Search

Objective: Since fibronectin fragments (Fn-fs) enhance cartilage damage through integrins, the objective was to investigate the role of integrin linked kinases, focal adhesion kinase (FAK) and a soluble form of FAK, proline rich tyrosine kinase 2 (Pyk2) and cellular src kinase (c-src) and the transcription factor, nuclear factor kappaB (NF-kappaB) in cartilage damage.Methods: Bovine chondrocytes were cultured with various concentrations of three different Fn-fs, an amino-terminal 29 kDa, a gelatin binding 50 kDa and a central 140-kDa Fn-fs, each with progressively weaker cartilage damaging activity, or with native fibronectin (Fn), and lysates probed for activation of the selected kinases. Confocal microscopy was used to visualize intracellular location of activated kinases and NF-kappaB. Various kinase inhibitors were tested for their effects on Fn-f mediated upregulation of matrix metalloproteinase (MMP)-3 and -13 and cartilage proteoglycan (PG) depletion.Results: The Fn-fs kinetically enhanced phosphorylation of FAK but did not show a clear dose-response effect. The 29-kDa and 50-kDa Fn-fs enhanced phosphorylation of Pyk2, c-src and NF-kappaB to a much greater extent than the 140-kDa Fn-f and native Fn and did so as a function of dose. The 29-kDa Fn-f enhanced the phosphorylation of nuclear Pyk2 as compared with no treatment or native Fn. Inhibitors of Pyk2, c-src, NF-kappaB and protein kinase Cdelta (PKCdelta) decreased MMP upregulation and decreased Fn-f mediated damage to cartilage.Conclusions: These studies enhance our knowledge of crucial factors in Fn-f mediated signaling in MMP upregulation and cartilage damage and because of the potential physiologic relevance of Fn-fs, provide a better knowledge of cartilage degeneration in general. [ABSTRACT FROM AUTHOR]

Post-traumatic osteoarthritis (PTOA) is characterized by progressive cartilage degeneration in injured joints. Since fibronectin-fragments (Fn-fs) degrade cartilage mainly through up-regulating matrix metalloproteinases (MMPs) and pro-inflammatory cytokines, we hypothesized that Fn-fs play a key role in PTOA by promoting chondrolysis in and around injured cartilage. To test this hypothesis, we profiled the catabolic events focusing on fibronectin fragmentation and proteinase expression in bovine osteochondral explants following a single blunt impact on cartilage with a drop tower device which created partial-thickness tissue damage. Injured and control explants were cultured for up to 14 days. The presence of Fn-fs, MMPs (-1, -3, -13), ADAMTS-5 in culture media and in cartilage was determined with immunoblotting. The daily proteoglycan (PG) depletion of cartilage matrix was assessed with DMMB assay. The effect of explant-conditioned media on chondrocytes was also examined with immunoblotting. Impacted cartilage released significantly higher amount of native Fn, three chondrolytic Fn-fs and PG than non-impacted controls did. Those increases coincided with up-regulation of MMP-3 both in culture media and in impacted cartilage. These findings support our hypothesis that PTOA may be propelled by Fn-fs which act as catabolic mediators through up-regulating cartilage-damaging proteinases., (© 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Objective: To determine the activation of Mitogen activated protein (MAP) kinases in and around cartilage subjected to mechanical damage and to determine the effects of their inhibitors on impaction-induced chondrocyte death and cartilage degeneration., Design: The phosphorylation of MAP kinases was examined with confocal microscopy and immunoblotting. The effects of MAP kinase inhibitors on impaction-induced chondrocyte death and proteoglycan (PG) loss were determined with fluorescent microscopy and 1, 9-Dimethyl-Methylene Blue (DMMB) assay. The expression of catabolic genes at mRNA levels was examined with quantitative real-time PCR., Results: Early p38 activation was detected at 20 min and 1h post-impaction. At 24h, enhanced phosphorylation of p38 and extracellular signal-regulated protein kinase (ERK)1/2 was visualized in chondrocytes from in and around impact sites. The phosphorylation of p38 was increased by 3.0-fold in impact sites and 3.3-fold in adjacent cartilage. The phosphorylation of ERK-1 was increased by 5.8-fold in impact zone and 5.4-fold in adjacent cartilage; the phosphorylation of ERK-2 increased by 4.0-fold in impacted zone and 3.6-fold in adjacent cartilage. Furthermore, the blocking of p38 pathway did not inhibit impaction-induced ERK activation. The inhibition of p38 or ERK pathway significantly reduced injury-related chondrocyte death and PG losses. Quantitative Real-time PCR analysis revealed that blunt impaction significantly up-regulated matrix metalloproteinase (MMP)-13, Tumor necrosis factor (TNF)-α, and ADAMTS-5 expression., Conclusion: These findings implicate p38 and ERK mitogen activated protein kinases (MAPKs) in the post-injury spread of cartilage degeneration and suggest that the risk of post-traumatic osteoarthritis (PTOA) following joint trauma could be decreased by blocking their activities, which might be involved in up-regulating expressions of MMP-13, ADAMTS-5, and TNF-α., (Copyright © 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Introduction: The present study examined the effect of collagen fragments on anabolic and catabolic activities by chondrocyte/agarose constructs subjected to dynamic compression., Methods: Constructs were cultured under free-swelling conditions or subjected to continuous and intermittent compression regimes, in the presence of the N-terminal (NT) and C-terminal (CT) telopeptides derived from collagen type II and/or 1400 W (inhibits inducible nitric oxide synthase (iNOS)). The anabolic and catabolic activities were compared to the amino-terminal fibronectin fragment (NH2-FN-f) and assessed as follows: nitric oxide (NO) release and sulphated glycosaminoglycan (sGAG) content were quantified using biochemical assays. Tumour necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) release were measured by ELISA. Gene expression of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), collagen type II and fibronectin were assessed by real-time quantitative polymerase chain reaction (qPCR). Two-way ANOVA and the post hoc Bonferroni-corrected t-test was used to examine data., Results: The presence of the NT or CT peptides caused a moderate to strong dose-dependent stimulation of NO, TNFalpha and IL-1beta production and inhibition of sGAG content. In some instances, high concentrations of telopeptides were just as potent in stimulating catabolic activities when compared to NH2-FN-f. Depending on the concentration and type of fragment, the increased levels of NO and cytokines were inhibited with 1400 W, resulting in the restoration of sGAG content. Depending on the duration and type of compression regime employed, stimulation with compression or incubation with 1400 W or a combination of both, inhibited telopeptide or NH2-FN-f induced NO release and cytokine production and enhanced sGAG content. All fragments induced MMP-3 and MMP-13 expression in a time-dependent manner. This effect was reversed with compression and/or 1400 W resulting in the restoration of sGAG content and induction of collagen type II and fibronectin expression., Conclusions: Collagen fragments containing the N- and C-terminal telopeptides have dose-dependent catabolic activities similar to fibronectin fragments and increase the production of NO, cytokines and MMPs. Catabolic activities were downregulated by dynamic compression or by the presence of the iNOS inhibitor, linking reparative activities by both types of stimuli. Future investigations which examine the signalling cascades of chondrocytes in response to matrix fragments with mechanical influences may provide useful information for early osteoarthritis treatments.

Objective and Design: We hypothesize that N-telopeptide (NT) and C-telopeptides (CT) of type II collagen can enhance proteinases and cause cartilage damage and have compared damaging activities to an extensively characterized potent fibronectin fragment (Fn-f)., Materials: NT and CT peptides were synthesized., Methods: Interaction of labeled peptides with chondrocytes was studied by fluorescence microscopy. Effects on the metalloproteinases (MMPs) MMP-3 and MMP-13 and on ADAMTS-5 were analyzed by western blotting. Cartilage damage was assayed by loss of proteoglycan (PG) from cultured explants., Results: NT and CT peptides penetrated cartilage, bound to chondrocytes and enhanced proteinase release and cartilage PG depletion. Peptides had detectable activity at 0.3 microM (1 microg/ml) and were comparable at 30 microM (100 microg/ml) to 1 microM Fn-f (29 microg/ml). However, while the Fn-f enhanced IL-1beta and TNF-alpha, the NT and CT peptides did not., Conclusions: Collagen peptides containing NT and CT regions were less active on a molar basis than Fn-fs but were still potent damaging agents. Since collagen fragments are found in OA cartilage at microg/ml, they have the potential to play a role in physiologic cartilage damage.

