Proteases involved in cartilage matrix degradation in osteoarthritis

Osteoarthritis is a common joint disease for which there are currently no disease-modifying drugs available. Degradation of the cartilage extracellular matrix is a central feature of the disease and is widely thought to be mediated by proteinases that degrade structural components of the matrix, primarily aggrecan and collagen. Studies on transgenic mice have confirmed the central role of Adamalysin with Thrombospondin Motifs 5 (ADAMTS-5) in aggrecan degradation, and the collagenolytic matrix metalloproteinase MMP-13 in collagen degradation. This review discusses recent advances in current understanding of the mechanisms regulating expression of these key enzymes, as well as reviewing the roles of other proteinases in cartilage destruction. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Paraoxonase-1 does not reduce or modify oxidation of phospholipids by peroxynitrite

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated esterase/lactonase implicated to play a role in protection against atherosclerosis. However, the exact mechanism(s) and substrates for PON1 are still uncertain. In this article, we review some of the evidence for PON1's antioxidant activity, as well as our efforts to identify the actual substrates and products for this activity. We originally reported that PON1 had phospholipase activity toward oxidized phosphatidylcholine (J. Biol. Chem. 276:24473-24481; 2001). Subsequently, Marathe et al. (J. Biol. Chem. 278:3937-3947; 2003) reported that this activity was due to a contaminating lipase. However, that article did not replicate the conditions used in our previous study. To address this controversy, we purified serum PON1 by a modified method that separates the paraoxonase activity from an activity detectable as platelet-activating factor acetyl hydrolase (PAF-AH) (Teiber et al., J. Lipid. Res. 2004; Epub ahead of print, PMID 15342686) and reexamined the oxidation of phosphatidylcholine by peroxynitrite using 3-morpholinosydnonimine as a peroxynitrite generator and apolipoprotein AI-phosphatidylcholine- PON1 complexes. The phosphatidylcholines were studied by electrospray ionization tandem mass spectrometry. PON1 preparations free of PAF-AH activity showed no phospholipase activity when reconstituted into apolipoprotein AI-phosphatidylcholine complexes. We conclude that PON1 does not affect the accumulation of phosphatidylcholine oxidation products. Further, we have no evidence that PON1 has an intrinsic phospholipase A2 activity toward oxidized phospholipids.     

Creatine supplementation does not reduce muscle damage or enhance recovery from resistance exercise

Previous studies have shown that creatine supplementation reduces muscle damage and inflammation following running but not following high-force, eccentric exercise. Although the mechanical strain placed on muscle fibers during high-force, eccentric exercise may be too overwhelming for creatine to exert any protective effect, creatine supplementation may protect skeletal muscle stressed by a resistance training challenge that is more hypoxic in nature. The purpose of this study was to examine the effects of short-term creatine supplementation on markers of muscle damage (i.e., strength, range of motion, muscle soreness, muscle serum protein activity, C-reactive protein) to determine whether creatine supplementation offers protective effects on skeletal muscle following a hypoxic resistance exercise test. Twenty-two healthy, weight-trained men (19-27 years) ingested either creatine or a placebo for 10 days. Following 5 days of supplementation, subjects performed a squat exercise protocol (5 sets of 15-20 repetitions at 50% of 1 repetition maximum [1RM]). Assessments of creatine kinase (CK) and lactate dehydrogenase activity, high-sensitivity C-reactive protein, maximal strength, range of motion (ROM), and muscle soreness (SOR) with movement and palpation were conducted pre-exercise and during a 5-day follow up. Following the exercise test, maximal strength and ROM decreased, whereas SOR and CK increased. Creatine and placebo-supplemented subjects experienced significant decreases in maximal strength (creatine: 13.4 kg, placebo: 17.5 kg) and ROM (creatine: 2.4 degrees , placebo: 3.0 degrees ) immediately postexercise, with no difference between groups. Following the exercise test, there were significant increases in SOR with movement and palpation (p < 0.05 at 24, 48, and 72 hours postexercise), and CK activity (p < 0.05 at 24 and 48 hours postexercise), with no differences between groups at any time. These data suggest that oral creatine supplementation does not reduce skeletal muscle damage or enhance recovery following a hypoxic resistance exercise challenge.     

