Lysophosphatidic acid provides a missing link between osteoarthritis and joint neuropathic pain.

OBJECTIVE: Emerging evidence suggests that osteoarthritis (OA) has a neuropathic component; however, the identity of the molecules responsible for this peripheral neuropathy is unknown. The aim of this study was to determine the contribution of the bioactive lipid lysophosphatidic acid (LPA) to joint neuropathy and pain. DESIGN: Male Lewis rats received an intra-articular injection of 50 µg of LPA into the knee and allowed to recover for up to 21 days. Saphenous nerve myelination was assessed by g-ratio calculation from electron micrographs and afferent nerve damage visualised by activation transcription factor-3 (ATF-3) expression. Nerve conduction velocity was measured electrophysiologically and joint pain was determined by hindlimb incapacitance. The effect of the LPA antagonist Ki-16425 was also evaluated. Experiments were repeated in the sodium monoiodoacetate (MIA) model of OA. RESULTS: LPA caused joint nerve demyelination which resulted in a drop in nerve conduction velocity. Sensory neurones were ATF-3 positive and animals exhibited joint pain and knee joint damage. MIA-treated rats also showed signs of demyelination and joint neuropathy with concomitant pain. Nerve damage and pain could be ameliorated by Ki-16425 pre-treatment. CONCLUSION: Intra-articular injection of LPA caused knee joint neuropathy, joint damage and pain. Pharmacological blockade of LPA receptors inhibited joint nerve damage and hindlimb incapacitance. Thus, LPA is a candidate molecule for the development of OA nerve damage and the origin of joint neuropathic pain.

Identification and pharmacological characterization of a novel inhibitor of autotaxin in rodent models of joint pain.

OBJECTIVE: Autotaxin is a secreted lysophospholipase that mediates the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA), a bioactive lipid mediator. Autotaxin levels in plasma and synovial fluid correlate with disease severity in patients with knee osteoarthritis (OA). The goal of this study was to develop and characterize a novel small molecule inhibitor of autotaxin to inhibit LPA production in vivo and determine its efficacy in animal models of musculoskeletal pain. DESIGN: Compound libraries were screened using an LPC coupled enzyme assay that measures the amount of choline released from LPC by the action of autotaxin. Hits from this assay were tested in a plasma assay to assess inhibition of endogenous plasma autotaxin and subsequently tested for their ability to lower plasma LPA levels upon oral dosing of rats. The best compounds were then tested in animal models of musculoskeletal pain. RESULTS: Compound screening led to the identification of compounds with nanomolar potency for inhibition of autotaxin activity. Studies in rats demonstrated a good correlation between compound exposure levels and a decrease in LPA levels in plasma. The leading molecule (compound-1) resulted in a dose dependent decrease in joint pain in the mono-sodium iodoacetate (MIA) and meniscal tear models and a decrease in bone fracture pain in the osteotomy model in rats. CONCLUSION: We have identified and characterized a novel small molecule inhibitor of autotaxin and demonstrated its efficacy in animal models of musculoskeletal pain. The inhibitor has the potential to serve as an analgesic for human OA and bone fracture.

A short-term pharmacodynamic model for monitoring aggrecanase activity: injection of monosodium iodoacetate (MIA) in rats and assessment of aggrecan neoepitope release in synovial fluid using novel ELISAs.

