Molecular Mechanisms Of Serum Amyloid A Facilitated Angiogenesis Through Toll-like Receptor 4 Signal Pathway In Rheumatoid Arthritis

Objective: Rheumatoid arthritis(RA) is a chronic inflammatory disease characterized by symmetrical polyarticular synovitis and abundant neovascularization. Serum amyloid A(SAA) plays an important role in its pathogenesis. Studies in past few years have found that TLR4 as a ligand of SAA may be related to the angiogenesis of RA. In our study, a variety of immunological techniques were used to analyze the expression of TLR4 in synovium in patients with RA. Furthermore, proangiogenic factors- VEGF, Ang-1and Ang-2 were detected to explore the effect of interaction between SAA and TLR4 on HUVECs. Finally, a three-dimension in vitro model of angiogenesis was constructed to explore the role of SAA and TLR4 mediated NFκB signaling pathway in the mechanisms of RA angiogenesis. The purpose of this study is to lay the foundation for the study that the role of SAA and TLR4 in the pathogenesis of RA and founding new targets for diagnosis and therapy.Methods: 1. Expression of TLR4 in synovium of RA and OA were detected by immunohistochemistry. 2. The role of interaction between SAA and TLR4 in SAA induced HUVECs migration and proliferation were detected by cell migration and MTT assay respectively. 3. The role of interaction between SAA and TLR4 in angiogenesis was detected by the three-dimension in vitro model of angiogenesis 4. The influence of SAA and TLR4 on HUVECs producing effector molecules was analyzed by ELISA. 5. The expression and activation of NFκB in HUVECs was analyzed by western blot.Results: 1. Pathology showed that SAA was widely expressed in the lining and sublining layers of synovium, endothelial cells, synovial fibroblasts, macrophages and perivascular areas of RA patients; while it weakly expressed in the lining layers of synovium and synovial fibroblasts of OA patients. It has significant differences both in quantity and location.2. Cell migration,MTT assay and in vitro model of angiogenesis respectively showed that after SAA stimulated, HUVECs could migrate, proliferate and connect to form new blood vessels, while after inhibiting TLR4, these functions of HUVECs were suppressed obviously. 3. ELISA showed that HUVECs could secrete VEGF, Ang-1 and Ang-2 after stimulating by SAA, after using TLR4 inhibitor, VEGF secretion decreased obviously. 4. Western blot showed that NFκB signaling pathway was activated after SAA stimulating HUVECs, while it was suppressed obviously after using TLR4 inhibitor. 5. Cell migration and in vitro model of angiogenesis respectively showed that after SAA stimulated, HUVECs could migrate and connect to form new blood vessels, while after inhibiting NFκB, these functions of HUVECs were suppressed obviously.Conclusions: 1. Pathology showed that SAA was widely expressed in synovium of RA patients compared with OA,which suggest that TLR4 may play an important role in inflammation reaction and joint destruction of RA. 2. TLR4 inhibitor can suppress migration, proliferation and angiogenesis of HUVECs Moreover, it can also suppress HUVECs to secrete angiogenic factor. 3. NFκB signaling pathway was activated after SAA stimulating HUVECs, while it was suppressed obviously after using TLR4 inhibitor, which indicate that SAA-TLR4-NFκB signaling pathway may be involved in RA pannus formation directly and result in bone and cartilage damage in RA patients indirectly.