Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis

Authors

  • Manas K. Majumdar,

    1. Wyeth Research, Cambridge, Massachusetts
    Current affiliation:
    1. GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania
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  • Roger Askew,

    1. Wyeth Research, Cambridge, Massachusetts
    Current affiliation:
    1. Merck Research Laboratories, Rahway, New Jersey
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    • Drs. Askew, Schelling, and Stedman, Ms Blanchet, Ms Hopkins, Dr. Morris, and Ms Glasson own stock in Wyeth.

  • Scott Schelling,

    1. Wyeth Research, Cambridge, Massachusetts
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    • Drs. Askew, Schelling, and Stedman, Ms Blanchet, Ms Hopkins, Dr. Morris, and Ms Glasson own stock in Wyeth.

  • Nancy Stedman,

    1. Wyeth Research, Cambridge, Massachusetts
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    • Drs. Askew, Schelling, and Stedman, Ms Blanchet, Ms Hopkins, Dr. Morris, and Ms Glasson own stock in Wyeth.

  • Tracey Blanchet,

    1. Wyeth Research, Cambridge, Massachusetts
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    • Drs. Askew, Schelling, and Stedman, Ms Blanchet, Ms Hopkins, Dr. Morris, and Ms Glasson own stock in Wyeth.

  • Bei Hopkins,

    1. Wyeth Research, Cambridge, Massachusetts
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    • Drs. Askew, Schelling, and Stedman, Ms Blanchet, Ms Hopkins, Dr. Morris, and Ms Glasson own stock in Wyeth.

  • Elisabeth A. Morris,

    1. Wyeth Research, Cambridge, Massachusetts
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    • Drs. Askew, Schelling, and Stedman, Ms Blanchet, Ms Hopkins, Dr. Morris, and Ms Glasson own stock in Wyeth.

    • Ms Glasson and Dr. Morris have a patent pending for ADAMTS small molecular inhibitors.

  • Sonya S. Glasson

    Corresponding author
    1. Wyeth Research, Cambridge, Massachusetts
    • Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140
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    • Drs. Askew, Schelling, and Stedman, Ms Blanchet, Ms Hopkins, Dr. Morris, and Ms Glasson own stock in Wyeth.

    • Ms Glasson and Dr. Morris have a patent pending for ADAMTS small molecular inhibitors.


Abstract

Objective

To phenotypically characterize ADAMTS-4– and ADAMTS-5–double-knockout mice, and to determine the effect of deletion of ADAMTS-4 and ADAMTS-5 on the progression of osteoarthritis (OA) in mice.

Methods

Mice lacking the catalytic domain of ADAMTS-4 and ADAMTS-5 were crossed to generate ADAMTS-4/5–double-knockout animals. Twelve-week-old and 1-year-old male and female ADAMTS-4/5–double-knockout mice were compared with age- and sex-matched wild-type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS-4/5–double-knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS-4/5–double-knockout mice were treated with interleukin-1 (IL-1) plus retinoic acid ex vivo, to examine proteoglycan degradation.

Results

There were no genotype-related phenotype differences between ADAMTS-4/5–double-knockout and WT mice through 1 year of age, with the exception that female ADAMTS-4/5–double-knockout mice had a lower mean terminal body weight at the 12-week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS-4/5–double-knockout mice compared with WT mice. Following stimulation of cartilage explants with IL-1 plus retinoic acid, aggrecanase-mediated degradation in ADAMTS-4/5–double-knockout mice was ablated, to a level comparable with that in ADAMTS-5–knockout mice.

Conclusion

Dual deletion of ADAMTS-4 and ADAMTS-5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS-4/5–double-knockout mice were comparable with those observed with deletion of ADAMTS-5 alone.

Articular cartilage is composed of chondrocytes embedded in an extracellular matrix (ECM), which provides the biomechanical characteristics that are essential during articular movement. One of the major ECM components of cartilage is aggrecan, a large proteoglycan that provides cartilage with the ability to resist compressive forces. Degradation of aggrecan is an important manifestation of osteoarthritis (OA) (1). Analysis of synovial fluid from patients with arthritis revealed that pathologic aggrecan cleavage occurred at the “aggrecanase” cleavage site, Glu373–Ala374, within the interglobular domain (2). Several members of the ADAMTS family of proteins have been shown to cleave aggrecan in vitro at the aggrecanase cleavage site (3–6). Of these, ADAMTS-4 and ADAMTS-5 are the most efficient aggrecanases (3, 4). Increased expression of these aggrecanases after stimulation of articular tissues with inflammatory cytokines or overexpression of these aggrecanases has resulted in cartilage matrix degradation (7–11). Both ADAMTS-4 and ADAMTS-5 are expressed in normal and OA cartilage as well as in OA synovium (9), and inhibition of these enzymes correlates with prevention of aggrecan degradation in vitro (12). However, there is minimal agreement in the literature as to which of the aggrecanases is more important in human OA.

