Molecular Mechanisms Of Protein Arginine Methyltransferases In Endothelial To Mesenchymal Transition Of Vascular Endothelial Cells

BackgroundAccording to the seventh national census,China has more than 260 million people aged 60 or above,indicating that the country has officially entered the stage of population aging.Cardiovascular diseases were the leading cause of death in urban and rural areas,46.66%in rural areas and 43.81%in urban areas.The occurrence and development of cardiovascular diseases experience a complex pathophysiological process,in which Endothelial to mesenchymal transition（EndMT）is involved in the occurrence of most cardiovascular diseases,including congenital heart disease,coronary heart disease,valvular disease,cardiomyopathy,etc.However,the process of EndMT is complex and its mechanism has not been clearly defined,so studies on the role of EndMT in related cardiovascular diseases have been widely concerned by scholars.As a widespread endothelial cell in the circulatory system,EndMT refers to the transformation of the endothelial state into the interstitial state,with the original function of the endothelium weakened and the interstitial effect enhanced.Endothelial cells,as the first barrier in blood vessels,have various stimuli that can induce EndMT changes.TGF-βis one of the proteins of the transforming growth factorβfamily.It is one of the most in-depth cytokines to regulate EndMT process at present,and it is also the most commonly used stimulating factor to establish EndMT process in vitro.Tgf-β-associated Smad-dependent and non-Smad-dependent pathways are intracellular mechanisms that regulate EndMT occurrence.Classical TGF-βsignaling is mainly transmitted through the Smad pathway,which activates Smad3 protein phosphorylation and promotes its transfer to the nucleus,and regulates the expression of specific genes under the synergistic action of other transcription factors.EndMT is then induced.In addition,protein post-translational modification is also closely related to the function regulation of endothelial cells.It has been reported that protein methylation modification can affect the function of epithelial cells,which are a type of epithelial cells.However,whether methylation affects the EndMT process of endothelial cells remains unclear.Protein arginine methyltransferases（PRMTs）can catalyze the transfer of methyl of S-adenosine methionine to the guanidine group of the corresponding substrate arginine,and are the main substances that catalyze the formation of methylated proteins in vivo.At present,11 PRMTs have been found and can be divided into 4 types according to their catalytic activity.Type Ⅰ includes PRMT1-4,PRMT6 and PRMT8.Asymmetric dimethylarginine（aDMA）and Monomethyl arginine（MMA）were formed as catalysts;Type Ⅱ includes PRMT5 and PRMT9,which catalyze the formation of MMA and Symmetric dimethylarginine（s DMA）,in which PRMT5 has a wide range of biological effects.Type Ⅲ,PRMT7,catalyzes the formation of MMA only,while type Ⅳ is currently found only in yeast.PRMTs can regulate biological activity through a variety of signaling pathways,but the current research on PRMTs is mainly focused on the field of cancer,and relatively few studies on the relationship between PRMTs and cardiovascular diseases.The discovery of PRMTs inhibitors has become a new target for tumor therapy.If it is clear that PRMTs is involved in EndMT process and its main action factors and mechanisms are identified,it may provide a very promising therapeutic strategy for cardiovascular diseases.Objectives1.To determine whether the modification of protein arginine methylation occurs during EndMT.2.To screen the PRMTs with the most prominent expression in EndMT.3.To explore the specific role and mechanism of PRMTs participating in EndMT processMethods1.To establish the EndMT model of vascular endothelial cells（1）EndMT induction of endothelial cells:In the vascular endothelial cell experiment,Human umbilical vein endothelial cells（HUVECs）were used to construct cell models.The umbilical vein endothelial cells were cultured with ECM containing 5%FBS,the experimental group was treated with ECM medium containing 10 ng/mL TGF-βtransforming growth factor for 6 days to induce EndMT in HUVECs,and the control group was cultured with ECM medium without TGF-βfor the same time.（2）Morphological changes of endothelial cells after EndMT:The morphological changes of cells were directly observed under the microscope.The microscopic morphology of normal HUVECs was paving-stone,and the cells with EndMT became loose and slender.（3）Phenotype marker changes after EndMT:Endothelial cell marker,platelet-endothelial cell adhesion factor（CD31）and VE-cadherin were selected.Interstitial cell markers,（-smooth muscle actin（-SMA）and Vimentin）,were compared by Western-blot（WB）assay to determine whether the EndMT cell model was successfully constructed.