Residual Aligner-based Network (RAN): Motion-separable structure for coarse-to-fine discontinuous deformable registration.

Deformable image registration, the estimation of the spatial transformation between different images, is an important task in medical imaging. Deep learning techniques have been shown to perform 3D image registration efficiently. However, current registration strategies often only focus on the deformation smoothness, which leads to the ignorance of complicated motion patterns (e.g., separate or sliding motions), especially for the intersection of organs. Thus, the performance when dealing with the discontinuous motions of multiple nearby objects is limited, causing undesired predictive outcomes in clinical usage, such as misidentification and mislocalization of lesions or other abnormalities. Consequently, we proposed a novel registration method to address this issue: a new Motion Separable backbone is exploited to capture the separate motion, with a theoretical analysis of the upper bound of the motions' discontinuity provided. In addition, a novel Residual Aligner module was used to disentangle and refine the predicted motions across the multiple neighboring objects/organs. We evaluate our method, Residual Aligner-based Network (RAN), on abdominal Computed Tomography (CT) scans and it has shown to achieve one of the most accurate unsupervised inter-subject registration for the 9 organs, with the highest-ranked registration of the veins (Dice Similarity Coefficient (%)/Average surface distance (mm): 62%/4.9mm for the vena cava and 34%/7.9mm for the portal and splenic vein), with a smaller model structure and less computation compared to state-of-the-art methods. Furthermore, when applied to lung CT, the RAN achieves comparable results to the best-ranked networks (94%/3.0mm), also with fewer parameters and less computation.

Accurate volume alignment of arbitrarily oriented tibiae based on a mutual attention network for osteoarthritis analysis.

Damage to cartilage is an important indicator of osteoarthritis progression, but manual extraction of cartilage morphology is time-consuming and prone to error. To address this, we hypothesize that automatic labeling of cartilage can be achieved through the comparison of contrasted and non-contrasted Computer Tomography (CT). However, this is non-trivial as the pre-clinical volumes are at arbitrary starting poses due to the lack of standardized acquisition protocols. Thus, we propose an annotation-free deep learning method, D-net, for accurate and automatic alignment of pre- and post-contrasted cartilage CT volumes. D-Net is based on a novel mutual attention network structure to capture large-range translation and full-range rotation without the need for a prior pose template. CT volumes of mice tibiae are used for validation, with synthetic transformation for training and tested with real pre- and post-contrasted CT volumes. Analysis of Variance (ANOVA) was used to compare the different network structures. Our proposed method, D-net, achieves a Dice coefficient of 0.87, and significantly outperforms other state-of-the-art deep learning models, in the real-world alignment of 50 pairs of pre- and post-contrasted CT volumes when cascaded as a multi-stage network.

In vivo optical imaging of early osteoarthritis using an antibody specific to damaged arthritic cartilage.

BACKGROUND: The lack of specific and sensitive serum and radiographic biomarkers for early diagnosis of osteoarthritis (OA) as well as for monitoring subtle changes in disease activity in clinical trials has hampered the development of treatments for OA. We previously showed that 1-11E, a human single chain fragment variable (scFv) specific to collagen type II that has been post-translationally modified by reactive oxidants (ROS-CII), binds exclusively to arthritic cartilage. Here we test the validity of 1-11E as a radiographic biomarker for early disease in experimental OA. METHODS: Murine OA was induced by destabilisation of the medial meniscus (DMM) in adult male mice. Immunohistochemistry of destabilised or sham-operated knees was performed from 2 to 8 weeks post-surgery with Cy5.5-labelled 1-11E and negative control scFv, C7. Prospective in vivo optical images were taken 4 and 8 weeks post-DMM following intra-articular injection of Cy5.5-labelled scFvs, or intravenous injection of Cy5.5-labelled full length monoclonal antibodies (mAbs). RESULTS: Specific cartilage staining with 1-11E was apparent as early as 4 weeks post-DMM at the time of earlier cartilage degradation assessed by histology. Prospective in vivo optical images taken 4 and 8 weeks post-DMM following local intra-articular injection of Cy5.5-labelled scFv (n = 7) showed specific in vivo retention of Cys5.5-1-11E scFv following local administration into the knee joint (tissue half-life >78 hours, n = 7, signal to noise ratio (SNR) > 2.1). Specific localization of Cys-5.5-1-11E-mAb to DMM knees (SNR >1.65) was also observed (p < 0.01, n = 8, SNR >1.65). In both cases the SNR increased with time post-DMM. CONCLUSIONS: 1-11E binds specifically to early osteoarthritic cartilage and can be used as a radiographic biomarker following local or systemic delivery to facilitate early diagnosis and monitor disease progression in OA.