To understand the role of ADAMTS-4 and ADAMTS-5 in OA, we challenged mice lacking ADAMTS-4 or ADAMTS-5 and exhibiting a normal phenotype by surgical induction of joint instability, leading to OA (13, 14). Those studies demonstrated that ADAMTS-5 deletion is protective against aggrecan degradation in OA or cytokine-stimulated articular cartilage. In order to investigate the effects of a complete absence of ADAMTS-4 and ADAMTS-5 aggrecanase activity, we generated mice with dual deletion of both genes. Adult ADAMTS-4/5–double-knockout animals underwent extensive phenotype analyses to examine the effect of dual deletion on development and physiology, and were also challenged in a surgically induced model of OA. Articular hip and nonarticular xiphisternum cartilage explants from wild-type (WT) and ADAMTS-4/5–double-knockout mice were treated with inflammatory agents ex vivo to ascertain whether deletion of both of these genes would provide additional protection compared with that in ADAMTS-5–knockout mice (14, 15).

MATERIALS AND METHODS

Generation of ADAMTS-4/5–double-knockout mice.

ADAMTS-4– and ADAMTS-5–knockout mice (on a 129SvEv-Brd background) underwent deletion of the exon encoding the majority of the catalytic site of the enzyme (13, 14). ADAMTS-4/5–double-knockout mice were generated by crossing single-knockout mice to produce compound heterozygotes and then crossing compound heterozygotes to either ADAMTS-4– or ADAMTS-5–knockout mice to produce reciprocal homozygous/heterozygous-null mice. Reciprocal homozygous/heterozygous-null mice were crossed to produce ADAMTS-4/5–double-knockout mice.

Phenotyping of ADAMTS-4/5–double-knockout mice.

Terminal body weights and organ weights were recorded, and bone density was measured by tissue densitometry, using the PIXImus mouse densitometer (Faxitron X-ray Corporation, Wheeling, IL), at both 12 weeks and 1 year of age (n = 40 mice [10 male WT, 10 female WT, 10 male double-knockout, and 10 female double-knockout]). The results of macroscopic evaluations were recorded, and 44 tissues were evaluated microscopically: liver, lung, trachea, pancreas, brain, adrenal cortex, adrenal medulla, ileum, gut-associated lymphatic tissue, mandibular lymph node, eye, optic nerve, sciatic nerve, esophagus, gall bladder, cervical and lumbar spinal cord, cecum, colon, tongue, Harderian gland, aorta, thyroid gland, spleen, pituitary gland, duodenum, mesenteric lymph node, urinary bladder, skin, bone marrow, thymus, parathyroid gland, kidney, skeletal muscle, stomach, jejunum, salivary gland, mammary gland, prostate, testis, epididymis, seminal vesicle, ovary, vagina, cervix, uterus, bone, and joint.

Surgical induction of OA.

All studies were performed with approval of the Wyeth Institutional Animal Care and Use committee. Male mice underwent surgical transection of the medial meniscotibial ligament, resulting in destabilization of the medial meniscus, as previously described (13). Eleven double-knockout and 9 WT male animals were killed 8 weeks postoperatively.

Assessment of progression and severity of OA.

Serial 6-μm frontal sections of 4% paraformaldehyde–fixed, decalcified, paraffin-embedded knee joints were stained with Safranin O–fast green and graded at 80–90-μm intervals through the joint, using a semiquantitative scoring system, with scores ranging from 0 (normal) to 6 (>80% loss of cartilage) (13). All quadrants of the joint (medial tibial plateau, medial femoral condyle, lateral tibial plateau, and lateral femoral condyle) were scored separately, on 12–16 levels, by 2 blinded observers. Scores were expressed as the maximum histologic score observed in each joint or the summed histologic score. The summed score represented the additive scores for each quadrant of the joint on each histologic section through the joint. This method of analysis enabled assessment of the severity of lesions as well as reflecting the surface area of cartilage affected with OA lesions.

Culture of cartilage explants.

Hip and xiphisternum cartilage explants were prepared according to previously reported procedures (13, 15). Briefly, femoral heads and xiphisternum cartilage were harvested from 3–4-week-old WT, ADAMTS-4–knockout, ADAMTS-5–knockout, and ADAMTS-4/5–double-knockout mice (n = 20–48 mice per group) and cultured as explants in Dulbecco's modified Eagle's medium (DMEM) containing 1% antibiotic/antimycotic solution (Sigma, St. Louis, MO), 2 mM glutamine, 10 mM HEPES, 50 mg/ml ascorbate, and 10% fetal bovine serum, for 48 hours. Explants were then washed and cultured for an additional 72 hours in serum-free DMEM, with and without 10 ng/ml mouse interleukin-1α (IL-1α) plus 10−5M retinoic acid (both from Sigma).

