The Role Of Alpha-2-macroglobulin In Posttraumatic Osteoarthritis

Part ?.A preliminary study of protein in the synovial fluid of the knee joint in patients with osteoarthritisObjective:To find a reliable biomarker to reflect traumatic osteoarthritis(PTOA)progression by comparison of protein content in synovial fluids(SF)between osteoarthritic and healthy knee joints.Methods:SF from age matched osteoarthritis(OA)and healthy people were determined by SDS-PAGE and coomassie blue staining.Potential biomarker was identified by mass spectrometry analysis,further validated by Western blot,ELISA and Immunohistochemistry.mRNA levels of potential biomarker in OA cartilage and adjacent normal cartilage within the same patients were determined with quantitative PCR.Degree of cartilage damage was determined under arthroscopy,with the corresponding SF was used to detect the level of potential biomarker.And the correlation between the degree of cartilage damage and the level of potential biomarker was assessed.Results:An 180kDa dark blue belt was found in the gel and alpha-2-macroglobulin(A2M)in the belt was recognized by mass spectrometry analysis.Further study showed that A2M level in OA SF was significantly higher than that in normal SF,The levels of A2M in both OA and normal SF were much lower than in plasma.Immunohistochernical staining showed positive staining of A2M in cartilage and synovium from OA patients and normal controls,but the amount of A2M was significantly increased in OA tissues.Quantification of mRNA from human knee joints with OA indicated that A2M mRNA levels were lower in the cartilage from the involved compartment compared to the uninvolved compartment of the joint,which we called "relatively normal"(RN)cartilage,from the same patients.However,the severity of cartilage damage was not correlated to the change of A2M levels in the SF.Conclusion:A2M was present at higher levels in OA SF,cartilage and synovium compared to age matched normal control indicating that A2M participated in the pathological changes of cartilage in OA development.However,there is no correlation between A2M and cartilage damage.A2M is not a suitable biomarker for monitoring PTOA progressionPart ?.The function and regulative mechanism of alpha-2-macroglobulin in cartilage degenerationObjective:To determine whether alpha-2-macroglobulin(A2M)plays a role in inhibiting cartilage degradation.Methods:A2M and MMP-13 levels in plasma and synovial fluid in patients with OA were compared by ELISA.The effects of supplemental A2M on expression of IL-1 induced cartilage catabolic cytokines and degrading enzymes,and MMP-13 activity were determined by Luminex Human Inflammatory 5-Plex Panel,Luminex Human MMP 3-Plex Panel,and ELISA in OA explants,human OA chondrocytes,and human chondrocyte cell line(C28/I2)cultures.Results:The concentration of A2M in OA plasma is much higher than that in the osteoarthritis(OA)synovial fluid,whereas the level of MMP-13 shows the opposite pattern.In vitro,A2M(200nM)inhibits the IL-lb induced expression of the cartilage catabolic cytokines IL-1b,IL-8,TNF-?,and GM-CSF and degradative enzymes MMP-3,-9,and-13 in human primary OA chondrocye cultures.A2M also inhibits IL-1b induced MMP-13 activity in a dose dependent manner.A similar result was also found in the OA explants and human chondrocyte cell line(C28/I2)cultures.Conclusion:A2M is a broad-spectrum protease inhibitor,and inhibits a variety of catabolic factors and cartilage degradation enzymes.And the relative shortage of A2M in the joint cavity may be one of the reasons for the occurrence of posttraumatic osteoarthritis after joint injury.Part ?.Early supplemental intra-articular injection of alpha-2-macroglobulin reduced the posttraumatic osteoarthritis pathogenesisObjective:To determine the protective role of exogenous alpha-2-macroglobulin(A2M)in inhibiting posttraumatic osteoarthritis(PTOA)cartilage degradation in vivoMethods:In vivo effects were evaluated in male rats(N=120)randomized to four treatments using anterior cruciate ligament transection(ACLT)model:(1)ACLT +saline,(2)ACLT + A2M(1IU/kg),(3)ACLT + A2M(2IU/kg)or(4)sham surgery +saline.Intra-articular injections were given immediately and 3 days after surgery,then once weekly for 6 weeks.Histological analyses and IHC were performed to assess cartilage damage.The concentration of MMP-13 in synovial fluid(SF)lavages was measured using ELISA.Gene expression was quantified by RT-qPCR.Results:Early supplemental intra-articular injection of A2M reduced the OA pathogenesis and concentration of MMP-13 in SF in the rat ACLT model.The mRNA levels of type ? collagen and aggrecan were increased in the ACLT+A2M(1IU/kg)group and ACLT+A2M(2IU/kg)group compared with the ACLT+Saline group,suggesting the positive impact of A2M on anabolic metabolism.In contrast,MMP-3,-13,Runx2 and type X collagen showed the opposite pattern.Conclusion:Our findings suggest that early supplemental intra-articular A2M could provide chondral protection for PTOA.