DOTAM derivatives as active cartilage-targeting drug carriers for the treatment of osteoarthritis.

Targeted drug-delivery methods are crucial for effective treatment of degenerative joint diseases such as osteoarthritis (OA). Toward this goal, we developed a small multivalent structure as a model drug for the attenuation of cartilage degradation. The DOTAM (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid amide)-based model structure is equipped with the cathepsin D protease inhibitor pepstatin A, a fluorophore, and peptide moieties targeting collagen II. In vivo injection of these soluble probes into the knee joints of mice resulted in 7-day-long local retention, while the drug carrier equipped with a scrambled peptide sequence was washed away within 6-8 h. The model drug conjugate successfully reduced the cathepsin D protease activity as measured by release of GAG peptide. Therefore, these conjugates represent a promising first drug conjugate for the targeted treatment of degenerative joint diseases.

In vivo visualization of osteoarthritic hypertrophic lesions.

Osteoarthritis (OA) is one of the most common diseases in the aging population. While disease progress in humans is monitored indirectly by X-ray or MRI, small animal OA lesions detection always requires surgical intervention and histology. Here we introduce bimodal MR/NIR probes based on cartilage-targeting 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid amide (DOTAM) that are directly administered to the joint cavity. We demonstrate applications in healthy and diseased rat joints by MRI in vivo. The same joints are inspected post-mortem by fluorescence microscopy, showing not only the precise location of the reagents but also revealing details such as focal cartilage damage and chondrophyte or osteophyte formation. This allows for determining the distinct pathological state of the disease and the regeneration capability of the animal model and will help to correctly assess the effect of potential disease modifying OA drugs (DMOADs) in the future.

Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency.

INTRODUCTION: We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis. METHODS: Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis. RESULTS: 1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10). CONCLUSIONS: Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.

In vivo imaging of matrix metalloproteinase 12 and matrix metalloproteinase 13 activities in the mouse model of collagen-induced arthritis.

OBJECTIVE: To develop enzyme-activatable Förster resonance energy transfer (FRET) substrate probes to detect matrix metalloproteinase 12 (MMP-12) and MMP-13 activities in vivo in mouse models of inflammatory arthritis. METHODS: Peptidic FRET probes activated by MMP-12 and MMP-13 were reverse designed from inhibitors selected from a phosphinic peptide inhibitor library. Selectivity of the probes was demonstrated in vitro using MMP-1, MMP-2, MMP-3, MMP-12, and MMP-13. In vivo activation of the probes was tested in the zymosan-induced mouse model of inflammation, and probe specificity was evaluated by the MMP inhibitor GM6001 and specific synthetic inhibitors of MMP-12 and MMP-13. The probes were used to monitor these enzyme activities in the collagen-induced arthritis (CIA) model in vivo. RESULTS: The MMP-12 and MMP-13 activity probes (MMP12ap and MMP13ap, respectively) discriminated between the activities of the 2 enzymes. The in vivo activation of these probes was inhibited by GM6001 and by their respective specific inhibitors. In the CIA model, MMP12ap activation peaked 5 days after disease onset and showed strong correlation with disease severity during this time (r = 0.85, P < 0.0001). MMP13ap activation increased gradually after disease onset and correlated with disease severity over a longer period of 15 days (r = 0.58, P < 0.0001). CONCLUSION: We generated two selective FRET probes that can be used to monitor MMP-12 and MMP-13 activities in live animals. MMP12ap follows the initial stage of inflammation in CIA, while MMP13ap follows the progression of the disease. The specificity of these probes is useful in monitoring the efficacy of MMP inhibitors.