OBJECTIVE: To develop a short-term in vivo model in rats, with an enzyme-linked immunosorbent assay (ELISA) readout for specific aggrecanase-cleaved aggrecan fragments, to facilitate testing of aggrecanase inhibitors. METHODS: Monosodium iodoacetate (MIA), a metabolic inhibitor, was injected into the right knee joint of male Lewis rats and the release of aggrecanase-cleaved fragments of aggrecan containing the NITEGE or ARGN neoepitope was measured in the synovial fluid at 7 days post MIA injection using novel ELISAs. The ELISAs utilize a commercial antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan, in combination with either an alpha-NITEGE antibody (NITEGE ELISA) or an alpha-ARGS/BC3 antibody (ARGS ELISA), to detect aggrecanase-cleavage of aggrecan within the interglobular domain (IGD). Aggrecan fragments present in in vitro digests, in cytokine-treated cartilage explant culture supernatants and in rat synovial fluid lavage samples were detected and quantified using the two ELISAs. Small molecule inhibitors of aggrecanase activity were dosed orally on days 3-7 to determine their ability to inhibit MIA-induced generation of the NITEGE and ARGN neoepitopes measured in the rat synovial fluid. RESULTS: The NITEGE assay was shown to specifically detect the N-terminal fragment of aggrecan comprising the G1 domain and the NITEGE neoepitope sequence. This assay can readily measure aggrecanase-cleaved bovine, human and rat aggrecan without the need for deglycosylation. The ARGS assay specifically detects C-terminal fragments of aggrecan comprising the ARGS/ARGN neoepitope and the G2 domain. Keratan sulfate (KS) residues of aggrecan interfere with this ELISA, and hence this assay works well with native rat articular cartilage aggrecan (that lacks KS residues) and with deglycosylated bovine and human aggrecan. Injection of MIA into the rat knee joints resulted in a time-dependent increase in the release of aggrecanase-cleaved aggrecan fragments into the synovial fluid and treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of the generation of these neoepitopes. CONCLUSIONS: We have established a short-term in vivo model in rats that involves measurement of synovial fluid biomarkers that are dependent on aggrecanase activity in the joint. The short duration of the model combined with the mechanistic biomarker readout makes it very useful for the initial in vivo screening of aggrecanase inhibitors prior to testing them in time and resource-intensive disease models of osteoarthritis (OA).

Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

OBJECTIVE: Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS: The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS: We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Analysis of early changes in the articular cartilage transcriptisome in the rat meniscal tear model of osteoarthritis: pathway comparisons with the rat anterior cruciate transection model and with human osteoarthritic cartilage.

OBJECTIVE: The purpose of this study was to use microarray technology to: (1) understand the early molecular events underlying the damage of articular cartilage initiated by this surgical procedure, and (2) determine whether these changes mimic those that are occurring in human osteoarthritic (OA) cartilage. DESIGN: Cartilage was harvested from both medial and lateral sides of the tibial plateaus and femoral condyles of both meniscal tear (MT) and sham surgery groups on days 3, 7 and 21 post-surgery. mRNA prepared from these rat cartilage samples was used for microarray analysis. RESULTS: Statistical analysis identified 475 genes that were differentially expressed between the sham and MT groups, at one or more of the time points that were analyzed. By integrating these genes with OA-related genes reported previously in a rat OA model and in human OA array studies, we identified 20 commonly changed genes. Six out of these 20 genes (Col5A1, Col6A2, INHBA, LTBP2, NBL1 and SERPINA1) were differentially expressed in two animal models and in human OA. Pathway analysis identified some key features of OA pathology, namely cartilage extracellular matrix remodeling, angiogenesis, and chondrocyte cell death that were recapitulated in the animal models. The rat models suggested increased inflammation and cholesterol metabolic pathways may play important role in early cartilage degeneration. CONCLUSION: We identified a large number of differentially expressed genes in the articular cartilage of the MT model. While there was lack of overall identity in cartilage gene expression between the rat models and human OA, several key biological processes were recapitulated in the rat MT OA model.

Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage.

Proteolysis of triple-helical collagen is an important step in the progression toward irreversible tissue damage in osteoarthritis. Earlier work on the expression of enzymes in cartilage suggested that collagenase-1 (MMP-1) contributes to the process. Degenerate reverse transcription polymerase chain reaction experiments, Northern blot analysis, and direct immunodetection have now provided evidence that collagenase-3 (MMP-13), an enzyme recently cloned from human breast carcinoma, is expressed by chondrocytes in human osteoarthritic cartilage. Variable levels of MMP-13 and MMP-1 in cartilage was significantly induced at both the message and protein levels by interleukin-1 alpha. Recombinant MMP-13 cleaved type II collagen to give characteristic 3/4 and 1/4 fragments; however, MMP-13 turned over type II collagen at least 10 times faster than MMP-1. Experiments with intact type II collagen as well as a synthetic peptide suggested that MMP-13 cleaved type II collagen at the same bond as MMP-1, but this was then followed by a secondary cleavage that removed three amino acids from the 1/4 fragment amino terminus. The expression of MMP-13 in osteoarthritic cartilage and its activity against type II collagen suggest that the enzyme plays a significant role in cartilage collagen degradation, and must consequently form part of a complex target for proposed therapeutic interventions based on collagenase inhibition.

