Hemoglobin stimulates the expression of ADAMTS-5 and ADAMTS-9 by synovial cells: a possible cause of articular cartilage damage after intra-articular hemorrhage

Hemoarthrosis or intra-articular bleeding caused by joint trauma such as ligament rupture or intra-articular fractures leads to the various long-term problems and disorders such as joint pain, joint swelling, stiffness and decreased range of motion, joint deformity, and dysfunction [1]. Synovial tissues play important and critical roles in the situation of blood-induced joint disorders have been reported [1, 2]. In experimentally model of hemoarthrosis, proliferation and hypertrophic change of synovial tissue, neovascularization, and a perivascular acute inflammatory reaction were confirmed at the initial stage. At the end-stage of hemoarthrosis, the joint is characterized by a breakdown of articular cartilage, joint effusion, formation of fibrous tissue, and secondary osteoarthritis. On the other hand, not only repeated hemoarthrosis, but also even a single issue of intra-articular hemorrhage following trauma that frequently occurs in the cases of sports injuries, may cause irreversible joint damage. Moreover, exposure of articular cartilage to low concentrations of blood for a period as short as 2 days has also been confirmed to induce the irreversible cartilage damage [3, 4]. However, the pathomechanisms of blood-induced joint damage is still unclear in detail.

Roosendaal et al. examined the effects and mechanisms of blood-induced joint damage in vivo and in vitro study; which blood components contribute to the cartilage damage. They reported that erythrocytes play critical and important roles in blood-induced joint disorder and this effect was independent of cytokine production [5‐7].

Articular cartilage is constituted of small numbers of chondrocytes which were embedded in large amount of extracellular matrix containing collagen and aggrecan. The extracellular matrix of the articular cartilage provides the essential biomechanical characteristics for joint motion. For example, the important function of resisting compressive force is provided by aggrecan, a large proteoglycan [8]. Degeneration of aggrecan is a critical event in articular cartilage destruction [9].

Aggrecanases are members of the ADAMTS family (A disintegrin and metalloprotease with thrombospondin motifs), and are the principal enzymes responsible for aggrecan cleavage in the initial events of cartilage turnover [9]. Aggrecanase-mediated aggrecan degeneration is also a key and critical event in initial-stage osteoarthritis. 19 ADAMTS genes numbered 1 to 20 were already identified in mammalian genomes; the designation ADAMTS-11 is excluded because it was already identified as ADAMTS-5, previously [10]. Many members, such as ADAMTS-1, −4, −5, 8, −9, −15, −16, and −18, have been reported to cleave aggrecan [8‐12].

Previously, it has been reported that hemoglobin (Hb) stimulates urokinase type plasminogen activator (uPA), matrix metalloproteinase (MMP)-2, and MMP-9 expression by cultured fibroblasts and synovial cells, indicating that Hb contributes to the cellular functions management [4, 13]. Based on these findings, Hb may play critical and important roles to enhance the expression of proteinases from synovial cells, leading to the destruction of the extracellular matrix of the cartilage. The present study was designed to verify the hypothesis described as above by evaluating the influence of Hb on ADAMTS family activity produced by cultured human synovial cells.

Recent studies have found that ADAMTS-5 and -9 play an important pathological role in matrix degeneration; for example, ADAMTS-5 contributes to intervertebral disc and articular cartilage degeneration [10, 11, 17‐20], and ADAMTS-9 contributes to arthritis, breast cancer metastasis, and central nervous system pathologies [10, 15, 21, 22]. Moreover, both ADAMTS-5 and -9 cause turnover of proteoglycans in liver fibrosis [23]. Previously, in an animal study, the combined proteolytic activities of ADAMTS-5 and -9 were found to be required keeping extracellular matrix proteolysis above the threshold required for web regression [10]. ADAMTS-8 can cleavage hyaluronan-binding chondroitin sulfate proteoglycan extracellular proteins [10], and that can also exert as an anti-angiogenic factor [10]. Because of such multiple functions and abilities of ADAMTS-8, we sought to investigate the role of ADAMTS-8 in the present study. In contrast, ADAMTS-10 has been reported to contribute to the connective tissue disorder including joint stiffness and cardiac valve stenosis [10]; this protein can bind to fibrillin-1 and -2 and promote microfibril formation in the extracellular matrix, and this function may be independent of protease activity was reported [10]. Therefore, we employed ADAMTS-10 as a contrast of aggrecanase in the present study.

Our preliminary examination found that Hb induced the secretion of ADAMTS-5 and -9 by cultured synovial cells. Dose-dependency studies indicated that Hb induced significant expression of these aggrecanases even at concentrations as low as 25 μg/ml [Fig. 3a and b]. Time-course studies showed that Hb significantly induced the expression of ADAMTS-5 and -9 at 6, 12, and 24 h [Fig. 4a and b]. These enzymes cleave the aggrecanase-specific Glu373-Ala374 bond in the interglobular domain region of aggrecan core protein, and are deeply involved in the pathology of arthritic joint diseases such as osteoarthritis [9‐11]. That ADAMTS-9 was observed even in control medium without Hb suggests that it may contribute to normal cartilage turnover. These findings indicate that Hb induce ADAMTS-5 and -9 expressions by human synovial cells at an early phase, even at low doses, after Hb-treatment. We could detect neither ADAMTS-8, which has been reported to cleave aggrecan core protein as well at Glu373-Ala374 [10, 12], nor ADAMTS-10 by Western blotting in the present study [Fig. 2a and b].

