Sulfated Glycosaminoglycan Release Research Articles

Inflammatory cytokines and mechanical injury induce post-traumatic osteoarthritis-like changes in a human cartilage-bone-synovium microphysiological system

BackgroundTraumatic knee injuries in humans trigger an immediate increase in synovial fluid levels of inflammatory cytokines that accompany impact damage to joint tissues. We developed a human in vitro cartilage-bone-synovium (CBS) coculture model to study the role of mechanical injury and inflammation in the initiation of post-traumatic osteoarthritis (PTOA)-like disease.MethodsOsteochondral plugs (cartilage-bone, CB) along with joint capsule synovium explants (S) were harvested from 25 cadaveric distal femurs from 16 human donors (Collin’s grade 0–2, 23–83years). Two-week monocultures (cartilage (C), bone (B), synovium (S)) and cocultures (CB, CBS) were established. A PTOA-like disease group was initiated via coculture of synovium explants with mechanically impacted osteochondral plugs (CBS+INJ, peak stress 5MPa) with non-impacted CB as controls. Disease-like progression was assessed through analyses of changes in cell viability, inflammatory cytokines released to media (10-plex ELISA), tissue matrix degradation, and metabolomics profile.ResultsImmediate increases in concentrations of a panel of inflammatory cytokines occurred in CBS+INJ and CBS cocultures and cultures with S alone (IL-1, IL-6, IL-8, and TNF-α among others). CBS+INJ and CBS also showed increased chondrocyte death compared to uninjured CB. The release of sulfated glycosaminoglycans (sGAG) and associated ARGS-aggrecan neoepitope fragments to the medium was significantly increased in CBS and CBS+INJ groups. Distinct metabolomics profiles were observed for C, B, and S monocultures, and metabolites related to inflammatory response in CBS versus CB (e.g., kynurenine, 1-methylnicotinamide, and hypoxanthine) were identified.ConclusionCBS and CBS+INJ models showed distinct cellular, inflammatory, and matrix-related alterations relevant to PTOA-like initiation/progression. The use of human knee tissues from donors that had no prior history of OA disease suggests the relevance of this model in highlighting the role of injury and inflammation in earliest stages of PTOA progression.

Clusterin secretion is attenuated by the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α in models of cartilage degradation.

The protein clusterin has been implicated in the molecular alterations that occur in articular cartilage during osteoarthritis (OA). Clusterin exists in two isoforms with opposing functions, and their roles in cartilage have not been explored. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone that prevents protein aggregation, enhances cell proliferation and promotes viability, whereas nuclear clusterin acts as a pro-death signal. Therefore, these two clusterin isoforms may be putative molecular markers of repair and catabolic responses in cartilage and the ratio between them may be important. In this study, we focused on sCLU and used established, pathophysiologically relevant, in vitro models to understand its role in cytokine-stimulated cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and isolated unpassaged chondrocytes was analyzed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α. Release of sulfated glycosaminoglycans (sGAG) was determined using the dimethylmethylene blue assay. Clusterin messenger RNA (mRNA) expression was quantified by quantitative real-time polymerase chain reaction. MMP-3, MMP-13, COMP, and sGAG release from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. sCLU release was attenuated with cytokine treatment in all models, potentially limiting its cytoprotective function. Clusterin mRNA expression was down-regulated 7-days post cytokine stimulation. These observations implicate sCLU in catabolic responses of chondrocytes, but further studies are required to evaluate its role in OA and its potential as an investigative biomarker.

In vitro chondroprotective potential of Senna alata and Senna tora in porcine cartilage explants and their species differentiation by DNA barcoding-high resolution melting (Bar-HRM) analysis.

