In vitro activation of the receptor EphB4 positively affects human osteoarthritis (OA) articular cell metabolism. However, the specific in vivo role of this ephrin receptor in OA remains unknown. We investigated in mice the in vivo effect of bone-specific EphB4 overexpression on OA pathophysiology.
Morphometric, morphologic, and radiologic evaluations were performed on postnatal day 5 (P5) mice and on 10-week-old mice. Knee OA was induced surgically by destabilization of the medial meniscus (DMM) in 10-week-old male EphB4 homozygous transgenic (EphB4-Tg) and wild-type (WT) mice. Medial compartment evaluations of cartilage were performed using histology and immunohistochemistry, and evaluations of subchondral bone using histomorphometry, osteoclast staining, and micro–computed tomography.
There was no obvious phenotype difference in skeletal development between EphB4-Tg mice and WT mice at P5 or at 10 weeks. At 8 and 12 weeks post-DMM, the EphB4-Tg mice demonstrated significantly less cartilage alteration in the medial tibial plateau and the femoral condyle than did the WT mice. This was associated with a significant reduction of aggrecan and type II collagen degradation products, type X collagen, and collagen fibril disorganization in the operated EphB4-Tg mice. The medial tibial subchondral bone demonstrated a significant reduction in sclerosis, bone volume, trabecular thickness, and number of tartrate-resistant acid phosphatase–positive osteoclasts at both times assessed post-DMM in the EphB4-Tg mice than in the WT mice.
This is the first in vivo evidence that bone-specific EphB4 overexpression exerts a protective effect on OA joint structural changes. The findings of this study stress the in vivo importance of subchondral bone biology in cartilage integrity.
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.
MATERIALS AND METHODS
Bone-specific EphB4 receptor–overexpressing mice.
The model used in this study was a transgenic mouse in which EphB4 is overexpressed under the control of the mouse Col1 promoter (17). Briefly, mouse Ephb4 complementary DNA was subcloned upstream of the osteoblast-specific promoter region of the mouse proα1(1) collagen gene. The Nar l–Sal l fragment containing the 2.3-kb proα1(1) promoter Ephb4 coding–region poly(A) signal was isolated and microinjected into pronuclei of fertilized eggs from B6C3F1 (C57BL/6 × C3H1/He) females.
We bred 2 transgenic EphB4/Col1 heterozygous couples. Routine genotyping was carried out on DNA from ear punch biopsy samples using specific primers. Genomic DNA was isolated with DirectPCR lysis reagent containing proteinase K (Viagen Biotech) according to the manufacturer's instructions. The polymerase chain reaction (PCR) was performed on the genomic DNA using the following primers: 5′-GCATGAGCCGAAGCTAACCC-3′ (Col1α1) and 5′-CTTTGATTTGCACCACCACCGGAT-3′ (primer of the vector to which EphB4 is attached, pNASSβ). GAPDH was used as the reference gene (5′-CACAGTCCATGCCATCAC-3′ and 5′-GATCCACGACGGACACATTG-3′). Zygosity was determined by comparing by real-time quantitative PCR the transgene expression to the reference gene (GAPDH) according to the method of Shitara H et al (24). Briefly, the relative quantification performed with the 2 method determined the zygosity, yielding 0 wild-type, ≥1 heterozygous, and ≥2 homozygous mice.
Transgene overexpression protein production was also confirmed by Western blotting. Briefly, osteoblasts were released from long bones by sequential enzymatic digestion at 37°C, and cells were seeded in Ham's F-12/Dulbecco's modified Eagle's medium (Wisent) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories) and an antibiotic mixture and incubated at 37°C in a humidified atmosphere until confluence. Cell lysates were solubilized in sample buffer (62.5 mM Tris HCl, 10% glycerol, 5% 2-mercaptoethanol, and 2% sodium dodecyl sulfate), and evaluation was performed by Western blotting, as previously described (25), using mouse EphB4 affinity-purified polyclonal goat antibody (1:5,000 dilution; R&D Systems). As a loading control, β-actin was used.
All procedures involving animals were performed according to regulations of the Canadian Council on Animal Care and were approved by the Animal Care Committee of the University of Montreal Hospital Centre. All mice were maintained under a 12-hour light/dark cycle. Food and water were available ad libitum.