Harvested explants were digested with proteinase K (Sigma) and centrifuged. The proteoglycan content in the media and digested cartilage supernatant was measured as sulfated glycosaminoglycan, by a colorimetric assay using dimethylmethylene blue. Results were expressed as the percentage of proteoglycan release relative to the total proteoglycan in the media and digested cartilage, and then normalized to the release in the WT group. Aggrecan fragments in conditioned media from WT and ADAMTS-4/5–double-knockout mouse cartilage, with and without IL-1 plus retinoic acid, were analyzed by Western blotting according to a previously reported procedure, using monoclonal antibody AGG-C1, which recognizes the neoepitope TEGE373 (13).

RESULTS

Phenotypic assessment of ADAMTS-4/5–double-knockout mice.

Analysis of the clinical appearance, tissue densitometry results, clinical pathology parameters (hematology and clinical chemistry), organ weights, and macroscopic and microscopic findings in multiple organs revealed no genotype-related differences between age- and sex-matched WT and ADAMTS-4/5–double-knockout animals at either 12 weeks or 1 year of age. A 15% lower mean terminal body weight in female ADAMTS-4/5–double-knockout mice compared with WT controls (P < 0.05 by one-way nonparametric analysis of variance) was observed at the 12-week but not the 1-year time point and had no significant correlates.

Histologic evaluation of OA.

WT mice developed moderate OA 8 weeks after surgical induction of joint instability. WT mice had a mean ± SEM maximal score of 3.1 ± 0.4 and a mean ± SEM summed score of 31.5 ± 4.8. In contrast, ADAMTS-4/5–double-knockout mice had mild OA, with a mean ± SEM maximal score of 1.6 ± 0.2 and a mean ± SEM summed score of 8.5 ± 1.4. The scores were significantly different (P = 0.007 and P = 0.0004 by unpaired t-test, respectively) and reflected a 47% lower maximal score and a 73% lower summed score in double-knockout mice (Figures 1A and B). Typical tibial plateau lesions consisted of fibrillation extending to the tidemark in WT mice (Figure 2A) and lesions restricted to the transitional zone in double-knockout mice (Figure 2B).

Figure 1.

Mean and SEM histologic scores of joints from 9 wild-type (WT) and 11 ADAMTS-4/5–double-knockout (DKO) mice 8 weeks after surgical induction of joint instability. A, Maximal osteoarthritis (OA) score. B, Summed OA score.

Figure 2.

Representative Safranin O–stained specimens from wild-type (A) and ADAMTS-4/5–double-knockout (B) mouse knee joints. Lesions on the medial tibial plateau (arrows) extend to the calcified cartilage in the joint from a wild-type mouse (score of 3) and the transitional zone in the joint from a double-knockout mouse (score of 2).

Characterization of aggrecan degradation in ADAMTS-4/5–double-knockout mice.

Hips from WT and ADAMTS-4–knockout mice treated with IL-1 plus retinoic acid showed a 2–3-fold increase in proteoglycan release compared with the levels in hips from mice treated with media only, while xiphisternum from WT and ADAMTS-4–knockout mice treated with IL-1 plus retinoic acid showed a 4–5-fold increase (results not shown). When release in all samples that were stimulated with IL-1 plus retinoic acid was normalized to release in WT samples treated with IL-1 plus retinoic acid (100%), a high level of reproducibility between hip and xiphisternum cartilage was observed (Figure 3). Cartilage from ADAMTS-4–knockout mice showed substantial release, slightly higher than, but comparable with, that in WT cartilage. Hip and xiphisternum cartilage from ADAMTS-5–knockout mice demonstrated 34% and 38% release, respectively, relative to WT, while hip and xiphisternum cartilage from ADAMTS-4/5–double-knockout mice showed 26% and 36% release, respectively, relative to WT. Release in the non–cytokine-stimulated ADAMTS-4/5–double-knockout group was slightly reduced compared with that in the WT group but was not significantly different (results not shown). Western blot analysis using the TEGE373 antibody revealed significant neoepitope generation in the WT hip and xiphisternum media following treatment with IL-1 plus retinoic acid but none in the ADAMTS-4/5–double-knockout hip and xiphisternum media (results not shown). These findings were comparable with previous results using hip (14, 16) and xiphisternum (15) cartilage from ADAMTS-5–knockout mice.

Figure 3.

Total proteoglycan release from cultured hip and xiphisternum cartilage after 72 hours of treatment with interleukin-1 plus retinoic acid. Release from cartilage from ADAMTS-4–knockout (KO) mice was comparable with that from cartilage from wild-type (WT) mice. Release from cartilage from ADAMTS-5–knockout mice and ADAMTS-4/5–double-knockout (DKO) mice was dramatically decreased compared with that from cartilage from wild-type (100%) or ADAMTS-4–knockout mice.

