is up-regulated in uremia environment.2.Effects of chronic kidney disease on the expression of Jagged1 and Endo MT in vascular endothelial cellsa.uremic serum on vascular endothelial cell Jagged1 expression and the influence of Endo MT:use 10%uremic serum stimulate HUVEC 24h,immune protein imprinting detection,and the results showed that with 10%fetal bovine serum,10%normal serum group,compared to 10%uremic serum groups of HUVEC Jagged1,Notch1 andα-SMA protein expression increased,VE-cadherin expression decrease,the difference statistically significant（P<0.05）;The results of scratch test showed that HUVEC mobility in 10%uremic serum group was significantly increased compared with 10%fetal bovine serum group and 10%normal serum group,and the difference was statistically significant（P<0.05）.It is suggested that uresis serum activates Notch pathway by upregulating the expression of Jagged1 in vascular endothelial cells,inducing the occurrence of Endo MT.b.Effect of silence of Jagged1 gene expression on Endo MT induced by uremia serum:after HUVEC Jagged1 gene expression was silenced by si RNA,uremia serum was incubated for 24h,and immunoblotting was performed.The results showed that,compared with si RNA-con control group,the expression of Jagged1 and legu-sma were decreased in the si RNA-JAG1 group,with statistically significant difference（P<0.05）.The results of scratch test showed that compared with si RNA-con control group,the HUVEC mobility of si RNA-JAG1 group decreased.It is suggested that silence of Jagged1 gene expression can counter the promotion of HUVEC Endo MT by uremia serum.c.The effect of Jagged1 on the expression of HUVEC NF-κB on Endo MT:after 10%uremia serum was used to stimulate HUVEC for 24h,the immunoblotting results showed that HUVEC NF-κB protein expression in 10%uremia serum group was increased compared with 10%fetal bovine serum and 10%normal serum group,with statistically significant difference（P<0.05）.After the HUVEC Jagged1 gene expression was silenced by si RNA,uremia serum was incubated for 24h.The results of immunoblot detection showed that,compared with the si RNA-con control group,NF-κB and glu-sma expression were significantly decreased in the si RNA-JAG1 group,while VE-cadherin expression was significantly increased in the si RNA-JAG1 group,with a statistically significant difference（P<0.05）.Conclusion1.The rat model of arteriovenous internal fistula in chronic kidney disease was successfully constructed by majority nephrectomy and distal and lateral anastomosis of common carotid artery and external jugular vein.2.In rats with chronic kidney disease,Endo MT and Jagged1 expression were observed common carotid artery-external jugular vein fistula within 8 weeks after anastomosis.3.The up-regulated expression of Jagged1 in vascular endothelial cells in patients with chronic kidney disease may regulate the expression ofα-sma through the NF-κB signaling pathway and promote the transformation of endothelial cells into mesenchymal cells,which is one of the mechanisms of intimal hyperplasia in patients with chronic kidney disease.