Evaluation of knee joint swelling.
The operated mice (see below) were examined daily. The knee diameter was measured in the mediolateral plane every 3 days, using digital calipers (model 2071M; Mitutoyo), as previously described (25).
Morphologic evaluation of the mouse skeleton was performed with alizarin red and Alcian blue staining on postnatal day 5 (P5). Samples were fixed in 70% ethanol (24 hours) and then placed in 100% acetone (24 hours). Skeletons were then stained with a mixture of 0.05% alizarin red, 0.015% Alcian blue, and 5% acetic acid, placed in 2% potassium hydroxide until clear, and stored in 70% ethanol and glycerol (1:1) as described previously (26, 27).
High resolution radiographs of 10-week-old mice were obtained with a Faxitron model MX-20 machine equipped with an FPX-2 imaging system (MedOptics/DALSA Life Sciences). Radiographs were used to qualitatively assess bone morphology.
Surgically induced OA mouse model.
OA was surgically induced in 10-week-old male EphB4 homozygous and wild-type (WT) mice by destabilization of the medial meniscus (DMM) in the right knee, as previously described (28). The mice were anesthetized with isoflurane and O2, and the right knee joint was destabilized by transection of the anterior attachment of the medial meniscus to the tibial plateau. A sham operation, which involved a similar incision to the knee without compromising the joint capsule, was also performed on the right knee of 10-week-old WT mice and 10-week-old mice overexpressing EphB4. Mice were observed daily to verify healing and to ensure that they were using their right limb.
Operated mice were euthanized at 8 and 12 weeks after surgery, nonoperated control mice at an equivalent age, and sham-operated mice at 12 weeks postsurgery. The right knee joints were dissected free of tissue, fixed in TissuFix (Chaptec), decalcified in RDO Rapid Decalcifier (Apex Engineering), and embedded in paraffin, as previously described (25). Sections (5 μm) were deparaffinized in xylene, followed by a graded series of alcohol washes, and stained with Safranin O–fast green (Sigma-Aldrich). Two independent observers who were blinded with regard to group allocation graded the severity of the OA lesions using the Osteoarthritis Research Society International (OARSI) scoring method (29). Three sections were prepared from each block, each slide was examined, and the final score was a consensus between the 2 observers.
Collagen disorganization was evaluated on 5-μm paraffin sections following sirius red staining, as described elsewhere (30, 31). Two independent observers who were blinded with regard to group allocation graded the severity of collagen disorganization under polarized light microscopy using a modified scale of 0–2, where 0 = normal cartilage, 1 = partial disorganization, and 2 = total disorganization. Three areas were evaluated, and the scores summed (maximum score 6).
The right knee joints were dissected, fixed in 4% paraformaldehyde for 16 hours at 4°C, decalcified in 10% EDTA for 14 days at 4°C, and embedded in paraffin. Immunohistochemical analysis was performed on 5-μm paraffin sections. Briefly, sections were pretreated with 0.25 units/ml of protease-free chondroitinase ABC in phosphate buffered saline (PBS; Sigma-Aldrich), and 1% hyaluronidase in 0.1M Tris acetate (Sigma-Aldrich) for 60 minutes at 37°C. The specimens were incubated for 18 hours at 4°C with the following primary antibodies: goat polyclonal anti-EphB4 (1:100 dilution; R&D Systems), rabbit polyclonal anti–C-terminal peptide of aggrecan G1 domain (VDIPEN, 1:800 dilution; provided by Dr. J. S. Mort, Shriners Hospital for Children, McGill University Hospital Centre, Montreal, Quebec, Canada) (32), a rabbit polyclonal antibody that represents a type II collagen primary cleavage site (Col2-3/4Cshort, 1:200 dilution; provided by Dr. A. R. Poole, Shriners Hospital for Children, McGill University Hospital Centre, Montreal, Quebec, Canada) (33), and mouse anti–type X collagen antibody (1:100 dilution; provided by Dr. E. Lee, Shriners Hospital for Children, McGill University Hospital Centre, Montreal, Quebec, Canada) (34).
