| BackgroundHemodialysis is the main treatment for patients with end-stage renal disease.The key to this method is to establish and maintain adequate blood circulation pathways.In the process of hemodialysis,continuous vascular access is an important part of ensuring that patients can receive timely treatment.About 70%of patients with end-stage renal disease currently undergo renal replacement therapy through dialysis.Dialysis was performed by autogenous arteriovenous fistula(AVF)puncture of the radial artery-cephalic vein.Compared with other dialysis channels,AVF can effectively reduce the risk of complications and infections,and the cost of treatment is relatively low,the puncture and compression operations are simple and easy,and the intervention rate is lower,so it is more and more accepted by the majority of hemodialysis patients.Good and mature AVF is the lifeline of hemodialysis patients,but so far there is still a lack of methods to effectively maintain the smoothness of the hemodialysis pathway.The main cause of loss of AVF function is severe venous intimal hyperplasia near the anastomosis,causing vascular stenosis.The occurrence and development of intimal hyperplasia is related to the excessive proliferation and migration of vascular smooth muscle cells,and is also affected by the synthesis of extracellular matrix.Vascular smooth muscle plays an important role in maintaining the stability of blood vessels.When damaged by the action of extravascular force,the vascular smooth muscle cells of the blood vessels are stimulated and proliferated,and the volume of the outer membrane is increased,eventually remodeling the blood vessels.In addition,the permeability increased after vascular injury,causing extravasation of neutrophils and monocytes,which stimulated the release of inflammatory factors and the expression of vascular endothelial cell adhesion factor,which ultimately promoted the proliferation of smooth muscle cells and the growth of new endothelial cells.The mechanism of fistula stenosis is caused by hemodynamic pressure,which belongs to a chronic development process,due to the long-term increase of hemodynamic stress at the anastomosis of veins and veins between arteries and veins or grafts,plus postoperative results.Intimal injury and inflammatory reaction promote the production and release of various inflammatory factors,which leads to endothelial cell damage,proliferation and migration of smooth muscle cells,migration and deposition of a large number of extracellular matrix,and eventually results in thickening of the blood vessel wall and shrinking of the lumen,causing thrombosis and vascular occlusion.Intimal hyperplasia caused by massive proliferation of vascular smooth muscle positively expressed by a-SMA is the main pathological manifestation of intimal stenosis.Basic fibroblast growth factor(bFGF)is a basic polypeptide.bFGF has biological functions such as promoting angiogenesis,inhibiting apoptosis and inducing cell migration,and thus participates in many physiological and pathological processes.Including organ development,tissue regeneration and damage repair.bFGF has a strong mitogenic effect on vascular endothelial cells and vascular smooth muscle cells.Endogenous bFGF can be produced and released after damage to vascular smooth muscle cells,and regulation of its own proliferation is achieved via endocrine and paracrine forms.Current research has confirmed that bFGF can participate in tissue proliferation and fibrosis by regulating the proliferation and migration of vascular smooth muscle cells.Other studies have shown that the rate of cell migration increases with the increase of bFGF concentration,suggesting that the vascular smooth muscle cell migration rate is positively correlated with bFGF concentration.This bFGF-induced migration effect is associated with the expression of fibroblast growth factor receptor on the surface of vascular smooth muscle cells,and is also closely related to the expression of downstream migration proteins.When endothelial cells are damaged or inflammatory,local endothelial cells are activated,which in turn produces and releases a large amount of bFGF,which causes mononuclear cells and neutrophils to move to the injured site via chemotaxis,causing an inflammatory reaction,and bFGF is also cells capable of chemotaxis including fibroblasts,endothelial cells,and the like are involved in the repair and reconstruction of local vascular damage.However,whether bFGF affects arteriovenous fistula stenosis in hemodialysis patients remains unknownThe study found that bFGF usually exerts its biological effects through the transforming growth factor-β1(TGF-β1)/Smad3 signaling pathway.TGF-β is a polypeptide cytokine that is widely involved in a variety of biological functions including cell growth,differentiation,migration,extracellular matrix secretion,and immune regulation.TGF-β1 can promote the proliferation of fibroblasts,thereby promoting the production of extracellular matrix components and promoting the abnormal proliferation of vascular cells.In arterial injury,TGF-β1 is secreted in a large amount to inhibit the expression of MMP and increase the expression of protease inhibitors,promote the secretion of collagen and eventually lead to intimal hyperplasia.The study found that up-regulation of TGF-β1 gene or protein expression by intraperitoneal injection can stimulate the proliferation of vascular smooth muscle cells and cause intimal hyperplasia;while TGF-β1 antibody can be acid-resistant when injected into the body.The TGF-β1 signaling pathway is broken,thereby reducing intimal hyperplasia and delaying vascular remodeling.There is a close relationship between the major signaling mechanisms of TGF-β1 and the Smad3 family.The TGF-β1/Smad3 signal transduction pathway is closely related to various biological processes in the body,including cell proliferation,immunosuppression,and inflammatory response.The most studied TGF-β1/Smad3 signal transduction pathways are currently available.The role of disease fibrosis remodeling is also related to ventricular remodeling therapy.However,there is no research on the relationship between TGF-β1/Smad3 signaling pathway and arteriovenous fistula stenosis.ObjectiveThis study examined the expression of a-SMA in arteriovenous fistulas and explored