The Role Of ADAMTS-7in Chondrogenesis And Osteoarthritis Development

Arthritis is a group of chronic diseases that characterized by the damage of the joints and inflammation of soft tissue around the joints. According to the NIH prevalence data for arthritis published in2005, the most common form of arthritis is osteoarthritis (OA), which is a degenerative joint disease and affects estimated27million people in the United States. The other common type is rheumatoid arthritis (RA), resulting from the autoimmune disorder that causes the chronic inflammation of joints. The impact of arthritic conditions is expected to grow as the population both increases and ages in the coming decades. Despite its prevalence, the precise etiology, pathogenesis, and progression of arthritis remain unknown. ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family has19identified members until now, they share the similar structure, and digest the extracellular matrix as substrate, they play a vital role in multiple biological processes such as embryogenesis, vasculogenesis and blood coagulation. It has been reported that ADAMTS-7was upregulated in the arthritic cartilage, but the specific function of it has to been elucidated. Herein, we generated ADAMTS-7transgenic mice and conditional knockdown mice to further study its role in osteoarthritis.ADAMTS-7, a metalloproteinase that belongs to ADAMTS family, associated with and degraded cartilage oligomeric matrix protein (COMP), and its level was significantly elevated in arthritis. In addition, subsequent findings also revealed a physiological, developmental role for ADAMTS-7in chondrogenesis in vitro. In order to better understand the biological function of ADAMTS-7in cartilage development and arthritis, we first generated ADAMTS-7transgenic mice, using a chondrocyte-specific Col2al promoter to drive the transgene expression, we found that newborn ADAMTS-7transgenic (Tg) mice were significantly smaller, X-ray images revealed remarkable reductions of skeletal length ad bone volume in transgenic mice. A double labeling assay indicated a reduced new born formation rate in Tg mice. Tg mouse growth plate was only the half the size of that in wild-type (WT) control. Whole mount staining revealed that Tg mice had hypoplastic limb skeletons and dramatically delayed endochondral bone formation compared with control mice. However, Tg mice reached normal size at about5weeks ostpartum and no defects were observed in articular cartilage and joints. Interestingly,8-months old Tg mice spontaneously developed an osteoarthritis (OA)-like phenotype. Micro-CT of the knee joints showed osteophyte formation in aged Tg mice, and Safranin O staining showed a remarkable loss of proteoglycan staining and meniscus ossification in Tg mice, in addition, clear chondrocyte clustering and migration of the irregular tidemark to the superficial zone were also observed, these are also the typical features of OA. To further determine the role of ADAMTS-7in OA development and progression, we established a surgically-induced OA model (DMM), histological analysis showed Tg mice a moderate loss of safranin O staining as early as4weeks after surgery, while the control mice joints showed moderate loss only after12weeks. Histological score grading indicated higher scores in the operated Tg mice knees, with a significant reduction of cartilage thickness and loss of proteoglycan. In addition, COMP degradative fragments were increased in the post-operative Tg mice. The proinflammatory cytokine TNFa, known to activate ADAMTS-7, was induced by ADAMTS-7in vitro as well as in Tg mice, demonstrating a positive feedback loop of ADAMTS-7and TNFa in OA.With regard to the early diagnosis of OA, the ability to diagnose arthritis and take corrective steps before marked loss of articular cartilage has begun would represent an important advance in arthritis treatment. Cartilage oligomeric matrix protein (COMP) is a prominent noncollagenous component of cartilage. Fragments of COMP have been detected in the diseased cartilage, synovial fluid, and serum of patients with knee injuries, posttraumatic and primary osteoarthritis (OA), and rheumatoid arthritis (RA). Although several commercial enzyme-linked immunosorbent assay (ELISA) kits for COMP are available for measuring COMP levels in human serum, the limitation for these kits is the lack of specificity for degraded COMP fragments, which dampens the use of COMP as a biomarker for the clinical monitoring of arthritis. A panel of murine mAbs against recombinant COMP fragments were generated and one of these clones,2127F5B6, was identified as a mAb preferentially recognizing the COMP C-terminal degradative fragments. A novel COMP fragment ELISA was then developed using mAb2127F5B6as a detection antibody. Compared to a commercial COMP ELISA kit that detected no significant difference in total COMP levels between symptomatic knee OA (SKOA) and non-OA control groups, a significant increase of the COMP fragments was noted in the serum of SKOA patients assayed by this COMP fragment ELISA. In addition, serum fragmentary COMP levels were well-correlated with Kellgren-Lawrence grade score in OA patients, and the progression of surgically-induced OA in murine models. Furthermore, the levels of COMP fragments in the serum of RA patients, mice with CIA, and TNF transgenic mice are significantly higher as well when compare with their corresponding controls. Interestingly, treatments with TNFa inhibitors and methotrexate led to significant decrease of serum COMP fragments in RA patients.In conclusion, targeted overexpression of ADAMTS-7in chondrocytes results in defects in chondrogenesis in "young" mice and OA-like phenotype in "aged" mice, and overexpression of ADAMTS-7also accelerates surgically-induced OA. ADAMTS-7regulates OA progression through a) degrading COMP and destabilizing the cartilage matrix and b) inducing TNFa that in turn activates cartilage degrading metaloproteinases, including MMP-9, MMP-13, ADAMTS-4and ADAMTS-5. These findings not only provide novel insights into the role of ADAMTS-7in cartilage and arthritis in vivo, but may also lead to the development of novel therapeutic intervention strategies for osteoarthritis.On the other hand, using recombinant COMP fragments we have generated a series of mAbs recognizes diverse regions of the molecule and one of these antibodies reactive with the C-terminal portion of the molecule was further exploited to establish a unique sandwich ELISA capable of reproducibly measure the levels of COMP fragments (COMP catabolism) in the body fluids of both arthritic patients and murine arthritis models. In addition, this system also provides a valuable means to exploit COMP fragments biomarker for monitoring the effects of therapeutic interventions.