Full-length WARS Affecting Intestinal Mucosal Angiogenesis By Promoting VEGFA Expression In Inflammatory Bowel Disease (IBD)

Background Inflammatory Bowel Disease(IBD)is a chronic inflammatory disease mainly involving the digestive tract.At present,IBD is mainly divided into ulcerative colitis,Crohn’s disease and unclassified.There is no cure.Studies have shown that in the occurrence and development of inflammatory bowel disease,the density of microvessels in patients with inflammatory bowel disease is increased compared with that in normal people,and the increase of vascular density may be related to the severity of IBD.The imbalance of positive and negative regulators may lead to pathological angiogenesis.It is characterized by the absence of pericytes,leakage,stenosis,hyperthrombotic edema,and increased permeability formation.Pathological angiogenesis may be one of the pathological bases of IBD.Therefore,finding the key regulatory mechanism of the initiation of neovascularization is expected to become a new treatment for inflammatory bowel disease.Vascular endothelial growth factor-A(VEGFA)is a typical vascular endothelial growth factor(VEGF).It is considered to be the most potent stimulator of angiogenesis and the main regulator of angiogenesis in IBD,which can promote the induction of angiogenesis by activating several pathways that regulate endothelial cell(EC)proliferation,migration and overall survival,while increasing vascular permeability and further promoting angiogenesis.It has been reported that the expression level of VEGFA in the intestinal mucosa of IBD is higher than that of normal people.Tryptophanyl-t RNA Synthetase(WARS)is a member of aminoacyl-t RNA synthetase(ARSs).It is abundantly expressed during the differentiation of human monocyte-derived macrophages and dendritic cells,can be induced by Interferon-γ(IFN-γ),and can also be rapidly secreted from immune cells during bacterial and viral infections.Secreted forms of WARS include Full-length WARS(FL-WARS)and truncated WARS,which are alternatively spliced or truncated into truncated variants such as mini-WARS,T1-WARS,and T2-WARS.Among them,T2-WARS has been shown to be effective in inhibiting VEGF-induced angiogenesis by binding to VE-cadherin.Secretory truncated WARS have been found to be involved in the regulation of angiogenesis in tumor development,but FL-WARS has a different structure.It does not have the effect of inhibiting angiogenesis.In the present study,FL-WARS has been mainly demonstrated to activate innate immunity in macrophages,which is related to the expression of Toll-like receptors-2(TLR2)and/or Toll-like receptors-4(TLR4)on macrophages.TLR4)interacted with each other to activate innate immunity,induce the production of tumor necro-sis factor(TNF-α)and chemokines,and enhance neutrophil infiltration and phagocytosis.However,it has not been reported that FL-WARS has an effect on angiogenesis.In our preliminary experiments,we used human recombinant WARS to mimic the function of FL-WARS in cell experiments,and the preliminary results suggested that FL-WARS could promote angiogenesis.Objective To explore the effect of FL-WARS on VEGFA expression and angiogenesis of intestinal mucosa in inflammatory bowel disease(IBD).Methods 1.Clinical samples were used to validate the correlation between VEGFA,WARS and IBD.Real-time PCR(q PCR)/ Immunofluorescence assay(IF)was used to detect the expression of WARS and VEGFA in the intestinal mucosa of IBD patients.2.To explore the effect of FL-WARS on angiogenesis.h CMEC cells were treated with different concentrations of hr WARS(0.25 μg/ml,0.5 μg/ml,1.0 μg/ml)for 48 hours,and matrigel was used for angiogenesis test.The number of nodes and the length of blood vessels in different groups were counted.3.To explore the effect of FL-WARS on the expression of VEGFA in endothelial cells.h CMEC cells were treated with different concentrations of Human Recombinant WARS protein(hr WARS)(0.25 μg/ml,0.5 μg/ml,1.0 μg/ml)to mimic the effect of full-length WARS for 48 hours.The expression level of VEGFA was detected by q PCR/IF /Western Blot.4.To explore the inhibitory effect of bevacizumab on hr WARS-induced VEGFA expression.The cells were treated with 1.0 μg/ml hr WARS,1.0 μg/ml hr WARS+150 mg/ml bevacizumab,150 mg/ml bevacizumab for 48 hours,respectively.The expression of VEGFA was detected by q PCR/Western Blot.Results 1.The IF and q PCR results of clinical intestinal mucosa samples showed that the expression levels of WARS and VEGFA in the intestinal mucosa of IBD patients were higher than those of non-IBD patients.2.FL-WARS increased the expression of VEGFA in vascular endothelial cells.3.FL-WARS can promote angiogenesis.4.The promoting effect of FL-WARS on angiogenesis was partially dependent on VEGFA.Conclusions FL-WARS is involved in the occurrence and development of IBD.FL-WARS can promote angiogenesis and increase the expression of VEGFA,and the effect of FL-WARS on angiogenesis is partly dependent on VEGFA.