The Role Of WDR1 In Vascular Smooth Muscle Cells Involved In Endometrial Remodeling Process After Vascular Injury

BackgroundPost-injury remodelling of blood vessels is a common vascular pathophysiological process in clinical practice,however this process often leads to Poor prognosis,such as arterial lumen stenosis,post-arterial graft hyperplasia and intimal hyperplasia after interventional procedures.Failure to address this process can indirectly lead to cardiovascular disease and other cardiovascular related complications.Common complications include atherosclerosis,myocardial infarction,and cerebral infarction.Large amounts of low-density lipoproteins（LDL）gradually accumulate in the damaged subendothelial space and are taken up by macrophages and smooth muscle cells（SMCs）in the vessel wall,which form foam cells and gradually evolve into atherosclerosis.Lipid-mediated inflammation induces massive proliferation and migration of smooth muscle cells to the vascular intima.Vascular smooth muscle cells（VSMCs）are the main cellular component of the vascular wall.After stimulation by external forces or inflammation,smooth muscle cells located in the middle layer of the vessel rapidly undergo phenotypic transformation,proliferate and then migrate towards the intima,leading to intimal hyperplasia,which is the process of neointima formation.The molecular mechanism of this process remains unclear.Previous studies have shown that the proliferation and migration of SMCs have a crucial role in the process of vascular endothelial formation.The actin cytoskeleton,as the support base of the cell,is involved in numerous cell biological processes,such as cell motility,phenotypic transition,cell proliferation and migration.The reconstitution of the cytoskeleton determines a series of biological processes,such as cell movement and proliferation.Since polymerized actin is relatively stable,specific actin regulatory proteins are required to assist and facilitate the disaggregation of actin filaments.The physiological significance of actin filament depolymerization lies not only in the removal of old actin filaments,but also in the formation of actin monomers after depolymerization that can replenish the actin monomer pool required for a new round of actin assembly.Thus,the balance of actin assembly and depolymerization is particularly important for the cytoskeleton.The actin depolymerizing factor（ADF/Cofilin）promotes actin depolymerization by severing actin filaments and dissociating actin monomers from the filaments.Although ADF/Cofilin usually accumulates in subcellular regions of the actin cytoskeleton dynamics,ADF/Cofilin alone,especially at high concentrations,is not very effective in driving rapid actin filament depolymerization.Therefore,when rapid actin turnover is required,other depolymerization factors need to be coupled with ADF/Cofilin to achieve rapid actin depolymerization.Actin interacting protein 1（AIP1）,also known as WD repeat domain 1（WDR1）,strongly enhances actin filament disaggregation even when ADF/Cofilin is saturated by actin filaments.As a major cofactor of ADF/Cofilin,WDR1-mediated actin dynamics is not only important for the regulation of cell polarity,but also has an integral role in the regulation of cell proliferation and migration processes.Previous studies have shown that migration and proliferation of VSMCs play a crucial role in the process of angiogenesis,but the mechanism of the role of WDR1 as a cofactor of cytoskeletal depolymerization factor in mediating vascular remodeling in VSMCs remains unclear.Signal Transducer and Activator of Transcription 3（STAT3）is one of the key factors of the Janus kinase/signal transducer and activator of transcription（JAK/STAT）signaling pathway,which is involved in inflammation and various cell biological processes such as cell division and cell proliferation.Previous studies have shown that the levels of the phosphorylated STAT3 are significantly elevated during the seventh day after vascular injury,suggesting that STAT3 is essential for the formation of vascular neointima,but it is unknown whether STAT3 is involved in the regulation of WDR1 expression in VSMCs.Activation and nuclear translocation of STAT3 is a prerequisite for its transcriptional function,and it has been shown that nuclear translocation of STAT3 is Ran-and importinβ-dependent.The cytoskeleton responds to alterations in the cellular microenvironment and mechanical stimuli that affect numerous cell biological processes.WDR1 is one of the important factors in cytoskeletal remodeling and it has been reported that deletion of WDR1 in breast cancer cells affects importinβexpression.Taken together,STAT3 may promote intimal formation by facilitating WDR1 transcription,and in turn WDR1-mediated actin dynamics may influence STAT3 nuclear trafficking,with WDR1-STAT3 forming a feedback loop that collectively regulates post-injury vascular remodeling.ObjectiveThis study investigates the mechanism of WDR1 in post-vascular injury remodeling at the molecular,cellular and animal levels,and elucidates the effect of the interaction between STAT3 and WDR1 on the process of post-vascular injury remodeling to further improve the molecular regulatory network of post-vascular injury remodeling and provide clues for the clinical treatment of peripheral vascular diseases.MethodsChapter 2:In this study,a mouse vascular injury model was constructed by means of common carotid artery ligation,and mice were executed by cervical dislocation method at different days of injury and collection of materials according to different experimental purposes.The expression of Wdr1 was measured by immunohistochemistry,RT-q PCR,Westren blot at different days of vascular injury.Meanwhile,the expression of proliferation marker PCNA and matrix metalloproteinase9 activity were detected by Western blot and Zymogram profiling assay,respectively.Chapter 3:Wdr1 conditional knockout mice were constructed by Cre-Loxp system,primers were designed and mice were identified genotypically by PCR.Knockout of Wdr1 was induced by intraperitoneal injection of tamoxifen（10mg/KG/day）in vivo,and Wdr1 expression was detected at the protein and RNA levels using Western blot and RT-PCR,respectively.The common carotid artery was then surgically injured and intimal thickening was detected by H&E staining.After