Induction of expression of c-fos and c-myc protooncogenes by basic calcium phosphate crystal: effect of beta-interferon.

Basic calcium phosphate (BCP) crystals control the traverse of cells from the G0-G1 to S-phase of the cell cycle and initiate proliferation by rendering fibroblasts competent to respond to insulin-like growth factors in plasma. The present study examines whether BCP crystals induce transcription of the protooncogenes c-fos and c-myc and the effect of beta-interferon (IFN-beta) on protooncogene transcription as well as BCP crystal-induced DNA synthesis. Stimulation of density-arrested BALB/c-3T3 cells with either BCP crystal or platelet-derived growth factor (PDGF) results in maximal accumulation of c-fos mRNA at 30 min after stimulation. Induction of c-myc transcription by BCP crystal or PDGF occurs within 1 h and is maximal at around 3 h after stimulation. Simultaneous addition of IFN-beta with either BCP crystals or PDGF had little effect on c-fos induction but delayed both c-myc message accumulation and entry into S phase. The delay in c-myc message induction after IFN-beta treatment cannot account for the observed delay in the onset of DNA synthesis, since IFN-beta can be added at up to 6 h after stimulation with either PDGF or BCP crystals, and a similar delay in the onset of DNA synthesis is still observed.

Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice.

BACKGROUND: Extracellular matrix synthesis and degradation contribute to the morphological changes that occur after myocardial infarction (MI). METHODS AND RESULTS: We tested the hypothesis that inhibition of matrix metalloproteinases (MMPs) attenuates left ventricular remodeling in experimental MI. Seventy-one male FVB mice that survived ligation of the left anterior coronary artery were randomized to a broad-spectrum MMP inhibitor (CP-471,474) or placebo by gavage. Echocardiographic studies were performed before randomization (within 24 hours of surgery) and 4 days later and included short-axis imaging at the midpapillary and apical levels. Infarction as defined by wall motion abnormality was achieved in 79% of the procedures (n=56), and mortality rate during the 4-day protocol was 23% (9 of 36 on treatment vs 7 of 35 on placebo; P=NS). Baseline end-diastolic and end-systolic dimensions and areas were similar (P=NS) between treated and placebo groups. At follow-up, infarcted mice allocated to MMP inhibitor had significantly smaller increases in end-systolic and end-diastolic dimensions and areas at both midpapillary and apical levels compared with infarcted mice allocated to placebo (all P<0.05). In addition, infarcted animals that received MMP inhibitor had no change in fractional shortening (-3+/-13%), whereas animals that received placebo had a decrease in fractional shortening (-12+/-12%) (P<0.05). In an analysis stratified by baseline end-diastolic area, the effects of MMP inhibition on the changes in end-systolic area and end-diastolic area were most prominent in animals that had more initial left ventricular dilatation (both P<0.05). CONCLUSIONS: -Administration of an MMP inhibitor attenuates early left ventricular dilation after experimental MI in mice. Further studies in genetically altered mice and other models will improve understanding of the role of MMPs in left ventricular remodeling.

Induction of stromelysin-1 and collagenase synthesis in fibrochondrocytes by tumor necrosis factor-alpha.

Stromelysin-1 and collagenase mRNA levels were assayed in fibrochondrocytes by Northern blot analysis at 0, 2, 4, 8 and 24 h after stimulation with tissue necrosis factor-alpha (TNF-alpha). Peak collagenase mRNA levels occurred 24 h after stimulation and were increased nine-fold over the level at time 0. Stromelysin-1 mRNA levels peaked 8 h after stimulation, with a five-fold increase over the level at time 0. A TNF-alpha dose-related response to both collagenase and stromelysin-1 mRNA accumulation was also demonstrated. Confirmation of the presence of secreted metallo-proteinases in the conditioned media was established by immunoprecipitation of stromelysin-1 and Western blotting of collagenase. Both enzymes were secreted in latent forms. Consistent with stromelysin-1 activity, substrate gels demonstrated a doublet of caseinase activity with molecular masses at 57 kDa and 59 kDa in TNF-alpha stimulated samples. Collagenase assays of conditioned media also demonstrated a significant increase in collagenase activity after stimulation by TNF-alpha. While epidermal growth factor had a minimal effect on stromelysin-1 and collagenase expression, transforming growth factor-beta, and insulin-like growth factor-1 did not induce either enzyme activity.