Halofuginone does not reduce fibrosis in bleomycin-induced lung injury

Halofuginone, a coccidiostatic alkaloid, has anti-fibrotic properties, and may be useful as a therapeutic agent in lung fibrosis. To test this hypothesis we investigated the effect of halofuginone on bleomycin-induced lung fibrosis in Sprague-Dawley rats. Treatment groups included: (1) a single intratracheal (IT) instillation of 1.2U bleomycin, and intraperitoneal (IP) injection of halofuginone (0.5 mg/dose), every other day; (2) IT 1.2U bleomycin and IP distilled water (D.W.), every other day; (3) IT 0.8U bleomycin and daily IP halofuginone (0.5 mg/dose); (4) IT 0.8U bleomycin and daily IP D.W.; (5) IT saline and IP halofuginone, every other day; (6) IT saline and daily IP D.W.; (7) IT 0.625U bleomycin and oral halofuginone (10 mg/kg rodent lab chow); (8) IT 0.625U bleomycin and standard lab chow. Animals were studied 14 days after IT instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage fluid, by a semi-quantitative morphological index of lung injury, and by biochemical analysis of lung hydroxyproline content. Overt signs of lung injury were apparent in bleomycin-treated rats by all measures. These changes were not affected by treatment with halofuginone, irrespective of the treatment regimen used. This study does not support the use of halofuginone to prevent or ameliorate lung fibrosis.     

Social play does not reduce aggression in wild meerkats

Of the numerous hypotheses advanced to explain the adaptive significance of play, several assert that social play increases social harmony, cementing alliances and reducing aggression between group members or littermates. These hypotheses are frequently cited, but their validity remains unknown. We examined the relation between social play and aggression in juvenile meerkats, Suricata suricatta, living in a wild population in the southern Kalahari Desert. We tested the hypothesis that social play reduces aggression, by examining rates of play, play partner choices, the structure of social play and rates of aggressive interactions during foraging. We found no relation between frequency of play and level of aggression, either between individuals or during the course of development. Pups that played together frequently were just as aggressive towards one another as pairs of pups that played infrequently, and play interactions had no subsequent effect on the likelihood of aggression. In contrast, aggressive interactions during foraging inhibited the subsequent likelihood of play, and high levels of aggression during foraging changed the structure of social play, with victimized pups avoiding play wrestling. We conclude that social play does not reduce aggression in young meerkats. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.      

Identification of catabolin,a protein from synovium which induces degradation of cartilage in organ culture

Explants of porcine synovium produce a factor which causes degradation of the matrix of live cartilage in organ culture. Cartilage degradation was measured as release of glycosaminoglycan from explants of bovine nasal septum. Fractionation of synovial culture medium showed the factor to be a protein of 20,000 mol.wt. and iso-electric point pH = 4.6. The factor has been named catabolin.     

Physiologically regulated transgenic ABCA1 does not reduce amyloid burden or amyloid-beta peptide levels in vivo