knocking down Wdr1,apoptosis was detected using TUNEL assay,while the protein levels of p-H3 and SMA was detected by immunofluorescence.The activity of MMP9after Wdr1 knockdown was detected by Zymogram profiling assay.Mouse primary vascular smooth muscle cells were isolated and knockdown of Wdr1 in primary cells was induced in vitro by addition of 4-OH tamoxifen.After testing the efficiency of cell knockdown as well as apoptosis,the effect of WDR1 on smooth muscle cell proliferation and migration was then confirmed by CCK8 and scratch assays.Chapter 4:Overexpression of Wdr1,the effect of WDR1 on migration and proliferation in smooth muscle cells was further confirmed by Transwell and CCK8assays.Activation of STAT3 was detected by Western blot in vessels at different days of injury,confirming the relationship between STAT3 and vascular injury repair.Smooth muscle cells were treated with Ang II and IL-6 in vitro to imitate the vascular injury in vivo,and the proliferation and migration ability after Ang II and IL-6treatment were examined using scratch assay and CCK8.The expression of Wdr1 after different cytokine treatments was detected by Western bloting and RT-PCR.The expression of Wdr1 was detected by knocking down Stat3 in smooth muscle cells,treating smooth muscle cells with AG490 and overexpressing Wdr1 on top of knocking down STAT3 to detect the proliferation and migration ability of the cells.Overexpression of Cofilin after Wdr1 knockdown,immunofluorescence assays,CCK8,Western blot and scratch assays were used to confirm the relationship between WDR1-mediated actin dynamics and smooth muscle cell proliferation and migration.Finally,the dual luciferase reporter gene assay and chromatin immunoprecipitation confirmed that STAT3 directly targets the promoter of Wdr1.Chapter 5:Detection the expression and activation of STAT3 after knockdown or overexpression of Wdr1 to investigate whether WDR1 can have an effect on Stat3expression and activation.The effect of WDR1 on nuclear translocation of STAT3 was confirmed by separation of nucleoplasm and cytoplasm,and the expression and nucleoplasmic distribution of Ntf2,Importinβ,Importinαand Ran,factors associated with nuclear translocation of STAT3,were investigated by Western blot and separation of nucleoplasm and cytoplasm.The effects of Ntf2 and Importinβoverexpression on proliferation and migration of smooth muscle cells were verified by Transwell and CCK8 assays.ResultsChapter 2:HE results confirm that common carotid artery ligation effectively induces intimal thickening,and immunohistochemical assays,Western blot and RT-PCR results show a time-dependent increase in Wdr1 expression at different days of injury,with peak expression at day 14 of injury,and a time-dependent increase in the expression of the proliferation marker PCNA.The results of gelatinase profiling suggested that the activity of MMP9 was significantly increased after arterial injury.Chapter 3:Genotype identification results show that Wdr1^f/f;ERT2Cre mice were successfully constructed.Western blot and RT-PCR results showed that tamoxifen injection was able to successfully induce Wdr1 knockdown in mouse arterial cells.Further results showed that Wdr1 knockdown effectively inhibited the neointima formation after vascular injury.The results of Immunofluorescence and Zymogram profiling assays showed that cell proliferation and migration were significantly inhibited in arteries after Wdr1 knockdown,and TUNEL assays showed that Wdr1knockdown did not affect cell apoptosis.Mouse primary vascular smooth muscle cells were isolated and the results of Western blot and RT-PCR showed successful knockdown of Wdr1 after 4-OH tamoxifen induction.The results of scratch and CCK8assays showed that Wdr1 knockdown inhibited the proliferation and migration of primary smooth muscle cells.Chapter 4:The results of Transwell assays and CCK8 experiments showed that overexpression of Wdr1 enhanced the proliferation and migration capacity of HAVSMCs cells.In the induced proliferation of HAVSMCs and damaged blood vessels,Western blot results showed that the phosphorylation level of STAT3 was significantly increased.Knockdown of Stat3 significantly inhibited proliferation and migration of HAVSMCs,and the same results were obtained by specific inhibition of HAVSMCs treated with JAK2.Western blot and immunofluorescence assays suggest that actin dynamics are required for WDR1-mediated smooth muscle cell proliferation and migration.Chapter 5:Western blot results suggest that WDR1 deficiency does not affect STAT3 expression and activation.Separation of nucleoplasm and cytoplasm results showed that WDR1 deficiency inhibited the nuclear translocation of activated STAT3.Further Western blot and separation of nucleoplasm and cytoplasm results showed that WDR1 deficiency inhibited the expression of importinβand the nucleoplasmic distribution of Ran,thereby inhibiting the nuclear translocation of activated STAT3.ConclusionVascular injury repair is a complex biological process that is influenced by multiple factors.Various transcription factors,inflammatory factors,etc.together form a regulatory network involved in the formation of the neointima of blood vessels.In this study,we have revealed the molecular mechanisms by which WDR1 regulates intima formation,mainly the expression of Wdr1 regulated by STAT3 and the effect of WDR1 deletion on nuclear translocation of STAT3,which is activated and translocated into the nucleus in response to altered haemodynamics and inflammatory factor stimulation,and binds to the promoter of the Wdr1 gene to promote its transcription.In turn,the deletion of WDR1 inhibited the expression of nuclear transport factor 2（NTF2）and importinβ,which in turn affected the nuclear transport of STAT3.Meanwhile,the nucleoplasmic ratio of Ran was significantly decreased in smooth muscle cells with Wdr1 knockdown,further indicating that WDR1 is essential for STAT3 nuclear translocation.Using the cytoskeletal depolymerization factor WDR1 as an entry point,this study focused on elucidating the molecular mechanisms by which WDR1 affects the formation of vascular neointima,providing new insights into the mechanisms underlying the development of endothelial hyperplasia and providing clues for the clinical treatment of postoperative vascular endothelial hyperplasia.