Each slide was washed 3 times in PBS (pH 7.4) and incubated with a secondary antibody using a Vectastain ABC kit (Vector) and following the manufacturer's instructions. The color was developed with 3,3′-diaminobenzidine containing hydrogen peroxide, and slides were counterstained with eosin and, for EphB4, with methyl green.
Control procedures were performed according to the same experimental protocol as follows: 1) omission of the primary antibody, 2) substitution of the primary antibody with a nonspecific IgG from the same host as the primary antibody (Santa Cruz Biotechnology), and 3) a third control for type X collagen was performed by adsorption with the peptide YNRQQHYDPRSGIFTCKIPGIYYFSYGGC (provided by Dr. E. Lee) at a 10-fold. Controls showed only background staining.
VDIPEN and Col2-3/4Cshort staining was graded on a scale of 0–3, where 0 = no staining, 1 = minor staining, 2 = marked staining, and 3 = maximal staining, as described previously (35). Each slide was examined and scored by 2 independent observers who were blinded with regard to group allocation. Type X collagen–expressing cells were quantified following determination of the total number of chondrocytes and the total number staining positive for the antigen. The final results were expressed as the percentage of chondrocytes staining positive for the antigen (cell score; maximum score 100%).
Tartrate-resistant acid phosphatase (TRAP).
The right knee joints were dissected free of tissue, fixed in 4% paraformaldehyde for 16 hours at 4°C, decalcified in 10% EDTA for 14 days at 4°C, and embedded in paraffin. Staining for TRAP enzyme activity was carried out as previously described (36). Briefly, the deparaffinized and rehydrated sections were incubated in the staining medium containing naphthol AS-TR phosphate as substrate, pararosaniline HCl as the coupler, and N,N-dimethylformamide tartrate solution. Counterstaining was performed with 0.4% methyl green. Negative staining was performed without substrate.
To determine the number of osteoclasts, the subchondral bone area that was evaluated comprised the cartilage–bone junction and the growth plate as the upper and lower limits. The analysis was performed on 1 section per specimen using a Leitz Diaplan microscope (Leica Microsystems) connected to a personal computer (Pentium IV based, using Bioquant Osteo II Image Analysis software), as previously described (11, 25, 37). Data are expressed as the number of cells expressing TRAP per subchondral bone surface (mm2).
Subchondral bone plate histomorphometry.
The right knee joints were fixed in TissuFix, decalcified in RDO Rapid Decalcifier, embedded in paraffin, and sections (5 μm) were deparaffinized in xylene followed by a graded series of alcohol washes and then stained with Safranin O–fast green as described above. Histomorphometry was done on the medial compartment, as described elsewhere (11), using a Leitz Diaplan microscope connected to a personal computer as above. From the digital image, a box with a fixed width (1,000 μm) and variable length was created with the upper limit at the calcified cartilage–subchondral bone junction and the lower limit at the subchondral bone–trabecular bone junction. The mean distance between the upper and lower limit was calculated automatically by the software. All measurements were made by a single experienced observer who was blinded with regard to the experimental conditions.
Micro–computed tomography (micro-CT).
Prior to sectioning, the right femoral distal metaphysis and tibial proximal metaphysis were scanned with a SkyScan 1176 in vivo micro-CT instrument. Image acquisition was performed at 72 keV and 142 μA, with a 0.6° rotation between frames through a total of 180°, and at 9 μm spatial resolution. Two dimensional (2-D) images were used to generate 3-D reconstructions of the trabecular subchondral bone. For the subchondral bone, 15 reconstructed grayscale images were selected from immediately above the tibial growth plate, and 3-D analysis was used to calculate morphometric parameters in the medial compartment, including the volume fraction (bone volume/total volume; %), trabecular thickness (mm), and trabecular separation (μm) at the same threshold with the 3D Creator software supplied with the SkyScan CT Analyzer.
Values are expressed as mean ± SEM. Statistical analysis was performed using the Mann-Whitney U test (GraphPad Prism software).
All data reported herein are from experiments performed on male mice. However, characterization of the zygosity done on the females revealed the same findings (data not shown).
Characterization of the EphB4-transgenic mice.