Senna species and anthraquinone derivatives generated by these organisms, rhein and aloe-emodin, exert anti-inflammatory effects. These species present a similar morphology but produce different ingredients when they are used as medicinal products. In this study, a DNA barcoding- (Bar-) high-resolution melting (HRM) technique was developed using internal transcribed sequence 2 (ITS2) to differentiate between Senna alata and Senna tora as a result of significant differences in their melting profiles. We used this approach for confirmation of S. alata and S. tora raw materials, and we examined the chondroprotective properties of the ethanolic extracts of S. alata and S. tora using a porcine model of cartilage degradation induced by a combination of interleukin-17A (IL-17A) and IL-1β. We found that both Senna ethanolic extracts, at a concentration of 25 μg/mL, effectively prevented cartilage degradation. Rhein and aloe-emodin were present in the extract of S. alata but not in that of S. tora. We observed a reduction in the release of sulfated glycosaminoglycans (S-GAGs) and hyaluronic acid (HA) into media in both treatments of Senna extracts, which indicated proteoglycan preservation in explant tissues. These results suggest that neither rhein nor aloe-emodin are the main factors responsible for cartilage-protecting properties. Taken together, results show that both S. alata and S. tora are promising for further development as anti-osteoarthritic agents and that Bar-HRM using ITS2 could be applied for species confirmation with Senna products.

Coculture of bovine cartilage with synovium and fibrous joint capsule increases aggrecanase and matrix metalloproteinase activity

BackgroundA hallmark of osteoarthritis is increased proteolytic cleavage of aggrecan. Cross talk between cartilage and the synovium + joint capsule (SJC) can drive cartilage degradation by activating proteases in both tissues. We investigated aggrecan proteolysis patterns in cartilage explants using a physiologically relevant explant model of joint injury combining cartilage mechanical compression and coincubation with SJC.MethodsBovine cartilage explants were untreated; coincubated with SJC; or subjected to mechanical injury and coincubated with SJC, mechanical injury alone, or mechanical injury and incubated with tumor necrosis factor-α (TNF-α). To compare the patterns of aggrecan proteolysis between 6 h and 16 days, release of sulfated glycosaminoglycans and specific proteolytic aggrecan fragments into medium or remaining in cartilage explants was measured by dimethylmethylene blue and Western blot analysis.ResultsAggrecanase activity toward aggrecan was observed in all conditions, but it was directed toward the TEGE↓ARGS interglobular domain (IGD) site only when cartilage was coincubated with SJC or TNF-α. Matrix metalloproteinase (MMP) activity at the aggrecan IGD site (IPES↓FFGV) was not detected when cartilage was exposed to TNF-α (up to 6 days), but it was in all other conditions. Compared with when bovine cartilage was left untreated or subjected to mechanical injury alone, additional aggrecan fragment types were released into medium and proteolysis of aggrecan started at an earlier time when SJC was present.ConclusionsIndicative of different proteolytic pathways for aggrecan degradation, the SJC increases both aggrecanase and MMP activity toward aggrecan, whereas TNF-α inhibits MMP activity against the IGD of aggrecan.

Chondroprotective Effect of Kartogenin on CD44-Mediated Functions in Articular Cartilage and Chondrocytes.

Objective:A recent report identified the small molecule kartogenin as a chondrogenic and chondroprotective agent. Since changes in hyaluronan metabolism occur during cartilage degeneration in osteoarthritis, we began studies to determine whether there was a connection between extracellular hyaluronan, CD44–hyaluronan interactions and the effects of kartogenin on articular chondrocytes.Methods:Chondrocytes cultured in monolayers, bioengineered neocartilages, or cartilage explants were treated with kartogenin with or without stimulation by IL-1β. Accumulation of matrix was visualized by a particle exclusion assay or by safranin O staining and release of sulfated glycosaminoglycans was determined. Production of aggrecanases and aggrecan G1-ITEGE neoepitope, fragmentation of CD44 and the SMAD1/5/8 signaling pathway were evaluated by western blotting.Results:Kartogenin treatment enhanced chondrocyte pericellular matrix assembly and retention in the presence of IL-1β. The chondroprotective effects of kartogenin on IL-1β-induced release of sulfated glycosaminoglycans from articular cartilage explants, reduction in safranin O staining of neocartilage discs as well as a reduction in aggrecan G1-ITEGE neoepitope in chondrocyte and explant cartilage cultures were observed. Kartogenin partially blocked the IL-1β-induced increased expression of ADAMTS-5. Additionally, kartogenin-treated articular chondrocytes exhibited a decrease in CD44 proteolytic fragmentation. However, kartogenin treatment did not enhance proteoglycan in control, non-IL-1β-treated cultures. Similarly, kartogenin enhanced the SMAD1 phosphorylation but only following pretreatment with IL-1β.Conclusion:These studies provide novel information on the chondroprotective function of kartogenin in adult articular cartilage. The effects of kartogenin are significant after activation of chondrocytic chondrolysis, which may occur following disruption of homeostasis maintained by hyaluronan–CD44 interactions.

