Metalloprotease inhibitor TIMP proteins control FGF-2 bioavailability and regulate skeletal growth.

Regulated growth plate activity is essential for postnatal bone development and body stature, yet the systems regulating epiphyseal fusion are poorly understood. Here, we show that the tissue inhibitors of metalloprotease (TIMP) gene family is essential for normal bone growth after birth. Whole-body quadruple-knockout mice lacking all four TIMPs have growth plate closure in long bones, precipitating limb shortening, epiphyseal distortion, and widespread chondrodysplasia.

Mice Exhibit Altered Aggrecan Proteolytic Profiles That Correlate With Ascending Aortic Anomalies.

Objective: Investigate the requirement of Aggrecan (Acan) cleavage during aortic wall development in a murine model with ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin-type motifs) 5 deficiency and bicuspid aortic valves.

Approach: Mice with altered extracellular matrix remodeling of proteoglycans will be examined for anomalies in ascending aortic wall development. Neo-epitope antibodies that recognize ADAMTS cleaved Acan fragments will be used to investigate the mechanistic requirement of Acan turnover, in aortic wall development.

ADAMTS-9 in Mouse Cartilage Has Aggrecanase Activity That Is Distinct from ADAMTS-4 and ADAMTS-5.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 are the principal aggrecanases in mice and humans; however, mice lacking the catalytic domain of both enzymes (TS-4/5∆cat) have no skeletal phenotype, suggesting there is an alternative aggrecanase for modulating normal growth and development in these mice. We previously identified aggrecanase activity that (a) cleaved at E↓G rather than E↓A bonds in the aggrecan core protein, and (b) was upregulated by retinoic acid but not IL-1α. The present study aimed to identify the alternative aggrecanase.

Glucocorticoids influence versican and chondroitin sulphate proteoglycan levels in the fetal sheep lung.

Background: Prenatal glucocorticoid treatment decreases alveolar tissue volumes and facilitates fetal lung maturation, however the mechanisms responsible are largely unknown. This study examines whether changes in versican levels or sulphation patterns of chondroitin sulphate (CS) side chains, are associated with glucocorticoid-induced reductions in peri-alveolar tissue volumes.

Methods: Lung tissue was collected from 1) fetal sheep at 131 ± 0.

An aggrecan fragment drives osteoarthritis pain through Toll-like receptor 2.

Pain is the predominant symptom of osteoarthritis, but the connection between joint damage and the genesis of pain is not well understood. Loss of articular cartilage is a hallmark of osteoarthritis, and it occurs through enzymatic degradation of aggrecan by cleavage mediated by a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS-4) or ADAMTS-5 in the interglobular domain (E373-374A). Further cleavage by MMPs (N341-342F) releases a 32-amino-acid aggrecan fragment (32-mer).

Cartilage MicroRNA Dysregulation During the Onset and Progression of Mouse Osteoarthritis Is Independent of Aggrecanolysis and Overlaps With Candidates From End-Stage Human Disease.

Objective: To identify candidate microRNAs (miRNAs) that potentially regulate the initiation and progression of osteoarthritis (OA).

Methods: OA was induced in 10-12-week-old male wild-type C57BL/6 mice and in mice resistant to aggrecanase cleavage (Acan p.374ALGS→374NVYS) by destabilization of the medial meniscus (DMM).

Wide bandwidth nanomechanical assessment of murine cartilage reveals protection of aggrecan knock-in mice from joint-overuse.

Aggrecan loss in human and animal cartilage precedes clinical symptoms of osteoarthritis, suggesting that aggrecan loss is an initiating step in cartilage pathology. Characterizing early stages of cartilage degeneration caused by aging and overuse is important in the search for therapeutics. In this study, atomic force microscopy (AFM)-based force-displacement micromechanics, AFM-based wide bandwidth nanomechanics (nanodynamic), and histologic assessments were used to study changes in distal femur cartilage of wildtype mice and mice in which the aggrecan interglobular domain was mutated to make the cartilage aggrecanase-resistant.