The offspring of the breeding animals were genotyped using PCR analysis. As illustrated in Figure 1A, the data showed the 193-bp band in homozygous mice, but not WT mice. The parent mice and their subsequent generations were further genotyped by quantitative PCR (Figure 1B). Significant differences in zygosity were obtained when heterozygous and homozygous mice were compared to the WT control mice (P ≤ 0.005). Additional Western blot experiments (Figure 1C) confirmed the overproduction of EphB4 protein in long bone osteoblasts from heterozygous mice, more so in those from homozygous mice, as compared to WT mice. Further experiments were carried out using homozygous transgenic EphB4 (EphB4-Tg) mice and their WT littermates (controls).
Morphometric assessment of 5-day-old (P5) mice showed that the EphB4-Tg mice were similar in body size and gross appearance to the WT mice (Figure 2A). Skeletal staining showed no significant differences in the growth and development of the mice (Figure 2B). The body weight of the male mice examined at P3 (25 WT and 29 EphB4-Tg mice), P10 (25 WT and 28 EphB4-Tg mice), P28 (28 WT and 23 EphB4-Tg mice), and P70 (8 WT and 8 EphB4-Tg mice) also showed no change across the genotype until at least 10 weeks of age (data not shown). Similarly, at the age of 10 weeks, mice demonstrated no difference in size or weight morphologically (Figure 2C). However, radiographic evaluation showed an increase in the femur density in the EphB4-Tg mice as compared to the WT mice (Figure 2D). Overexpression of EphB4 in transgenic osteoblasts was confirmed by immunohistochemistry, in which the staining per osteoblast was more enhanced in the EphB4-Tg mice (Figure 2E). However, when compared to WT mice, no significant differences in osteoblasts per bone surface were seen in subchondral bone from EphB4-Tg mice by 10 weeks of age.
Knee joint swelling.
Knee joint swelling was determined by the diameter of the operated (right) knee in WT and EphB4-Tg mice at 8 (WT, n = 10; EphB4-Tg, n = 10) and 12 (WT, n = 20; EphB4-Tg, n = 20) weeks post-DMM surgery. Mice were examined at baseline (day 0) and every 3 days following surgery. Data showed that the initial swelling following surgery receded similarly in both WT and EphB4-Tg mice; this was true for mice sacrificed at 8 and 12 weeks postsurgery (data not shown).
Significant resistance of EphB4-Tg mice to OA cartilage alterations post-DMM surgery.
The cartilage integrity in the medial tibial plateau and femoral condyle at 8 and 12 weeks post-DMM surgery was assessed histologically and scored according to the OARSI scoring system (29). First, we compared the histology scores for the medial tibial plateau and femoral condyle from the sham-operated EphB4-Tg mice (mean ± SEM 0.7 ± 0.3 and 1.0 ± 0.0, respectively; n = 3) and the WT control mice (1.3 ± 0.6 and 1.3 ± 0.6, respectively; n = 3) to those from 22-week-old nonoperated mice from both groups (1.0 ± 0.0 and 0.7±0.6, respectively, in EphB4-Tg mice [n = 3]; 1.0 ± 0.0 and 0.7 ± 0.3, respectively, in WT mice [n = 6]). The data revealed no significant differences among these control groups.
As illustrated in Figures 3A–D, the medial tibial plateaus and femoral condyles of DMM-operated WT mice at both times postsurgery showed an increased loss of cartilage integrity, including loss of Safranin O–fast green staining, accompanied by reduced cellularity, thinning of the cartilage, and increased fibrillation as compared to the DMM-operated EphB4-Tg mice. These observations were corroborated by the OARSI scores (Figures 3E–H), which showed significantly lower histologic scores in the DMM-operated EphB4-Tg mice at 8 weeks (P ≤ 0.006 and P ≤ 0.01, respectively) and 12 weeks (P ≤ 0.01 and P ≤ 0.03, respectively) postsurgery.
Protective effect of some cartilage markers in EphB4-Tg mice.
