Study On The Role Of HMGB1 In Angiogenesis Of Temporomandibular Joint Osteoarthritis

Part 1.The expression of high mobility group box protein 1 in synovium and disc perforation temporomandibular joint osteoarthritisBackground:High mobility group box protein 1(HMGB1),a potent promoter of inflammation,was believed to be a potential trigger of osteoarthritis(OA).The aim of this part was to investigate the expression of HMGB1 expression in synovium and perforated disc of temporomandibular joint osteoarthritis(TMJOA).Methods:Human TMJ synovial membrane and perforated disc tissues were collected from TMJOA patients during surgical procedures.The control group of TMJ disk specimens and synoviums were collected from patients with condyle fractured(CF).The expression of HMGB1 was detected using immunohistochemistry,real-time quantitative PCR and Western blot.Results:HMGB1 expressed strongly in cytoplasm and nucleus of lining layer cells and endothelial cells in TMJ osteoarthritic synovium.Staining of HMGB1 was found intensive in the frontier tissue of the perforation in the perforated discs.HMGB1 expression was also upregulated in osteoarthritic synovial cells and disc cells according to Real-time quantitative PCR and Western blott analysis.Conclusion:HMGB1 expression was upregulated in TMJ OA and might promote the progression of TMJ OA.Part 2.HMGB1-induced angiogenesis in perforated disc cells of human temporomandibular jointObjective:HMGB1,a highly conserved nuclear DNA-binding protein and inflammatory mediator,has been recently found to be involved in angiogenesis.Our previous study has demonstrated the elevation of HMGB1 in the tissue of perforated disc of temporomandibular joint(TMJ).This study aims to investigated a novel mediator of HMGB1 in regulating HIF-1? and VEGF to mediate angiogenesis in perforated disc cells of TMJ.Methods:Perforated disc cells were stimulated with HMGB1 for various concentrations(0-500ng/ml)or different times(0-24h),the expression of HIF-1? and VEGF in these cells were detected by WB,quantitative-real-time PCR and IF,The tube formation and cell migration in human umbilical vein endothelial cells(HUVECs)were also investigated.The expression level of HIF-la and VEGF were detected by Western blot in HMGB1-induced disk cells cultured with Erk inhibitor(U0126)and JNK inhibitor(SP600125),respectively.Results:HMGB1 increased the expression of HIF-1? and VEGF in a dose-and time-dependent manner in these cells.Moreover,immunofluorescence assay exhibits that the HIF-1? were activated by HMGB1.In addition,HMGB1 activated extracellular signal-related kinase 1/2(Erkl/2),Jun N-terminal kinase(JNK),but not P38 in these cells.Furthermore,both U0126(ErK inhibitor)and SP600125(JNK inhibitor)significantly suppressed the enhanced production of HIF-la and VEGF induced by HMGB1.Tube formation of human umbilical vein endothelial cells(HUVECs)was significantly increased by exposure to conditioned medium derived from HMGB1-stimulated perforated disc cells,while attenuated with pretreatment of inhibitors for VEGF,HIF-1?,Erk and JNK,individually.Conclusion:Abundance of HMGB1 mediates activation of HIF-1? in disc cells via Erk and JNK pathway and then,initiates VEGF secretion,thereby leading to disc angiogenesis and accelerating degenerative change of the perforated disc.Part 3.HMGB1-induced angiogenic and inflammatory responses in synoviocyte of human temporomandibular jointObjective:This study was undertaken to examine the role of HMGB1 in up-regulating expression or secretion of the angiogenic and inflammatory factors in TMJOA synoviocyte,and the underlying signaling mechanisms involved.Methods:We used Real-time PCR,Western blott,Enzyme-linked immunosorbent assays(Elisa),IF,Tube formation and Transwell assay to assess the role of HMGB1 in TMJOA synoviocyte.Results:HMGB1 significantly enhanced the expression of VEGF,HIF-1?,MMP13,ADAMTS5 and production of VEGF,IL-1?,IL-6 in a dose-and time-dependent manner.In addition,Immunofluorescence assay exhibits that the HIF-1? were activated by HMGB1.Moreover,Tube formation of HUVECs was significantly increased by exposure to conditioned medium derived from HMGB1-stimulated TMJOA synoviocyte,while attenuated with pre-treatment of inhibitors for VEGF.Additionally,HMGB1 induced phosphorylation of Erk and JNK in TMJOA synoviocyte in a concentration-and time-dependent manner.HMGB1 induced VEGF,HIF-la,MMP13,ADAMTS5 up-regulation was dependent on Erk and JNK signaling pathways,and while HMGB1-induced secretion of VEGF,IL-1? and IL-6 in TMJOA synoviocyte was also dependent on Erk and JNK signaling pathways.Concusion:Our data suggest that HMGB1 may contribute to inflammation and angiogenesis by triggering production of inflammatory and angiogenic cytokines in TMJOA synoviocyte.