Objective: To define the role of mitogen activated protein (MAP) kinases in fibronectin fragment (Fn-f) mediated matrix metalloproteinase (MMP) upregulation and damage to bovine cartilage and to compare activities of three Fn-fs with native fibronectin (Fn), which is inactive in terms of cartilage damage., Methods: Bovine chondrocytes were cultured with three Fn-fs, an amino-terminal 29-kDa, a gelatin-binding 50-kDa and a central 140-kDa Fn-f or native Fn at concentrations from 0.01 to 1 microM, concentrations lower than those found in osteoarthritis synovial fluids. Lysates were probed for activation of MAP kinases, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and stress activated protein kinase/c-jun N-terminal kinase (SAPK/JNK). Confocal fluorescent microscopy was used to visualize movement of activated kinases. Kinase inhibitors were tested for their abilities to block Fn-f mediated protein upregulation of MMP-3 and MMP-13 and Fn-f induced depletion of cartilage proteoglycan (PG) from cultured explants., Results: The 29-kDa, the most potent Fn-f in terms of cartilage damage, enhanced phosphorylation of ERK1/2, p38 and JNK1/2 within a 1-h incubation while the 50 and 140-kDa Fn-fs required up to 4 h for maximal activity and native Fn was only minimally active toward p38 and JNK, but did strongly activate ERK1/2. The activated kinases displayed a distribution toward the nuclear membrane and within the nucleus. MAP kinase inhibitors markedly decreased Fn-f mediated upregulation of MMP-3 or MMP-13 and Fn-f mediated cartilage PG depletion., Conclusions: These results suggest that Fn-fs upregulate MMP-3 and MMP-13 in bovine chondrocytes through MAP kinases and that kinase inhibitors afford protection against this degenerative pathway.

Objective: To test the effectiveness of glucosamine (GluNH(2))-HCl, chondroitin sulfate (CS) and mixtures in protecting cartilage exposed to fibronectin fragments (Fn-fs), an exposure known to enhance catabolic cytokines and matrix metalloproteinases (MMPs)., Methods: Pharmacologic formulations of GluNH(2) (FCHG49) and CS (TRH122) (Nutramax Laboratories, Inc.) were added at 1, 10 or 100 microg/ml singly or in mixtures to bovine cartilage cultures in serum or serum-free conditions with or without Fn-f. Proteoglycan (PG) release into media and remaining cartilage PG content were measured by dye binding analysis and effects on PG synthesis by assays of 35-sulfate incorporation. Effects on MMP-3 and -13 expression were measured by Western blotting of conditioned media., Results: In serum-free conditions, the agents singly or as mixtures did not block Fn-f mediated matrix degradation. In serum, single agents were weakly effective at 100 microg/ml, while the mixture of each agent at 0.1 microg/ml decreased PG loss by about 50% by day 7 and at 1 microg/ml restored nearly 50% of the PG after 7 days in Fn-f pretreated cartilage. However, both agents singly and as mixtures at 0.1-100 microg/ml decreased MMP release. In serum, the single agents at 1-10 microg/ml weakly reversed Fn-f mediated PG synthesis suppression, while the mixtures were 100% effective at 1 microg/ml., Conclusions: GluNH(2) and CS act synergistically in reversing damage and promoting repair at concentrations found in plasma after oral ingestion of these agents. Reversal of PG synthesis suppression correlates more with these activities than suppression of MMP-3 or -13 expression.

Objective and Design: To determine whether the ability of high molecular weight hyaluronan (HA) to reverse cartilage damage caused by specific catabolic mediators of cartilage damage, fibronectin fragments (Fn-fs), occurs through a low grade of enhanced catabolic events such as enhanced matrix metalloproteinase (MMP) expression or cytokine activities., Material: HA from 6.8-kDa to 2 million daltons was studied., Treatment: The ability of HA to enhance matrix metalloproteinase-3 (MMP-3) epitopes and cartilage proteoglycan (PG) degradation neoepitopes was tested in bovine cartilage, as well as the ability of recombinant human interleukin-1 receptor antagonist protein (rhIRAP) to reverse PG depletion in cartilage first exposed to Fn-f., Results: All HA forms enhanced MMP-3 epitopes and PG degradation in normal undamaged cartilage and in the case of HA800, the degradation was not sufficient to decrease steady state levels of cartilage PG. When HA800 was added to Fn-f damaged cartilage, restoration of PG occurred, but this was blocked by rhIRAP., Conclusions: These results collectively suggest that some of the repair activity of HA800 is through proteolytic activity which is not sufficient to decrease matrix PG content, but is nonetheless elevated above levels in cartilage not treated with HA800.

Objective and Design: To test whether hyaluronan (HA) restores proteoglycan (PG) in fibronectin fragment (Fn-f) damaged cartilage through activities of insulin-like growth factor 1 (IGF-1)., Material: Bovine cartilage explants were studied., Methods: Cartilage PG content was measured by a dye binding assay. PG synthesis was measured by incorporation of radiolabeled sulfate. Release of IGF-1 from explants was measured by ELISA., Results: Higher mass HA forms were more reparative than smaller and enhanced PG synthesis in explants, but not in monolayer cultures. Higher mass HA enhanced PG synthesis and retention of IGF-1 within the explants while IGF-1 binding proteins decreased activities of HA., Conclusions: The reparative effects of HA appear to involve enhanced PG synthesis through IGF-1 and requires cartilage matrix., (Copyright 2004 Birkhäuser Verlag, Basel)

Objective and Design: To determine whether fibronectin fragments (Fn-f) upregulate nitric oxide (NO) and inducible NO synthetase (iNOS) levels in explants and whether iNOS inhibitors block Fn-f mediated cartilage damage or promote repair., Material: Bovine cartilage explants were studied., Treatment: Explants were cultured with Fn-f and involvement of NO pathway investigated., Methods: Induction of iNOS was tested by RT-PCR. Upregulation of iNOS protein was visualized by Western blotting. Proteoglycan (PG) content of cartilage treated with iNOS inhibitors was tested by dye binding assays of cartilage digests., Results: Fn-fs elevated NO release in explants and upregulated iNOS protein and mRNA while native Fn had only weak activity. Three different iNOS inhibitors blocked Fn-f mediated PG depletion from explants and facilitated restoration of PG in pre-damaged cartilage., Conclusions: The Fn-f cartilage damaging pathway involves upregulation of iNOS. Interestingly, iNOS inhibitors not only blocked damage but repaired damaged cartilage. This may be the first report of reparative activities of iNOS inhibitors., (Copyright 2004 Birkhäuser Verlag, Basel)

Objective: To compare the response of knee and ankle cartilages to fibronectin fragments (Fn-f) in terms of kinetics of matrix proteoglycan (PG) degradation and synthesis, since previous data had shown that knee was more sensitive to Fn-f in terms of steady-state PG content., Design: Human knee and ankle cartilage explants were treated with the 29kDa Fn-f, and its effects on PG-degradation kinetics, on the half-lives of 35S-sulfate-labeled PG, on PG synthesis suppression and on matrix metalloproteinase -3 (MMP-3) were compared. Cultures were also treated with the interleukin (IL) receptor antagonist protein (IRAP) in order to determine whether IL-1 is involved in the Fn-f effect., Results: The Fn-f enhanced PG-degradation rates in both human knee and ankle cartilages. Knee cartilage showed a greater effect of Fn-f on half-lives of newly synthesized 35S-labeled PG than ankle. The extent of release of MMP-3 was similar for human ankle and knee cartilages. However, PG synthesis in knee cartilage was sensitive to 10- to 100-fold lower concentrations of Fn-f than was ankle cartilage. IRAP partially reversed Fn-f activity in ankle cartilages., Conclusions: The role of Fn-f in proteolysis leading to cartilage damage appears to be minor in human cartilages than had previously been shown for bovine. This decreased proteolysis is true for both knee and ankle. The major difference between human ankle and knee cartilage appears to be greater sensitivity to PG synthesis suppression in knee cartilage. A further indication that IL-1 is involved in the pathway was provided by the partial reversal with IRAP.

