Rapid-onset clinical and mechanistic effects of anti-C5aR treatment in the mouse collagen-induced arthritis model.

Preclinical evidence supports targeting the C5a receptor (C5aR) in rheumatoid arthritis (RA). To support ongoing clinical development of an anti-C5aR monoclonal antibody, we have investigated for the first time the mechanism of action and the pharmacodynamics of a blocking anti-murine C5aR (anti-mC5aR) surrogate antibody in mouse collagen-induced arthritis (CIA). First, efficacy was demonstrated in a multiple-dose treatment study. Almost complete inhibition of clinical disease progression was obtained, including reduced bone and cartilage destruction in anti-mC5aR-treated mice. Then, the mechanism of action was examined by looking for early effects of anti-mC5aR treatment in single-dose treatment studies. We found that 48 h after single-dose treatment with anti-mC5aR, the neutrophil and macrophage infiltration into the paws was already reduced. In addition, several inflammatory markers, including tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-17A were reduced locally in the paws, indicating reduction of local inflammation. Furthermore, dose-setting experiments supported a beneficial clinical effect of dosing above the C5aR saturation level. In conclusion, these preclinical data demonstrated rapid onset effects of antibody blockade of C5aR. The data have translational value in supporting the Novo Nordisk clinical trials of an anti-C5aR antibody in rheumatoid arthritis patients, by identifying potential biomarkers of treatment effects as well as by providing information on pharmacodynamics and novel insights into the mechanism of action of monoclonal antibody blockade of C5aR.

Mice over-expressing salmon calcitonin have strongly attenuated osteoarthritic histopathological changes after destabilization of the medial meniscus.

OBJECTIVE: Calcitonin is well-known for its inhibitory actions on bone-resorbing osteoclasts and recently potential beneficial effects on cartilage were shown. We investigated effects of salmon calcitonin (sCT) on the articular cartilage and bone, after destabilization of the medial meniscus (DMM) in normal and sCT over-expressing mice. DESIGN: Bone phenotype of transgenic (TG) C57Bl/6 mice over-expressing sCT at 6 months and 12 months was investigated by (1) serum osteocalcin and urinary deoxypyridinoline and (2) dynamic and normal histomorphometry of vertebrae bodies. In subsequent evaluation of cartilage and subchondral bone changes, 44 10-week old TG or wild-type (WT) mice were randomized into four groups and subjected to DMM or sham-operations. After 7 weeks animals were sacrificed, and knee joints were isolated for histological analysis. RESULTS: Trabecular bone volume (BV/TV) increased 150% after 6 months and 300% after 12 months in sCT-expressing mice when compared to WT controls (P<0.05). Osteoblast number, bone formation rate and osteocalcin measurements were not affected in TG mice over-expressing sCT. In WT animals, a 5-fold increase in the quantitative erosion index was observed after DMM, and the semi-quantitative OARSI score showed over 400% (P<0.001) increase, compared to sham-operated WT mice. DMM-operated TG mice were protected against cartilage erosion and showed a 65% and 64% (P<0.001) reduction, respectively, for the two histopathological evaluation methods. CONCLUSIONS: sCT over-expressing mice had higher bone volume, and were protected against cartilage erosion. These data suggest that increased levels of sCT may hamper the pathogenesis of osteoarthritis (OA). However more studies are necessary to confirm these preliminary results.

MAPKs are essential upstream signaling pathways in proteolytic cartilage degradation--divergence in pathways leading to aggrecanase and MMP-mediated articular cartilage degradation.

OBJECTIVES: Matrix metalloproteinases (MMPs) and aggrecanases are essential players in cartilage degradation. However, the signaling pathways that results in MMP and/or aggrecanase synthesis and activation are not well understood. We investigated the molecular events leading to MMP- and aggrecanase-mediated cartilage degradation. METHODS: Cartilage degradation was induced in bovine articular cartilage explants by oncostatin M (OSM) and tumor necrosis factor (TNF), in the presence or absence of specific inhibitors of the mitogen-activated protein kinases (MAPKs) P38, P44/42 and Src family. Toxicity was followed by the AlamarBlue colorimetric assay. MMP-activity was assessed using a fluorescent substrate assay and MMP-9 and -2 activities by gelatinase zymography. MMP-mediated collagen type II degradation and MMP as well as aggrecanase-mediated aggrecan degradation was investigated with specific ELISA and hydroxyproline release by standard methods. The findings were verified by immunohistochemistry and histology. RESULTS: Stimulation of cartilage degradation by OSM+TNF resulted in 100-fold induction of CTX-II release (P<0.01). This was dose-dependently inhibited by MAPK P38 inhibitors and by the MAPK P44/42 inhibitors. MMP-activity and expression was significantly decreased, as evaluated by cleavage of fluorescence MMP-substrate and zymography. Immunohistochemistry confirmed these findings. Interestingly, only the P44/42 inhibitors abrogated aggrecanase-mediated aggrecan degradation. CONCLUSION: We found that inhibition of MAPK P38, P44/42 and Src family abrogated proteolytic cartilage degradation by blocking MMP synthesis and activity. However, only MAPK P44/42 was essential for aggrecanase-mediated aggrecan degradation. These data suggest that various aspects of cartilage degradation can be targeted independently by inhibiting specific upstream signaling pathway.

