Levels of chondroitin 4-sulfate, chondroitin 6-sulfate and carboxy-terminal type II procollagen peptide in knee synovial fluid after injury to the anterior cruciate ligament

Changes in the metabolism of articular cartilage associated with injury to the anterior cruciate ligament (ACL) is known to be one of the important factors for the progression to secondary osteoarthritis. To investigate the efficacy of biochemical markers for monitoring the cartilage metabolism after injury to ACL, we measured the levels of chondroitin 4-sulfate (C-4S), chondroitin 6-sulfate (C-6S) and carboxy-terminal type II procollagen peptide (pCOL II-C) in knee synovial fluid (SF) from the patients with ACL rupture and compared with those in knee osteoarthritis (OA). Within 10 days after ACL rupture, levels of C-6S and C-4S in SF were significantly higher than those in early stage of OA. Both levels decreased gradually and became to the same levels as those in early stage of OA at 30 days after the injury. In contrast, pCOL II-C levels in SF just after the injury were observed to be lower than those in early stage of OA. Then they increased gradually to the levels of those in early stage of OA at 30 days after the injury. High levels of C-6S and C-4S in SF just after ACL rupture seemed to reflect the increased release of matrix fragments caused by cartilage destruction associated with the injury. pCOL II-C levels in SF seemed to reflect the repairing process that increased slowly after the cartilage destruction. Measurement of these cartilage derived macromolecules in SF could be useful tools for monitoring the metabolism in articular cartilage after injury.     

Evidence for altered synthesis of type II collagen in patients with osteoarthritis

There is evidence to suggest that the synthesis of type II collagen is increased in osteoarthritis (OA). Using an immunoassay, we show that the content of the C-propeptide of type II procollagen (CPII), released extracellularly from the newly synthesized molecule, is directly related to the synthesis of this molecule in healthy and osteoarthritic articular cartilages. In OA cartilage, CPII content is often markedly elevated (mean 7.6-fold), particularly in the mid and deep zones, reaching 29.6% of the content in newborn. Synthesis is also directly related to total collagen II content in OA, suggesting its importance in maintaining collagen content and cartilage structure. The release of CPII from cartilage is correlated directly with cartilage content. However, the increase in CPII in OA cartilage is not reflected in serum, where a significant reduction is observed. Together these studies provide evidence for alterations in procollagen II synthesis in vivo in patients with OA.     

Integrated radiation hybrid and yeast artificial chromosome map of chromosome 9p

OBJECTIVES: To determine concentrations of chondroitin sulphate (CS) and keratan sulphate (KS) epitopes, glycosaminoglycans (GAGs) and hyaluronan (HA) in knee synovial fluid (SF) from normal subjects and patients with osteoarthritis (OA) or rheumatoid arthritis (RA), to test whether these variables may be used as markers of the OA process. METHODS: OA was subdivided into large joint OA (LJOA), nodal generalised OA (NGOA), and OA with calcium pyrophosphate crystal deposition (CPA). Clinical assessment of inflammation (0-6) was undertaken on OA and RA knees. Knee SF was examined by enzyme linked immunosorbent assay for: CS epitopes, using monoclonal antibodies 3-B-3 and 7-D-4; KS epitope using monoclonal antibody 5-D-4; and HA, using biotinylated HA binding region of cartilage proteoglycan. Total sulphated GAGs were measured by dye binding with 1:9 dimethylmethylene blue. RESULTS: Increased SF 3-B-3 concentrations and 3-B-3/GAG ratio were found in OA, compared with RA or normal knees, with higher 3-B-3 and 3-B-3/GAG in LJOA and NGOA than in CPA. SF 7-D-4 and 7-D-4/GAG were reduced in RA, compared with normal and OA; SF 5-D-4 was reduced in OA compared with normal. GAG and HA concentrations were decreased in both OA and RA. No correlations with radiographic scores were observed, but SF 7-D-4 was lower in 'inflamed' compared with 'non-inflamed' RA and OA knees. In patients with bilateral samples there were strong correlations between right and left knees for all SF variables. CONCLUSIONS: Changed concentrations of SF CS and KS can be detected in OA with a profile that differs from that seen in RA. Clinical subgrouping and local joint inflammation may influence these measures, supporting different pathogenesis within OA subgroups and requirement for careful patient characterisation in SF studies.     

Progressive polyarthritis induced in BALB/c mice by aggrecan from normal and osteoarthritic human cartilage

OBJECTIVE: To find an "unlimited" source of antigenic material (aggrecan) for arthritis induction in BALB/c mice; to analyze the specificities of immune reactions to aggrecan and type II collagen in 2 arthritis-susceptible murine strains, BALB/c mice for proteoglycan (aggrecan)-induced arthritis and DBA/1j mice for collagen-induced arthritis; to compare the histopathologic features of arthritis induced by purified aggrecans or total extracts of osteoarthritic (OA) cartilage; and to determine arthritis susceptibility in various BALB/c colonies. METHODS: Aggrecans from total extracts of human fetal, normal adult, OA, and rheumatoid cartilage samples and from osteophytes were isolated, purified by gradient centrifugation, deglycosylated, characterized, and tested for arthritis induction. Purified type II collagen and salt-soluble collagens from OA cartilage were denatured, stromelysin treated, and used for immunization and arthritis induction in arthritis-susceptible (DBA/1j and BALB/c) murine strains. RESULTS: Chondrocytes from OA cartilage synthesize predominantly fetal-type aggrecan, which is the most efficient antigenic material for arthritis induction in BALB/c mice. The critical autoimmune/arthritogenic T cell epitopes of aggrecan are located in the G1 domain. Although most of the aggrecan molecules are heavily degraded and lost from OA cartilage, the G1 domain-containing fragments accumulate in OA cartilage. The amount of G1-containing fragments is approximately twice as much in OA than in normal adult articular cartilage, and the arthritogenic epitope(s) remains intact in G1-containing fragments retained in cartilage. Thus, total extracts of OA cartilage (without additional purification), if deglycosylated appropriately, can be used as arthritogenic material in BALB/c mice. CONCLUSION: Predominantly G1 domain-containing fragments of aggrecan accumulate in OA cartilage, and these are the fragments which induce arthritis in BALB/c mice. Arthritis induction is highly specific for aggrecan epitopes and dictated by the genetic background of the BALB/c strain.     

