Osteoarthritis (OA) is the most common form of arthritis and a leading cause of long-term disability. With increasing life expectancy, OA is a major socioeconomic and clinical concern, as no curative treatment yet exists. While considerable advancement has been made toward a better understanding of the pathophysiology of the disease process, there is still much to be accomplished before a disease-modifying OA drug is developed that can effectively reduce or stop the disease progression. It is therefore of the utmost importance to identify new candidates that can contribute to the development of therapeutic agents to prevent or arrest the disease process.
Although the hallmark of OA is the progressive degeneration of articular cartilage, the subchondral bone is also suggested to be an active component of the OA process in humans (1–4). The rationale is that because the subchondral bone plate is in direct contact with the cartilage, it influences not only mechanical effects, but also cartilage degradation by providing catabolic factors to this overlying tissue, thus promoting abnormal cartilage metabolism. The presence of clefts or channels in the tidemark during the OA process, as well as microcracks between the subchondral bone region and the uncalcified cartilage and vascularization in the subchondral bone, could favor a diffusion of factors from the subchondral bone region to the basal layer of cartilage and be responsible for the remodeling in the deep zone of OA articular cartilage.
The concept that the subchondral bone and cartilage should be considered an interdependent functional unit is gaining strong support, as illustrated by in vitro studies in which human OA subchondral bone osteoblasts demonstrated abnormal metabolism, including elevated levels of some bone markers and factors involved in bone biology (5–8). These findings are also consistent with the in vivo observations in animals (2, 9–11) and in knee OA patients (12–16), demonstrating such interdependence between the loss of cartilage and the deterioration of the subchondral bone structure. These and other findings strengthen the hypothesis that changes in subchondral bone play a key role in the genesis of cartilage lesions during OA.
In the musculoskeletal system, recent studies suggest the involvement of the receptor erythropoietin-producing hepatocellular B4 (EphB4) and its specific ligand, ephrin B2, in bone biology (17–21). The Eph receptors and their ephrin ligands constitute the largest subfamily of membranous receptor tyrosine kinases. The ephrin/Eph signaling depends on their expression/production and on the nature of the interacting/targeting cell types. Ephrins were originally identified as axon guidance molecules that mediate neuron repulsion during central nervous system development. They were since shown to regulate a variety of tissues and cell types and to act on cells, resulting in a myriad of biologic functions. Interestingly, a major common role is the control of extracellular matrix remodeling. The first member of the Eph family was identified and cloned in 1987 and, to date, 14 receptors and 8 ligands have been described in mammals. Eph receptors are grouped into two subclasses (A and B) according to their ligand (ephrin A or B) specificity.
In bone, osteoclasts express ephrin B1 and ephrin B2 without any detectable EphB receptors (17), while osteoblasts express both ephrin B2 and EphB4 receptors (18, 22). Of note, ephrin B2 is the sole ligand for the EphB4 receptor. The ephrin B2/EphB4 system was also recently reported to be present in another articular tissue, the cartilage (23). In vitro data revealed that ephrin B2 activation positively affects some abnormal metabolism in human OA subchondral bone osteoblasts and chondrocytes (22, 23). Briefly, these in vitro studies suggest that in human OA, EphB4 receptor activation could act at two different levels: by limiting the extent of matrix degradation in both cartilage and subchondral bone, and by regulating the abnormal osteoclastogenesis process in the subchondral bone and anabolism in cartilage, indicating that this system could be an interesting therapeutic target for OA. Collectively, these data suggest that enhancing the activation of this system could impart a protective effect on the structural changes in these articular tissues. Further in vivo studies are therefore essential to complement our understanding of the role of ephrin B2/EphB4 in articular tissues.
This study thus aimed to determine the in vivo effect of the EphB4 receptor in the pathophysiology of OA. As evidence suggests the subchondral bone to be an active component of the OA process, we investigated the in vivo effect of bone-specific overexpression of EphB4 receptors on OA development in mice.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
This study is the first to delineate in vivo the role of the EphB4 receptor in articular tissues during the development of OA, using bone-specific EphB4 receptor–overexpressing mice. Our data demonstrated that bone-specific overexpression of EphB4 exerted a protective effect in OA not only on the subchondral bone, but also on the cartilage structure as well as on some tissue markers of the disease.
The findings of this in vivo study support the hypothesis that protecting the subchondral bone prophylactically reduces the severity of cartilage lesions during the OA process. Indeed, although it was shown that during the OA process, there is a remodeling of the subchondral bone that results in sclerosis, recent studies reported in the literature showed that this pathologic tissue demonstrates abnormal mineralization and, consequently, hypomineralization associated with a lower tissue modulus, which adversely affects the capacity of adjacent articular cartilage to adapt to mechanical loads (38–42). In turn, this will lead to cartilage damage, and be at least partly responsible for the evolution of cartilage lesions during the disease.
This study first showed that the EphB4-Tg mice have normal skeletal development and body weight at birth. Our data are also consistent with the characterization reported by Zhao et al (17), in that although the EphB4-Tg mice cannot be differentiated morphologically, radiographic evaluation showed an increase in long bone density, a decrease in TRAP-positive cells in subchondral bone, an enhanced staining of the EphB4 receptor in bone, and no significant differences in osteoblasts per bone surface by 10 weeks of age as compared to WT mice.
The OA model chosen was surgical DMM of the right knee, which induces mild-to-moderate OA lesions (28). Only males were used, since this sex develops better characteristics of the disease (28). The data demonstrated in the DMM-operated mice a transient joint swelling following surgery, possibly reflecting wound healing, with a further similar decline in the WT controls and EphB4-Tg mice. These findings are strongly indicative that bone EphB4 overexpression has little involvement in the process of inflammation during OA. However, as this OA model did not show appreciable synovitis as reported by Glasson et al (28), the implication of EphB4 in synovial inflammation requires additional studies in which another OA model with more synovitis is used.