the relationship between bFGF and TGF-β1/Smad3 signaling pathways in arteriovenous fistulas in hemodialysis patients.At the same time,the animal model of carotid arteriovenous fistula of bFGF knockout mice was established to further verify the mechanism of bFGF in arteriovenous fistula stenosis,and provide a prevention and treatment for functional insufficiency and stenosis of clinical AVF.It aims to improve the patency rate of AVF and quality of life of patients with end-stage renal disease and prolong their survival time.Methods(1)Twenty-four patients with stage 5 chronic kidney disease admitted to the Department of Nephrology/Blood Purification of Jinan Central Hospital affiliated to Shandong University were selected as subjects.All patients were selected for arteriovenous fistula with severe stenosis or occlusion and unable to maintain normal hemodialysis.The patient needs to be admitted to the hospital for arteriovenous fistula,and the above patients are listed as the AVFS group.General information about the patient was collected,including the minimum internal diameter of the vessel,age,gender,serum albumin(ALB),hemoglobin(Hb),and cholesterol(CHO).Blood vessel segments in which all of the above patients developed AVF stenosis during the reconstruction period were collected.In addition,8 patients who underwent vascular resection due to peripheral vascular disease into the vascular surgery were selected as the control group.The venous blood vessel samples of AVF stenosis were collected from each group.The expression of a-SMA and bFGF mRNA in blood vessel samples were detected by real-time PCR.The expression of TGF-β1 and Smad3 protein was detected by western blot.(2)C57BL/6 mice and C57BL/6 background bFGF knockout mice were randomly divided into 3 groups,10 in each group:control group:C57BL/6 mice were routinely reared without any treatment;AVF Group:C57BL/6 mice established an arteriovenous fistula model of carotid-jugular vein,followed by routine feeding;AVF+sibFGF group:C57BL/6 background bFGF knockout mice established carotid-cervical vein The arteriovenous fistula model was followed by routine feeding.The above groups of mice were sacrificed at the 4th week after the corresponding treatment,and the external jugular vein samples were collected for the next experimental study.Venous side specimens were collected after AVF,and histological changes of the venous intima were studied by HE staining.The expressions of TGF-β1 and Smad3 mRNA and protein in the intima of AVF stenosis were detected by real-time PCR and Western blot.Mouse inferior vena cava vascular smooth muscle cells were isolated and randomly divided into 2 groups,with 3 replicates in each group:control group:control plasmid was transfected into C57BL/6 mouse vascular smooth muscle cells;bFGF group:bFGF overexpression plasmid was transferred C57BL/6 mouse vascular smooth muscle cells were stained,and the proliferation activity and migration ability of the cells were examined.Result(1)Compared with the control group,the expression levels of a-SMA and bFGF mRNA in the vascular tissues of the AVFS group were significantly increased(P<0.001),indicating that there was a large amount of smooth muscle cell proliferation in the vascular segment of the AVFS.Compared with the control group,the expression of TGF-β1 protein(P = 0.003)and Smad3 protein(P =0.001)in the AVFS group was significantly increased,indicating that there was a large amount of smooth muscle cell proliferation in the vascular segment of AVFS,Activation of theTGF-β1/Smad3 signaling pathway may be involved in the process by which bFGF promotes smooth muscle cell proliferation.(2)The results of HE staining showed that the vein structure of the Control group was almost normal,and the endothelium of the control group was a single layer of endothelial cells.The appearance was flat,the arrangement was tight and the edges were flat,the long axis of the cells was parallel to the lumen,and there was no internal elastic membrane under the endothelium.The middle membrane is longitudinal smooth muscle and circular smooth muscle,which contains 2-3 layers of elastic membrane;the outer membrane is loose connective tissue,and its composition is collagen fiber.The intimal hyperplasia was most prominent in the venous lumen of the AVF group.The local intima showed papillary hyperplasia and extended to the contralateral wall.The structure was disordered.A large number of inflammatory cells infiltrated,localized proliferative endometrial necrosis was observed.Smooth muscle cells increased,collagen fibers proliferated and mixed with the muscle layer;the elastic membrane was significantly damaged,showing a small amount of fibroblasts.In the AVF+sibFGF group,the venous wall of the mice was obviously proliferated,the endothelial cells were disordered,the intima was thickened,and smooth muscle hyperplasia,local endothelial cell shedding,extracellular matrix hyperplasia,and scattered inflammatory cell infiltration were observed.However,compared with the AVF group,the degree of stenosis of arteriovenous fistula in mice after bFGF gene knockout was significantly relieved compared with non-knockout mice.The results of real-time PCR showed that the expression levels of TGF-β1 and Smad3 mRNA and protein in the AVF group and AVF+sibFGF group were significantly higher than those in the control group(P<0.05);Compared with the AVF group,the expression levels of TGF-β1 and Smad3 mRNA and protein in the AVF+sibFGF group were significantly decreased(P<0.05).The results of cell proliferation assay showed that the proliferation activity of bFGF cells on day 3 and day 5 was significantly higher than that of control group(P<0.05),indicating that bFGF can promote the proliferation of vascular smooth muscle cells.The results of cell migration assay showed that compared with the control group,the number of migrated cells in the bFGF group was significantly increased(P<0.05),indicating that bFGF can promote the migration of vascular smooth muscle cells.Conclusion(1)Massive proliferation of smooth muscle cells exists in AVF stenotic vessels;(2)bFGF promotes proliferation and migration of vascular smooth muscle by regulating TGF-β1/Smad3 signaling pathway,which leads to intimal stenosis after AVF. |
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