Objective: The addition of exogenous high molecular weight hyaluronic acid (HA) reverses cartilage damage caused by fibronectin fragments (Fn-fs) added to explant cultures of bovine and human cartilage and by Fn-fs in an experimental in vivo model of rabbit knee joint damage. Our objective was to test whether HA was also effective in an IL-1 damage model and whether this repair was stable and occurred in older bovine cartilage., Design: Bovine cartilage explants from 18-month-old or 6-year-old bovines in 10% serum/Dulbecco's modified Eagle's medium were exposed to Fn-f or to IL-1 and the ability of 1mg/ml HA of 800 kDa to block damage or promote restoration of proteoglycan (PG) after the damage was measured. The damage phase as well as the exposure to HA were varied., Results: Exposure of exogenous HA decreased Fn-f-mediated damage, but did not decrease IL-1 beta-induced cartilage damage. If explants from 18-month-old bovines were damaged by a 7-day exposure to Fn-f or IL-1 beta and then exposed for 7 days to HA, PG was restored. This reparative activity persisted up to 4 weeks after the removal of HA from the culture medium. The restoration of PG did not occur in 0.1% serum-free cultures, was less when the exposure to the Fn-f was doubled and failed when exposure to IL-1 beta was doubled. In explants from 6-year-old bovines damaged with IL-1 beta for 7 days, HA fully restored PG content to normal levels., Conclusions: The reparative activities of HA occur not only in a Fn-f damage model, but also in an IL-1 damage model and occur with older bovine cartilage.

Objective: Intraarticular Na-Hyaluronate (HA) exerts a beneficial effect on adolescent rabbits after fibronectin fragment (Fn-f) mediated cartilage injury. We extended our studies to a population of rabbits which have reached full skeletal maturity., Design: Adult male NZW rabbits received an injury with Fn-f and no further treatment; an injection of HA followed by Fn-f injury, or Fn-f injury followed by a single or weekly intraarticular injection of HA. All animals were sacrificed 38 days after receiving the Fn-f injury. After sacrifice, proteoglycan (PG) content was determined from articular cartilage from the medial femoral condyles and tibial plateaus. The patellae were processed for histology., Results: Cartilage PG contents were significantly reduced after Fn-f injection (P=0.0167) and were only slightly improved with HA pre-treatment. However, post-treatment with HA resulted in significant improvements in cartilage PG content when compared to Fn-f only (single HA, P=0.01; weekly HA,P =0.01). Loss of Safranin-O staining, cell loss, osteophyte formation and inflammation were present in the patellae following Fn-f injection. Pre-treatment with HA reduced these changes. More significant protection of cartilage and restoration of Fn-f injury were noted in animals receiving post-treatment with HA., Conclusions: These results suggest that 38 days after Fn-f injury the lost PG content induced by Fn-f injection is substantially restored by weekly post-treatment with intraarticular HA in rabbits that have reached full skeletal maturity., (Copyright 2003 Published by Elsevier Science Ltd on behalf of OsteoArthritis Research Society International.)

Objective: To determine whether fibronectin fragments (Fn-f) known to enhance cartilage matrix degradation and to alter chondrocyte metabolism, bind on the chondrocyte cell surface close enough to the alpha(5)beta(1) fibronectin (Fn) receptor to be chemically cross-linked to it., Design: Biotinylated Fn-fs were added to chondrocytes, followed by cross-linking with dithiobissulfosuccinimidyl propionate, and the resultant alpha(5) complexes trapped on to antialpha(5)-agarose. Adherent material was analysed by probing with avidin-HRP. In a more specific approach in which only proximal targets could be cross-linked, photoaffinity labeled Fn-fs or Fn were added to cells, the derivatives activated and the cross-linked material analysed. Interaction of biotinylated Fn-fs and Fn with insolubilized alpha(5)beta(1) receptor was also visualized and quantified., Results: Biotinylated Fn-fs and Fn, but not a control of BSA, were cross-linked to alpha(5) protein in the presence of the propionate. Photoaffinity label Fn-f and Fn, but not BSA, were cross-linked to alpha(5) protein as well. Interaction was decreased by addition of an excess of unlabeled Fn-f or Fn. Fn-fs bound to alpha(5)beta(1)-agarose, although the affinity was 30-fold weaker and the stoichiometry 20-fold greater when the smallest Fn-f was compared to native Fn., Conclusions: These data are consistent with a role for the alpha(5) subunit in Fn-f activities and suggest that the Fn-fs bind proximal or directly to alpha(5) receptors. The weaker, higher stoichiometry interaction of Fn-fs with receptor suggests that fragmentation has allowed de novo interactions not possible in native Fn., (Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd.)

Unlabelled: Addition of fibronectin fragments (Fn-fs) to cultured cartilage explants has been shown to mediate extensive cartilage matrix degradation followed by anabolic responses., Objective: To determine whether specific Fn-fs regulate cartilage metabolism through a mechanism, in part, involving insulin-like growth factor (IGF) and insulin-like growth factor binding proteins (IGFBPs)., Methods: Primary bovine articular chondrocyte cultures were treated with Fn-fs. mRNA from the cultures was analysed by Northern blotting. Changes in the levels of IGFBPs in cellular extracts and conditioned media were analysed by Western ligand blotting. Explant cultures of bovine articular cartilage were used to assay release of exogenous IGF-I and IGFBP-2. An analog of IGF-I with altered affinity for IGFBPs was used to assay the effect of IGFBPs on proteoglycan synthesis., Results: The Fn-fs increased protein levels of IGFBPs-2, -3 and -5 in conditioned media and of IGFBP-2 in cell extracts by as much as nine-fold. Conversely, the protein level of constitutively expressed IGBP-4 was decreased in conditioned medium. Northern blot analysis reflected increased IGFBP-3 mRNA but not decreased IGFBP-4 mRNA. The IGF-I analog was more effective at restoring PG synthesis suppression by Fn-fs than was wild type IGF-I., Conclusions: The Fn-fs increased levels of IGFBPs in cultures of bovine articular chondrocytes and elicited release of IGFBP-2 and IGF-I from articular cartilage. The increased level of IGFBPs may trap IGF-I and account in part for the initial suppression of PG synthesis. Induced proteinases may subsequently liberate IGF-I and cause greatly enhanced anabolic processes, contributing to cartilage repair., (Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.)

Objective: To investigate involvement of the integrin alpha(5) subunit of the classical fibronectin receptor in cartilage chondrolytic activities of fibronectin fragments (Fn-f)., Design: Bovine chondrocytes and cartilage explants were cultured in the presence of antisense oligonucleotide (ASO), or sense (SO) or scrambled sequence oligonucleotide (SCO) corresponding to the bovine alpha(5) subunit. The effects of the oligonucleotides on mRNA and protein expression of the alpha(5) subunit were analysed by rtPCR and Western blotting, respectively. To test effects on Fn-f activities, three different Fn-f were first added to serum or serum-free cultures, followed by addition of oligonucleotides and the effects on Fn-f mediated proteoglycan (PG) degradation, cartilage PG depletion and PG and general protein synthesis suppression were tested., Results: The ASO decreased alpha(5) mRNA and protein expression to 69% and 55%, respectively, in monolayer cultures and decreased protein expression 67% in cartilage explants, while SO and SCO were ineffective. The ASO partially reversed the ability of the Fn-fs to suppress PG and general protein synthesis in cartilage explant and high density chondrocyte cultures. Concentrations of ASO from 1 nM to 5 microM effectively suppressed Fn-f activities in particular assays and the effects were reversible, while SO and SCO were not significantly effective. ASO also suppressed, in a dose-dependent and reversible fashion, the ability of the Fn-fs to enhance degradation and release of PG from cartilage explants. The ASO were also effective in suppressing the ability of an antibody to the alpha(5) subunit to enhance PG degradation, but were ineffective in blocking endotoxin or IL-1beta enhanced degradation., Conclusions: These data implicate the alpha(5) integrin subunit in Fn-f mediated activities, consistent with a role for the alpha(5)beta(1) integrin in this pathway., (Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.)