It is well known that during the OA process in humans, the subchondral bone becomes sclerotic. The same was seen in the DMM-operated WT mice, in which sclerosis of the subchondral bone was found at the medial tibial plateau by histologic assessment and micro-CT analysis. This finding is consistent with data from a more advanced stage of the disease using the DMM model of OA (25, 43) and other OA animal models (44–47). However, our data on the micro-CT features of the subchondral bone in control mice (sham), which showed no significant differences in bone volume between EphB4-Tg and WT mice, are in contrast to those of Zhao et al (17), which showed an increase in bone volume in the trabecular bone. This difference could be explained by the fact that the subchondral bone and the trabecular bone respond differently, since they are both structurally and functionally different.
In the DMM-operated EphB4-Tg mice, the subchondral bone plate thickness, trabecular bone volume and thickness, and osteoclast numbers were significantly decreased compared to the DMM-operated WT, indicating that the EphB4-Tg mice demonstrated a better preservation of the subchondral bone structure following surgery. These findings suggest a role of EphB4 in preserving subchondral bone during OA and are consistent with the findings of an in vitro study of human OA subchondral bone osteoblasts, in which ephrin B2–activating EphB4 receptors inhibited various catabolic mediators that may have acted to limit the abnormal metabolism of this tissue (22).
These changes in the subchondral bone were associated with significantly less cartilage damage in the DMM-operated EphB4-Tg mice than in the WT mice at both times examined. These data strongly support the hypothesis that preserving the subchondral bone properties positively affects the cartilage structure. Indeed, by preserving the subchondral bone structure, the tissue will be less prone to microcracks and microfractures, thus preventing the vascular invasion of the cartilage and diffusion of factors from the remodeling subchondral bone. This is corroborated by the immunologic data showing that the DMM-operated EphB4-Tg mice demonstrated significantly less aggrecan and type II collagen degradation products as well as collagen disorganization.
Of note, DMM-operated EphB4-Tg mice showed a significant decrease in aggrecan cleavage in both the tibial plateau and the femoral condyle as compared to the DMM-operated WT mice, while the type II collagen degradation products as well as the decrease in collagen disorganization occurred only on the tibial plateau. A possible explanation could be that aggrecans are affected before the collagen during disease development (48–50), combined with the fact that the region at which degradation of these macromolecules takes place may vary due to differences in the mechanical stress (28, 51). Indeed, DMM surgery involves the medial displacement of the medial meniscus, resulting in weight-bearing load redistribution in a small area, leading to increased local mechanical stress. Since the mouse knee is flexed during weight bearing, there is consequently greater stress predominantly in the tibial plateau on the medial side (28).
Our findings on type X collagen further validate cartilage protection in the DMM-operated EphB4-Tg mice, as the implication of the recapitulation of growth plate–like hypertrophic differentiation of chondrocytes has been well described in the pathogenesis of cartilage degradation (52). This study clearly showed that the DMM-operated EphB4-Tg mice displayed a significantly lower level of type X collagen, thus less chondrocyte hypertrophic differentiation, than the WT mice, which is indicative of a prevention of the terminal differentiation of chondrocytes during OA in these mice. Moreover, our data showing both the reduction in type X collagen and fewer collagen degradation products, as determined with the Col2-3/4Cshort antibody, which in turn, is associated with less type II collagen, are also consistent with data suggesting that the proteolytic generation of collagen peptides may drive chondrocyte hypertrophy (53) and that proteolytically derived type II collagen fragments regulate terminal hypertrophic chondrocyte differentiation (54).
A particular feature of ephrin/Eph biology is its capacity for bidirectional signaling in which the EphB4 receptor induces a forward signaling and the ligand ephrin B2 a reverse signaling. Thus, one could question whether the effects seen in the OA subchondral bone and cartilage in this transgenic model are due to the forward and/or reverse signaling and whether this occurs through osteoclast–osteoblast and/or osteoblast–osteoblast interactions, as EphB4 was found only on the osteoblasts, but ephrin B2 on both osteoclasts and osteoblasts (18, 21, 22). With the use of the same transgenic mouse model, Zhao et al (17) demonstrated in vivo that both EphB4 forward signaling on osteoblasts and ephrin B2 reverse signaling on osteoclasts could occur, whereby the former will enhance the Dlx5, Osx, and Runx2 genes and the latter will inhibit the Fos and Nfatc1 genes. In addition, that group of investigators (17) also showed in vivo the osteoblast–osteoclast interaction leading to both forward and reverse signaling, as well as in vitro the possibility of the osteoblast–osteoblast interaction leading to EphB4 forward signaling. In studies using human OA subchondral bone osteoblasts, our group reported the presence of EphB4 forward signaling, resulting in the inhibition of the catabolic factors interleukin-1β (IL-1β), IL-6, matrix metalloproteinase 1 (MMP-1), MMP-9, MMP-13, and RANKL (22).
In conclusion, in addition to showing a protective effect of bone-specific EphB4 overexpression on subchondral bone and cartilage during OA and defining this receptor as a potential novel therapeutic avenue for the treatment of the disease, this study also provides evidence to the effect that the in vivo integrity of the overlying articular cartilage is related to the subchondral properties and that changes in the metabolism of the subchondral bone are an integral part of the OA disease process.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Martel-Pelletier had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Valverde-Franco, Pelletier, Fahmi, Matsuo, Kapoor, Martel-Pelletier.
Acquisition of data. Valverde-Franco, Hum, Lussier.
Analysis and interpretation of data. Valverde-Franco, Pelletier, Matsuo, Kapoor, Martel-Pelletier.