Up-regulation Of Calreticulin Expression In Patients With Rheumatoid Arthritis In Vivo And The Role Of Calreticulin On Endothelial Cell Migration

Objective:Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitis, inflammation reaction and pannus formation. Its pathogenesis is not very clear. Studies have reported that Calreticulin(CRT)may be related to the pathogenesis of RA. In order to investigate the potential role of CRT in RA pathogenesis, a variety of immunological techniques were used to analyze the expression of CRT in serum, synovial fluid and synovium in patients with RA in this study. In view of NO as an important part in the inflammatory response in the pathogenesis of RA. We study whether CRT play a key role in the NO-pathway in the pathogenesis of RA in vitro.Methods:1. Levels of CRT in sera from patients with RA, osteoarthritis (OA), systemic lupus erythematosus (SLE), other autoimmune diseases and health control (HC) were detected by ELISA and Western blot.2. CRT levels in synovial fluid from RA and OA patients were measured by ELISA.3. Pathological methods were employed to analyze the expression and localization of CRT in synovial membrane.4. HUVECs were cultured in vitro. CRT act on HUVECs, and we detect whether CRT influence the secretion of NO.5. The role of CRT in HUVECs proliferation and migration were detected by MTT assay and Wound-healing assay.Results:1. CRT was found to be significantly up-regulated in sera from RA [(4.817±2.423) ng/ml] than that in OA [(3.574±0.923) ng/ml], SLE [(4.013±1.524) ng/ml], other autoimmune diseases [(3.882±0.842) ng/ml] and HC [(3.726±0.619) ng/ml].2. There was only one form of CRT (monomer) in serum.3. Pathological results showed that CRT was mainly expressed in the lining and sublining layers of synovium, endothelial cells and perivascular areas; while in OA, only weak staining of CRT was seen in perivascular areas and the synovial lines.4. HUVECs were cultured in high glucose DMEM containing TNF-a, the level of NO was significantly higher than that in the control group. And20ng/ml of TNF-a was effective in stimulating the production of NO. HUVECs in different concentrations of CRT in high glucose DMEM medium with10%fetal bovine serum were cultured by24hours, the levels of released NO were significantly increased compared with the control, and the level of NO was dose-dependent with CRT concentration.5. MTT assay showed that CRT has no effect on the proliferation of HUVECs and Wound-healing assay demonstrated that CRT can stimulate the migration of HUVECs.Conclusions:1. In RA, increased levels of CRT were detected in sera. Moreover, high expression of CRT was observed in synovium and the locations were different from OA. Our results indicated that CRT may be involved in the RA joint inflammation reaction and pannus formation. CRT may become a potential serological marker in the diagnosis of RA.2. The concentrations of CRT which stimulated NO production were significantly increased compared with the control. NO-production was increased with increasing concentration of CRT. CRT can promote the inflammatory response and angiogenesis in RA by stimulating the secretion of NO. CRT may be directly involved in RA angiogenesis by inducing endothelial cell migration.