Objective: To investigate effects on cartilage metabolism and degeneration of injection of fibronectin fragments (Fn-fs) into rabbit knee joints., Design: The knees of adolescent New Zealand white rabbits were intraarticularly injected with rabbit Fn-fs. Cartilage sections from both injected and non-injected joints were treated with Safranin-O, with antibodies to the VDIPEN and NITEGE neoepitopes of degraded aggrecan and to matrix metalloproteinase-3 (MMP-3). Proteoglycan (PG) content of cartilage was measured by a dimethylmethylene blue assay of papain digests. PG synthesis rates were measured by(35)S-sodium sulfate incorporation into explanted cartilage., Results: In the injected joint cartilage, the Fn-fs bound cells in the upper superficial zone maximally between 6 and 24 h. By day 2, MMP-3 protein was enhanced and cartilage PG content and PG synthesis rates were reduced 40% and 70%, respectively. MMP-3 epitope and VDIPEN and NITEGE neoepitopes were also enhanced. The PG content then increased to supernormal levels from days 14 to 35 and then declined to normal levels by day 70, as did PG synthesis rates. In the non-injected joint cartilage, Fn-fs were not detected. Although MMP-3 expression was enhanced between days 2 and 21 as well as VDIPEN neoepitope, the PG content was never reduced but rather enhanced to supernormal levels from days 21 to 35. This was associated with enhanced PG synthesis by day 7, which decreased to control levels by day 70., Conclusions: In this cartilage degeneration model, loss of cartilage PG is followed by supernormal anabolic responses that facilitate PG restoration. Further, the damage causes a systemic effect of enhanced PG synthesis and content in the non-injected joint cartilage., (Copyright 2001 OsteoArthritis Research Society International.)

Catabolic cytokines play a major role in cartilage degradation not only in rheumatoid arthritis but also in osteoarthritis. Although the major source in rheumatoid arthritis may be mononuclear cells and synovial tissue and the cause of release may be multifactorial, the source of cytokines in osteoarthritis would be mostly from chondrocytes. However, there are few explanations of how upregulation of the cytokines might occur in osteoarthritis. One possibility is that degradation products of the extracellular matrix arising from elevated protease levels, substrate, or both, might regulate cytokine activities. Fragments of the extracellular matrix protein, fibronectin, upregulate cytokine expression and induce the events of suppressed matrix synthesis and upregulation of matrix metalloproteinases, characteristic of osteoarthritis. The catabolic aspects of this system are short term, subsequently serve to enhance anabolic processes above untreated levels, and condition the tissue against additional insult. It will be necessary to determine whether in vivo these degradation products precede cytokine expression and act early and are targets for intervention or instead are a consequence of cytokine damage. Whether they regulate anabolism and catabolism, blocking of their activities may not be ideal.

Hyaluronic acid, recently renamed hyaluronan, has been used as a therapeutic intervention in the treatment of osteoarthritis. We have reported that high-molecular-weight (800 kDa) hyaluronan is effective in blocking the catabolic action of fibronectin fragments in explant cultures of bovine cartilage and in an experimental in vivo model of damage to the rabbit knee joint. The fibronectin fragments induce catabolic cytokines in human cartilage, which, in turn, suppress proteoglycan synthesis and induce matrix metalloproteinases to decrease the proteoglycan content. Since the clinical target of high-molecular-weight hyaluronan is human cartilage, which may differ in certain ways from bovine cartilage, we tested the effect on human knee cartilage. We found that 1 mg/ml hyaluronan completely blocked fibronectin fragment-mediated decreases in proteoglycan content in five of five specimens of cartilage from the human knee. This was associated with binding of exogenous hyaluronan to the superficial surface, suppressed penetration of the fibronectin fragment into the cartilage, decreased expression for the first week in culture of one of the matrix metalloproteinases involved in cartilage degradation, matrix metalloproteinase-3, and proteoglycan synthesis rates that increased to supernormal levels. However, the appearance of the NITEGE and VDIPEN neoepitopes, indices of cartilage degradation, was not blocked but was delayed by 1 week. The addition of hyaluronan to cartilage previously damaged by the fibronectin fragments or to osteoarthritic cartilage fully restored the proteoglycan content to control levels. We conclude that hyaluronan blocked damage at least partly by blocking penetration of the fibronectin fragments and slowing matrix metalloproteinase expression. However, the major effect on blocking damage and promoting repair may be through enhanced proteoglycan synthesis, a mechanism that requires further study. Nonetheless, these data clearly demonstrate that hyaluronan completely protected human cartilage in explant culture and facilitated a full restoration of proteoglycan in damaged cartilage.

There are few candidates for biochemical pathways that either initiate or amplify catabolic processes involved in osteoarthritis (OA). Perhaps, one of the most likely sources for such pathways may be within the extracellular matrix itself. This review focuses on an example of how specific degradation products of the extracellular matrix of cartilage, produced during proteolytic damage, have the potential to enhance OA-like processes. In this example, these products can induce or activate other factors, such as catabolic cytokines, that amplify the damage. The damage, in turn, enhances levels of the degradation products themselves, as in a positive feedback loop. Since these products are derived from the cartilage matrix, they could be considered barometers of the health of the cartilage that signal to the chondrocyte, through outside to inside signaling, the health or status of the surrounding matrix. The best example and most characterized system is that of fragments of the matrix protein, fibronectin (Fn), although as discussed later, other recently discovered fragment systems may also have the potential to regulate cartilage metabolism. In the case of Fn fragments (Fn-fs), the Fn-fs enhance levels of catabolic cytokines as in OA and, thus, are potentially earlier damage mediators than catabolic cytokines. The Fn-fs up-regulate matrix metalloproteinase (MMP) expression, significantly enhance degradation and loss of proteoglycan (PG) from cartilage and temporarily suppress PG synthesis, all events observed in OA. However, this Fn-f system may be involved in normal cartilage homeostasis as well. For example, low concentrations of Fn-fs enhance anabolic activities and could play a role in normal homeostasis. This system may also be involved in not only amplifying damage but also coupling damage to repair. For example, high concentrations of Fn-fs that might arise in OA temporarily offset the anabolic response of lower Fn-f concentrations and cause short-term enhanced catabolic events that are followed by slowly increasing anabolic responses. Such effects would be expected for mediators with roles in regulation of metabolism in both normal or diseased cartilage. Other products of matrix degradation have also been shown to regulate cartilage metabolism. A common mechanistic theme to these systems may be that they perturb the cartilage matrix and directly or indirectly alter function of specific receptors involved in metabolism. These concepts illustrate the potential of the cartilage matrix to regulate its composition in both health and disease.

Objective and Design: The abilities of osteogenic protein-1 (OP-1) and TGF-beta1 to affect cartilage damage caused by fibronectin fragments (Fn-fs) that are known to greatly enhance cartilage proteoglycan (PG) degradation were compared., Material: Articular cartilage was obtained from 18 month old bovines., Treatment: To test blocking of damage, cartilage was cultured with or without OP-1 or TGF-beta in the presence of 100 nM Fn-fs. To test restoration of PG, cartilage was first cultured with Fn-fs and the cartilage then treated with factors., Methods: Cartilage PG content was measured in papain digests using the dimethylmethylene blue assay. PG synthesis was measured by incorporation of 35S labeled sulfate., Results: OP-1 blocked damage and restored PG in damaged cartilage, apparently due to enhanced PG synthesis. However, TGF-beta1 alone decreased PG content., Conclusions: These results clearly demonstrate differences between OP-1 and TGF-beta1, both members of the TGF-beta superfamily and illustrate the efficacy of OP- in blocking Fn-f mediated damage.