Although the pathomechanisms of blood-induced joint degeneration and degradation are still elusive, several pathomechanisms have been suggested via both enzymatic and non-enzymatic processes, such as by expression of neutrophil elastase [24], uPA [4, 25], gelatinases [26], cytokines [27], calpain [28], and hydroxyl radicals [29] produced by inflamed synovial cells and inflammatory cells. Hb is a potent inflammatory substance that can produce a tissue response [30]. It is well known that hydroxyl radicals which can destroy cell membranes were produced by catalytically active iron from Hb via Fenton reaction [31]. On the other hand, there are other pathways that can enzymatically degrade the extracellular matrix of articular cartilage. Of the components of Hb, only the globin chain stimulates fibrinolytic activity, while protoporphyrin IX and heme do not [13]. Once aggrecan was degraded, elasticity of the articular cartilage was disappeared, and the joint shows similar condition to the initial stage of osteoarthritis [8, 9]. The enzymatic depletion of aggrecan is caused by members of the MMPs and ADAMTS family proteins [10, 11, 32]. Previously, ADAMTS-5 was also shown to play an important role in proteoglycan turnover and cartilage degeneration [33, 34]. uPA is able to convert plasminogen to plasmin, which can also cleavage extracellular matrix components [4]. In addition, plasminogen-plasmin system including uPA are also able to activate several MMP family enzymes. Therefore, the combination of the plasminogen-plasmin system, MMPs, and ADAMTS family enzymes could strongly degrade extracellular matrix. Previously, Hb was found to induce the expressions of uPA, MMP-2, and MMP-9 by fibroblasts [4]. Increased levels of aggrecanases, gelatinolytic and fibrinolytic enzymes following Hb stimulation may, at least in part, contribute to degradation of extracellular matrix of articular cartilage.

It has been reported that exposure of human cartilage tissue to low concentrations of blood for as little as 2 days leads to prolonged cartilage damage [3]. Our results suggest that the deleterious effects of Hb on cartilage may occur in an acute phase at 6 h after exposure to blood due to the presence of ADAMTS-5 and -9. The present study aimed to gather information regarding the thresholds of Hb concentration and the time that is responsible for ADAMTS gene expression. We already reported that the peak expressions of gelatinases MMP-2, MMP- 9, and uPA by Hb-treated human synovial cells occurred at 24–48 h [4]. Combined with the results of the present study, it appears that Hb stimulates the expression of MMP-2 and -9, ADAMTS-5 and -9, and uPA from synovial cells in the early stage. The combination of these enzymes may contribute to articular cartilage degeneration after intra-articular hemorrhage.

Our results demonstrated that Hb acutely induced ADAMTS-9 mRNA without enhancing the mRNA level of ADAMTS-5 [Fig. 5a and b]. Although there was a slight time lag when we compared the results of the peak immunoreactivity against ADAMTS-9 with peak mRNA expression, the mRNA expression of ADAMTS-9 at 12 and 24 h after Hb-treatment was significantly higher than that of the control [Fig. 5b]. Previously, Yoshida et al. reported that Hb enhances the synthesis of uPA through post-transcriptional regulation of mRNA [13]. Moreover, ADAMTS-5 is expressed constitutively in synovium with no or little transcriptional regulation when activated by recombinant human interleukin-1 alpha [35]. Thus, it is possible that separate stimulation pathways and mechanisms could exist between ADAMTS-5 and -9 when they are treated by Hb. Further investigations to clarify the mechanism by which Hb stimulates the expression of ADAMTS family members in vivo and in vitro are required.

It is well known that two major proteinases in the ADAMTS family are the capital enzymes contributed in the pathogenesis of arthritis: ADAMTS-4 and ADAMTS-5 called aggrecanase-1 and aggrecanase-2, respectively [8‐11, 36]. Recently, synovial fibroblasts have been reported to provide the higher levels of ADAMTS-4 in osteoarthritis [37]. Although these enzymes are the principal aggrecanases those present in cartilage, previous in vitro study showed ADAMTS-5 is 1000-times more effective than ADAMTS-4 [38]. In animal model of arthritis, ADAMTS-5 alone is the critical enzyme to contribute for joint destruction were also reported [39, 40]. Thus, ADAMTS-5 may be an important target for therapeutic inhibition to prevent articular cartilage degeneration.

New therapies based on reducing or inhibiting the levels of ADAMTS-5 using an anti-ADAMTS-5 antibody in animal models of osteoarthritis have been reported [34, 41, 42]. Given the results of the present study, we suggest that provision of both anti-ADAMTS-5 and -9 antibodies into the joint at the early phase after intra-articular hemorrhage could be a superior therapeutic option to prevent the cartilage degeneration.

Our results suggest a possible role for Hb in joint damage after intra-articular hemorrhage. Hb induces the expression of ADAMTS family proteinases such as ADAMTS-5 and -9 by synovial cells at low doses even at an acute phase. These findings suggest that a single instance of intra-articular hemorrhage during trauma or sports injury could deleteriously affect articular cartilage metabolism. Immediate treatment for intra-articular hemorrhage, such as washing out and removing the blood from the joint cavity, may be required regardless of whether it occurs once or multiple times. Moreover, it could be possible that inhibiting the activities of ADAMTS-5 and -9 together may serve as a superior therapeutic strategy to prevent cartilage damage after intra-articular hemorrhage.