In vitro inhibition of compression-induced catabolic gene expression in meniscal explants following treatment with IL-1 receptor antagonist

BackgroundDamage to the knee meniscus may result in tears that are difficult or unable to heal, and are often treated by partial removal of the damaged tissue. In vitro, 20% dynamic compressive strains on meniscal tissue explants have resulted in an increase in the release of sulfated glycosaminoglycans (GAG) and nitric oxide (NO) from the tissue explants and increased expression of matrix metalloproteinases (MMP) and interleukin-1α (IL-1α). The objective of this study was to explore the efficacy of IL-1 blockade on the expression of a wide range of genes, as well as NO and GAG release, following dynamic compression of porcine meniscal explants. MethodsExplants were dynamically compressed for 2 h at 1 Hz to 0, 10, or 20% strain with and without a pretreatment of 500 ng/ml interleukin-1 receptor antagonist (IL-1RA). Relative changes in gene expression of IL-1α, MMP-1, -3, -13, A Disintegrin and Metalloproteinase with ThromboSpondin 4 (ADAMTS-4), ADAMTS-5, iNOS, aggrecan, and COX-2, as well as changes in NO and GAG release, were measured with standard biochemical assays. ResultsExpression of IL-1α, MMP-3, MMP-13, and ADAMTS-4 in superficial explants was significantly downregulated at 20% dynamic strain compared to 10% strain following treatment with IL-1RA. GAG and NO release were not significantly influenced by IL-1RA treatment. ConclusionsTreatment of meniscal explants with IL-1RA inhibited the expression of many catabolic genes following a single bout of high dynamic strain. IL-1RA may therefore be a potential therapy option during the acute phase of meniscal tear or meniscectomy treatment.

Cartilage viability and catabolism in the intact porcine knee following transarticular impact loading with and without articular fracture

Posttraumatic arthritis commonly develops following articular fracture. The objective of this study was to develop a closed joint model of transarticular impact with and without creation of an articular fracture that maintains the physiologic environment during loading. Fresh intact porcine knees were preloaded and impacted at 294 J via a drop track. Osteochondral cores were obtained from the medial and lateral aspects of the femoral condyles and tibial plateau. Chondrocyte viability was assessed at days 0, 3, and 5 postimpact in sham, impacted nonfractured, and impacted fractured joints. Total matrix metalloproteinase (MMP) activity, aggrecanase (ADAMTS-4) activity, and sulfated glycosaminoglycan (S-GAG) release were measured in culture media from days 3 and 5 posttrauma. No differences were observed in chondrocyte viability of impacted nonfractured joints (95.9 ± 6.9%) when compared to sham joints (93.8 ± 7.7%). In impacted fractured joints, viability of the fractured edge was 40.5 ± 27.6% and significantly lower than all other sites, including cartilage adjacent to the fractured edge (p < 0.001). MMP and aggrecanase activity and S-GAG release were significantly increased in specimens from the fractured edge. This study showed that joint impact resulting in articular fracture significantly decreased chondrocyte viability, increased production of MMPs and aggrecanases, and enhanced S-GAG release, whereas the same level of impact without fracture did not cause such changes.

