Sue A. Yocum

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.

Similarities and discrepancies in subchondral bone structure in two differently induced canine models of osteoarthritis

In osteoarthritis (OA), cartilage degradation is accompanied by subchondral bone changes. The pathogenesis and physiology of bone changes in OA are still unclear. The changes in subchondral bone architecture and cartilage damage were compared in differently induced experimental models of OA. Experimental OA was induced bilaterally by anterior cruciate ligament transection (ACLT) or by cartilage trauma (Groove model); bilateral sham surgery served as control. Lysylpyridinoline (LP, bone resorption) and C‐telopeptide of type II collagen (CTX‐II, cartilage breakdown) were measured over time. At 20 weeks after surgery, the subchondral cortical plate and trabecular bone of the tibia were analyzed by micro–computed tomography (µCT) and cartilage degeneration was analyzed histologically and biochemically. In both models, cartilage degeneration and cortical subchondral plate thinning were present. CTX‐II levels were elevated over time in both models. Subchondral trabecular bone changes were observed only in the ACLT model, not in the Groove model. Correspondingly, LP levels were elevated over time in the ACLT model and not in the Groove model. Interestingly, the trabecular bone changes in the ACLT model were extended to the metaphyseal area. The early decrease in plate thickness, present in both models, as was cartilage damage, suggests that plate thinning is a phenomenon that is intrinsic to the process of OA independent of the cause/induction of OA. On the other hand, trabecular changes in subchondral and metaphyseal bone are not part of a common pathway of OA development and may be induced biomechanically in the destabilized and less loaded ACLT joint.

Inhibitors of MMP-1: an examination of P1′ Cα gem-disubstitution in the succinamide hydroxamate series

Abstract The effect of P 1 ′ C α gem-disubstitution in a series of succinamide hydroxamate inhibitors of MMP-1 has been investigated. While in all cases P 1 ′ gem-disubstitution led to loss of potency relative to the corresponding P 1 ′ isobutyl and phenyl compounds 1 and 3 , respectively, the loss of activity was less pronounced in certain instances, e.g., the P 1 ′ gem-cyclohexyl analogue 12 IC 50 = 0.15 μM).

The canine bilateral groove model of osteoarthritis

In studies aimed at local treatment of experimental osteoarthritis (OA) it is optimal to have an internal (untreated) OA control. Such an approach excludes interanimal variation, and allows paired statistical evaluation of treatment efficacy. For this purpose, we developed and characterized a bilateral version of the canine Groove model. We hypothesized that the bilateral version of the canine Groove model would show consistent and clear development of features of OA similar to those found in the unilateral version. In six Beagle dogs, grooves were surgically made in the articular cartilage of the femoral condyles of both knee joints. Six additional dogs underwent bilateral sham surgery. The degree of OA was quantified 20 weeks after surgery and was compared in retrospect to 23 animals that undergone the same procedure in a single knee joint with the contralateral knee serving as a non‐OA control. Bilateral groove surgery resulted in OA. This was based on the observed ineffective repair response in which an increase in proteoglycan synthesis, a diminished retention of these newly formed proteoglycans, and an enhanced loss of resident proteoglycans resulted in a decreased cartilage proteoglycan content. These biochemical effects were corroborated by clear histological features of OA. All these effects were found in femor as well as in the (surgically untouched) tibia. Interestingly, features of OA were slightly more severe in the bilateral model than in the unilateral variant. The bilateral canine Groove model showed consistent and clear development of features of OA, comparable to the unilateral model.

Complement 1s is the serine protease that cleaves IGFBP-5 in human osteoarthritic joint fluid

UNLABELLED Insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) are trophic factors for cartilage and have been shown to be chondroprotective in animal models of osteoarthritis (OA). IGFBP-5 is degraded in joint fluid and inhibition of IGFBP-5 degradation has been shown to enhance the trophic effects of IGF-I. OBJECTIVE To determine the identity of IGFBP-5 protease activity in human OA joint fluid. METHOD OA joint fluid was purified and the purified material was analyzed by IGFBP-5 zymography. RESULTS Both crude joint fluid and purified material contained a single band of proteolytic activity that cleaved IGFBP-5. Immunoblotting of joint fluid for complement 1s (C1s) showed a band that had the same Mr estimate, e.g., 88 kDa. In gel tryptic digestion and subsequent peptide analysis by LC-MS/MS showed that the band contained human C1s. A panel of protease inhibitors was tested for their ability to inhibit IGFBP-5 cleavage by the purified protease. Three serine protease inhibitors, FUT175 and CP-143217 and CB-349547 had IC50s between 1 and 6 microM. Two other serine protease inhibitors had intermediate activity (e.g., IC50s 20-40 microM) and MMP inhibitors had no detectible activity at concentrations up to 300 microM. CONCLUSION Human OA fluid contains a serine protease that cleaves IGFBP-5. Zymography, immunoblotting and LC-MS/MS analysis indicate that C1s is the protease that accounts for this activity.

Inhibitors of MMP-1: an examination of P1′ Cα gem-disubstitution in the N-carboxyalkylamine and glutaramide carboxylate series

Abstract Modification of the N-carboxyalkylamine 3 by independent replacement of the P1′ NH group for CH2 and introduction of P1′ gem-cyclohexyl substitution affords compounds 5 and 6a which retain appreciable activity against MMP-1 (IC50s = 0.023 μM and 0.09 μM, respectively). The glutaramide 7a which incorporates both these structural changes also retains potent activity (IC50 = 0.038 μM).

IGFBP-5 Metabolism Is Disrupted in the Rat Medial Meniscal Tear Model of Osteoarthritis

Insulin-like growth factor binding protein 5 (IGFBP-5) has been proposed to promote cartilage anabolism through insulin-like growth factor (IGF-1) signaling. A proteolytic activity towards IGFBP-5 has been detected in synovial fluids from human osteoarthritic (OA) joints. The purpose of this study was to determine if protease activity towards IGFBP-5 is present in the rat medial meniscal tear (MMT) model of OA and whether inhibition of this activity would alter disease progression. Sprague-Dawley rats were subject to MMT surgery. Synovial fluid lavages were assessed for the presence of IGFBP-5 proteolytic activity. Treatment animals received intra-articular injections of vehicle or protease inhibitor peptide PB-145. Cartilage lesions were monitored by India ink staining followed by macroscopic measurement of lesion width and depth. The MMT surgery induced a proteolytic activity towards IGFPB-5 that was detectable in joint fluid. This activity was stimulated by calcium and was sensitive to serine protease inhibitors as well as peptide PB-145. Significantly, intra-articular administration of PB-145 after surgery protected cartilage from lesion development. PB-145 treatment also resulted in an increase in cartilage turnover as evidenced by increases in serum levels of procollagen type II C-propeptide (CPII) as well as synovial fluid lavage levels of collagen type II neoepitope (TIINE). IGFBP-5 metabolism is disrupted in the rat MMT model of OA, potentially contributing to cartilage degradation. Inhibition of IGFBP-5 proteolysis protected cartilage from lesion development and enhanced cartilage turnover. These data are consistent with IGFBP-5 playing a positive role in anabolic IGF signaling in cartilage.