Expression Of Aggrecanase And Matrix Metalloproteinase In Synovial Fluid Of Patients With Different Stages Of Osteoarthritis

Objective Osteoarthritis (OA) is a progressive degenerative disease of synovial joints characterized by erosion of articular cartilage in which loss of aggrecan is a crucial initial pathological event. Recent studies have shown that aggrecanase and matrix metalloproteinase (MMP) are the two key families of enzymes responsible for the degradation of aggrecan from articular cartilage matrix, however, there is a dispute over which group of enzymes play the major role in human under pathological circumstance. Several studies have been performed to investigate the expression of these enzymes in the synovial joints of OA in recent years, however, no study has been reported on the different expression of aggrecanase or MMPs in the synovial fluid (SF) from patients with different stages of OA. The objective of the present study was to detect and compare the expression of aggrecanase (ADAMTS-4and ADAMTS-5) and MMPs (MMP-1and MMP-3) as well as the expression of their products in synovial fluid (SF) of patients with different stages of osteoarthritis to explore the respective effects of these two groups of enzymes in human cartilage destruction in different stages of OA.Materials and Methods A total of144patients with unilateral knee osteoarthritis were enrolled from June2008to May2011, including38males and106females and aging18to86years with an average age of58.5years. All of them were divided into early-, middle-and late-stage OA groups according to the degree of knee articular cartilage degeneration, which was classified by Recht’s grading standard by MRI image and Outerbridge classification system. Early-stage OA group (65cases) contains the patients who were in both grade1 and grade2following the radiological grading standard, while middle-stage OA group (25cases) and advanced-stage OA group (54cases) was made up of patients in grade3and4respectively. Enzyme-linked immunosorbent assay (ELISA) was applied to investigate the expression levels of ADAMTS-4, ADAMTS-5. MMP-1, MMP-3and the proteolytic products of aggrecan ARGxx and FFGxx in synovial fluids from144cases of OA patients, and the results of ELISA were represented by the optical densities (OD). Western blot was applied to quantify the protein expression levels of ADAMTS-4. ADAMTS-5, MMP-1, MMP-3, ARGxx and FFGxx in the synovial fluid samples from patients with early-stage OA (n=8), middle-stage OA (n=8) and late-stage OA (n=8), and the results were expressed as proteins levels relative to GAPDH protein levels. The one-way ANOVA and Kruskal-Wallis test were applied to test for expression difference of aggrecanase, matrix metalloproteinase as well as the proteolytic products of aggrecan among the three groups, while the LSD test was applied to compare the expression difference between two groups. Association of aggrecanase, matrix metalloproteinase and the proteolytic products of aggrecan was analyzed using correlation analysis.Results1. The result of ELISA showed ADAMTS-4and ADAMTS-5had higher expression in early-and late-stage OA group than those in middle-stage group, while the expression of ADAMTS-4in SF of patients with early-stage OA was significantly higher than those in SF of patients with late-stage OA(P<0.01). Compared with the middle-stage OA group, MMP-1had higher expression in late-stage OA group and MMP-3had higher expression in early-stage OA group (P<0.05). ARGxx had higher expression level in SF of patients in early-stage OA than those in SF of patients in middle-and late-stage group (P<0.01). Conversely, FFGxx had higher expression level in late-stage group than those in early-and middle-stage group (P<0.01).2. The expression levels of ADAMTS-5, MMP-3and ARGxx in three groups were significantly higher than the levels of ADAMTS-4, MMP-1and FFGxx, respectively (P<0.01).3. Both ADAMTS-4and ADAMTS-5expression levels were correlated with ARGxx levels, and ADAMTS-4was more closely related to it (r=0.530versus r=0.285). Besides, the expression level of MMP-3was correlated with that of FFGxx in OA synovial fluid (r=0.475, P<0.01), and there was no correlations between MMP-1and FFGxx level.4. Western blot analysis revealed that ADAMTS-4had higher expression level in early-stage group, while ADAMTS-5had higher expression level in both early-stage and late-stage group (P<0.05). The protein expression levels of ARGxx in early-and late-stage OA group were significantly higher than those in middle-stage group (P<0.05). MMP-1and FFGxx had higher expression in late-stage group (P<0.05), while the protein expression of MMP-3was significantly higher in early-stage than that in middle-stage group (P<0.05).Conclusions1. Aggrecanases, especially ADAMTS-4, may play the major role in aggrecan degradation in human OA progression.2. Aggrecanases are primarily involved in the cartilage destruction in the early and late stages of OA while matrix metalloproteinases mainly performed in the late stage of OA in human which reveal that inhibition of aggrecanases may not only prevent the pathogenesis of osteoarthritis but also prevent its progression.