The Expression And Pathogenic Role Of Aggrecanase And MMP In Joints Of OA And RA

Background and Objectives Degradation of extracellular matrix(ECM)is main feature for destruction of articular cartilage,in which aggrecan is seriously cleaved by matrix metalloproteinase(MMP)and aggrecanase at Asn³⁴¹-Phe³⁴²and Glu³⁷³-Ala³⁷⁴site of the aggrecan core protein within the interglobular domain(IGD), respectively.The process generates the new peptides of FFGxx and ARGxx.Up to now,the ADAMTS-4 and ADAMTS-5 have been widely recognized as aggrecanases. For MMPs family,MMP-2 has many substrates including aggrecan and typeâ…¡collagen,whereas MMP-3 is mainly responsible for degradation of aggrecan among the MMPs family.Though both aggrecanase and MMP can cleave aggrecan at core protein region,it is augmented which group of enzymes plays the major role under pathological condition.The in vitro cartilage explant system has confirmed that aggrecanase appears to be the primary enzyme to degrade aggrecan in the first several weeks and then MMP in the later weeks.However,the proteolytic activity of aggrecanase at the late stage of cartilage degradation remains unknown.The mediators which regulate the expression of aggrecanases in chondrocytes are much complicated,even reports describing the in vitro studies are not consistent.For example,Flannery et al reported that the treatment of cultured normal human cartilage with IL-1 had no effect on the expression of ADAMTS-4 and ADAMTS-5.In contrast,others reported that IL-1 could increase the expression of ADAMTS-4,but not for ADAMTS-5. Both osteoarthritis(OA)and rheumatoid arthritis(RA)have common manifestation of the destruction of articular cartilage,but the characters of these two diseases are different.OA is a degenerative arthritis without inflammation,while RA is an autoimmune with inflammatory response,in which IL-1 plays an important role. Thus,the comparative study with these two diseases is not only helpful to understand the roles of aggrecanase and MMP,but also important to elucidate the regulatory mechanism for expression of aggrecanase under pathological conditions.In the current study,we studied the expression of ADAMTS-4,ADAMTS-5,MMP-2, MMP-3 and their products in the damaged joint cartilage,synovial tissue and synovial fluid in the patients with OA and RA to investigate pathogenic function of aggrecanase and MMP and the possible regulatory mechanisms for the two diseases.Materials and Methods Synovial tissues,joint cartilages and synovial fluids were collected from 21,21 and 32 patients with OA in that order;Joint cartilages, synovial tissues and synovial fluids were collected from 9,12 and 28 patients with RA,respectively.Three joint cartilages were also collected from femoral condyle from accident injury as controls of the normal.Parts of OA cartilage were conducted explant cultures for 5 days with stimulation of IL-1βat 10ug/ml on the first day of culture.Immunohistochemistry was applied to observe the expression and distribution of ADAMTS-4 in surface zone, middle zone and deep zone of cartilage in samples of cartilagesfrom OA cartilages being stimulated or not.Semi-quantitative reverse transcriptase- polymerase chain reaction(RT-PCR)was applied to examine the expression levels of ADAMTS-4 mRNA in those samples.Immunohistochemistry was also applied to detect the expressions and distribution of ADAMTS-4 and ADAMTS-5 in cartilages and synovial tissues from OA and RA.The levels of ADAMTS-4,ADAMTS-5,MMP-2,MMP-3,ARGxx and FFGxx in synovial fluids of OA patients and with RA were investigated by enzyme-linked immunosorbent assay(ELISA),and the optical densities(OD)were used to represent the concentrations.The results were analyzed by T test and correlation analysis. Results In the immunohistochemistry slices of 3 normal cartilages,very few chondrocytes expressed ADAMTS-4.In OA cartilages,there were many ADAMTS-4 positive cells which was mainly located in the superficial zone,but less in the middle and very little in the deep zone.When the superficial zone was destroyed,the ADAMTS-4 positive cells were mainly distributed in the area of the middle zone close to the articular surface and few was found in the area near the deep zone, whereas no positive expression in the deep zone.When OA cartilages were further damaged and the middle zone wore off as well,cells in the deep zone were proliferated and began to express ADAMTS-4.After stimulated with IL-1β, ADAMTS-4 increased its expression in the cartilage.Statistic analysis showed that the ADAMTS-4 positive cells in the superficial and middle zones of OA and cultivated OA cartilages were significantly increased than that of normal control. There was no significant difference of the ADAMTS-4 positive cells in the superficial zone in OA cartilage before and after cultivation,while the positive cells were increased in the middle zone of OA cartilage after cultivation.RT-PCR showed that the expression of ADAMTS-4 mRNA was significantly elevated in OA cartilage than that in normal control,and the expression in the cartilage stimulated by IL-1βwere significantly increased than that in the OA cartilage.The immunohistochemistry also found that both OA and RA cartilages had the expressions of ADAMTS-4 and ADAMTS-5.In OA cartilages,ADAMTS-4 and ADAMTS-5 were mainly present in the superficial zones,while the RA superficial zones were replaced by pannus which strongly expressed aggrecanases together with the middle zone below.The ratio of middle zone expressing aggrecanase in RA cartilage was higher than in OA.The expressions of ADAMTS-4 and ADAMTS-5 in OA synovial tissues were 52%(11/21)and 43%(9/21)respectively,which had no differences with those in RA, which were 50%(6/12)and 58%(7/12).The result of ELISA indicated that ADAMTS-4,ADAMTS-5,MMP-2, MMP-3,ARGxx and FFGxx had higher levels in the RA synovial fluids than those in OA synovial fluids(P＜0.01).In both RA and OA synovial fluids,ARGxx had significantly higher contents than FFGxx,ADAMTS-4 higher than ADAMTS-5,and MMP-3 higher than MMP-2(P＜0.01).In OA synovial fluids,ADAMTS-4 expression was positively correlated with ARGxx expression(r=0.38,P＜0.05),and the expression of ADAMTS-5 was not significantly correlated with that of ARGxx, and MMP-2,MMP-3 expression not with FFGxx,either.In RA synovial fluids, ATAMTS and MMP enzymes detected had no significant correlations with their products.Conclusions1.ADAMTS-4 is significantly expressed in severe wore cartilages,suggesting that ADAMTS-4 not only plays an important role in the early development stage of OA,but also in the middle and late stage.Therefore,the inhibition to ADAMTS-4 may be helpful for the treatment of OA on the middle and late stages.2.Aggrecanase was always expressed in the area close to the articular surface, which strongly supports the mechanical stress theory of OA pathogenesis and suggests that mechanical stress is not a simple mechanical wearing factor but may activate the aggrecanases.These are the possible factors to cause cartilage to be thinner.3.The concentrations of ARGxx were higher than that of FFGxx in the synovial fluids,suggesting that aggrecanases may play more important role than MMPs for the loss of aggrecan in OA and RA cartilage.4.The main source of aggrecanase and MMP in OA is chondrocytes,and in RA joints are chondrocytes and pannus overlying the cartilage surface.Synovium only expressed in minute amounts of aggrecanases and MMPs.5.IL-1βcould up-regulate the gene expression of ADAMTS-4.Compared with the mechanical friction,IL-1βhad more effects on different areas,suggesting that the synovitis in OA can accelerate the destruction of cartilage.6.Aggrecanase,MMP and their metabolic products had higher expression in the synovial fluids of RA than those of OA,indicating the inflammatory cytokines not only contribute to up-regulate the expression of MMPs,but also to mediate aggrecanases during the development of human arthritis.