Prolonged monitoring of the blood-aqueous barrier with fluorescein-labeled albumin.

Aqueous fluorophotometry has proved to be a useful indicator of changes in the blood-aqueous barrier after surgical, immunologic, or laser manipulations. Previously used fluorescent tracers have been unable to follow rapid changes continuously in the blood-aqueous barrier. Fluorescein-labeled homologous serum albumin, however, provides extremely stable and high plasma levels of fluorescence due to active renal reabsorption with very low levels in the normal aqueous because of its high molecular weight. This feature allows prolonged, continuous, and highly sensitive monitoring of the blood-aqueous barrier before, during, and after a manipulation. The usefulness of this technique is demonstrated in a model that has been well studied with other methods: the response to argon laser iris photocoagulation.

Cataract and age-related maculopathy: the Blue Mountains Eye Study.

PURPOSE: To assess the relationship between different types of cataract or past cataract surgery and late or early age-related maculopathy (ARM) in an older population. METHODS: A population-based survey examined 3,654 people aged >/=49 years, 82% of permanent residents of an area near Sydney, Australia. Participants had a detailed eye examination, including standardised dilated lens and stereo macular photographs. Presence of cataract and ARM was diagnosed from masked photographic grading using the Wisconsin Cataract and Age-related Maculopathy Grading Systems. Generalized Estimating Equation (GEE) and logistic regression models were used in the statistical analysis. RESULTS: Higher prevalence was found for both late and early ARM in eyes with cataract or a past history of cataract surgery. However, after adjusting for age, sex and other ARM risk factors, no consistent association was found between presence of cortical, nuclear or posterior subcapsular (PSC) cataract or past history of cataract surgery and either late or early ARM. In the GEE model, the only statistically significant association found was between PSC and late ARM. Non-significant increased odds were found for late ARM in eyes with cortical cataract. However, detailed analyses of cortical cataract location failed to show a relationship. CONCLUSIONS: The co-existence of cataract and ARM was almost entirely explained by the age-related increase in prevalence of both conditions. We found no evidence of a consistent relationship between cortical, nuclear or PSC cataract or history of past cataract surgery and either late or early ARM, after adjusting for age and other potential ARM risk factors. The possibility of a relationship between PSC and late ARM or between cortical cataract and any ARM was not excluded. Long-term follow up data from this population will be useful.

Diabetes, fasting blood glucose and age-related cataract: the Blue Mountains Eye Study.

AIMS: To examine the relationship between diabetes and the presence of cortical, nuclear and posterior subcapsular (PSC) cataract in a defined older population, while controlling for known cataract risk factors. METHODS: Slit-lamp and retroillumination lens photographs were taken on 3654 participants attending the population-based Blue Mountains Eye Study during 1992-94. Masked grading of the photographs was performed using the Wisconsin Cataract Grading System. RESULTS: 217 subjects (5.9% of the population) had previously diagnosed diabetes and 66 (1.8%) had diabetes diagnosed from fasting blood glucose measurements. Cortical cataract, PSC and past cataract surgery were associated with known diabetes in age-sex adjusted models. However, only PSC (odds ratio (OR) 1.8, 95% confidence interval (CI) 1.0-3.1) and past cataract surgery (OR 2.5, CI 1.5-4.2) remained statistically significantly associated with diabetes after further adjustment for other known cataract risk factors. Increasing therapy, as an index of diabetes severity (oral or insulin treatment, compared to treatment by diet alone), was associated with a markedly increased risk of PSC (OR 5.4). CONCLUSIONS: These findings support previous research showing that diabetes has a harmful effect on the lens. The markedly increased risk for PSC may also have been reflected in the association found between diabetes and past cataract surgery. Contrary to findings from the Beaver Dam Eye Study, we found only a weak association with cortical cataract, which was not statistically significant after adjusting for other known cataract risk factors.

Direct determination of metals in milligram masses and microlitre volumes by direct-current argon-plasma emission spectrometry with sample introduction by electrothermal vaporization.