（4）Changes in endothelial cell migration function after EndMT:Changes in cell migration function in HUVECs after EndMT were detected by cell scratch assay and Transwell assay.2.To clarify the methylation level changes of cell proteome during EndMTTo detect changes in the methylation level of cell proteome in HUVECs after EndMT:symmetric dimethylarginine（s DMA）antibodies and asymmetric dimethylarginine（aDMA）antibodies were used to detect the overall difference in the methylation level of proteins after EndMT by WB assay,so as to determine whether PRMTs participated in the EndMT process.3.To screen PRMTs with the most significant expression changes during EndMT and clarify their specific role（1）Detection of PRMTs expression changes in EndMT process:After clarifying the correlation between PRMTs and EndMT,WB experiment was used to detect the difference in PRMTs expression levels,and the PRMTs with the most obvious changes was screened out.Previous studies have found that PRMT5 is a methyltransferase with the most obvious expression change during EndMT.In order to further clarify the role of PRMT5 in EndMT,the following experiments were mainly completed:（2）The role of PRMT5 in EndMT was determined in vitro:PRMT5 inhibitors were used to inhibit the expression of PRMT5 in endothelial cells.TGF-βtreatment group,TGF-β+specific PRMT5 inhibitor treatment group and control group were set up to determine the role of PRMT5 in the process of EndMT in vitro by detecting the EndMT phenotype changes of HUVECs cells.（3）Construction of vascular endothelial injury model in mice:High-fat diet can induce activation of TGF-βpathway by increasing oxidative low-density lipoprotein infiltration,leading to EndMT changes in mouse aorta.Therefore,high-fat feeding was used to obtain EndMT animal model.Eight-week-old male ApoE-/-mice were selected for the experiment.The experimental group was fed a high-fat diet,while the control group was fed a normal diet.（4）Detection of EndMT changes in mouse vascular endothelium:Phenotypic protein changes in mouse vascular endothelium（CD31）and interstitium（Vimentin）were detected by immunofluorescence co-staining assay.（5）The role of PRMT5 in EndMT was determined in vivo:three groups of 8-week-old male ApoE-/-mice were set up and given high-fat diet,high-fat diet+intraperitoneal injection of PRMT5 inhibitor and ordinary diet control group,respectively.The aorta vessels of mice were taken 4 weeks later,and the changes of CD31 and PRMT5 were detected by immunofluorescence co-staining assay.4.To clarify the mechanism by which PRMT5 facilitates the EndMT process（1）To explore the relationship between TGF-β/Smad pathway and EndMT process:TGF-βtreatment group,TGF-β+specific Smad3 pathway inhibitor treatment group and control group were set up,WB test was used to detect the changes of Smad3 protein and endothelial and interstitial phenotypic protein expression.（2）To detect the effect of PRMT5 on Smad3 protein expression in the nucleus:TGF-βtreatment group,TGF-β+specific PRMT5 inhibitor treatment group and control group were set up to extract the intracellular protein,and the change of Smad3 protein expression in the nucleus was detected by WB assay and immunofluorescence assay.（3）To detect the effect of PRMT5 on Smad3 protein phosphorylation:TGF-βtreatment group,TGF-β+specific Smad3 pathway inhibitor treatment group,TGF-β+specific PRMT5 inhibitor treatment group and control group were set up.WB test was used to detect the expression of Smad3 protein and phosphorylated Smad3 protein（p-Smad3）.（4）It was clear that PRMT5 was involved in EndMT by regulating phosphorylated Smad3 protein:TGF-βtreatment group,TGF-β+specific Smad3 pathway inhibitor treatment group,TGF-β+specific PRMT5 inhibitor treatment group and control group were set up.WB assay was used to detect changes in endothelial and interstitial phenotypic protein expression levels.（5）To verify the effects of PRMT5 and Smad3 on cell migration function:PRMT5inhibitor treatment group,Smad3 inhibitor treatment group and control group were set up.WB assay was used to detect the change of PRMT5 and Smad3 protein expression,and cell scratch assay and Transwell assay were used to further clarify the effect on cell migration function.5.Statistics and analysis of experimental dataAll test results were repeated at least 3 times.The calculated experimental data were processed by SPSS 20.0,and the paired sample t test or wilcoxon rank sum test were used to compare the difference between the sample mean and the population represented by each,and it was concluded that P<The result of 0.05 means that the difference is statistically significant,specifically expressed as^nsp>0.05,*p<0.05,**p<0.01.Results1.EndMT occurs in HUVECs stimulated by TGF-β（1）The cell morphology of HUVECs treated with TGF-βshowed significant interstitial transformation.Under microscope,HUVECs in the control group showed a compact arrangement structure,similar to pebbles or paving stones.In the TGF-βtreatment group,HUVECs became significantly looser in arrangement and became elongated and fusiform,similar to interstitial smooth muscle cells,suggesting that EndMT transformation occurred in HUVECs after TGF-βtreatment.（2）Changes in endothelial and interstitial phenotypic proteins were observed in HUVECs treated with TGF-β.The results of WB experiment showed that the expressions of CD31 and VE-cadherin in TGF-βtreatment group were significantly decreased.