ABCA1-deficient mice have low levels of poorly lipidated apolipoprotein E (apoE) and exhibit increased amyloid load. To test whether excess ABCA1 protects from amyloid deposition, we crossed APP/PS1 mice to ABCA1 bacterial artificial chromosome (BAC) transgenic mice. Compared with wild-type animals, the ABCA1 BAC led to a 50% increase in cortical ABCA1 protein and a 15% increase in apoE abundance, demonstrating that this BAC supports modest ABCA1 overexpression in brain. However, this was observed only in animals that do not deposit amyloid. Comparison of ABCA1/APP/PS1 mice with APP/PS1 controls revealed no differences in levels of brain ABCA1 protein, amyloid, Abeta, or apoE, despite clear retention of ABCA1 overexpression in the livers of these animals. To further investigate ABCA1 expression in the amyloid-containing brain, we then compared ABCA1 mRNA and protein levels in young and aged cortex and cerebellum of APP/PS1 and ABCA1/APP/PS1 animals. Compared with APP/PS1 controls, aged ABCA1/APP/PS1 mice exhibited increased ABCA1 mRNA, but not protein, selectively in cortex. Additionally, ABCA1 mRNA levels were not increased before amyloid deposition but were induced only in the presence of extensive Abeta and amyloid levels. These data suggest that an induction of ABCA1 expression may be associated with late-stage Alzheimer's neuropathology.     

Chronic rapamycin treatment or lack of S6K1 does not reduce ribosome activity in vivo

Reducing activity of the mTORC1/S6K1 pathway has been shown to extend lifespan in both vertebrate and invertebrate models. For instance, both pharmacological inhibition of mTORC1 with the drug rapamycin or S6K1 knockout extends lifespan in mice. Since studies with invertebrate models suggest that reducing translational activity can increase lifespan, we reasoned that the benefits of decreased mTORC1 or S6K1 activity might be due, at least in part, to a reduction of general translational activity. Here, we report that mice given a single dose of rapamycin have reduced translational activity, while mice receiving multiple injections of rapamycin over 4 weeks show no difference in translational activity compared with vehicle-injected controls. Furthermore, mice lacking S6K1 have no difference in global translational activity compared with wild-type littermates as measured by the percentage of ribosomes that are active in multiple tissues. Translational activity is reduced in S6K1-knockout mice following single injection of rapamycin, demonstrating that rapamycin’s effects on translation can occur independently of S6K1. Taken together, these data suggest that benefits of chronic rapamycin treatment or lack of S6K1 are dissociable from potential benefits of reduced translational activity, instead pointing to a model whereby changes in translation of specific subsets of mRNAs and/or translation-independent effects of reduced mTOR signaling underlie the longevity benefits.     

Subchondral bone and ligament changes precede cartilage degradation in guinea pig osteoarthritis

There is increasing recognition that osteoarthritis (OA) is a complex disease involving the whole synovial joint, rather than the articular cartilage alone, however its aetiology and pathogenesis is not understood. Our initial studies revealed elevated turnover of bone and ligament collagen in human and mouse OA, respectively. To investigate the relative appearance of pathology in cartilage, bone and ligament, we studied the progression of spontaneous OA in the Dunkin-Hartley (DH) guinea pig knee, and compared with age-matched control Bristol Strain 2 (BS2) knees. The classical radiographic OA score of the DH knees compared to BS2 knees was 2-fold higher at 24 weeks of age. The patella perimeter and subchondral bone density was significantly greater in the DHs at 24 and 36 weeks compared to BS2. The femoral intercondylar notch width was found to be significantly lower in the DHs at 24 and 36 weeks, compared to BS2, indicating bone remodelling at the cruciate ligament (CL) insertion site. We found significantly greater laxity of the DH anterior CL at 12, 16 and 20 weeks compared to BS2. This elevated laxity was associated with increased remodelling of the CLs, based on markers of collagen turnover, and occurred prior to bone and cartilage pathology. We propose that the laxity of the CL leads to remodelling of the subchondral bone, and intercondylar notch, due to a change in load through the joint. Remodelling of the CLs and bone occurs prior to and concomitant with histopathological changes in the articular cartilage respectively, demonstrating the fundamental role of the ligament and subchondral bone in the aetiology of knee OA.     