Novel Elements of the Chondrocyte Stress Response Identified Using an in Vitro Model of Mouse Cartilage Degradation.

The destruction of articular cartilage in osteoarthritis involves chondrocyte dysfunction and imbalanced extracellular matrix (ECM) homeostasis. Pro-inflammatory cytokines such as interleukin-1α (IL-1α) contribute to osteoarthritis pathophysiology, but the effects of IL-1α on chondrocytes within their tissue microenvironment have not been fully evaluated. To redress this we used label-free quantitative proteomics to analyze the chondrocyte response to IL-1α within a native cartilage ECM.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood.

Brief Report: JNK-2 Controls Aggrecan Degradation in Murine Articular Cartilage and the Development of Experimental Osteoarthritis.

Objective: The pathogenesis of osteoarthritis (OA) is poorly understood. Loss of the proteoglycan aggrecan from cartilage is an early event. Recently, we identified a role for the JNK pathway, particularly JNK-2, in human articular chondrocytes in vitro in regulating aggrecan degradation.

3D Bioprinting of Cartilage for Orthopedic Surgeons: Reading between the Lines.

Chondral and osteochondral lesions represent one of the most challenging and frustrating scenarios for the orthopedic surgeon and for the patient. The lack of therapeutic strategies capable to reconstitute the function and structure of hyaline cartilage and to halt the progression toward osteoarthritis has brought clinicians and scientists together, to investigate the potential role of tissue engineering as a viable alternative to current treatment modalities. In particular, the role of bioprinting is emerging as an innovative technology that allows for the creation of organized 3D tissue constructs via a "layer-by-layer" deposition process.

Bioactivity in an Aggrecan 32-mer Fragment Is Mediated via Toll-like Receptor 2.

Objective: To determine whether an aggrecan 32-mer fragment derived from dual ADAMTS and matrix metalloproteinase (MMP) cleavage in the aggrecan interglobular domain was bioactive and, if so, to elucidate its mechanism of action.

Methods: Mouse primary chondrocytes, synovial fibroblasts, or peritoneal macrophages, human primary chondrocytes, and cells or cell lines from myeloid differentiation factor 88 (MyD88)-deficient and Toll-like receptor 2 (TLR-2)-deficient mice were stimulated with synthetic mouse 32-mer peptide, human 32-mer peptide, a 32-mer scrambled peptide, or native, glycosylated 32-mer peptide. Cells stimulated with 32-mer peptide were analyzed for changes in messenger RNA (mRNA) expression by quantitative polymerase chain reaction.

Mast cell-restricted, tetramer-forming tryptases induce aggrecanolysis in articular cartilage by activating matrix metalloproteinase-3 and -13 zymogens.

Mouse mast cell protease (mMCP)-6-null C57BL/6 mice lost less aggrecan proteoglycan from the extracellular matrix of their articular cartilage during inflammatory arthritis than wild-type (WT) C57BL/6 mice, suggesting that this mast cell (MC)-specific mouse tryptase plays prominent roles in articular cartilage catabolism. We used ex vivo mouse femoral head explants to determine how mMCP-6 and its human ortholog hTryptase-β mediate aggrecanolysis. Exposure of the explants to recombinant hTryptase-β, recombinant mMCP-6, or lysates harvested from WT mouse peritoneal MCs (PMCs) significantly increased the levels of enzymatically active matrix metalloproteinases (MMP) in cartilage and significantly induced aggrecan loss into the conditioned media, relative to replicate explants exposed to medium alone or lysates collected from mMCP-6-null PMCs.

Transcriptomics of wild-type mice and mice lacking ADAMTS-5 activity identifies genes involved in osteoarthritis initiation and cartilage destruction.

Objective: To identify changes in gene expression in mice with osteoarthritis (OA) in order to explore the mechanisms of the disease.