According to numerous cadaveric, radiographic, and clinical studies, ankle and knee joints differ in susceptibility to osteoarthritis. To test for biochemical differences in susceptibility to damage, a chondrocytic chondrolysis system has been utilized. In this system, fibronectin fragments are added to cultured cartilage explants, resulting in enhanced release of catabolic cytokines, induction of matrix metalloproteinases, temporary suppression of proteoglycan synthesis, and consequently, severe loss of cartilage proteoglycan. We found that the addition of an amino-terminal thrombin-generated 29-kDa fibronectin fragment to cultured knee cartilage from 14 donors (average age: 53 years) usually caused a 30-50% decrease in proteoglycan content by day 7. However, of the ankle cartilage specimens examined from 21 donors (average age: 50 years), only three showed damage by day 7, one by day 14, and six by day 21, and 11 were not damaged until day 28. For eight of the donors (average age: 44 years), both knee and ankle cartilages were obtained: this allowed comparison between tissues from the same donor. The analysis showed that the ankle cartilage was much more refractory to damage than was the knee cartilage from the same donor. These data clearly show differences between ankle and knee cartilage in susceptibility to the fibronectin fragments and suggest the feasibility of use of these fragments for discerning differences in homeostasis of the ankle and knee cartilage.

Objective: To investigate whether fibronectin fragments (Fn-fs), shown to damage cultured cartilage, can be found in cartilage from patients with osteoarthritis (OA) or rheumatoid arthritis, or can be generated from fibronectin (Fn) within synovial fluids or from Fn in the matrix of cultured cartilage. To also determine whether cartilage or synovial fluid Fn-fs are active and, thus, could contribute to cartilage damage in vivo., Methods: Fn-fs were immunochemically identified in cartilage extracts from patients with OA or rheumatoid arthritis or in bovine cartilage cultured with IL-1 alpha or in bovine synovial fluids treated with stromelysin-1 (MMP-3). The effect of removal of Fn-fs from OA synovial fluids was tested by passing fluids over an anti-Fn column and adding the resultant fluids to bovine cartilage cultures to measure proteoglycan (PG) degradation. Gelatin-Sepharose purified Fns from bovine plasma, synovial fluid or cartilage were digested with MMP-3 and the Fn-fs tested for degradation of PG in cultured cartilage., Results: Extracts of cartilage from patients with rheumatoid arthritis or with OA contained a range of Fn-fs. Removal of Fn-fs from OA synovial fluids significantly reduced the resultant damage when the fluids were added to cultured cartilage. Addition of IL-1 alpha to cultured cartilage or of MMP-3 to synovial fluids enhanced generation of Fn-fs. Fn-fs, whether derived from bovine plasma or synovial fluid or cartilage Fns, damaged cartilage., Conclusions: These data demonstrate that although Fn-fs could be generated in vivo within synovial fluids and Fn-fs found in OA synovial fluid may contribute to cartilage damage in vivo, Fn-fs could also be generated within cartilage and amplify cartilage damage. Thus, Fn-fs may be both autocrine and paracrine regulators of cartilage metabolism.

The addition of fibronectin fragments to cultured cartilage causes an initial suppression of proteoglycan synthesis, induction of matrix metalloproteinases, and resultant decrease in proteoglycan content by about 50% during the first few days in culture. Because the proteoglycan loss appears to be limited, we investigated whether the fibronectin fragments induce anabolic responses that might counter the damage. The effects of various lengths of exposure of cultured cartilage to the fibronectin fragment on proteoglycan content, proteoglycan synthesis rates, stromelysin-1 release, and tumor necrosis factor-alpha, interleukin-1alpha, and interleukin-6 release were investigated. The results showed that about 7 days of exposure of cultured cartilage to the fibronectin fragment was required for maximal cytokine release, proteoglycan depletion, and stromelysin-1 release. However, nearly maximal suppression of proteoglycan synthesis occurred within 1 day of the addition of the fibronectin fragment and, after its removal, the rates increased to supernormal levels. Decreasing exposure to 3 days caused only a small decrease in cartilage proteoglycan content, although stromelysin-1 release still occurred. Decreasing exposure to 1 day caused an immediate increase in proteoglycan synthesis and an increase to supernormal proteoglycan contents. The effect of first treating cartilage with the fibronectin fragment for various periods and then allowing a recovery was to make the cartilage more resistant to secondary exposures. This study shows that cartilage damage can be caused by short exposures to the fibronectin fragment and that exposures either optimal or suboptimal for damage additionally amplify anabolic processes to make the cartilage resistant to further damage and, thus, condition it against pending amplification of damage.

Objective and Design: The objective was to determine if agents that suppress catabolism might also enhance repair of irreversibly damaged cartilage., Material: Articular cartilage from bovine metacarpophalangeal joints was studied in explant culture., Treatment: Fibronectin fragments or IL-1 alpha, which potently cause proteoglycan (PG) loss from cartilage, were added to cultures also containing the catabolism-blocking agents: insulin-like growth factor-1, or N-acetylcysteine, or Arg-Gly-Asp-Ser peptide, and the effects of these agents on blocking PG loss determined. To test for repair or restoration of PG, cartilage was first damaged, damage agents removed and inhibitory agents added., Methods: Each mean and SD value for cartilage PG content was determined by assays of papain digests of cartilage from three similar cultures., Results: The agents either partially or fully blocked PG loss and promoted repair., Conclusions: Normally irreversible cartilage damage was reversed by slowing ongoing catabolic processes during attempted repair. Thus, catabolic inhibitors have reparative potential.

A commercial preparation of 800-kDa hyaluronic acid (HA), (ARTZ from Seikagaku, Inc.), has been used as a therapeutic intervention in the treatment of osteoarthritis (OA). We tested the effect of this HA form, HA/800, in an in vitro cartilage chondrolytic system in which a specific amino-terminal 29-kDa fragment of fibronectin (Fn-f) penetrates cartilage tissue to activate chondrocytes to amplify two major chondrolytic activities: suppression of proteoglycan (PG) synthesis and induction of matrix metalloproteinases. We report that HA/800 did not block damage by Fn-f in serum free cartilage cultures. However, HA/800 was effective in blocking the ability of 100 nM Fn-f to cause the degradation and release of half of the total cartilage PG from cartilage in 10% serum/DMEM cultures. While the Fn-f caused a half-time for PG release of 3 days, continuous exposure to 0.1 or 1 mg/ml HA/800 slowed the half-time to 12 days. Further, a single 1 day pre-incubation with 0.1 or 1 mg/ml HA/800 was sufficient to decrease the half-time of 100 nM Fn-f mediated PG depletion to 7 and 12 days, respectively. HA/800 completely blocked the effect of 10 nM Fn-f. Blocking of Fn-f-mediated cartilage PG depletion was associated with a decreased concentration of Fn-f on the superficial cartilage surface and decreased penetration into the cultured cartilage tissue. Further, the two major chondrolytic activities of the Fn-f, suppression of synthesis of PG and enhanced release of stromelysin-1, were suppressed by HA/800. HA/800 also partially restored PG in cartilage first damaged with the Fn-F. We conclude that HA/800 slows Fn-f-mediated cartilage chondrolysis in vitro and has some reparative potential. The damage blocking activity appears to be associated with the ability of HA/800 to block penetration of the Fn-f, rather than with direct effects on cartilage tissue.

Intra-articular sodium hyaluronic acid (HA) has been used as a treatment intervention in the management of osteoarthritis. It has been observed that HA can coat the articular surface, and thus, has been suggested to provide a possible prophylactic barrier for the articular cartilage. In an accompanying manuscript (Homandberg et al.), we report that a commercially available high-molecular-weight HA (approximately 800-kDa, ARTZ, Seikagaku Corp.) can partially block fibronectin fragment (Fn-f)-mediated cartilage injury in vitro. Herein we report a study of the effects of intra-articular HA on an in vivo animal model of Fn-f-mediated cartilage injury. Rabbit knees were injected with Fn-f, and after 1 week, the cartilage proteoglycan (PG) content had decreased to 59 +/- 8% of control. In sharp contrast, PG content in knees receiving pre-treatment with HA followed by Fn-f injection had only decreased to 85 +/- 27% of control (P < 0.01). Similarly, the PG content in knees receiving an injection of Fn-f, followed by an injection of HA were significantly higher (74 +/- 18% of control) than Fn-f injured knees with no treatment (P < 0.02). Intra-articular HA alone had no effect on cartilage PG content. The results in this study suggest that HA is effective in partially preventing Fn-f mediated cartilage injury, most likely by coating the articular surface. Further, HA treatment after Fn-f injury may facilitate restoration of matrix components.

