The Mechanism Of Calreticulin Promoting Survival Of Fibroblast-like Synoviocytes And Angiogenesis In Rheumatoid Arthritis

Objective: Rheumatoid arthritis(RA) is a chronic immune-mediated inflammatory disease characterized by synovitis and pannus formation, which leading to the destruction of articular cartilage and bone. The etiology and pathogenesis of RA is not very clear. Studies have reported that calreticulin(CRT) may be involved in the pathogenesis of RA. Our preliminary findings showed high expression of CRT in RA patients. Furthermore, a significant positive correlation was observed between serum levels of CRT and disease activity in RA. In addition, previous studies also showed that CRT may be involved in the pathogenesis of RA by inhibiting T cell apoptosis. The present study was undertaken to explore the mechanism of CRT to promote fibroblast-like synoviocytes(FLS) survival and angiogenesis in RA. This study not only helps to understand the role of CRT in the pathogenesis of RA, it can also provide therapeutic targets for the treatment of RA.Methods: 1. Serum CRT levels were measured by enzyme-linked immnuosorbent assay(ELISA) in 106 patients with established RA, 75 osteoarthritis(OA), and 80 healthy controls(HC). CRT levels in synovial fluid(SF) were also measured in 25 RA and 22 OA patients. 2. The expression of CRT, Bcl-XL and Mcl-1 in RA and OA synovium was detected by immunohistochemistry. Correlations of synovial CRT expression with Bcl-XL and Mcl-1 were analyzed. 3. FLS were isolated by enzymatic digestion of synovial tissue specimens obtained from RA and OA patients and cultured in vitro. The expression of Bcl-XL and Mcl-1 in FLS was detected by q RT-PCR, Western Blot and immunofluorescence. 4. RA and OA FLS were cultured with different concentrations of recombinant human CRT, the expression of Bcl-XL and Mcl-1 was detected by q RT-PCR and Western Blot. 5. RA FLS were cultured with CRT in the absence or presence of PI3K/Akt or STAT3 inhibitor, the expression of Bcl-XL and Mcl-1 was assessed by q RT-PCR and WesternBlot. The activation and expression of PI3K/Akt and STAT3 signaling following CRT stimulation was analyzed by Western Blot. 6. The proliferation of RA FLS following CRT stimulation was examined by MTT assay; the ability of CRT to inhibit RA FLS apoptosis was assessed by flow cytometry. 7. HUVECs were isolated by enzymatic digestion of newborn’s umbilical cord specimen and cultured in vitro. NO production by HUVECs following CRT stimulation was detected. The expression and activation of PI3K/Akt-e NOS signaling following CRT stimulation was also detected. 8. The role of PI3K/Akt-e NOS-NO signaling pathway in CRT mediated proliferation, migration and tube formation of HUVECs was detected by MTT assay, scratch wound healing assay and tube formation assay, respectively.Results: 1. Serum CRT levels [(6.4±3.1) ng/ml)] of RA were significantly higher compared to that of OA [(3.7±0.9) ng/ml] and HC [(3.4±1.0) ng/ml](P<0.001); and significantly higher CRT in SF [(6.9±3.4) ng/ml] of RA vs OA [(3.9±0.7) ng/ml](P<0.001). No significant difference was found between serum and SF CRT levels in RA(P=0.478). 2. Pathology results showed high expression of CRT, Bcl-XL and Mcl-1 in the lining and sublining, endothelial cells, inflammatory cells and perivascular areas of RA synovium. While, weak staining of CRT, Bcl-XL and Mcl-1 was observed in the OA synovium. There was a signigficant positive correlation between synovial CRT expression and local expression of Bcl-XL and Mcl-1. 3. QRT-PCR, Western Blot and immunofluorescence results showed higher expression of Bcl-XL and Mcl-1 in RA FLS compared with OA FLS. 4. CRT stimulation increased the expression of Bcl-XL and Mcl-1 in RA FLS both in m RNA and protein levels. However, neither Bcl-XL nor Mcl-1 was changed in OA FLS. 5. Treatment with PI3K/Akt or STAT3 inhibitor inhibited CRT-mediated Bcl-XL and Mcl-1 upregulation in RA FLS. Furthermore, increased levels of p-Akt and p-STAT3 in RA FLS were observed following CRT stimulation. 6. CRT protected RA FLS from Fas L induced apoptotic death as detected by flowcytometry. MTT results showed that CRT had no significant effect on the proliferation of RA FLS. 7. CRT increased the production of NO by HUVECs at a concentration-dependent manner. Elevated levels of p-e NOS were observed in HUVECs after treatment with CRT, while total e NOS levels were not significantly changed both in m RNA and protein levels. Use of a specific PI3K/Akt inhibitor reduced CRT-mediated elevated expression of p-e NOS. 8. CRT promoted the proliferation, migration and tube formation of HUVECs, which were significantly inhibited by a specific e NOS inhibitor.Conclusions: 1. Increased expression of CRT was detected in RA(including serum, synovial fluid and synovial tissue). PI3K/Akt and STAT3 signaling pathways were involved in CRT-mediated Bcl-XL and Mcl-1 upregulation in RA FLS and promoted the survival of RA FLS, which leading to synovial hypertrophy and pannus formation. 2. PI3K/Akt-e NOS-NO signaling pathway was involved in CRT-induced proliferation, migration and tube formation of HUVECs, which indicate that CRT may be involved in synovitis and pannus formation by promoting angiogenesis in RA.