A. Robin Poole

MT1-MMP-Deficient Mice Develop Dwarfism, Osteopenia, Arthritis, and Connective Tissue Disease due to Inadequate Collagen Turnover

MT1-MMP is a membrane-bound matrix metalloproteinase (MT-MMP) capable of mediating pericellular proteolysis of extracellular matrix components. MT1-MMP is therefore thought to be an important molecular tool for cellular remodeling of the surrounding matrix. To establish the biological role of this membrane proteinase we generated MT1-MMP-deficient mice by gene targeting. MT1-MMP deficiency causes craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity that is essential for modeling of skeletal and extraskeletal connective tissues. Our findings demonstrate the pivotal function of MT1-MMP in connective tissue metabolism, and illustrate that modeling of the soft connective tissue matrix by resident cells is essential for the development and maintenance of the hard tissues of the skeleton.

Composition and structure of articular cartilage: a template for tissue repair.

The authors review the structure and composition of articular cartilage. This tissue is composed of an extensive extracellular matrix synthesized by chondrocytes. It contains different zones with respect to depth from the articular surface and has a regional organization around the chondrocytes. Its composition varies regionally and zonally in its collagen and proteoglycan contents and those of other matrix molecules. There is a macrofibrillar collagen network and a microfilamentous network about which other noncollagenous molecules are organized. Its structure and composition are reflective of its special mechanical properties that primarily reflect its tensile strength (collagens) and compressive stiffness (proteoglycan aggrecan) and cell-matrix interactions (noncollagenous proteins).

Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice

It has been suggested that increased collagenase-3 (MMP-13) activity plays a pivotal role in the pathogenesis of osteoarthritis (OA). We have used tetracycline-regulated transcription in conjunction with a cartilage-specific promoter to target a constitutively active human MMP-13 to the hyaline cartilages and joints of transgenic mice. Postnatal expression of this transgene resulted in pathological changes in articular cartilage of the mouse joints similar to those observed in human OA. These included characteristic erosion of the articular cartilage associated with loss of proteoglycan and excessive cleavage of type II collagen by collagenase, as well as synovial hyperplasia. These results demonstrate that excessive MMP-13 activity can result in articular cartilage degradation and joint pathology of the kind observed in OA, suggesting that excessive activity of this proteinase can lead to this disease.

Evidence for altered synthesis of type II collagen in patients with osteoarthritis.

There is evidence to suggest that the synthesis of type II collagen is increased in osteoarthritis (OA). Using an immunoassay, we show that the content of the C-propeptide of type II procollagen (CPII), released extracellularly from the newly synthesized molecule, is directly related to the synthesis of this molecule in healthy and osteoarthritic articular cartilages. In OA cartilage, CPII content is often markedly elevated (mean 7.6-fold), particularly in the mid and deep zones, reaching 29.6% of the content in newborn. Synthesis is also directly related to total collagen II content in OA, suggesting its importance in maintaining collagen content and cartilage structure. The release of CPII from cartilage is correlated directly with cartilage content. However, the increase in CPII in OA cartilage is not reflected in serum, where a significant reduction is observed. Together these studies provide evidence for alterations in procollagen II synthesis in vivo in patients with OA.

Spontaneous air space enlargement in the lungs of mice lacking tissue inhibitor of metalloproteinases-3 (TIMP-3)

Tissue inhibitors of metalloproteinases regulate ECM degradation by matrix metalloproteinases (MMPs). We have developed a mouse line deficient for tissue inhibitor of metalloproteinases-3 (TIMP-3), the only TIMP known to reside within the ECM. Homozygous Timp-3-null animals develop spontaneous air space enlargement in the lung that is evident at 2 weeks after birth and progresses with age of the animal. As early as 13 months of age animals become moribund. Lung function, measured by carbon monoxide uptake, is impaired in aged null animals. Lungs from aged null animals have reduced abundance of collagen, enhanced degradation of collagen in the peribronchiolar space, and disorganization of collagen fibrils in the alveolar interstitium, but no increase in inflammatory cell infiltration or evidence of fibrosis in comparison with controls. Using in situ zymography, we show that lungs from aged null animals have heightened MMP activity over wild-type and heterozygotic animals. Finally, TIMP-3-null fibroblast cultures demonstrate enhanced destruction of ECM molecules in vitro. We propose that the deletion of TIMP-3 results in a shift of the TIMP/MMP balance in the lung to favor ECM degradation, culminating in incapacitating illness and a shorter life span.