Fibronectin fragments have both catabolic and anabolic activities toward articular cartilage explants in vitro. Whereas a 1 nM concentration of an N-terminal 29 kDa fibronectin fragment (Fn-f) increases the proteoglycan (PG) content of cartilage without induction of matrix metalloproteinases (MMPs), 0.1-1 microM Fn-f temporarily suppresses PG synthesis and enhances MMP release. The higher concentrations cause an initially rapid PG depletion during the first week of culture, followed by much slower PG loss and gradually increasing rates of PG synthesis. To test for the involvement of mediators, human articular cartilage was cultured with Fn-f, and conditioned media were assayed for selected cytokines and factors. With 1 nM Fn-f, the release of the anabolic factors, insulin growth factor-I and transforming growth factor beta1, from cultured cartilage was enhanced by 50-100% during the entire 28-day culture period and this was associated with both supernormal rates of PG synthesis and PG content. However, the higher concentrations of Fn-f additionally enhanced release, by at least 10-fold, of the cytokines, tumour necrosis factor alpha, interleukin-1alpha, interleukin-1beta and interleukin-6 while causing depletion of cartilage PG. Release of tumour necrosis factor alpha, interleukin 1beta and interleukin 1alpha peaked at days 2, 3 and 9 during or slightly after the period of maximal PG depletion and decreased to control levels by days 7, 7 and 21 respectively, whereas release of interleukin 6 was enhanced throughout the culture period. Neutralizing antibodies to the catabolic cytokines reduced Fn-f-mediated MMP-3 release and suppression of PG synthesis. The temporal aspects of this interplay between catabolic and anabolic factors are consistent with the kinetics of Fn-f-mediated cartilage damage and attempted repair and may be relevant to cartilage damage and repair in vivo.

In an accompanying manuscript, it was shown that the cartilage chondrolytic activities of fibronectin fragments (Fn-f), which are mediated through catabolic cytokines such as TNF-alpha, IL-1 and IL-6, could be suppressed by anti-oxidants (AOs). The AOs neutralized reactive oxygen species (ROS) which are known to mediate catabolic cytokine action. The objective in this work was to test whether AOs would promote restoration of proteoglycan (PG) in Fn-f treated cartilage, since under normal culturing conditions, PG is not restored after removal of the Fn-f. Cartilage was first cultured with an amino-terminal 29-kDa Fn-f to cause loss of about half of the total PG and then treated with NAC (1 and 10 mM) or glutathione (10 microM) or DMSO (0.1 or 1%). Treatment with NAC and glutathione maximally caused restoration of PG within 14 days to normal or supernormal levels, while DMSO was less effective. Catalase, but not superoxide dismutase, enhanced PG content to a small but significant extent. The restoration of PG in Fn-f treated cartilage occurred throughout the full depth of the cartilage slices as shown by histochemical analysis. However, removal of the AO allowed a subsequent decrease in PG content suggesting that the AOs had not blocked cytokine expression but had merely suppressed cytokine activities. Addition of NAC to IL-1 treated cartilage promoted a restoration of PG, while addition to chymopapain or trypsin treated cartilage was not very effective, suggesting that the effect of AOs requires a cytokine driven damage system. We conclude that the AOs promote a restoration of PG in the Fn-f treated cartilage by suppressing the effects of catabolic cytokines. The data suggest a potential for AOs in reversing tissue damage caused by cytokines.

Fibronectin fragments damage cartilage in vitro by greatly enhancing metalloproteinases and suppressing proteoglycan (PG) synthesis which results in severe cartilage PG depletion. Since reactive oxygen species (ROS) have been implicated in catabolic cytokine action and preliminary data suggested that catabolic cytokines such as TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 are responsible for fibronectin fragment mediated damage, selected anti-oxidants (AOs) were tested as inhibitors of cytokine. ROS and fibronectin fragment activity. Damage was measured by depletion of cartilage PG during tissue culture. The AO, N-acetylcysteine (NAC), decreased the extent of cartilage PG depletion caused by TNF-alpha and IL-1 alpha and by the ROS, hydrogen peroxide and superoxide anion, confirming that the cytokines operate through ROS and that ROS can initiate cartilage PG depletion. NAC at 0.1 and 1 mM, totally suppressed PG depletion caused by a highly potent amino-terminal 29-kDa fibronectin fragment (Fn-f) for 14 days in culture. NAC at 10 mM totally blocked Fn-f mediated PG depletion for 21 days and increased the cartilage PG content by 30% above normal levels. Glutathione (10 microM) and DMSO (1%) were also totally effective while catalase and superoxide decreased Fn-f mediated damage only during the first week and superoxide dismutase alone caused damage after 1 wk. The AOs caused protection by reducing the major catabolic activities of the Fn-f: enhanced release of stromelysin-1 (MMP-3) and suppression of PG and protein synthesis. NAC also decreased normal rates of PG degradation and increased the half-lives of labeled PG in both control and Fn-f treated cartilage. We conclude that the Fn-f mediates cartilage chondrolysis through ROS, consistent with the involvement of catabolic cytokines in the Fn-f mechanism, and that AOs greatly reduce Fn-f mediated cartilage chondrolysis. In an accompanying manuscript we also report that AOs promote reparative responses in Fn-f and cytokine treated cartilage.

Addition of fibronectin fragments to bovine articular cartilage explant cultures results in enhanced release of metalloproteinases and rapid cartilage proteoglycan (PG) degradation and loss. The chondrolysis begins with rapid PG degradation which markedly slows after 1 week. Preliminary observations suggest that catabolic cytokines mediate chondrolytic activities of the fibronectin fragments. The objectives of this work were to investigate the correlations between: (a) release of specific cytokines; (b) release of the metalloproteinase (MMP), stromelysin-1 (MMP-3); (c) release of the tissue inhibitor of MMPs, TIMP-1, and; (d) degradation and release of PG from cultured cartilage. We report that human articular cartilage cultured with an amino-terminal 29-kDa fragment (Fn-f) at 0.1 microM, released enhanced levels of TNF-alpha, IL-1beta, and IL-1alpha with peaks at Days 2, 3, and 9, respectively. MMP-3 release was elevated with a peak at Day 6 and a profile similar to that for the Fn-f-induced cartilage PG depletion. IL-6 release was enhanced within 2 days and continued at the same level throughout the culture period but this did not lead to enhanced release of TIMP-1, a known activity of IL-6. These data suggest that in the early chondrolytic events induced in cultured cartilage by Fn-f, enhanced MMP-3 release and maximal degradation and release of PG from cultured cartilage are kinetically associated with elevated release of the catabolic cytokines, TNF-alpha, IL-1beta, and IL-1alpha. Further, a later period of slowing PG loss and slowing MMP-3 release is associated with greatly slowed release of these cytokines, but prolonged release of IL-6. This model of cartilage damage may be useful for studies of the interplay between cytokines and the effects of combinations of cytokines on cartilage homeostasis.