In DMM-operated mice at 8 and 12 weeks postsurgery, the effect of EphB4 overexpression on the degradation products of aggrecan (Figure 4A) and on type II collagen (Figure 4B) and type X collagen (Figure 4C) was examined using specific antibodies, and the effect on collagen disorganization (Figure 4D) was examined using the sirius red method. Data are reported only for 12 weeks post-DMM surgery, but similar data were found at 8 weeks post-DMM surgery. DMM-operated EphB4-Tg mice demonstrated significantly reduced levels of aggrecan fragments (P ≤ 0.04), in the α1 chain of type II collagen (P ≤ 0.008), and collagen disorganization in the medial tibial plateau (P ≤ 0.0008) at 12 weeks postsurgery (Figures 4A, B, and D). In addition, the medial femoral condyle exhibited a significant decrease in aggrecan degradation products (P ≤ 0.04) (Figure 4A); however, data obtained for the type II collagen degradation products (Figure 4B) and for collagen disorganization (Figure 4D) showed no significant difference. Type X collagen (Figure 4C), which is associated with chondrocyte hypertrophy, also showed significantly lower levels in both the tibial plateau (P ≤ 0.009) and femoral condyle (P ≤ 0.02) in DMM-operated EphB4-Tg mice as compared to the DMM-operated WT mice.
Better preserved subchondral bone in EphB4-Tg mice.
Representative histology sections of the subchondral bone plate thickness from the sham-operated WT mice and the EphB4-Tg mice at 12 weeks postsurgery are shown in Figures 3A–D. Histomorphometric evaluation of the subchondral bone plate thickness demonstrated similar values for the sham-operated WT and EphB4-Tg mice at both 8 weeks (data not shown) and 12 weeks post-DMM surgery (Figure 5A). Compared to sham-operated mice at 12 weeks postsurgery, the subchondral bone plate thickness at 8 weeks and 12 weeks postsurgery was significantly increased in the WT (P ≤ 0.01) and EphB4-Tg (P ≤ 0.05) DMM-operated mice (Figure 5A). However, the DMM-operated EphB4-Tg mice demonstrated a statistically significant decrease in subchondral bone plate thickness compared to the DMM-operated WT mice at both 8 weeks (P ≤ 0.007) and 12 weeks (P ≤ 0.003) postsurgery (Figure 5A).
TRAP analysis revealed significantly lower numbers of osteoclasts in the EphB4-Tg mice than in the WT mice (P ≤ 0.009) at 12 weeks postsurgery (Figures 5B–F).
The 3-D rendering and reconstruction scans of WT mice at 12 weeks postsurgery (Figure 6A) showed alterations of the knee joint, including the thickness of the medial subchondral plate and pronounced sclerosis of the subchondral bone, while the EphB4-Tg mice (Figures 6B) demonstrated fewer changes. Comparison of the WT and EphB4-Tg sham-operated mice 12 weeks postsurgery revealed similar findings for the subchondral bone parameters (Figures 6C–E). However, DMM-operated WT mice had a significant increase in bone volume (P ≤ 0.02) and trabecular thickness (P ≤ 0.02) compared to the sham-operated mice. Moreover, at 12 weeks post-DMM surgery, the EphB4-Tg mice exhibited significantly reduced bone volume and trabecular thickness (P ≤ 0.0007 and P ≤ 0.02, respectively) compared to the WT mice (Figures 6C and D). There were similar values for trabecular separation (Figure 6E) in both the operated WT and EphB4-Tg mice at 12 weeks post-DMM surgery, and no difference was found as compared to their sham-operated controls.
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.
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.
The authors wish to express their gratitude to Frédéric Paré, Stéphane Tremblay, and François Mineau for their expert technical support and to Virginia Wallis for her assistance with the manuscript preparation. We also wish to acknowledge the professionalism of the animal care technicians at the University of Montreal Hospital Research Centre (CRCHUM). The authors are grateful to Dr. Janet E. Henderson (Director of Orthopaedic Research, McGill University, Montreal, Quebec, Canada) for sharing her expertise, and to Dr. A. Robin Poole, Dr. John S. Mort, and Dr. Eunice Lee (Shriners Hospital for Children, McGill University Hospital Centre, Montreal, Quebec, Canada) for generously providing some of the antibodies and peptides used in this project.