BAG3 Promotes Proliferation And Migration Of Arterial Smooth Muscle Cells By Regulating STAT3 Phosphorylation In Diabetic Vascular Remodeling

Background:A large number of previous clinical and basic studies have confirmed the correlation between diabetes mellitus and macrovascular and microvascular diseases.Chronic hyperglycemia can regulate the occurrence and development of cardiovascular complications of diabetes mellitus through various mechanisms,including increased formation of advanced glycation end products（AGEs）and activation of AGEs receptor.As the most important pathological basis of diabetic cardiovascular complications,diabetic vascular remodeling is mainly manifested as the increase of AGEs caused by elevated blood sugar leads to the proliferation and migration of vascular smooth muscle cells（VSMCs）,which contributes to arterial wall thickening and narrowing,and finally vascular remodeling.Therefore,abundant proliferation and migration of VSMCs,as the main pathological basis for the occurrence and development of diabetic vascular complications,is an important therapeutic target for diabetic vascular remodeling.However,due to the lack of breakthrough in basic experiments,there is currently a lack of clinical treatment for the excessive proliferation and migration of VSMCs in diabetic vascular remodeling.Bcl-2-associated athanogene 3（BAG3）protein is a multifunctional protein highly expressed in skeletal muscle and myocardium.Previous studies have confirmed that BAG3can not only promotes cell survival and inhibits cell apoptosis,but also participates in the regulation of cell proliferation and migration.Since excessive proliferation and migration of VSMCs is an important pathogenesis of vascular remodeling in diabetes mellitus,the role of BAG3 in the excessive proliferation and migration of VSMCs and its molecular mechanism deserve further investigation.Signal transducer and activator of transcription 3（STAT3）is a multifunctional protein involved in many biological processes such as cell proliferation,differentiation and apoptosis.Studies have confirmed that activation of STAT3 not only plays a vital role in embryonic development,hematopoietic and immune system regulation,but also promotes the occurrence,angiogenesis and metastasis of a variety of tumors.Therefore,this study focuses on whether BAG3 is involved in the excessive proliferation and migration of VSMCs in diabetic vascular remodeling by regulating the phosphorylation of STAT3.Objective:In this study,BAG3 gene was knocked out in smooth muscle to aquire SM22αCre;BAG3^FL/FL mice and the role of BAG3 protein in the hyperproliferation and migration of VSMCs during vascular remodeling in diabetic mice was studied,and the molecular mechanism was further explored.Methods:In the first part,streptozotocin（STZ）was used to induce diabetes in SM22αCre;BAG3^FL/FLmice,and the differences in aortic vessel thickness between each group were compared.Subsequently,human aortic smooth muscle cells（HASMC）were stimulated with different concentrations of AGEs,and the expression of proteins and the proliferation and migration status of HASMC were detected.Then,we up-regulated or down-regulated BAG3 expression in HASMC and used AGEs aiming to determine the role of BAG3 in the AGEs-induced proliferation and migration of HASMC by detecting the expression of proteins and HASMC proliferation and migration status.In the second part,we first detected the expression of different proteins in aorta of SM22αCre;BAG3^FL/FLmice.Subsequently,BAG3 expression was up-regulated and down-regulated in HASMC respectively and AGEs were given.The downstream signaling pathway regulated by BAG3 was analyzed by detecting the expression of different proteins in HASMC.On this basis,we first up-regulated or down-regulated BAG3 expression and gave AGEs stimulation,and then gave inhibitors targeting the two downstream signaling pathways of BAG3.By detecting the expression of different proteins in HASMC,the two downstream signaling pathways regulated by BAG3 were confirmed again.Finally,CO-IP were conducted in the aorta of SM22αCre;BAG3^FL/FLmice and HASMC in order to analyze the influence of BAG3 on the interaction between downstream proteins during the occurrence of diabetic vascular remodeling,and to identify the interaction domains of BAG3 and STAT3 by constructing plasmids that knocked out different BAG3 domains.Results:Through the first part of functional experiments,we confirmed that diabetic vascular remodeling was alleviated in SM22αCre+;BAG3^FL/FLmice.It was also observed that up-regulation of BAG3 expression in HASMC promoted the proliferation and migration of AGEs-induced HASMCs,while down-regulation of BAG3 expression inhibited the proliferation and migration of AGEs-induced HASMCs.In the second part of the experiment,we conducted an in-depth exploration of the specific mechanism of BAG3 protein promoting VSMCs migration in diabetic vascular remodeling.First,it was observed that BAG3 was involved in regulating the expression and phosphorylation of STAT3 protein in aorta of SM22αCre+;BAG3^FL/FLmice.Subsequently,we demonstrated in HASMC that upregulation of BAG3 further upregates AGEs-induced STAT3 protein expression and its phosphorylation at tyrosine 705 and inhibits its phosphorylation at serine 727.In addition,by up-regulating or down-regulating BAG3 in HASMC and then administering inhibitors of both pathways respectively,we confirmed that BAG3 can promote AGEs-induced HASMC migration by regulating STAT3 phosphorylation at tyrosine 705 and serine 727.Finally,we demonstrated that BAG3 can interact with STAT3 protein through its PXXP domain,and AGEs can enhance this interaction.BAG3 enhances the interaction between JAK2 and STAT3 by interacting with STAT3 and JAK2,thus promoting the phosphorylation of p-JAK2 to STAT3 tyrosine705 site.At the same time,BAG3 weakens the interaction between ERK1/2 and STAT3by interacting with STAT3 and ERK1/2,thus inhibiting p-ERK1/2 phosphorylation of serine 727 site of STAT3.Conclusion:BAG3 promotes the proliferation and migration of VSMCs in diabetic vascular remodeling.The induction of AGEs on BAG3 expression and VSMCs proliferation and migration is concentration-dependent.BAG3 promotes VSMCs migration during diabetic vascular remodeling by interacting with STAT3.AGEs promote the interaction between BAG3 and STAT3,while BAG3 enhances the interaction between STAT3 and JAK2 and weakens the interaction between STAT3 and ERK1/2,promoting the phosphorylation of STAT3 at tyrosine 705and inhibiting the phosphorylation of STAT3 at serine 727.Subsequently,the expression of MMP2 is upregulated,thus promoting the migration of VSMCs.