Yetunde Olabisi Taiwo

Gene expression of matrix metalloproteinases 1, 3, and 9 by chondrocytes in osteoarthritic human knee articular cartilage is zone and grade specific.

OBJECTIVES Matrix metalloproteinases (MMPs) are thought to be major mediators of cartilage destruction. Osteoarthritis (OA) is characterised by cartilage degradation. This study explores gene expression of three MMPs in articular chondrocytes during the histological development of the cartilage lesion of OA. METHODS Biopsy specimens of human normal and OA cartilage, classified into four grades on the basis of histology, were probed for MMPs 1, 3, and 9 using 35S-labelled cDNA probes. The signal was measured at four different depths (zones) using an automated image analyser and compared with signal from sections probed with λDNA. Rheumatoid synovium was used as a positive control for MMP gene expression. RESULTS Rheumatoid tissue contained mRNA for all three MMPs. Expression in chondrocytes varied with the depth of the chondrocyte in the cartilage and the histomorphological extent of the OA changes. There was no detectable mRNA signal for these three MMPs in normal cartilage. In general, in OA, MMP-1 gene expression was greatest in the superficial cartilage in established disease. By contrast mRNAs for MMP-3 and 9 were expressed deeper in the cartilage, MMP-9 early in disease and MMP-3 with a biphasic pattern in early and late stage disease, most pronounced in the latter. This was a consequence of differential expression in single cells and chondrocyte clusters in late disease. CONCLUSION The data indicate that expression of genes for MMPs 1, 3, and 9 is differentially regulated in human articular chondrocytes and, in individual cells, is related to the depth of the chondrocyte below the cartilage surface and the nature and extent of the cartilage lesion.

Matrix metalloproteinases are involved in C-terminal and interglobular domain processing of cartilage aggrecan in late stage cartilage degradation.

Monoclonal antibody (MAb) technology was used to examine aggrecan metabolites and the role of aggrecanases and matrix metalloproteinases (MMPs) in proteolysis of the interglobular domain (IGD) and C-terminus of aggrecan. An in vitro model of progressive cartilage degradation characterized by early proteoglycan loss and late stage collagen catabolism was evaluated in conjunction with a broad-spectrum inhibitor of MMPs. We have for the first time demonstrated that IGD cleavage by MMPs occurs during this late stage cartilage degeneration, both as a primary event in association with glycosaminoglycan (GAG) release from the tissue and secondarily in trimming of aggrecanase-generated G1 metabolites. Additionally, we have shown that MMPs were responsible for C-terminal catabolism of aggrecan and generation of chondroitin sulfate (CS) deficient aggrecan monomers and that this aggrecan truncation occurred prior to detectable IGD cleavage by MMPs. The onset of this later stage MMP activity was also evident by the generation of MMP-specific link protein catabolites in this model culture system. Recombinant MMP-1, -3 and -13 were all capable of C-terminally truncating aggrecan with at least two cleavage sites N-terminal to the CS attachment domains of aggrecan. Through analysis of aggrecan metabolites in pathological synovial fluids from human, canine and equine sources, we have demonstrated the presence of aggrecan catabolites that appear to have resulted from similar C-terminal processing of aggrecan as that induced in our in vitro culture systems. Finally, by developing a new MAb recognizing a linear epitope in the IGD of aggrecan, we have identified two novel aggrecan metabolites generated by an as yet unidentified proteolytic event. Collectively, these results suggest that C-terminal processing of aggrecan by MMPs may contribute to the depletion of cartilage GAG that leads to loss of tissue function in aging and disease. Furthermore, analysis of aggrecan metabolites resulting from both C-terminal and IGD cleavage by MMPs may prove useful in monitoring different stages in the progression of cartilage degeneration.

In situ zymographic localisation of type II collagen degrading activity in osteoarthritic human articular cartilage