Production of the chemokine eotaxin-1 in osteoarthritis and its role in cartilage degradation

The expression of the chemokine, eotaxin-1, and its receptors in normal and osteoarthritic human chondrocytes was examined, and its role in cartilage degradation was elucidated in this study. Results indicated that plasma concentrations of eotaxin-1 as well as the chemokines, RANTES, and MCP-1alpha, were higher in patients with osteoarthritis (OA) than those in normal humans. Stimulation of chondrocytes with IL-1beta or TNF-alpha significantly induced eotaxin-1 expression. The production of eotaxin-1 induced expression of its own receptor of CCR3 and CCR5 on the cell surface of chondrosarcomas, suggesting that an autocrine/paracrine pathway is involved in eotaxin-1's action. In addition, eotaxin-1 markedly increased the expressions of MMP-3 and MMP-13 mRNA, but had no effect on TIMP-1 expression in chondrocytes. However, pretreatment of anti-eotaxin-1 antibody significantly decreased the MMP-3 expression induced by IL-1beta. These results first demonstrate that human chondrocytes express the chemokine, eotaxin-1, and that its expression is induced by treatment with IL-1beta and TNF-alpha. The cytokine-triggered induction of eotaxin-1 further results in enhanced expressions of its own receptor of CCR3, CCR5, and MMPs, suggesting that eotaxin-1 plays an important role in cartilage degradation in OA.     

Overexpression of apolipoprotein O does not impact on plasma HDL levels or functionality in human apolipoprotein A-I transgenic mice

Apolipoprotein (apo) O is a newly discovered apolipoprotein preferentially contained within HDL; however, currently, no data are available on the (patho)physiological effects of apoO. Therefore, the present study assessed the impact of apoO overexpression on (i) plasma lipids and lipoproteins as well as on (ii) HDL functionality. Human apoO was overexpressed by means of recombinant adenovirus (AdhapoO) in human apoA-I transgenic mice, a humanized mouse model of HDL metabolism. AdhapoO substantially increased apoO in plasma and within HDL. However, plasma triglycerides, phospholipids, total cholesterol and HDL cholesterol did not change. HDL size distribution, lipid composition and the apoA-I and the apoO distribution over the different HDL fractions separated by FPLC remained unaltered. Furthermore, enrichment of HDL with apoO did not impact on HDL functionality assessed in four independent ways, namely (i) stimulation of cholesterol efflux from macrophage foam cells, (ii) protection against LDL oxidation, (iii) anti-inflammatory activity on endothelial cells, and (iv) induction of vasodilation in isolated aortic rings ex vivo as a measure of stimulating vascular NO production. These results demonstrate that although overexpression of apoO results in a substantial enrichment of HDL particles with this novel apolipoprotein, apoO does not impact the plasma lipoprotein profile or HDL functionality.     

The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class II (MHC II) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC II chaperone Ii, and hence in the formation of MHC II-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, Ii processing and MHC II peptide loading proceeded similarly in all three DC populations. We then analyzed MHC II localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.     

Plasma storage at -80 degrees C does not protect matrix metalloproteinase-9 from degradation

Recently, matrix metalloproteinase-9 (MMP-9) has been identified as a cardiovascular risk marker and is increasingly being determined in clinical studies. Among other matrix metalloproteinases, MMP-9 is known to be self-activable, as the cleavage of the propeptide leads to the formation of an active enzyme. In such a case the issue of storage of biological samples such as plasmas is of outstanding importance, as an enzymatic activity, although minimal, may remain at common storage temperature, i.e., -80 degrees C. Since 2000 our institute has created a plasma library from patients presenting with acute myocardial infarction. Recently, the evaluation of the MMP-9 led to the surprise of finding a dramatically low level of detectable enzyme in the oldest plasma samples. By using zymography, enzyme-linked immunosorbent assay and Western blots, we evaluated new and old samples and found that MMP-9 degrades over time. After 2 years, the detectable total MMP-9 dropped by 65%, and the asymptotic profile of the curve reached a residual 1% level after 43 months. These results were confirmed by zymography and western blotting. TIMP-1, the natural inhibitor of MMP-9 and MMP-2, remained rather stable over time. The results suggest that human plasma MMP-9 levels should be determined as soon as possible after sampling.