Dynamic loading enhances integrative meniscal repair in the presence of interleukin-1

Meniscal tears are a common knee injury and increased levels of interleukin-1 (IL-1) have been measured in injured and degenerated joints. Studies have shown that IL-1 decreases the shear strength, cell accumulation, and tissue formation in meniscal repair interfaces. While mechanical stress and IL-1 modulate meniscal biosynthesis and degradation, the effects of dynamic loading on meniscal repair are unknown. The purpose of this study was to determine the effects of mechanical compression on meniscal repair under normal and inflammatory conditions. Explants were harvested from porcine medial menisci. To simulate a full-thickness defect, a central core was removed and reinserted. Explants were loaded for 4h/day at 1 Hz and 0%-26% strain for 14 days in the presence of 0 or 100 pg/mL of IL-1. Media were assessed for matrix metalloproteinase (MMP) activity, aggrecanase activity, sulfated glycosaminoglycan (S-GAG) release, and nitric oxide (NO) production. After 14 days, biomechanical testing and histological analyses were performed. IL-1 increased MMP activity, S-GAG release, and NO production, while decreasing the shear strength and tissue repair in the interface. Dynamic loading antagonized IL-1-mediated inhibition of repair at all strain amplitudes. Neither IL-1 treatment nor strain altered aggrecanase activity. Additionally, strain alone did not alter meniscal healing, except at the highest strain magnitude (26%), a level that enhanced the strength of repair. Dynamic loading blocked the catabolic effects of IL-1 on meniscal repair, suggesting that joint loading through physical therapy may be beneficial in promoting healing of meniscal lesions under inflammatory conditions.

Effect of Impaction Sequence on Osteochondral Graft Damage: The Role of Repeated and Varying Loads

Background Osteochondral autografts and allografts require mechanical force for proper graft placement into the defect site; however, impaction compromises the tissue. This study aimed to determine the effect of impaction force and number of hits to seat the graft on cartilage integrity. Hypothesis Under constant impulse conditions, higher impaction load magnitudes are more detrimental to cell viability, matrix integrity, and collagen network organization and will result in proteoglycan loss and nitric oxide release. Study Design Controlled laboratory study. Methods Osteochondral explants, harvested from fresh bovine trochleae, were exposed to a series of consistent impact loads delivered by a pneumatically driven device. Each plug received the same overall impulse of 7 Ns, reflecting the mean of 23 clinically inserted plugs. Impaction loads of 37.5 N, 75 N, 150 N, and 300 N were matched with 74, 37, 21, and 11 hits, respectively. After impaction, the plugs were harvested, and cartilage was analyzed for cell viability, histology by safranin-O and picrosirius red staining, and release of sulfated glycosaminoglycans (GAGs) and nitric oxide. Data were compared with nonimpacted controls. Results Impacted plugs had significantly lower cell viability than nonimpacted plugs. A dose-response relationship in loss of cell viability with respect to load magnitude was seen immediately and after 4 days but lost after 8 days. Histological analysis revealed intact cartilage surface in all samples (loaded or control), with loaded samples showing alterations in birefringence. While the sulfated GAG release was similar across varying impaction loads, release of nitric oxide increased with increasing impaction magnitudes and time. Conclusion Impaction loading parameters have a direct effect on the time course of the viability of the cartilage in the graft tissue. Clinical Relevance Optimal loading parameters for surgical impaction of osteochondral grafts are those with lower load magnitudes and a greater number of hits to ensure proper fit.