OBJECTIVES Chondrocytic matrix metalloproteinases (MMPs) are believed to be important in osteoarthritic cartilage degradation. The cartilage lesion of osteoarthritis (OA) is focal and often progressive. During its development chondrocytes differentially up and down regulate production of mRNA for individual MMPs. This observation has potential implications for understanding the disease processes that lead to progressive cartilage loss in OA and designing appropriate targeted treatment. The complex regulation of MMP mediated effects means there is a pressing need to establish whether visualisation of MMP mRNA or protein equates to enzyme activity. The technique of in situ zymography (ISZ) offers a way of examining diseased human tissue for in vivo production of an excess of degrading enzyme over inhibitor. The primary objective of this study was to assess, and if positive follow, collagen II degrading activity in cartilage during development of the OA lesion. A secondary objective was to assess whether there was any correlation between sites of collagen II degrading activity and expression of the collagenase (MMP-13), recently implicated in type II collagen degredation in this lesion. METHODS Biopsied human normal and osteoarthritic cartilage, showing various degrees of damage, was examined by in situ zymography, with and without enzyme inhibitors, to establish sites of type II collagenase activity. Paired samples were probed for MMP-13 mRNA using 35S-labelled oligonucleotide probes. Comparative analyses were performed. RESULTS In situ zymography showed collagen II degrading activity over chondrocytes only in osteoarthritic cartilage. Distribution and amount varied with the extent of cartilage damage and position of chondrocytes, being greatest in deep cartilage and in cartilage lesions where fissuring was occurring. The enzyme causing the degradation behaved as a matrix metalloproteinase. MMP-13 mRNA expression codistributed with the type II collagenase activity. CONCLUSION In OA, chondrocytes can degrade type II collagen. The type II collagen degrading activity varies in site and amount as the cartilage lesion progresses and throughout codistributes with MMP-13 mRNA expression.

P38 MAP kinase inhibitors as potential therapeutics for the treatment of joint degeneration and pain associated with osteoarthritis

BackgroundEvaluate the potential role of p38 inhibitors for the treatment of osteoarthritis using an animal model of joint degeneration (iodoacetate-induced arthritis) and a pain model (Hargraeves assay).MethodsP38 kinase activity was evaluated in a kinase assay by measuring the amount of phosphorylated substrate ATF2 using a phosphoATF2 (Thr71) specific primary antibody and an alkaline phosphate coupled secondary antibody and measuring the OD at 405 nm. TNFα and IL-1β secretion from LPS stimulated THP-1 monocytic cells and human peripheral blood mononuclear cells were measured by ELISA. Rats treated with vehicle or p38 inhibitor were injected intra-articularly in one knee with iodoacetate and damage to the tibial plateau was assessed from digitized images captured using an image analyzer. The effect of p38 inhibitors on hyperalgesia was evaluated in rats given an intraplantar injection of carrageenan and 4 h later the paw withdrawal time to a radiant heat source was measured.ResultsSB-203580 and VX-745 are both potent inhibitors of p38 with IC50s of 136 ± 64 nM and 35 ± 14 nM (mean ± S.D.), respectively. Similarly, SB-203580 and VX-745 potently inhibited TNF release from LPS stimulated human THP-1 cells with IC50s of 72 ± 15 nM; and 29 ± 14 nM (mean ± S.D.) respectively. TNF release from LPS stimulated human peripheral blood mononuclear cells was inhibited with IC50s 16 ± 6 nM and 14 ± 8 nM, (mean ± S.D.) for SB-203580 and VX-745 and IL-1 was inhibited with IC50s of 20 ± 8 nM and 15 ± 4 nM (mean ± S.D.), respectively. SB-203580 and VX-745 administered orally at a dose of 50 mg/kg resulted in the significant (p < 0.05) inhibition of joint degeneration in the rat iodoacetate model of 45% and 31%, respectively. SB-203580 demonstrated a dose related inhibition of joint degeneration of 30, 25, 12 and 8% at 50, 25, 10 and 5 mg/kg p.o. b.i.d. in the rat iodoacetate model. Similarly, both p38 inhibitors significantly (p < 0.05) attenuated the pain response (paw withdrawal time) in the Hargraeves hyperalgesia assay when administered orally at 30, 10 and 3 mg/kg.ConclusionSB203580 and VX-745 demonstrated attenuation of both cartilage degeneration and pain in animal models and suggest that p38 inhibitors may be a useful approach for the treatment of osteoarthritis.

The Next Generation of MMP Inhibitors: Design and Synthesis

ABSTRACT: Since their inception during the eighties, MMP inhibitors (MMPIs) have gone through several cycles of metamorphosis. The design of early MMPIs was based on the cleavage site of peptide substrates. The second generation contained a substituted succinate scaffold (e.g., marimastat) coupled to a modified amino acid residue. The lower molecular weight analogs with multiple substitution possibilities produced a series of MMP inhibitors with varying degrees of selectivity for various MMPs. The introduction of sulfonamides in the midnineties added a new dimension to this field. The simplicity of synthesis coupled with high potency (e.g., CGS‐27023A, AG‐3340) produced a number of clinical candidates. This review highlights some of the key features that contributed to the discovery of this novel series of MMP inhibitors.

