Aggrecanases in the human synovial fluid at different stages of osteoarthritis

Abstract

In vivo and in vitro aggrecanases degrade proteoglycan aggrecan in articular cartilage. However, the expression of aggrecanases in patients in different stages of osteoarthritis (OA) has not been investigated. This study detected the expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) and ADAMTS-5 and their proteolytic products, ARGxx, in the synovial fluid (SF) of patients in different stages of OA. This study aimed to evaluate the expression of aggrecanases and to explore the respective roles of these enzymes in human cartilage degradation. A total of 144 patients with knee OA were divided into early-, middle-, and late-stage OA groups according to the degree of cartilage degradation using Recht's MRI grading standard and the modified Outerbridge classification system. Expression levels of ADAMTS-4, ADAMTS-5, and ARGxx in the SF from these patients were measured using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Our findings showed that ADAMTS-4 and ARGxx expression levels in the early-stage group were significantly higher than in the other two groups. ADAMTS-5 in the early-stage group and ADAMTS-4, ADAMTS-5, and ARGxx in the late-stage group were significantly higher than those in the middle-stage OA group. Both ADAMTS-4 and ADAMTS-5 levels were correlated with ARGxx levels (P < 0.05). The correlation coefficients of ADAMTS-4 and ADAMTS-5 were 0.236 and 0.068, 0.729 and 0.479, and 0.675 and 0.257 in the early-, middle-, and late-stage groups, respectively, and 0.530 and 0.258 in the total SF samples. Western blot analysis revealed that the ADAMTS-4 and ADAMTS-5 in SF were 50 kDa proteins and that ARGxx in SF had at least two molecular masses, 55 kDa and 70 kDa. The expression levels of all three proteins were consistent with the ELISA results. These results suggested that aggrecanases were involved in all stages of human OA aggrecan degradation, especially in the early and late stages. ADAMTS-4 levels were higher in early- compared with middle- or late-stage OA and were also more correlated with ARGxx than ADAMTS-5; thus, ADAMTS-4 might be the principal aggrecanase of aggrecan degradation in human OA.

Healthy and Osteoarthritic Synovial Fibroblasts Produce a Disintegrin and Metalloproteinase with Thrombospondin Motifs 4, 5, 7, and 12

“…Western blot analysis of ADAMTS 4 and 5 in SF culture supernatants revealed the presence of the active forms of both enzymes, between 48 and 74 kDa, similar to those reported in OA synovial fluids. 30 We also observed a major band between 74 and 114 kDa, which has been described as the ADAMTS-4 proenzyme, in cartilage from OA patients, 28 as well as in SF. 9 Regarding ADAMTS-5, other authors have also reported a 70-kDa form of ADAMTS-5 in cartilage, as well as in SF from OA patients, 9,28 that could represent different degradation fragments.…”

Aggrecanase-2 inhibitors based on the acylthiosemicarbazide zinc-binding group

“…Compound 35 was then tested in a panel of metalloproteases and was found selective of aggrecanases versus matrix metalloproteases (MMPs) or TNF-a-converting enzyme (TACE) ( Table 7). This is consistent with the fact that the S 0 1 pocket, is defined in the metalloproteinase family, as the specificity pocket [26]. Indeed, in ADAMTS-5, there is a compact S 0 1 loop counting for half of the pocket [26] while in MMPs the S 0 1 pocket is mostly entirely enclosed in the S 0 1 loop.…”

“…This is consistent with the fact that the S 0 1 pocket, is defined in the metalloproteinase family, as the specificity pocket [26]. Indeed, in ADAMTS-5, there is a compact S 0 1 loop counting for half of the pocket [26] while in MMPs the S 0 1 pocket is mostly entirely enclosed in the S 0 1 loop. This difference result in decreased activity on MMPs for compounds displaying a longer P 0 1 group [13f].…”

“…Though ADAMTS-5 has been shown to be involved in the disease progression in mice [4,25] the relative contribution of ADAMTS-4 and -5 in the human pathology is not fully defined. Recently, ADAMTS-4 levels were shown to be higher in early-compared with middleor late-stage OA in humans [26]. Interestingly, in our series, all compounds inhibit the two major enzymes responsible for aggrecan cleavage at the Glu 373 -Ala 374 site, with little selectivity.…”

“…Interestingly, substitution of the methylene group in gem-dimethyl analog 23 and cyclic derivative 24 led to slightly less active compounds than 1 (IC 50 6.9 and 3.3 mM respectively). Noteworthy, introduction of an NH in ortho position of the phenoxy group (24 vs 1) was tolerated, whereas isosteric replacement of the oxygen by an NH led to a decrease in activity (26). Shortening the chain by directly linking the aromatic ring to the acylthiosemicarbazide motif, increased activity as exemplified by the submicromolar compound 25.…”