Enhanced Tissue Integration During Cartilage Repair In Vitro Can Be Achieved by Inhibiting Chondrocyte Death at the Wound Edge

Experimental wounding of articular cartilage results in cell death at the lesion edge. The objective of this study was to investigate whether inhibition of this cell death results in enhanced integrative cartilage repair. Bovine articular cartilage discs (6 mm) were incubated in media containing inhibitors of necrosis (Necrostatin-1, Nec-1) or apoptosis (Z-VAD-FMK, ZVF) before cutting a 3 mm inner core. This core was left in situ to create disc/ring composites, cultured for up to 6 weeks with the inhibitors, and analyzed for cell death, sulfated glycosaminoglycan release, and tissue integration. Creating the disc/ring composites resulted in a significant increase in necrosis. ZVF significantly reduced necrosis and apoptosis at the wound edge. Nec-1 reduced necrosis. Both inhibitors reduced the level of wound-induced sulfated glycosaminoglycan loss. Toluidine blue staining and electron microscopy of cartilage revealed significant integration of the wound edges in disc/ring composites treated with ZVF. Nec-1 improved integration, but to a lesser extent. Push-out testing revealed that ZVF increased adhesive strength compared to control composites. This study shows that treatment of articular cartilage with cell death inhibitors during wound repair increases the number of viable cells at the wound edge, prevents matrix loss, and results in a significant improvement in cartilage-cartilage integration.

Induction of increased cAMP levels in articular chondrocytes blocks matrix metalloproteinase–mediated cartilage degradation, but not aggrecanase‐mediated cartilage degradation

Calcitonin has been suggested to have chondroprotective effects. One signaling pathway of calcitonin is via the second messenger cAMP. We undertook this study to investigate whether increased cAMP levels in chondrocytes would be chondroprotective. Cartilage degradation was induced in bovine articular cartilage explants by 10 ng/ml oncostatin M (OSM) and 20 ng/ml tumor necrosis factor (TNF). In these cultures, cAMP levels were augmented by treatment with either forskolin (4, 16, or 64 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 4, 16, or 64 microM). Cartilage degradation was assessed by 1) quantification of C-terminal crosslinking telopeptide of type II collagen fragments (CTX-II), 2) matrix metalloproteinase (MMP)-mediated aggrecan degradation by (342)FFGV- G2 assay, 3) aggrecanase-mediated degradation by (374)ARGS-G2 assay, 4) release of sulfated glycosaminoglycans (sGAG) into culture medium, 5) immunohistochemistry with a monoclonal antibody recognizing the CTX-II epitope, and 6) toluidine blue staining of proteoglycans. MMP expression and activity were assessed by gelatin zymography. OSM and TNF induced an 8,000% increase in CTX-II compared with control (P < 0.001). Both forskolin and IBMX dose-dependently inhibited release of CTX-II (P < 0.001). OSM and TNF induced a 6-fold increase in (342)FFGV-G2, which was abrogated by forskolin and IBMX (by >80%). OSM and TNF stimulated MMP expression as visualized by zymography, and MMP expression was dose-dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine-induced release of sGAG by 72%. Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently strongly inhibited cartilage degradation. Specific cAMP modulators in chondrocytes may be potential treatments for cartilage degenerative diseases.

The influence of mechanical compression on the induction of osteoarthritis-related biomarkers in articular cartilage explants

Macromolecules of the articular cartilage extracellular matrix released into synovial fluid, blood, or urine can serve as potentially useful biomarkers of the severity of osteoarthritis (OA). Biomechanical factors play an important role in OA pathogenesis, yet their influence on biomarker production is not well understood. The goal of this study was to examine the hypothesis that dynamic mechanical stress influences the release of these biomarkers from articular cartilage. Explants of porcine cartilage were subjected to dynamic compression at 0.5 Hz for 24h at stresses ranging from 0.006 to 0.1 MPa. The concentrations of cartilage oligomeric matrix protein (COMP), keratan sulfate (KS measured as the 5 D 4 epitope), total sulfated glycosaminoglycan (S-GAG), and the KS (keratanase-digestible) and chondroitin sulfate (CS) (chondroitinase-digestible) fractions of S-GAG were measured. Radiolabel incorporation was used to determine the rates of proteoglycan and protein synthesis. The magnitudes of mechanical stress applied in this study induced nominal tissue strains of 4-23%, consistent with a range of physiological to hyperphysiologic strains measured in situ. COMP release increased in proportion to the magnitude of dynamic mechanical stress, while KS, CS and total S-GAG release increased in a bimodal pattern with increasing stress. Protein and proteoglycan synthesis were significantly decreased at the highest level of stress. Mechanical stress differentially regulates the turnover of distinct pools of cartilage macromolecules. These findings indicate that mechanical factors, independent of exogenous cytokines or other stimulatory factors, can influence the production and release of OA-related biomarkers from articular cartilage.

