Mechanism Of Arteriovenous Fistula Stenosis With Growth Differentiation Factor 8 Promoting Mesenchymal Stem Cell Activation

Background:Arteriovenous fistula（AVF）patency is a prerequisite for the smooth progress of dialysis in hemodialysis patients,and neointima and stenosis caused by vascular access dysfunction are important factors in increased patient mortality.Currently,there are no effective preventive and therapeutic measures in clinical practice.Therefore,how to prevent and treat AVF neointima and stenosis and improve patency has become a hot and difficult issue in the field of blood purification in recent years.In basic research,there is an increasing number of studies on the mechanism of AVF neointima,but there is still no scientific and effective means to prevent and treat AVF stenosis.The reasons for this include the lack of specific targets,targeted intervention drugs,and limited clinical medication methods.So far,interventions targeting vascular endothelial cell and vascular smooth muscle cell proliferation have not completely alleviated endothelial stenosis,and mesenchymal stem cells（MSCs）with multi-differentiation functions in the vascular adventitia may be involved in the process of vascular remodeling,but the specific mechanism is still unclear and requires further exploration of the molecular regulatory mechanisms of MSCs in the process of vascular remodeling.GDF-8（growth differentiation factor 8）,also known as myostatin,is a member of the TGF-βsuperfamily and is mainly produced in skeletal muscle cells.Its expression is upregulated in CKD,promoting the degradation of skeletal muscle fibers.In recent years,it has been found that the GDF-8 family plays an important regulatory role in cardiovascular diseases,CKD,chronic malignant diseases,and other aging and metabolism-related diseases.However,the role of GDF-8 in CKD AVF neointima and stenosis has not been determined,and further exploration is needed to elucidate its molecular regulatory patterns,especially for MSCs with multi-differentiation functions.Research purpose:To explore the changes of GDF-8 in MSCs proliferation and activation and vascular remodeling,and further explore the role of the key molecule YAP in AVF neointima and stenosis and the related molecular regulatory mechanisms.Research methods:First,using in vitro cell culture and wild-type mice,exogenous GDF-8 was given to observe the activation and proliferation of MSCs and the pathological changes of mouse muscle and vascular adventitia.Further,a 5/6 nephrectomy CKD mouse model was established,and an end-to-side anastomosis of the cervical artery and jugular vein in CKD mice was performed to simulate the clinical autologous arteriovenous fistula model.GDF-8 antagonist was used for intervention to observe its effect on AVF neointima and stenosis.The molecular mechanisms of GDF-8 activation of YAP signal-regulated MSCs activation and CKD AVF stenosis were revealed by cell experiments,immunofluorescence staining,and western blotting.Finally,a TK-hb PPE gel containing reactive oxygen species（ROS）sensitive YAP inhibitor was wrapped around the AVF anastomosis,and far-infrared light was applied to the local irradiation area to achieve controlled remote targeting of the YAP signal in the AVF adventitia MSCs and observe the potential clinical efficacy of this drug delivery system.Research Results:1,GDF-8 stimulates the activation and proliferation of adventitial mesenchymal stem cells（1）In CKD mice,body weight decreased,skeletal muscle mass declined,muscle fiber area reduced,and the expression of GDF-8 in skeletal muscles increased.Aortic adventitial fibrosis worsened,and adventitial cell proliferation was more active.（2）After administration of exogenous GDF-8 in normal mice,effects similar to CKD occurred,namely weight loss,decline in skeletal muscle mass（in gastrocnemius,tibialis anterior,and solenus muscles）,and reduction in muscle fiber area.In mice treated with GDF-8,ECM deposition,such as type III collagen,in the aortic adventitia intensified,PDGFRα-positive MSCs in the adventitia became more active,and the expression of YAP protein in MSCs increased.In AVF,after GDF-8 treatment,the lumen of the AVF narrowed,neointimal hyperplasia worsened,adventitial fibrosis of AVF intensified,and the proliferation marker（increased Ki-67 signal）co-stained with PDGFRα-positive MSCs increased,indicating that adventitial MSCs of AVF were more proliferative after GDF-8 treatment.（3）In vitro experiments revealed that after stimulating MSCs with GDF-8,the expression of MSC’s fibronectin,αSMA,YAP and its downstream protein CTGF increased.However,GDF-8 antagonist ATA842 could mitigate these effects.Nevertheless,ATA842 could not reduce the stimulating effect of TGF-β,a member of the GDF-8 family,on MSCs.2 GDF-8 promotes neointimal hyperplasia and narrowing in arteriovenous fistulas in chronic kidney disease（1）The GDF-8 antagonist ATA842 can alleviate the weight loss and muscle atrophy caused by CKD in mice,reduce the expression of GDF-8 in skeletal muscle fibers,mitigate the neointimal formation in AVF in CKD mice,reduce the activation of adventitial MSCs in AVF,and decrease their proliferative and differentiative abilities（as evidenced by a reduction in the expression of Ki-67,YAP,and FSP-1 proteins compared to the control group）.3 The Mechanism of GDF-8 activating the YAP Signaling Pathway to Promote Activation and Proliferation of MSCs.（1）In in vitro cell experiments,we overexpressed the YAP gene in MSCs endogenously,which resulted in effects similar to those seen when MSCs are stimulated with GDF-8,i.e.,the expression of Fn,collagen I,αSMA increased in MSCs.Conversely,knocking down YAP resulted in the downregulation of Fn,αSMA,and the downstream protein of YAP,CTGF,while similar results were obtained when the YAP inhibitor verteporfin was used exogenously.（2）Under the stimulating effect of GDF-8,the expression of early response protein p ERK1in MSCs increased.The use of ERK1 inhibitor U0126 or YAP inhibitor verteporfin can suppressαSMA protein expression,however,verteporfin cannot inhibit p ERK1 expression.SGK1inhibitor had similar results to verteporfin,being able to suppress YAP protein expression but unable to inhibit p ERK1.Conversely,U0126 can downregulate SGK1 expression.From this,we believe that the activation of YAP protein expression by GDF-8 is through the ERK/SGK1pathway,and the expression of YAP plays a significant regulatory role in the activation of MSCs stimulated by GDF-8.4 The intervention effect of drug-loaded hydrogel system on inhibiting the overproliferation of neointima in arteriovenous fistulas in chronic kidney disease by suppressing the YAP signaling pathway in MSCs（1）In in vitro experiments,upon irradiation,the ^TKhb PPE/VER@PPP system could successfully control the release of verteporfin,thereby inhibiting the activation of MSCs induced by GDF-8.（2）In the mouse AVF model,the ^TKhb PPE/VER@PPP system,upon irradiation,could successfully control the release of verteporfin,inhibit the YAP protein expression in adventitial MSCs of AVF,attenuate the overactivation of MSCs,thus mitigating the lumen narrowing and neointimal hyperplasia of AVF.Research conclusion:We first discovered that skeletal muscle communicates with the arteriovenous fistula（AVF）outer membrane through GDF-8,demonstrating that GDF-8 is a important factor in AVF outer membrane vascular remodeling and AVF intimal hyperplasia.GDF-8 activates MSCs by upregulating YAP protein expression,promoting MSC proliferation and differentiation.Inhibiting YAP protein in MSCs suppresses their activation,thereby inhibiting AVF intimal hyperplasia and stenosis.For the first time,we used nanoparticles loaded with Verteporfin to remotely control and inhibit YAP protein expression in vascular outer membrane MSCs,thereby suppressing the activation of outer membrane MSCs,alleviating AVF intimal hyperplasia and stenosis,and providing potential strategies for the clinical prevention and treatment of AVF intimal hyperplasia and stenosis.