Pharmacological effect of tetracyclines on proteoglycanases from interleukin-1 -treated articular cartilage

Based on previous in vivo and in situ studies showing that tetracyclines possess antidegenerative effects on cartilage in conjunction with a reduced proteoglycan (PG) loss from the extracellular matrix, we investigated the effects of doxycycline, minocycline and tetracycline on the degradation and biosynthesis of PGs by bovine articular cartilage explants, both in vitro and in situ. Doxycycline, minocycline and tetracycline dose dependently, although weakly, inhibited PG degrading matrix metalloproteinases (MMPs) in vitro, when tested at concentrations ranging from 1 to 100 microM. Ro 31-4724 proved to be a potent inhibitor of MMP proteoglycanases (IC50 value 1.5 nM). Only at a concentration of 100 microM did doxycycline and minocycline significantly inhibit the interleukin-1 (IL-1)-induced augmentation of PG loss from cartilage explants into the nutrient media. The tetracyclines did not modulate the IL-1-mediated reduced aggregability of PGs, whereas 10 microM Ro 31-4724 partially restored the aggregability of PGs ex vivo. Tetracycline even at this high concentration was ineffective. Compared to the effects of the MMP inhibitor Ro 31-4724, treatment with tetracyclines at therapeutic serum levels of 1 or 10 microM was minimal, with little or no effect on cartilage proteoglycanases and PG biosynthesis. In our experiments, tetracyclines and Ro 31-4724 at doses evaluated had no cytotoxic effects on chondrocytes.

Design and synthesis of conformationally-constrained MMP inhibitors

A novel series of conformationally constrained matrix metalloprotease inhibitors was identified. The potencies observed for these inhibitors were highly dependent upon the substitution pattern on the caprolactam ring as well as the succinate moiety.

Isolation and quantification of fluoroacetate in rat tissues, following dosing of Z-Phe-Ala-CH2-F, a peptidyl fluoromethyl ketone protease inhibitor.

Peptidyl fluoromethyl ketones (PFMK) are irreversible inhibitors of cathepsin B, a cysteine proteinase thought to be involved in the degradation of cartilage. It has been speculated that PFMK inhibitors may metabolize in rodents to form fluoroacetate (FAC), an extremely toxic poison. A highly selective and sensitive separation and detection scheme was developed to measure trace levels of FAC in rat tissues following PFMK dosing. The procedure consisted of extracting FAC from tissue and spiking the extract with [18O]2-fluoroacetate (18O-FAC) as an internal standard. FAC and 18O-FAC were further isolated from matrix components using ion-exchange, solid-phase extraction. The pentafluorobenzyl esters of FAC and 18O-FAC were formed to facilitate the chromatographic separation. Two-dimensional gas chromatography coupled with selected-ion-monitoring detection provided the final measurement. The assay had a limit of detection of 2 ng FAC per g tissue, and was capable of accurately quantitating as little as 10 ng FAC per g tissue with a S/N ratio of 40:1. Linearity was established over two orders of magnitude, from 2-500 ng ml-1, with 5 microliters injected on-column. The method was used to demonstrate that FAC was formed in rats following dosing with Z-Phe-Ala-CH2-F, a PFMK cathepsin enzyme inhibitor.

Opioid receptor subtype mediating peripheral antinociception

T he accompanying focus article on the peripheral analgesic effects of opioids by Hargreaves and Joris is a timely review, prepared by authors who themselves have contributed significantly to knowledge in this field. Evidence accumulating over the past decade, and substantially reviewed in the focus article, has suggested that opioid receptors in the central nervous system cannot be the exclusive target for the antinociceptive actions of systemically administered opiate analgesics. While peripheral antinociceptive effects of opioids are by now sufficiently recognized, several aspects, including the anatomical localization of these receptors, are only beginning to be addressed. Since these issues were not a major subject of the focus article, this commentary will discuss some recent reports in which these aspects of mechanism have been targets of study. Although the focus article describes the elegant studies of Stein et al. s2 in which the intraplantar injection of mu, delta, and kappa opioid agonists produced receptor-selective and antagonizable antinociception, it does not discuss the possible peripheral anatomic localization of such actions. The bipolar primary afferent synthesizes its receptors in the cell body located in the dorsal root ganglion and thereafter transports these receptors both to its central terminals in the spinal dorsal horn, as well as to its peripheral terminals innervating skin, muscle, and joints. Autoradiography and receptor binding studies have demonstrated a dense localization of mu, kappa, and delta opioid receptors in the dorsal horn