Functional characterization of adherent synovial fluid cells in rheumatoid arthritis: destructive potential in vitro and in vivo.

To characterize the morphologic and immunologic features of adherent synovial fluid cells derived from patients with rheumatoid arthritis (RA), and to explore their potential function in vitro and in vivo by focusing on cartilage destruction. Synovial fluid adherent cells obtained from patients with RA and from control subjects were characterized by immunohistochemistry, flow cytometry, and electron microscopy. In vitro, these cells were cultured in the presence of cartilage particles. Cartilage destruction was monitored by the release of sulfated glycosaminoglycans (sGAG) into the medium, and the level of matrix metalloproteinase 1 (MMP-1) in the cell culture supernatant was measured by enzyme-linked immunosorbent assay. To inhibit cartilage destruction in vitro, the MMP inhibitor marimastat was tested in this system. In vivo, in the SCID mouse coimplantation model, RA synovial fluid adherent cells and RA synovial fibroblasts (as positive controls) were coimplanted with human cartilage under the kidney capsule and maintained there for 60 days. In vitro, the synovial fluid adherent cells consisted of 2 subpopulations, large round-shaped macrophage-like cells (CD68+) and spindle-shaped fibroblast-like cells (Thy-1+). When passaged, the latter cells proliferated and organized themselves into 3-dimensional formations. This allowed them to reach collagen particles fixed with agarose. Fibroblasts derived from synovial tissues could not be used in this assay because they grew only in monolayers and not on agarose. The majority (>90%) of passaged RA synovial fluid adherent cells expressed the Thy-1+,CD45-,CD68-,CD86- phenotype. Electron microscopy did not reveal important morphologic differences between the 2 types of fibroblasts, those from synovial tissue or those from synovial fluid. However, synovial fluid adherent cells expressed lower levels of adhesion molecules, including CD54 and galectin 3, as well as the complement-regulatory molecule CD55. The in vitro release of sGAG associated with cell activity was 2.5-fold higher from RA synovial fluid adherent cells in comparison with that from negative control cells. The release of sGAG correlated with the concentration of MMP-1 and was inhibited by the broad-range MMP inhibitor marimastat in a dose-dependent manner. RA synovial fluid adherent cells coimplanted with cartilage in SCID mice showed the same invasive behavior as that displayed by tissue-derived RA synovial fibroblasts. Similar to tissue-derived RA synovial fibroblasts, RA synovial fluid adherent cells, which contain "floating" anchorage-independent fibroblast-like cells, mediate cartilage destruction independent of the hyperplastic synovial tissue.

4′-Hydroxy aceclofenac suppresses the interleukin-1-induced production of promatrix metalloproteinases and release of sulfated-glycosaminoglycans from rabbit articular chondrocytes

This study demonstrates the novel actions of a non-steroidal anti-inflammatory drug aceclofenac, which is frequently used for rheumatoid arthritis and osteoarthritis. 4′-Hydroxy aceclofenac, a main metabolite of aceclofenac in humans, down-regulated the production of promatrix metalloproteinase-1/procollagenase 1 and promatrix metalloproteinase-3/prostromelysin 1 along with a decrease in their mRNAs in rabbit articular chondrocytes and synoviocytes, and interfered with the release of sulfated-glycosaminoglycans (proteoglycans) from the chondrocytes. 4′-Hydroxy aceclofenac also suppressed the proliferation of rabbit synoviocytes. In contrast, aceclofenac itself and its other metabolites, diclofenac and 4′-hydroxy declofenac, did not exert obvious actions on cellular functions. Therefore, it is suggested that the therapeutic effects of aceclofenac on rheumatoid arthritis and osteoarthrits are, at least in part, due to the novel chondroprotective effect of 4′-hydroxy aceclofenac via the suppression of promatrix metalloproteinase production and proteoglycan release. There is also evidence that inhibition of synoviocyte proliferation and the known inhibitory action on prostaglandin E 2 production play a role.