Changes in joint cartilage aggrecan after knee injury and in osteoarthritis.

OBJECTIVE To determine the concentrations of aggrecan fragments in synovial fluid from patients with knee joint injury, osteoarthritis (OA), or acute pyrophosphate arthritis (PPA; pseudogout), and to test their relative reactivity with the 846 epitope, a putative marker of cartilage aggrecan synthesis. METHODS Samples of knee joint fluid from 385 patients and 9 healthy-knee volunteers were obtained in a cross-sectional study. Study groups were acute PPA/ pseudogout (n = 60), anterior cruciate ligament (ACL) rupture (n = 159), meniscus lesion (n = 129), and primary knee OA (n = 37). The 846 epitope on aggrecan was assayed by competitive solution-phase radioimmunoassay. Aggrecan fragments were assayed by enzyme-linked immunosorbent assay using a monoclonal antibody (1-F21). Cartilage oligomeric matrix protein (COMP), C-propeptide of type II collagen (CPII), bone sialoprotein, matrix metalloproteinases 1 and 3, and tissue inhibitor of metalloproteinases 1 were previously quantified by immunoassays. RESULTS Reactivity of the 846 epitope was increased in all study groups compared with the reference group, and was highest in patients with primary OA. The median levels (in microg fetal aggrecan equivalents/ml) of the epitope were 0.28 (range 0.24-0.47) in the reference group, 0.48 (range 0.26-1.32) in PPA/pseudogout, 0.61 (range 0.12-2.87) in ACL rupture, 0.53 (range 0.22-3.02) in meniscus lesion, and 0.68 (range 0.31-4.31) in primary OA. The 846 epitope reactivity per microg aggrecan fragments in the joint fluid was higher in late-stage OA than in early-stage OA. Epitope 846 reactivity correlated positively with several markers of matrix turnover, particularly with COMP (r(s) = 0.421) and CPII (r(s) = 0.307). CONCLUSION The observed differences in 846 epitope reactivity in synovial fluid, and its concentration in relation to aggrecan and other markers of matrix turnover, were consistent with marked ongoing changes in aggrecan turnover after joint injury and in the development of OA. OA is thus a disease characterized by dynamic changes in tissue macromolecule turnover, which is reflected by measurable changes in aggrecan epitopes in the synovial fluid.

Rehabilitation After Anterior Cruciate Ligament Reconstruction A Prospective, Randomized, Double-Blind Comparison of Programs Administered Over 2 Different Time Intervals

Background There are adverse effects associated with immobilization of the knee after anterior cruciate ligament reconstruction, yet very little is known about how much activity will promote adequate rehabilitation without permanently elongating the graft, producing graft failure, or creating damage to articular cartilage. Hypothesis Rehabilitation with either an accelerated or nonaccelerated program produces no difference in anterior-posterior knee laxity, clinical assessment, patient satisfaction, functional performance, and the synovial fluid biomarkers of articular cartilage metabolism. Study Design Randomized controlled clinical trial; Level of evidence, 1. Methods Twenty-five patients who tore their anterior cruciate ligament were enrolled and underwent anterior cruciate ligament reconstruction. Patients were randomized to accelerated rehabilitation or nonaccelerated rehabilitation. At the time of surgery and 3, 6, 12, and 24 months later, measurements of anterior-posterior knee laxity, clinical assessment, patient satisfaction, functional performance, and cartilage metabolism were completed. Results At the 2-year follow-up, there was no difference in the increase of anterior knee laxity relative to the baseline values that were obtained immediately after surgery between the 2 groups (2.2-mm vs 1.8-mm increase relative to the normal knee). The groups were similar in terms of clinical assessment, patient satisfaction, activity level, function, and response of the bio-markers. After 1 year of healing, synthesis of collagen and turnover of aggrecan remained elevated in both groups. Conclusion Anterior cruciate ligament reconstruction with a bone-patellar tendon-bone graft followed by either accelerated or nonaccelerated rehabilitation produces the same increase of anterior knee laxity. Both programs had the same effect in terms of clinical assessment, patient satisfaction, functional performance, and the biomarkers of articular cartilage metabolism. There is concern that the cartilage biomarkers remained elevated for an extended period.

Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1)

OBJECTIVE To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). METHODS Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. RESULTS As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. CONCLUSION These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.

Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin‐1 and the selective inhibition of type II collagen cleavage by collagenase

OBJECTIVE To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and type II collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of collagenase 2 and collagenase 3 on these catabolic processes. METHODS Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of collagenase 2 and collagenase 3 (matrix metalloproteinase 8 [MMP-8] and MMP-13, respectively) but not of collagenase 1 (MMP-1). Immunoassays were used to measure collagenase-generated type II collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total type II collagen content (antibody COL2-3/4m) in articular cartilage and culture media. A colorimetric assay was used to measure total proteoglycan concentration (principally of aggrecan) as sulfated glycosaminoglycans (sGAG). RESULTS IL-1alpha initially induced a decrease in tissue proteoglycan content in nasal cartilage. A progressive loss of proteoglycan was noted during culture in articular cartilages, irrespective of the presence of IL-1alpha. In both cartilages, proteoglycan loss was followed by IL-1alpha-induced cleavage of type II collagen by collagenase, which was often reflected by increased denaturation. The inhibitor RS102,481 had no clear effect on the reduction in proteoglycan content (measured by sGAG) and collagen denaturation in either cartilage, but at 10 nM it inhibited the enhanced cleavage of type II collagen, partially in nasal cartilage and completely in articular cartilage. CONCLUSION IL-1alpha-induced cleavage and denaturation of type II collagen is observed in both hyaline cartilages and is secondary to proteoglycan loss. It probably involves different collagenases, since there is no evidence of a rate-limiting role for collagenase 1 in articular cartilage, unlike the case for nasal cartilage. Inhibitors of this kind may be of value in the treatment of cartilage damage in arthritis. Also, the ability to detect the release of type II collagen collagenase-generated fragments from degraded cartilage offers the potential to monitor cartilage collagen damage and its control in vivo.

The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone.

The osteocyte is the terminally differentiated state of the osteogenic mesenchymal progenitor immobilized in the bone matrix. Despite their numerical prominence, little is known about osteocytes and their formation. Osteocytes are physically separated in the bone matrix but seemingly compensate for their seclusion from other cells by maintaining an elaborate network of cell processes through which they interact with other osteocytes and bone-lining cells at the periosteal and endosteal surfaces of the bone. This highly organized architecture suggests that osteocytes make an active contribution to the structure and maintenance of their environment rather than passively submitting to random embedding during bone growth or repair. The most abundant matrix protein in the osteocyte environment is type-I collagen and we demonstrate here that, in the mouse, osteocyte phenotype and the formation of osteocyte processes is highly dependent on continuous cleavage of type-I collagen. This collagenolytic activity and formation of osteocyte processes is dependent on matrix metalloproteinase activity. Specifically, a deficiency of membrane type-1 matrix metalloproteinase leads to disruption of collagen cleavage in osteocytes and ultimately to the loss of formation of osteocyte processes. Osteocytogenesis is thus an active invasive process requiring cleavage of collagen for maintenance of the osteocyte phenotype.