Methods: Gene expression profiling was performed in cartilage from mice with surgically induced OA. We used wild-type (WT) mice and Adamts5Δcat mice, in which ADAMTS-5 activity is lacking and aggrecan loss and cartilage erosion are inhibited, to distinguish gene expression changes that are independent of ADAMTS-5 activity and cartilage breakdown.

Evidence for lysosomal exocytosis and release of aggrecan-degrading hydrolases from hypertrophic chondrocytes, in vitro and in vivo.

The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca(2+)-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development.

Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the aggrecan interglobular domain but robust at the aggrecan C-terminus.

Objective: To investigate aggrecan degradation in juvenile idiopathic arthritis (JIA).

Methods: The pattern and abundance of aggrecan fragments in synovial fluid (SF) aspirates from JIA patients were analyzed and compared with aggrecan fragments in SF from patients with other arthritides, children with knee injury, and a knee-healthy reference group. Concentrations of sulfated glycosaminoglycan (sGAG) in SF were measured by Alcian blue precipitation assay.

Emerging Frontiers in cartilage and chondrocyte biology.

Articular cartilage is a uniquely ordered tissue that is designed to resist compression and redistribute load, but is poorly equipped for self-repair. The chondrocyte is the only resident cell type, responsible for maintaining a specialised and extensive matrix that is avascular and lacks innervation. These attributes, as well as the slow turnover rate of aggrecan and type II collagen in mature articular cartilage, present a considerable challenge to the tissue engineer.

Proteoglycan degradation by the ADAMTS family of proteinases.

Proteoglycans are key components of extracellular matrices, providing structural support as well as influencing cellular behaviour in physiological and pathological processes. The diversity of proteoglycan function reported in the literature is equally matched by diversity in proteoglycan structure. Members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family of enzymes degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular function.

Matrilin-4 is processed by ADAMTS-5 in late Golgi vesicles present in growth plate chondrocytes of defined differentiation state.

The two aggrecanases ADAMTS-4 and ADAMTS-5 have been shown to not only play roles in the breakdown of cartilage extracellular matrix in osteoarthritis, but also mediate processing of matrilins in the secretory pathway. The matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion.

Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage.

Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix.

Internalization of aggrecan G1 domain neoepitope ITEGE in chondrocytes requires CD44.

Degradation of the cartilage proteoglycan aggrecan is one of the earliest events that occurs in association with osteoarthritis. Little is known concerning the fate of the residual N-terminal G1 domains of cleaved aggrecan; domains that remain bound to hyaluronan. In this study, 68-72-kDa bands representative of aggrecan G1 domains containing ITEGE(373) neoepitope were detected within a hyaluronidase-sensitive pool at the cell surface of bovine articular chondrocytes and within a hyaluronidase-insensitive, intracellular pool.

Cytokine-induced increases in ADAMTS-4 messenger RNA expression do not lead to increased aggrecanase activity in ADAMTS-5-deficient mice.

Objective: To compare the regulation of aggrecanase messenger RNA (mRNA) and enzyme activity by proinflammatory cytokines in primary mouse chondrocytes.

Methods: Primary chondrocytes were isolated from knee epiphyses of 6-8-day-old mice and cultured as monolayers. The cells were incubated with tumor necrosis factor α (TNFα), oncostatin M (OSM), or interleukin-6 (IL-6)/soluble IL-6 receptor, and mRNA levels were measured by quantitative polymerase chain reaction at various time points.

Is cartilage matrix breakdown an appropriate therapeutic target in osteoarthritis--insights from studies of aggrecan and collagen proteolysis?

Progressive cartilage degradation is considered a hallmark of osteoarthritis (OA), and as such methods to inhibit this process have been extensively investigated as potential disease-modifying therapies. However, all tissues of the joint are affected by disease in OA, and it is likely that the pain and disability which are the major clinical symptoms of OA, arise predominantly from pathology in these extra-cartilaginous structures. It is unclear therefore, whether specifically targeting inhibition of cartilage matrix breakdown will ameliorate global joint pathology and thereby affect the clinically-relevant OA-related disability.