Plasmin stimulates the release of dermatan sulfate from vascular smooth muscle cells in culture

We investigated the effect of plasmin on the release of sulfated glycosaminoglycans (GAG) from cultured vascular smooth muscle cells. Confluent cultures of vascular smooth muscle cells from bovine aorta were labelled with [ 35S]sulfate and incubated at 37 °C for principally 60 min in a serum-free medium in the presence of plasmin. Plasmin at 10 mU/ml (approximately 2.7 μg/ml) and above significantly increased the release of [ 35S]sulfate-labeled GAG ( 35S-GAG) from the cell layer after a 60 min incubation. A time course study showed that plasmin at 10 mU/ml significantly increased the 35S-GAG release after 20 min and longer. However, plasminogen at 100 mU/ml and below did not cause a significant change of the 35S-GAG release after a 90 min incubation. A characterization of 35S-GAG revealed that plasmin increased both dermatan sulfate and the other 35S-GAG in the medium. Plasmin at 100 mU/ml enhanced the cell detachment significantly but only slightly. From these results, it was suggested that a endogenous thrombin inhibitor heparin cofactor II may be activated in the liquid phase by dermatan sulfate released from plasmin-stimulated vascular smooth muscle cells when the vascular is disrupted and plasma is exposed to extravessel.

Catabolin/interleukin-1 regulation of cartilage and chondrocyte metabolism.

Catabolin/interleukin-1 effects on metabolism were studied in bovine nasal cartilage organ culture and articular chondrocyte cell culture. Keratan sulfate (KS) and hyaluronic acid (HA) were determined by an ELISA; prostaglandin E2 by RIA, sulfated glycosaminoglycan using dimethylmethylene blue and proliferation by incorporation of tritiated thymidine. Gel filtration of untreated 4-day organ culture media indicated that large sulfated and KS-containing proteoglycans were released and eluted in the void volume. Catabolin/interleukin-1 increased release of sulfated glycosaminoglycans and these were of lower molecular weight with an altered distribution of KS. Catabolin/interleukin-1 treatment of chondrocytes caused a decrease in KS production and proliferation but an increase in HA and in prostaglanding E2 production. Alterations of the chondrocyte metabolism by catabolin/interleukin-1 causing proteoglycan matrix degradation and modulation of chondrocyte glycosaminoglycan biosynthesis and proliferation may play a role in cartilage erosion and failure to repair in arthritic diseases.

Dibutyryl cyclic amp affects hyaluronate synthesis and macromolecular organization in normal adult articular cartilage in vitro

When normal adult dog articular cartilage was cultured in the presence of dibutyryl cyclic AMP a higher proportion than normal of newly synthesized 35S-labeled glycosaminoglycans was released from the tissue into the culture medium, although their net synthesis was not affected. In conjunction with this release of sulfated glycosaminoglycans, 24 times more [ 3H]glucosamine-labeled hyaluronic acid was released from the cartilage into the medium, and net hyaluronate synthesis was enhanced 3-fold. Virtually all of the newly synthesized hyaluronic acid in the medium was associated with proteoglycans. The proteoglycans in the medium of the dibutyryl cyclic AMP treated cultures were normal in hydrodynamic size and interacted normally with hyluronic acid to form large aggregates. These results suggest that the increase in hylarunate synthesis caused by dibutyryl cyclic AMP may have destabilized the interaction of protoeglycans with the collagen meshwork of the cartilage. The changes seen in normal adult articular cartilage after incubation with dibutyryl cyclic AMP, therefore, are similar to those whic are observed in cartilage of osteoarthritic joints.