Addition of proteolytically generated fibronectin fragments (Fn-f) to cultured cartilage tissue causes greatly enhanced release of metalloproteinases (MMPs), such as pro-stromelysin-1 (proSln-1), and suppression of proteoglycan (PG) synthesis, through release of catabolic cytokines, while native fibronectin is ineffective. We have investigated whether enhanced release of proSln-1 was due to up-regulation of pro-Sln-1 mRNA. We report the addition of a 29-kDa (amino-terminal heparin-binding Fn-f) or a 140-kDa (central cell-binding Fn-f) to bovine chondrocytes in monolayer culture causes a dose dependent increase in the expression of pro-Sln-1 mRNA and the greatly enhanced release of pro-Sln-1 protein into the culture media. Up to 700 nM pro-Sln-1 was found in the conditioned media and metabolic labeling showed that it constituted a major portion of newly synthesized protein. A potential activator of pro-Sln-1, urokinase (u-PA), was released at elevated levels in the presence of the Fn-f while other activators, tissue plasminogen activator (t-PA) and plasmin activities were not detected. Addition of these activators to conditioned media did not allow conversion of pro-Sln-1 to active Sln-1. However, aminophenyl mercuric acid activated pro-Sln-1 to a 48-kDa Sln-1 form capable of degrading PG when added to cartilage suspensions. Gelatinase A mRNA was also enhanced, suggesting that the Fn-f may induce MMPs in general. However, the major regulator of Sln-1 activity, tissue inhibitor of MMPs form 1 (TIMP-1), was not induced at the gene level. Thus, a major effect of Fn-f on chondrocytes is to up-regulate pro-Sln-1 expression at the gene level, resulting in pro-Sln-1 as a major protein product.

We reported earlier that Fn fragments (Fn-f) added to bovine articular cartilage cultured in serum-free DMEM cause marked elevated release of protease activity within a few days. This results in greatly elevated rates of release of proteoglycan (PG). We have now extended our studies to 4-week cultures of cartilage in the presence of 10% serum. We report here that cartilage cultured with 0.01, 0.1, and 1 microM of an amino terminal 29-kDa Fn-f in 10% serum accelerated the loss of PG from the tissue during the first few days in a concentration-dependent manner. However, beyond this period PG content decreased much more slowly. During this early period, the 29-kDa Fn-f decreased rates of protein and PG synthesis up to 50% in a concentration-dependent fashion. Beyond this period, the synthesis rates began to increase in a mode inversely related to 29-kDa Fn-f concentration, up to 135% of Fn-f free control values. However, during the entire culture period, cartilage cultured with 1 nM 29-kDa Fn-f had higher PG contents and had enhanced rates of protein and PG synthesis. Since 1 nM 29-kDa Fn-f stabilized cartilage against decreases in PG content, we tested its ability to block the activity of higher 29-kDa Fn-f concentrations. Cartilage was preincubated for 7 days with 1 nM 29-kDa Fn-f, and then the culture adjusted to 100 nM 29-kDa Fn-f to cause PG depletion. The preincubated cartilage showed markedly enhanced resistance to PG depletion. Since the protective effect was similar to known properties of IGF-1, the ability of 20 ng/ml IGF-1 to block against the effects of 100 nM Fn-f was tested and shown to be similar in magnitude to that of 1 nM 29-kDa Fn-f. We propose that the initial catabolic effects of higher concentrations of Fn-f, followed by the later anabolic effects, may aid in tissue repair. Also, the continuous anabolic effects of lower concentrations may be involved in tissue homeostasis.

We have reported that three different Fn fragments (Fn-f) added to bovine articular cartilage cultured in serum-free DMEM cause marked elevation of proteoglycan (PG) degradation and release into the culture media. We report here that the PG release required the continual presence of Fn-f, that PG release still occurred when serum-free cultures were switched to bovine synovial fluid media, and that addition of recombinant IGF-1, TGF-beta, and recombinant interferon gamma to cultures did not affect Fn-f-mediated PG release. The Fn-f caused a 25-fold enhanced release of stromelysin-1 protein from cartilage by Day 1 and up to 120-fold by Day 3. The stromelysin form released was 43 kDa, the activated form of pro-stromelysin-1. This stromelysin form apparently played a major role in Fn-f-mediated PG release, since addition of Sepharose-bound anti-stromelysin-1 to cartilage cultures greatly slowed rates of PG release. Potential activators of pro-stromelysin-1, plasmin, and u-PA (urinary plasminogen activator), were also detected in conditioned media of Fn-f-treated cartilage. u-PA levels were increased in the presence of the Fn-f but by only a few fold. Addition of alpha-1-antiproteinase inhibitor, which can block enzymatic activity of u-PA, was found to inhibit about half the PG-releasing activity of the Fn-f. Levels of TIMP-1, the 30-kDa tissue inhibitor of metalloproteinases, which can inhibit stromelysin, doubled within 24 h when a Fn-f was added to culture. These data suggest that stromelysin-1 may be a major mediator of Fn-f-mediated PG release from cartilage.

We have reported that Fn fragments (Fn-f), which have been detected in synovial fluids of osteoarthritis and rheumatoid arthritis patients, can potently cause cartilage chondrolysis and depress proteoglycan (PG) synthesis in cartilage tissue cultured as explants. Amino-terminal 29-kDa, gelatin-binding 50-kDa, and integrin-binding 140-kDa Fn-f are active. In order to investigate the mode of action and devise means of blocking the damage mediated by all Fn-f, we have tested the effects of various analogs resembling the integrin binding sequence, Arg-Gly-Asp-Ser, on blocking Fn-f-mediated chondrolysis. The analog peptides, Gly-Arg-Ala-Asp-Ser-Pro-Lys and Arg-Phe-Asp-Ser, at concentrations as low as 1 microM, blocked the effects of all three Fn-f on cartilage degradation, while the native sequence peptide, Arg-Gly-Asp-Ser, had very low Fn-f-blocking activity and by itself caused cartilage damage. Random sequence peptides dissimilar to the analog sequences were inactive as inhibitors as well as was a sequence analog, Phe-Asp-Arg-Ser, related to the Arg-Phe-Asp-Ser inhibitor. The analog inhibitory peptides decreased rates of Fn-f-mediated PG degradation and release from cartilage and decreased Fn-f-mediated PG synthesis depression. The analog inhibitory peptides alone had no detectable effect on cartilage PG degradation or PG synthesis rates. These data show that the chondrolytic activities of integrin-binding and nonbinding Fn-f can be blocked by synthetic peptide analogs of the Arg-Gly-Asp-Ser sequence and suggest that these peptides may be useful for blocking other activities of Fn-f.

We reported earlier that fibronectin fragments (Fn-f) added to bovine articular cartilage cultured in serum-free culture causes marked protease expression with resultant proteoglycan (PG) degradation and release into the culture media. We have further characterized the effects of Fn-f by studies of the effects on proteoglycan, collagen, general protein, and DNA synthesis and reversibility of the cartilage damage. We report here that the most active Fn-f, a 29-kDa amino-terminal Fn-f, when added to a 1 microM concentration, depressed PG and general protein synthesis in cartilage by over 50% within 24 h, as measured by sulfate and methionine/cysteine incorporation, respectively. This same Fn-f decreased PG synthesis throughout the full thickness cartilage section as shown by autoradiography. PG and general protein synthesis were significantly depressed within 24 h by 29-kDa Fn-f concentrations as low as 10 nM. Synthesis rates were effected by 100-fold lower Fn-f concentrations than was induction of proteinases. Removal of the 29-kDa Fn-f allowed a gain to supernormal levels of PG and protein synthesis. Cartilage damaged to the extent of removal of over 50% of the total PG did not replace PG after over 4 weeks in 10% serum-Dulbecco's modified Eagle minimum with or without added TGF-b1 and rIGF-a. These data show that while the effects of Fn-f on elevating protease expression and depressing PG synthesis are reversible, the resultant cartilage damage is apparently irreversible in vitro. Therefore, if Fn-f-mediated cartilage damage occurs as part of cartilage disease processes, the pathologic effects would be quite significant.