Generation of a mouse line harboring a Bi-transgene expressing luciferase and tamoxifen-activatable creER(T2) recombinase in cartilage.

We have used an aggrecan gene enhancer to generate a transgenic murine line (Acan-CreER-Ires-Luc) expressing firefly luciferase and tamoxifen activatable Cre recombinase (Cre-ER(T2) ). The expression and efficiency of the inducible Cre recombinase activity were tested in double transgenic mice created by crossing the Acan-CreER-Ires-Luc line with a Rosa26-lacZ reporter mouse. The expression pattern of the transgene of our line was restricted to cartilage from embryonic to adult stages. ß-galactosidase staining was observed in growth plate, articular cartilage, as well as fibrocartilage of meniscus, trachea, and intervertebral discs. Similar staining was observed in a previously described Agc1 (tm(IRES-creERT2)) murine line. The presence of luciferase in our transgene allows the visualization of the transgene expression in live animals. Weekly measurements from 2 to 8 weeks of age showed a reduction in luminescence in knee joints between 2 and 4 weeks of age, but stabilization thereafter. Following the surgical induction of osteoarthritis at 12 weeks of age, the level of luminescence remained the same in the knee joints for 8 weeks. This Acan-CreER-Ires-Luc murine line allows indirect monitoring of the transcriptional activity of the Acan gene via expression of luciferase, while the inducible Cre recombinase activity facilitates studies involving gain or loss of gene expression in cartilage.

In vivo fluorescence imaging of E-selectin: quantitative detection of endothelial activation in a mouse model of arthritis.

OBJECTIVE: In vivo optical imaging can delineate at the macroscopic level processes that are occurring at the cellular and molecular levels. E-selectin, a leukocyte adhesion molecule expressed on endothelium, is induced by tumor necrosis factor α (TNFα) and other cytokines involved in the pathogenesis of rheumatoid arthritis (RA). Collagen-induced arthritis (CIA) in mice is widely used to study the disease mechanisms and identify new treatments for RA. The purpose of this study was to demonstrate E-selectin-targeted fluorescence imaging in vivo in a mouse model of paw edema generated by local injection of TNFα as well as in mice with CIA. METHODS: Animals with either CIA or TNFα-induced paw edema were injected with anti-E-selectin or control antibodies labeled with a DyLight 750-nm near-infrared (NIR) probe. In vivo imaging studies were undertaken using an NIR optical imaging system, and images were coregistered with plain radiographic images. RESULTS: The mean fluorescence intensity measured over the time-course of TNFα-induced edema demonstrated a 1.97-fold increase (P<0.001) in signal in inflamed paws at 8 hours following injection of anti-E-selectin antibody, as compared to that in the isotype control. In the CIA model, a 2.34-fold increase in E-selectin-targeted signal was demonstrated (P<0.01). Furthermore, significant E-selectin-targeted signal was observed in the paws of animals immunized with collagen that did not display overt signs of arthritis. CONCLUSION: E-selectin-targeted fluorescence in vivo imaging is a quantifiable method of detecting endothelial activation in arthritis and can potentially be applied to the quantification of disease and the investigation of the effects of new therapies. Importantly, this approach may also be useful for the detection of subclinical disease in RA.

Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation.

Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of 'Reverse Design' represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation. AW-091 showed higher signal-to-background ratios at earlier time points than the commercially available polymer-based ProSense680 (VisEn Medical) and thus represents an efficient new tool for studying early proteolytic processes leading to various diseases, including inflammation, cancer, and rheumatoid arthritis. In addition, the fluorescent signal originating from the cleaved AW-091 was shown to be reduced by the administration of an anti-inflammatory drug, dexamethasone and by the cathepsin inhibitor E-64, providing a valuable system for the evaluation of small-molecule inhibitors of cathepsins.