The development of an electrothermal vaporization and direct-current argon-plasma emission spectrometric system which allowed the determination of metals in microlitre volumes and milligram masses is described. For the five metals investigated, the concentration detection limits were comparable to those for conventional pneumatic nebulization of solutions, and the mass detection limits were superior. The use of an ashing stage was found to reduce enhancement by sodium in the determination of copper and manganese by the plasma method. The system was shown to give accurate results for complex biological, nutritional, water and geological samples.

Protein kinase regulation of tumor necrosis factor alpha stimulated collagenase and stromelysin message levels in chondrocytes.

TNF stimulated transcription and secretion of the metalloproteinases collagenase and stromelysin in porcine articular chondrocytes. TNF induced metalloproteinase transcription could be inhibited with either protein kinase inhibitors (H7 or staurosporine) or by raising intracellular cAMP levels. HA1004, a protein kinase inhibitor structurally related to H7 but with a higher Ki for protein kinase C had no effect on TNF induced message levels. TNF treatment of chondrocytes did not induce membrane associated PKC or increase intracellular cAMP levels. Our results are consistent with the involvement of a staurosporine and H7 sensitive protein kinase distinct from PKC in TNF signal transduction in chondrocytes.

Tumor necrosis factor alpha and epidermal growth factor regulation of collagenase and stromelysin in adult porcine articular chondrocytes.

Chondrocyte-derived metalloproteases have been postulated to play a role in the degradation of articular cartilage during the development of chronic arthritic disorders. TNF alpha (tumor necrosis factor alpha), an inflammatory mediator released by activated macrophages, has been detected in the synovial fluid of patients with rheumatoid diseases. We have found that TNF alpha is a potent stimulator of collagenase and stromelysin mRNA accumulation, collagenase activity, and immunoprecipitable stromelysin in monolayer cultures of adult porcine articular chondrocytes. In contrast EGF (epidermal growth factor), which stimulates collagenase and/or stromelysin synthesis in fibroblast systems, stimulated minimal amounts of these enzymes at both the message and protein levels. Nuclear run-on transcription analysis demonstrated that the TNF alpha-stimulated increase in stromelysin and collagenase message levels was, at least partially, due to increased transcription. Elevated transcription of these genes, in response to TNF alpha, was apparent by at least 2 hours post-stimulation. The degree of c-fos and c-jun stimulation by TNF alpha or EGF did not correlate with the levels of collagenase and stromelysin message stimulated by these factors. EGF stimulated significant accumulation of both c-fos and c-jun mRNAs while only very low amounts of these messages were stimulated by TNF alpha. Our data suggests that TNF alpha may contribute to articular cartilage degradation by stimulating chondrocyte-derived matrix metalloproteases. In addition the regulation of metalloprotease genes in chondrocytes may be different from their regulation in fibroblasts.

Misoprostol, a prostaglandin E1 analogue, inhibits basic calcium phosphate crystal-induced mitogenesis and collagenase accumulation in human fibroblasts.

Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe destructive arthropathy. BCP crystals induce the secretion of matrix-degrading enzymes such as collagenase. No prophylactic or therapeutic agents are recognized to ameliorate the cartilage damage associated with BCP deposits in joints. As a chondroprotective effect of prostaglandins (PG) has been suggested, we studied the effect of misoprostol, a PGE1 analogue, on BCP crystal-induced mitogenesis and collagenase messenger RNA (mRNA) accumulation in human fibroblasts (HF). Mitogenesis was determined by 3H-thymidine incorporation assays and collagenase mRNA accumulation by Northern blot analysis, in HF stimulated with BCP crystals in the presence or absence of misoprostol. Misoprostol caused concentration-dependent inhibition of BCP crystal-induced mitogenesis. The inhibition of BCP-stimulated mitogenesis was not specific as misoprostol also inhibited the mitogenic response to 10% serum. There was only 50 (+/-5)% inhibition of serum-induced mitogenesis by misoprostol at 500 ng/ml, the concentration that completely inhibited BCP crystal-induced mitogenesis. Misoprostol also inhibited the accumulation of collagenase mRNA in BCP-stimulated HF by 63%. These data suggest that misoprostol may inhibit the synovial proliferation and cartilage degradation that accompany BCP crystal deposition.