Cartilage formation measured by a novel PIINP assay suggests that IGF-I does not stimulate but maintains cartilage formation ex vivo.

OBJECTIVES: The aim of this study was to investigate the time-dependent effect of insulin-like growth factor-I (IGF)-I on cartilage, evaluated by a novel procollagen type II N-terminal propeptide (PIINP) formation assay. This was performed in a cartilage model. METHODS: Bovine articular cartilage explants were cultured in Dulbecco's modified Eagle's medium (DMEM):F12 in the presence of 0, 0.01, 0.1, 1, 10, or 100 ng/mL of IGF-I. The viability of the chondrocytes was measured by the colorimetric Alamar blue assay. Collagen formation was assessed from the conditioned medium by the PIINP assay. Proteoglycan levels retained in the explants after 22 days of culture were extracted and measured by the sulfated glycosaminoglycan (sGAG) assay. RESULTS: In the absence of stimulation, PIINP markedly decreased as a function of time (99.4%, p < 0.001). IGF-I dose-dependently stimulated collagen formation and more than 3000% (p < 0.0005) at 100 ng/mL IGF-I at day 20 compared to vehicle control (W/O). IGF-I maintained PIINP at levels comparable to that of day 1. IGF-I dose-dependently protected against proteoglycan loss. CONCLUSION: IGF-I dose-dependently maintained cartilage formation. The current developed techniques aid the model to represent a more physiologically relevant model to test novel anabolic drugs for osteoarthritis (OA).

In vitro, ex vivo, and in vivo methodological approaches for studying therapeutic targets of osteoporosis and degenerative joint diseases: how biomarkers can assist?

Although our approach to the clinical management of osteoporosis (OP) and degenerative joint diseases (DJD)-major causes of disability and morbidity in the elderly-has greatly advanced in the past decades, curative treatments that could bring ultimate solutions have yet to be found or developed. Effective and timely development of candidate drugs is a critical function of the availability of sensitive and accurate methodological arsenal enabling the recognition and quantification of pharmacodynamic effects. The established concept that both OP and DJD arise from an imbalance in processes of tissue formation and degradation draws attention to need of establishing in vitro, ex vivo, and in vivo experimental settings, which allow obtaining insights into the mechanisms driving increased bone and cartilage degradation at cellular, organ, and organism levels. When addressing changes in bone or cartilage turnover at the organ or organism level, monitoring tools adequately reflecting the outcome of tissue homeostasis become particularly critical. In this context, bioassays targeting the quantification of various degradation and formation products of bone and cartilage matrix elements represent a useful approach. In this review, a comprehensive overview of widely used and recently established in vitro, ex vivo, and in vivo set-ups is provided, which in many cases effectively take advantage of the potentials of biomarkers. In addition to describing and discussing the advantages and limitations of each assay and their methods of evaluation, we added experimental and clinical data illustrating the utility of biomarkers for these methodological approaches.

An update review of cellular mechanisms conferring the indirect and direct effects of estrogen on articular cartilage.

OBJECTIVE: To review cellular mechanisms that have been proposed to mediate the indirect and direct effects of estrogen on articular cartilage, and to outline the remaining clinical questions that need to be clarified before utilizing the beneficial effects of estrogen for the prevention of osteoarthritis in early postmenopausal women. DESIGN: Summary of original research papers and reviews listed in Pubmed (1980-2007). RESULTS: Estrogen receptors have been identified in articular chondrocytes from various animals and humans. Molecular studies showed that estrogen can elicit genomic and rapid non-genomic effects on various cell types, including chondrocytes, and the latter effects are only inducible in females. In addition to direct effects, estrogen can also affect the homeostasis of articular cartilage by modulating the expression/production of different molecules such as various growth factors, inflammatory cytokines, matrix metalloproteinases, and reactive oxygen species. Moreover, in vivo observation argues for the notion that inhibition of subchondral bone turnover is also part of the mechanisms by which estrogen (and antiresorptive agents in general) can protect against joint degradation. Published studies undertaken at cellular, tissue, and in vivo levels illustrate that the effect of estrogen on cartilage may depend on the dose applied, the administration route, the time of initiation, and whether it is combined with a progestin. CONCLUSIONS: The herein reviewed direct and indirect effects of estrogen on articular cartilage further corroborate the due consideration of estrogen therapy for maintaining not only bone but also cartilage health in postmenopausal women. Future studies in postmenopausal women are needed to clarify whether the efficacy of estrogen therapy can be further optimized by using other forms of estrogen, other progestins, or by initiating the therapy in the peri- or early postmenopausal period.