Role of the netrin-like domain of procollagen C-proteinase enhancer-1 in the control of metalloproteinase activity.

The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.

In vivo optical imaging in arthritis--an enlightening future?

In vivo molecular optical imaging has significant potential to delineate and measure, at the macroscopic level, in vivo biological processes that are occurring at the cellular and molecular level. Optical imaging has already been developed for in vitro and ex vivo applications in molecular and cellular biology (e.g. fluorescence confocal microscopy), but is still at an early stage of development as a whole-animal in vivo imaging technique. Both sensitivity and spatial resolution remain incompletely defined. Rapid advances in hardware technology and highly innovative reporter probes and dyes will be expected to deliver significant insight into perturbations of molecular pathways that occur in disease, ultimately with the potential of translating into future molecular imaging techniques for patients with arthritis. This review will focus on currently available technologies for live in vivo animal optical imaging, including fluorescence reflectance imaging, potential novel tomographic techniques, bioluminescence reporter technology and potential novel labelling techniques, highlighting in particular the potential application of in vivo fluorescence imaging in arthritis.

A new role for TIMP-1 in modulating neurite outgrowth and morphology of cortical neurons.

BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) displays pleiotropic activities, both dependent and independent of its inhibitory activity on matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMP-1 is strongly upregulated in reactive astrocytes and cortical neurons following excitotoxic/inflammatory stimuli, but no information exists on its effects on growth and morphology of cortical neurons. PRINCIPAL FINDINGS: We found that 24 h incubation with recombinant TIMP-1 induced a 35% reduction in neurite length and significantly increased growth cones size and the number of F-actin rich microprocesses. TIMP-1 mediated reduction in neurite length affected both dendrites and axons after 48 h treatment. The effects on neurite length and morphology were not elicited by a mutated form of TIMP-1 inactive against MMP-1, -2 and -3, and still inhibitory for MMP-9, but were mimicked by a broad spectrum MMP inhibitor. MMP-9 was poorly expressed in developing cortical neurons, unlike MMP-2 which was present in growth cones and whose selective inhibition caused neurite length reductions similar to those induced by TIMP-1. Moreover, TIMP-1 mediated changes in cytoskeleton reorganisation were not accompanied by modifications in the expression levels of actin, betaIII-tubulin, or microtubule assembly regulatory protein MAP2c. Transfection-mediated overexpression of TIMP-1 dramatically reduced neuritic arbour extension in the absence of detectable levels of released extracellular TIMP-1. CONCLUSIONS: Altogether, TIMP-1 emerges as a modulator of neuronal outgrowth and morphology in a paracrine and autrocrine manner through the inhibition, at least in part, of MMP-2 and not MMP-9. These findings may help us understand the role of the MMP/TIMP system in post-lesion pre-scarring conditions.

N-O-isopropyl sulfonamido-based hydroxamates: design, synthesis and biological evaluation of selective matrix metalloproteinase-13 inhibitors as potential therapeutic agents for osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a key enzyme implicated in the degradation of the extracellular matrix in osteoarthritis (OA). For this reason, MMP-13 synthetic inhibitors are being sought as potential therapeutic agents to prevent cartilage degradation and to halt the progression of OA. Herein, we report the synthesis and in vitro evaluation of a new series of selective MMP-13 inhibitors possessing an arylsulfonamidic scaffold. Among these potential inhibitors, a very promising compound was discovered exhibiting nanomolar activity for MMP-13 and was highly selective for this enzyme compared to MMP-1, -14, and TACE. This compound acted as a slow-binding inhibitor of MMP-13 and was demonstrated to be effective in an in vitro collagen assay and in a model of cartilage degradation. Furthermore, a docking study was conducted for this compound in order to investigate its binding interactions with MMP-13 and the reasons for its selectivity toward MMP-13 versus other MMPs.

Synovial fluid is a site of citrullination of autoantigens in inflammatory arthritis.