(The expression levels of-SMA and Vimentin protein were significantly up-regulated,indicating that EndMT transformation occurred in HUVECs after TGF-βtreatment.（3）After EndMT occurred in HUVECs,migration ability was enhanced.The results of cell scratch and Transwell experiments showed that the migration function of HUVECs in TGF-βtreated group was enhanced,which further indicated the occurrence of EndMT in HUVECs.2.The level of symmetric methylation of the proteome as a whole is increased during EndMTThe level of symmetric methylation in the proteome increases during EndMT.The results of WB experiment showed that the expression level of symmetric dimethylarginine（s DMA）protein in TGF-β-induced EndMT was significantly higher than that in the control group,suggesting that type II PRMTs was the main catalyst for this process.3.PRMT5 is a PRMTs that plays a major role in promoting EndMT in HUVECs（1）The expression level of PRMT5 changed most significantly during EndMT.WB experiment results showed that the expression level of PRMTs changed during TGF-β-induced EndMT,and the increase of PRMT5 was the most obvious,and PRMT5 mainly catalyzed the generation of s DMA,suggesting that PRMT5 may play a major role in the process of EndMT.（2）PRMT5 promotes EndMT in HUVECs in vitro.After TGF-β-induced EndMT in HUVECs was treated with PRMT5 inhibitor,the results of WB experiment showed that the expression of interstitial phenotypic protein was significantly down-regulated,indicating that PRMT5 could promote EndMT in HUVECs.（3）EndMT is present in high fat diet-induced vascular endothelial injury.Immunofluorescence co-staining results showed that the expression of endothelial marker CD31 was significantly decreased and the expression of interstitial marker Vimentin was significantly up-regulated in the aorta of high-fat diet-induced vascular endothelial injury model mice,indicating the presence of EndMT changes during the process of vascular endothelial injury.（4）PRMT5 promotes EndMT in the body that damages blood vessels.Compared with ApoE-/-mice fed a high fat diet alone,the expression of endothelial marker CD31 in the aorta of ApoE-/-mice fed a high fat diet combined with intraperitoneal injection of PRMT5 inhibitor was significantly down-regulated by immunofluorescence co-staining results,indicating that PRMT5 can promote vascular endothelial EndMT in vivo.4.PRMT5 promotes EndMT by enhancing Smad3 phosphorylation（1）TGF-βpromotes EndMT through the Smad pathway.The results of WB experiment showed that the expression of Smad3 protein was significantly up-regulated during the induction of EndMT by TGF-β,while the expression of interstitial phenotypic protein was significantly decreased after the simultaneous use of Smad3 inhibitor,indicating that TGF-βpromoted EndMT through Smad3 pathway.（2）PRMT5 promotes the expression of Smad3 protein in the nucleus.The results of WB experiment showed that the expression level of Smad3 protein in the nucleus was significantly increased after TGF-βtreatment,while the expression level of Smad3 protein in the nucleus was significantly decreased after PRMT5 inhibitor was added,which had no significant difference with the control group.The results of immunofluorescence experiment were consistent with the results of WB experiment,indicating that PRMT5could promote the expression of Smad3 protein in the nucleus.（3）PRMT5 induced increased phosphorylation of Smad3 protein.WB experiment results showed that the expression of p-Smad3 protein increased after TGF-βtreatment,but significantly decreased after the addition of PRMT5 inhibitor,and there was no significant difference in the change of Smad3 protein expression,indicating that PRMT5 can promote Smad3 protein phosphorylation.（4）PRMT5 promotes Smad3 protein phosphorylation and thus induces EndMT.The results of WB experiment showed that the expression of endothelial phenotypic protein was decreased and the expression of interstitial phenotypic protein was upregulated after TGF-βtreatment.However,the interstitial transformation was significantly weakened after the addition of PRMT5 inhibitors and Smad3 inhibitors,indicating that PRMT5 promoted the phosphorylation of Smad3 protein and thus promoted EndMT.（5）PRMT5 can directly promote Smad3 phosphorylation and affect endothelial cell migration.In order to eliminate the interference caused by TGF-βtreatment,the expression levels of PRMT5 and p-Smad3 proteins were detected after the addition of only PRMT5inhibitor and Smad3 inhibitor.WB experiment showed that inhibition of PRMT5 could significantly inhibit the expression of p-Smad3 protein.Cell scratch and Transwell experiments showed that inhibition of PRMT5 could inhibit the migration ability of cells,suggesting that PRMT5 could directly promote Smad3 phosphorylation and thus affect the migration ability of endothelial cells.ConclusionThe protein methylation level of EndMT in vascular endothelial cells is significantly increased,and PRMT5 is the main methyltransferase promoting this process.The specific mechanism is to enhance the phosphorylation level of Smad3 protein through its methyl-transfer effect,and induce the intraconuclear transfer of Smad3 protein to generate EndMT.