MMP and non-MMP-mediated release of aggrecan and its fragments from articular cartilage: a comparative study of three different aggrecan and glycosaminoglycan assays.

OBJECTIVE: Aggrecan is the major proteoglycan in articular cartilage and is known to be degraded by various proteases, including matrix metalloproteinases (MMPs). The present study was undertaken to develop immunoassays detecting aggrecan and its fragments generated by MMP and non-MMP-mediated proteolysis. METHODS: Two immunoassays were developed: (1) the G1/G2 sandwich assay employing a monoclonal antibody (F-78) both as a capturing and a detecting antibody, and (2) the 342-G2 sandwich assay substituting the capturing antibody in the G1/G2 test with a monoclonal antibody, AF-28 recognizing the 342FFGVG neo-epitope generated by MMP cleavage. These assays were compared to the commercially available glycosaminoglycan (GAG) assay. RESULTS: In supernatants of Oncostatin M and Tumor Necrosis Factor alpha (OSM/TNFalpha) stimulated explants, high levels of G1/G2 fragments and GAGs were released in the initial phase (days 2-5), followed by low levels in the intermediate (days 9-12) and late phase (days 12-21). MMP-generated fragments were detected in the late phase only. In the presence of the general MMP inhibitor GM6001, 342-G2 was not detected, whereas the G1/G2 profile remained virtually unchanged. In patients with rheumatoid arthritis (RA), the release of G1/G2 molecules was decreased (27.3%), and that of the 342-G2 fragments increased compared to healthy controls (33.3%). CONCLUSION: The stimulation of bovine articular cartilage explants with OSM/TNFalpha released aggrecan fragments both in an MMP and non-MMP-mediated route. These immunoassays carry a potential as diagnostic tools for the quantitative assessment of the cartilage turnover in RA patients in addition to their utility in ex vivo explant cultures.

Anabolic and catabolic function of chondrocyte ex vivo is reflected by the metabolic processing of type II collagen.

OBJECTIVE: The aim of the present study was to investigate collagen metabolism after anabolic and catabolic stimulation of chondrocytes ex vivo. DESIGN: Metabolic activities in ex vivo bovine cartilage explants were stimulated with insulin-like growth factor I (IGF-I) or a combination of tumor necrosis factor alpha (TNFalpha) and oncostatin M (OSM). Supernatants were assessed for changes in biochemical markers, N-terminal propeptide of type II (PIINP) collagen and fragments of C-telopeptide of type II collagen (CTX-II). Matrix metalloproteinases (MMP) were added to metabolic inactivated cartilage and evaluated by the two biochemical markers for formation or degradation, respectively. Finally, urinary CTX-II and PIINP were evaluated for assessment of type II collagen turnover in patients with rheumatoid arthritis (RA). RESULTS: In the bovine articular cartilage explants, IGF-I induced an increase in PIINP level up to 4.8+/-1.1[ng/ml]/mg cartilage whereas CTX-II remained below 0.1+/-0.1[ng/ml]/mg cartilage. In the catabolic stimulated explants both PIINP and CTX-II were released to the supernatant, reaching concentrations of 9.0+/-1.4 and 9.1+/-2.2[ng/ml]/mg cartilage, respectively. RA patients had significantly lower serum concentrations of PIINP (3.4+/-3.7 ng/ml) compared with those healthy individuals (18.7+/-12.41 ng/ml, P<0.001). In contrast, RA patients had significantly higher urinary CTX-II (0.8+/-0.8 mg/mmol) compared to the healthy controls (0.1+/-0.08 mg/mmol, P=0.004). CONCLUSIONS: This study is the first to demonstrate that precursors and degradation products of type II collagen released into the supernatant can effectively reflect the anabolic and catabolic activities of stimulated cartilage explants.

Application of biomarkers in the clinical development of new drugs for chondroprotection in destructive joint diseases: a review.