OBJECTIVE: To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated alpha-enolase, in rheumatoid arthritis (RA). METHODS: Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, alpha-enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD-2) and PAD-4. Proteins from an RA synovial fluid sample were separated by 2-dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated alpha-enolase peptide 1 (CEP-1) and cyclic citrullinated peptide 2 were measured by enzyme-linked immunosorbent assay. RESULTS: Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha-enolase was detected in all of the samples, with mean levels of 6.4 ng/microl in RA samples, 4.3 ng/microl in SpA samples, and <0.9 ng/microl in OA samples. Two-dimensional electrophoresis provided evidence that the alpha-enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD-4 was detected in samples from all 3 disease groups. PAD-2 was detected in 18 of the RA samples, in 16 of the SpA samples, and in none of the OA samples. Antibodies to CEP-1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in 1 OA sample. CONCLUSION: These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.

Functional differences of the catalytic and non-catalytic domains in human ADAMTS-4 and ADAMTS-5 in aggrecanolytic activity.

ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) are multidomain metalloproteinases belonging to the ADAMTS family. We have previously reported that human ADAMTS-5 has much higher aggrecanolytic activity than human ADAMTS-4. To investigate the different proteolytic activity of the two enzymes, we generated a series of chimeras by exchanging various non-catalytic domains of the two proteinases. We found that the catalytic domain of ADAMTS-5 has higher intrinsic catalytic ability than that of ADAMTS-4. The studies also demonstrated that the non-catalytic domains of ADAMTS-5 are more effective modifiers than those of ADAMTS-4, making both catalytic domains more active against aggrecan, an Escherichia coli-expressed interglobular domain of aggrecan and fibromodulin. Addition of the C-terminal thrombospondin type I motif of ADAMTS-5 to the C terminus of ADAMTS-4 increased the activity of ADAMTS-4 against aggrecan and fibromodulin severalfold. In contrast to previous reports (Kashiwagi, M., Enghild, J. J., Gendron, C., Hughes, C., Caterson, B., Itoh, Y., and Nagase, H. (2004) J. Biol. Chem. 279, 10109-10119 and Gao, G., Plaas, A., Thompson, V. P., Jin, S., Zuo, F., and Sandy, J. D. (2004) J. Biol. Chem. 279, 10042-10051), our detailed investigation of the role of the C-terminal spacer domain of ADAMTS-4 indicated that full-length ADAMTS-4 is approximately 20-times more active against aggrecan than its spacer domain deletion mutant, even at the Glu373-Ala374 site of the interglobular domain. This discrepancy is most likely due to selective inhibition of full-length ADAMTS-4 by heparin, particularly for cleavage at the Glu373-Ala374 bond. However, removal of the spacer domain from ADAMTS-4 greatly enhanced more general proteolytic activity against non-aggrecan substrates, e.g. E. coli-expressed interglobular domain, fibromodulin, and carboxymethylated transferrin.

Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4.

Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0-9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.

X-ray structure of human proMMP-1: new insights into procollagenase activation and collagen binding.

Vertebrate collagenases, members of the matrix metalloproteinase (MMP) family, initiate interstitial fibrillar collagen breakdown. It is essential in many biological processes, and unbalanced collagenolysis is associated with diseases such as arthritis, cancer, atherosclerosis, aneurysm, and fibrosis. These metalloproteinases are secreted from the cell as inactive precursors, procollagenases (proMMPs). To gain insights into the structural basis of their activation mechanisms and collagen binding, we have crystallized recombinant human proMMP-1 and determined its structure to 2.2 A resolution. The catalytic metalloproteinase domain and the C-terminal hemopexin (Hpx) domain show the classical MMP-fold, but the structure has revealed new features in surface loops and domain interaction. The prodomain is formed by a three-helix bundle and gives insight into the stepwise activation mechanism of proMMP-1. The prodomain interacts with the Hpx domain, which affects the position of the Hpx domain relative to the catalytic domain. This interaction results in a "closed" configuration of proMMP-1 in contrast to the "open" configuration observed previously for the structure of active MMP-1. This is the first evidence of mobility of the Hpx domain in relation to the catalytic domain, providing an important clue toward the understanding of the collagenase-collagen interaction and subsequent collagenolysis.