We have reported that fibronectin (Fn) fragments added to bovine articular cartilage slices in culture causes marked cartilage damage by enhancing proteinase expression and resultant degradation and release of proteoglycan (PG). Several different non-overlapping Fn fragments, an amino-terminal 29-kDa, gelatin-binding 50-kDa and integrin-binding 140-kDa Fn fragment, representing nearly all of the polypeptide chain, were compared in terms of ability to cause PG release from cartilage and to bind cartilage. The most active fragment, the 29-kDa fragment, was able to enter cartilage in an intact metacarpophalangeal joint in culture and cause PG release at the same rate as with surgically cut cartilage. Further, when radiolabelled 29-kDa fragment was added to cartilage, a large proportion bound the intact articular surface, while a lesser amount diffused throughout the tissue matrix and concentrated in clusters near the mid-section of full thickness cartilage. The 29-kDa, 50-kDa, 140-kDa Fn fragments and Fn, respectively, showed PG degradation activities 9-, 6-, 2- and 1.1-fold that of control levels and bound cartilage to the extent of 180, 20, 18 and 2 pmol/100 mg cartilage, respectively. Therefore, the PG degradation activities were greatest for the smaller fragments, which bound to the greatest extent. The apparent Kd values for interaction of the 29-kDa, 50-kDa, 140-kDa fragments and Fn for cartilage tissue were about 1.2, 0.3, 0.1 and 0.02 microM, respectively, and the order was inversely related to PG degradation activities. We conclude that the smaller the Fn fragment, the greater the degradation activity and extent of binding to cartilage tissue, but the weaker the affinity.

We have reported that fibronectin fragments (Fn-f) cause cartilage damage in vitro by causing enhanced release of proteases. In order to determine whether the Fn-f can damage cartilage in vivo, we have injected native fibronectin (Fn) and Fn-f into adolescent rabbit knee joints. After 7 days, tissue was analyzed by histochemical and biochemical techniques and remaining proteoglycans quantified. Injection of 0.6 or 3 microM Fn-f caused up to a 70% loss in total cartilage proteoglycan while native Fn, rabbit serum albumin or an Arg-Gly-Asp-Ser synthetic peptide, derived from the cell-binding domain of Fn, did not cause damage. Our results suggest that this Fn-f/damage model may be useful for generating cartilage damage in vivo for other studies. Since Fn-f have been detected in synovial fluids from joints of patients with rheumatoid arthritis and osteoarthritis, our results are consistent with the notion that Fn-F mediated damage may occur in vivo.

Fibronectin is an adhesive multifunctional glycoprotein found in the extracellular matrix of most types of cells and that exerts growth factor, differentiative and chemotactic activities toward many types of cells, including those cells found in knee joint tissue. Since fibronectin levels in the synovial fluid (SF) and on the cartilage surface of patients with osteoarthritis (OA) have been shown to be greatly increased over normal levels and since protease levels are also enhanced in diseased cartilage, we have investigated the presence of fibronectin fragments in the SF of patients with OA. We report that concentrations of at least 1 microM of 100 to 200 kDa fragments were found in all OA fluids examined. Since we have recently shown that fibronectin fragments can cause cartilage to release metalloproteinases, resulting in severe proteoglycan depletion, and others have shown that fragments also enhance metalloproteinase expression in synovial fibroblasts, the presence of these fragments suggests pathologic consequences in arthritis.

Elevated fibronectin (Fn) and Fn fragment concentrations are found in the synovial fluid of osteoarthritic and rheumatoid arthritic patients. Fn has been shown to affect expression of chondrocytic matrix proteins, and Fn fragments have been shown to elevate gene expression of neutral proteinases in synoviocytes. For these reasons, we tested the effects of Fn fragments on protease release and resultant proteoglycan release from cartilage in serum-free bovine articular cartilage explant cultures. We have found that 1 microM amino-terminal 29- and 50-kDa gelatin-binding Fn fragments caused over a 50-fold enhancement of gelatinolytic and collagenolytic proteinase release with a 23-fold enhancement of proteoglycan (PG) release. Release was significant at fragment concentrations as low as 20 nM. An integrin-binding 140-kDa fragment mixture was the least active fragment, whereas native Fn had little activity. The relative activities of the fragments correlated with their relative abilities to bind to cartilage. The RGDS integrin-recognition peptide also caused release, although sequence mutants did not. PG release was blocked by actinomycin D, cycloheximide, and deoxyglucose. Fn fragment-mediated PG release was decreased in 10% serum by over 10-fold but was still 2-fold greater than in controls. In the presence of insulin-like growth factor-1, PG release was as great as without serum. We suggest that Fn fragments, as found in diseased synovial fluid, may contribute to protease-mediated damage to cartilage.

Extracts of cartilage have been reported to inhibit many serine proteinases and metalloenzymes. Such inhibition may be important in protecting cartilage against degradation by chondrocytic proteinases such as collagenase, stromelysin and by leukocytic proteases, such as elastase. We report here isolation and partial characterization of a 17-kD elastase inhibitor from 0.5 M NaCl extracts of both nasal septum cartilage and articular cartilage, which inhibits elastase and represents 0.08% of the weight of nasal cartilage and 0.002% of the weight of articular cartilage. The protein was highly specific for elastase and did not inhibit cartilage metalloproteinases, suggesting that it may be mainly directed toward protecting cartilage against leukocytic proteases. The inhibitor had a blocked amino-terminus, was high in serine and glycine and lacked carbohydrate. The ease with which the inhibitor was extracted from cartilage suggests that it may function in vivo as a highly abundant elastase inhibitor which is secreted into synovial fluid from cartilage. The inhibitor was shown to be synthesized by bovine articular cartilage in explant culture and nearly all of the metabolically labeled material was secreted into the culture media. The inhibitor cross-reacted with polyclonal antibodies to bovine neck ligament alpha-elastin and antibodies to the inhibitor reacted with bovine neck ligament elastin. The properties of this inhibitor are different than those of any other reported cartilage derived inhibitor.

Neutrophil elastases are serine proteinases released during acute and chronic inflammatory states. We have developed a novel isolation method for neutrophil elastase, involving conventional gel chromatography followed by adsorption of protein at low ionic strength on a high-performance liquid chromatography gel permeation column. The bound elastase is then eluted by application of higher ionic strength. This adsorption step at low ionic strength, a step to be avoided in most purification methods, was used to advantage here to allow isolation of homogeneous material. This purification procedure should be useful for quick, simple bulk preparation of the enzyme.

Low molecular weight two-chain urokinase is a 33-kD plasminogen activator, which has no innate affinity for fibrin and consequently, its use to facilitate lysis of blood clots may lead to systemic activation of plasminogen. In order to impart clot affinities to this urokinase form (UK) we have generated two novel fibrin-binding derivatives by partially reducing UK and exchanging the native disulfide-linked peptide A with peptide A analogs. The peptide A analogs contained the fibrin-adherent fibrin-derived sequences, GPRP (derived from positions 17-20 of the fibrinogen alpha chain) or QAGDV (407-411 sequence of the fibrinogen gamma chain), each coupled through amino-hexanoic acid to a synthetic peptide, LKFQCGQK, containing the Leu 144-Lys 158 sequence of the urinary plasminogen activator A Chain. The resultant derivatives contained about 0.4 moles peptide analog/mole UK, were 75% active toward synthetic UK substrates, and were recovered in a nearly 80% yield. The two fibrin peptide derivatives had a five-fold greater affinity for the clots.

Abbokinase is a commercially available two-chain lower molecular weight urinary plasminogen activator (u-PA) with low affinity for fibrin. Therefore, therapeutic use of this u-PA form (UK) may cause activation of not only clot-bound plasminogen but also systemic plasminogen. Since this form of UK contains a 13-residue peptide remnant of the A chain disulfide-linked to the catalytic B chain, disulfide-exchange could allow replacement of this native peptide with peptides of desired clot-directed properties. We report here a method in which native peptide was partially removed by subjecting UK to 1-100 mM dithiothreitol (DTT), in the presence of arginine. Arginine (250 mM) was found to slow the rate of release of the peptide by 2-fold, to slow the loss in enzymatic activity by about 9-fold and to limit the extent of disulfide reduction. Upon dialysis to remove DTT from the reduced UK mixture, the disulfides reformed and enzymatic activity was regained. Synthetic peptide added at this point became incorporated to the level of about 0.4 mol/mol UK. This method should be very useful for developing UK derivatives with altered affinities toward fibrin clots.