Emerging evidence supports the concept that biochemical markers are clinically useful non-invasive diagnostic tools for the monitoring of changes in cartilage turnover in patients with destructive joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Epidemiological studies demonstrated that measurements of different degradation products of proteins in the extracellular matrix of hyaline cartilage in urine or serum samples are (1) increased in OA or RA patients compared with healthy individuals, (2) correlate with disease activity, and (3) are predictive for the rate of changes in radiographic measures of cartilage loss. The present review provides an updated list of available biomarkers and summarize the research data arguing for their clinical utility. In addition, it addresses the question whether or not the monitoring of biomarkers during different treatment modalities could be a useful approach to characterize the chondro-protective effects of approved and candidate drugs. Finally, it briefly reviews the in vitro/ex vivo experimental settings - isolated chondrocyte cultures and articular cartilage explants - that can assist in the verification of novel markers, but also studies assessing direct effects of drug candidates on chondrocytes. Collectively, biomarkers may acquire a function as established efficacy parameters in the clinical development of novel chondro-protective agents.

Calcitonin directly attenuates collagen type II degradation by inhibition of matrix metalloproteinase expression and activity in articular chondrocytes.

OBJECTIVE: Calcitonin was recently reported to counter progression of cartilage degradation in an experimental model of osteoarthritis, and the effects were primarily suggested to be mediated by inhibition of subchondral bone resorption. We investigated direct effects of calcitonin on chondrocytes by assessing expression of the receptor and pharmacological effects on collagen type II degradation under ex vivo and in vivo conditions. METHODS: Localization of the calcitonin receptor on articular chondrocytes was investigated by immunohistochemistry, and the expression by reverse transcriptase polymerase chain reaction (RT-PCR). In bovine articular cartilage explants, cartilage degradation was investigated by release of C-terminal telopeptides of collagen type II (CTX-II), induced by tumor necrosis factor-alpha (TNF-alpha) [20 ng/ml] and oncostatin M (OSM) [10 ng/ml], with salmon calcitonin [0.0001-1 microM]. In vivo, cartilage degradation was investigated in ovariectomized (OVX) rats administered with oral calcitonin [2 mg/kg calcitonin] for 9 weeks. RESULTS: The calcitonin receptor was identified in articular chondrocytes by immunohistochemistry and RT-PCR. Calcitonin concentration-dependently increased cAMP levels in isolated chondrocytes. Explants cultured with TNF-alpha and OSM showed a 100-fold increase in CTX-II release compared to vehicle-treated controls (P<0.001). The degradation of type II collagen in these explants was concentration-dependently inhibited by calcitonin, 65% protection at 10 nM calcitonin (P<0.01). TNF-alpha and OSM induced a pronounced increase in matrix metalloproteinase (MMP) activity, which was strongly inhibited by calcitonin. In vivo, administration of salmon calcitonin to OVX rats resulted in significant (P<0.001) decrease in CTX-II levels. CONCLUSION: These results are the first evidence of calcitonin receptor expression on articular chondrocytes and that the chondroprotective effects of calcitonin might involve the inhibition of MMP expression.

Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation.

OBJECTIVE: Both matrix metalloprotease (MMP) activity and cathepsin K (CK) activity have been implicated in cartilage turnover. We investigated the relative contribution of MMP activity and CK activity in cartilage degradation using ex vivo and in vivo models. METHODS: Bovine articular cartilage explants were stimulated with oncostatin M (OSM) 10 ng/ml and tumor necrosis factor-alpha (TNF-alpha) 20 ng/ml in the presence or absence of the broad-spectrum MMP inhibitor GM6001 and the cysteine protease inhibitor, E64. Cartilage degradation was evaluated in the conditioned medium by glycosaminoglycans (GAG), hydroxyproline, and cross-linked C-telopeptide fragments of type II collagen (CTX-II), which were compared to immunohistochemical evaluations of proteoglycans and CTX-II. We assessed MMP expression by gelatine zymography and CK expression by immunohistochemistry. In vivo, CTX-II release was measured from CK-deficient mice. RESULTS: OSM and TNF-alpha combined induced significant (P<0.01) increase in cartilage degradation products measured by hydroxyproline and CTX-II compared to vehicle control. The cytokines potently induced MMP expression, assessed by zymography, and CK expression investigated by immunohistochemistry. Inhibition of MMP activity completely abrogated hydroxyproline and CTX-II release (P<0.01) and GAG release (P<0.05). In contrast, E64 resulted in increased CTX-II release by 100% (P<0.05) and inhibited GAG release by 30%. Up-regulation of CTX-II fragments was confirmed in vivo in CK null mice. CONCLUSION: Inhibition of MMP activity reduced both proteoglycan loss and type II collagen degradation. In contrast, inhibition of cysteine proteases resulted in an increase rather than a decrease in MMP derived fragments of collagen type II degradation, CTX-II, suggesting altered collagen metabolism.