The Role Of Annexin A2 In Promoting The Development Of Rheumatoid Arthritis

Objective: Annexin a2(AX ?)is an important mediating agent that induces angiogenesis in vascular diseases.The neovascularization network of pannus formation plays a crucial role in the development of rheumatoid arthritis(RA).The correlation between AX? and pannus formation has not been studied.The purpose of this study is to determine whether AX? contributes to pannus formation in RA,and to investigate the specific mechanisms involved in the effect.Methods:We looked for the Gene Expression Omnibus(GEO)datasets to compare the AX? and annexin A2 receptor(AX?R)expression of synovial tissues among in patients with RA and osteoarthritis(OA)patients and normal healthy people.Enzyme-linked immunosorbent assays(ELISA)were used to detect the differential expression levels of soluble AX ? inserumbetweenpatients with RA and common inflammatory diseases.Judged whether AX? affected arthritis development in DBA/1 mice with collagen-induced arthritis(CIA)by exogenously added AX?,and assessed the severity of arthritis by comparing the joint score,Paws thickness and the morbidity of CIA.Collected DBA/1 mice joint tissues to determine the changes in inflammation of joints by hematoxylin-eosin(H&E)staining and destruction of bone tissue in the joint by X-ray and Micro-CT examinations.To detect neovascularization,immunohistochemistry(IHC)and immunofluorescence(IF)were used to analyze the expression ofCD31,which are the surface marker of microvascular endothelial cells,and performed quantitative analysis of CD31.The expression levels of CD31 represented density of neovascularization.In cytological studies,on one hand,knock-downed AX?R mRNA expression on human umbilical vein endothelial cells(HUVEC)relied on technology of plasmid transfection to observe the relationship between AX?/AX?R and hedgehogs(HH)signaling pathway;On the other hand,HUVEC were co-cultured with cyclopamine(CYC),which is the specific inhibitor of HH,to determine the correlation between HH and cell proliferation.Compared the differential expression of the key node elements of the HH signaling pathway and the pro-angiogenic factors by qRT-PCR after the knockdown of AX?R on HUVEC.Results:Compared to OA patients and normal healthy people,the expression of AX?and AX?R in synovial tissues of patients with RA were increased,and the expression levels of soluble AX?in serum of RA were significantly higher than in patients with common inflammatory diseases.Joint swelling and neovascularization were increased significantly in CIA micethat were exogenously added AX?.Cell experiments showed that high expression of AX ? promoted HUVEC proliferation and angiogenesis by binding AX?R on HUVEC.Further mechanistic researches indicated thatAX?could up-regulate the expression of Ihh and Gli,which were the key molecules of HH.Besides,expression of Ihh,Ptc,Smo and Gli significantly reduced;and expression of angiogenic growth factor of HH signaling downstream matrix metalloproteinase-2(MMP-2),vascular endothelial growth factor(VEGF)and angiogenic factors-2(Ang-2)were also down-regulated after inhibition of expression AX?R on HUVEC.Conclusion:Compared to patients with common inflammatory diseases and normal health people,AX?and AX?R were highly expressedin the synovial tissuesof RA patients.Our researchdefinitely observed that AX?could promote the development of CIA for the first time.It played a crucial role in the progression of arthritis,especially during the process of pannus formation.The effect depended on AX?combined AX?R to activate and regulate HH signaling and the expression of its downstream VEGF,Ang-2 and MMP-2 to promote HUVEC proliferation,and eventuallycaused to angiogenesis.Therefore,study the role of AX?is instructive for understanding the development of RA,suppress the effect of AX?might to provide a new potential measure for treatment of RA.