DISCUSSION

ADAMTS-4 and ADAMTS-5 are considered the primary enzymes responsible for the cleavage of aggrecan and are potential targets for therapeutic intervention in OA (3, 4). In mice, ADAMTS-5 but not ADAMTS-4 was found to be responsible for disease progression in a surgically induced model of OA (13, 14). ADAMTS-4 and ADAMTS-5 are expressed in normal and OA cartilage (9), and inhibition of these enzymes correlates with reduced aggrecan degradation in vitro (12). However, there is still debate as to which aggrecanase is more important in the pathogenesis of human OA. Due to this ambiguity, one potential therapeutic approach is systemic inhibition of both ADAMTS-4 activity and ADAMTS-5 activity. We attempted to address this hypothesis by generating a mouse with dual deletion of the zinc-binding catalytic portion of both ADAMTS-4 and ADAMTS-5 genes and analyzing the gross and histologic appearance of the animals. The effect of deletion of both ADAMTS-4 and ADAMTS-5 was examined both ex vivo and in a surgically induced model of OA.

The ADAMTS-4/5–double-knockout mice were physiologically normal. Extensive histologic evaluation of 12-week-old and 1-year old ADAMTS-4/5–double-knockout mice revealed no genotype-related abnormalities, similar to observations in mice with either ADAMTS-4 or ADAMTS-5 deletion (13, 14). Knockin mice with aggrecan resistant to metalloproteinase or aggrecanase cleavage in the interglobular domain also have no skeletal abnormalities (17), supporting the hypothesis that ADAMTS-4/5–mediated aggrecanase activity, or aggrecan cleavage generating TEGE373 or DIPEN 341, is not required for normal development and physiology. The finding of lower mean terminal body weight of female (but not male) ADAMTS-4/5–double-knockout mice (at the early time point only) with no clinical or histopathologic correlates is intriguing. Further studies are required to evaluate whether possible transient effects occur earlier in development or whether this is a spurious result, especially in light of the lack of weight difference in 1-year-old mice.

The ADAMTS-4/5–double-knockout mice showed a decrease in the progression of OA after surgically induced joint instability. At the 8-week time point, the 47% and 73% reductions in maximal and summed OA scores, respectively, observed in ADAMTS-4/5–double-knockout mice were comparable with the 46% and 65% reductions observed in ADAMTS-5–knockout mice (14). No decrease in the severity of OA was observed in the ADAMTS-4–knockout mice (13). The destabilization of the medial meniscus model of surgical instability provides a model of progressive OA that increases in severity through 2, 4, 8, and 26 weeks postoperatively (Glasson S, et al: unpublished observations). Protection that was observed at the 8-week time point in ADAMTS-5–knockout mice has also been observed at 2, 4, and 26 weeks (ref.14, and Glasson S, et al: unpublished observations), and would also be expected in ADAMTS-4/5–double-knockout mice.

The in vivo observations were also reflected by the ex vivo cartilage explant studies of both the hip and xiphisternum. A significant reduction in proteoglycan release and TEGE373 neoepitope generation was observed in cartilage from ADAMTS-4/5–double-knockout mice, comparable with that observed in ADAMTS-5–knockout mice (14, 16), and contrasted with the lack of protection observed in ADAMTS-4–knockout mice (13, 15, 16). Given the normal physiology of ADAMTS-4/5–double-knockout mice, our results suggest that dual inhibition of ADAMTS-4 and ADAMTS-5 may be a reasonable strategy for inhibiting aggrecanase activity in human OA.

In summary, the results of this study show the importance of aggrecanases in the progression of disease in a mouse model of progressive OA. Due to the lack of convincing evidence identifying the specific aggrecanase responsible for human OA, inhibition of both ADAMTS-4 and ADAMTS-5 may be a reasonable therapeutic strategy for OA. Our results suggest that inhibiting both ADAMTS-4 and ADAMTS-5 would provide protection against the disease without affecting normal physiology. The answer to the central question of which enzyme or enzymes are responsible for human OA will require the development of extremely potent and selective therapeutic agents.

AUTHOR CONTRIBUTIONS

Ms Glasson 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 design. Majumdar, Askew, Morris, Glasson.

Acquisition of data. Majumdar, Schelling, Blanchet, Hopkins, Glasson.

Analysis and interpretation of data. Majumdar, Askew, Schelling, Stedman, Blanchet, Hopkins, Glasson.

Manuscript preparation. Majumdar, Askew, Schelling, Stedman, Glasson.

Statistical analysis. Glasson.

Management of animal production. Askew.

Acknowledgements

We acknowledge the valued input and efforts by Diane Peluso, Katy Wallace, Donna Gavin, Janet Golden, Wen Kuang, Jennifer Tavares, Exploratory Drug Safety, GMAP, and Bioresources.

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