Circulating levels of matrix metalloproteinases MMP-3 and MMP-1, tissue inhibitor of metalloproteinases 1 (TIMP-1), and MMP-1/TIMP-1 complex in rheumatic disease. Correlation with clinical activity of rheumatoid arthritis versus other surrogate markers

OBJECTIVE: To investigate whether plasma levels of matrix metalloproteinases 3 (MMP-3, stromelysin), MMP-1 (collagenase), tissue inhibitor of metalloproteinases 1 (TIMP-1), and MMP1/TIMP-1 complex (MT complex) are specifically elevated in erosive joint diseases compared to nonerosive rheumatic diseases, and to assess how these markers reflect the clinical activity of rheumatoid arthritis (RA) compared to circulating cytokines and markers of connective tissue turnover as well as established variables [C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and rheumatoid factor titer]. METHODS: Plasma levels of MMP-3, MMP-1, TIMP- 1, and MT complex were determined by ELISA. One hundred fifteen patients with RA, 20 with osteoarthritis (OA), 28 with psoriasis arthritis (PsA), 24 with ankylosing spondylitis (AS), 3 groups with systemic autoimmune diseases, and 30 healthy controls were analyzed. In patients with RA routine laboratory variables, circulating inflammatory cytokines [interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-6], collagen degradation products, and markers of bone formation were determined in parallel and were correlated to 4 variables of clinical activity. RESULTS: MMP-3 levels were markedly elevated in RA compared to controls and OA, but also in all other groups, including 26 patients with systemic lupus erythematosus (SLE). MMP-1 levels were significantly elevated in RA, but also in OA, PsA, SLE, and mixed connective tissue disease. In contrast, MT complex was elevated in RA only. TIMP-1 was not different from controls. CRP levels, MMP-3, and ESR correlated best with clinical activity of RA. In contrast, there was no correlation of IL-1 and TNF-alpha and only a weak correlation of IL-6 with clinical measures. Among variables of connective tissue turnover, only pyridinoline and deoxypyridinoline crosslinks were weakly correlated with disease activity. CONCLUSION: Elevated MMP-3 and MMP-1 levels are not specific for RA or for erosive joint diseases in general. In contrast, elevated MT complex levels were observed in patients with RA. However, the correlation of MT-1 with clinical data was weaker than that of MMP-3. Elevated MMP-3 levels reflected disease activity of RA better than cytokine levels or markers of connective tissue turnover. However, MMP-3 levels do not exceed the association of CRP with clinical activity.     

Cellular immunity to the G1 domain of cartilage proteoglycan aggrecan is enhanced in patients with rheumatoid arthritis but only after removal of keratan sulfate

OBJECTIVE: To determine whether patients with rheumatoid arthritis (RA) express cellular immunity to the purified G1 globular domain of cartilage proteoglycan (PG) aggrecan and whether it is influenced by the removal of keratan sulfate (KS) chains from the molecule. METHODS: The G1 globular domain of PG was purified from mature bovine articular cartilage, digested with keratanase, and used in proliferation assays with peripheral blood lymphocytes (PBL) isolated from 43 patients with RA, 11 patients with nonarticular rheumatism (NAR), including soft tissue rheumatism and mechanical back pain, and 13 healthy age- and sex-matched control subjects. RESULTS: Removal of KS chains from the G1 globular domain resulted in significantly increased prevalence and values of cellular immune responses to G1 in RA patients compared with the control and NAR groups. In the majority of RA patients, KS chains on G1 significantly inhibited its immune recognition by PBL. There was no significant effect of KS removal on the immunity to G1 in patients with NAR and in the healthy control group. CONCLUSION: These results reveal that immune reactivity to the G1 globular domain of the cartilage PG aggrecan is enhanced in patients with RA but only when KS chains are removed. Thus, KS chains inhibit immune responses to this domain of aggrecan. Since immunity to the G1 globular domain of aggrecan induces an erosive polyarthritis in BALB/c mice after removal of KS chains, immunity to the G1 globular domain, cleaved by proteases to remove KS chains, may play a role in the pathogenesis of RA.     

Effect of irradiation with a low-intensity diode laser on the metabolism of equine articular cartilage in vitro

OBJECTIVE: To determine whether irradiation with a low-intensity diode laser, which produces radiation at a wavelength of 810 nm, will induce nonthermal enhancement of chondrocyte metabolism. SAMPLE POPULATION: 144 grossly normal articular cartilage explants aseptically harvested from the femoral condyles of 6 adult horses. PROCEDURE: Treated cartilage explants were irradiated with a diode laser at 1 of 7 fluence levels that ranged from 8 to 1,600 J/cm2. Explants were incubated for 24 or 72 hours, labeled for 24 hours with [35S]Na2SO4, and assayed for newly synthesized sulfated glycosaminoglycan (GAG; measured incorporation of 35SO4) and endogenous GAG, chondroitin 6-sulfate (CS), and keratan sulfate (KS) content, using a dimethylmethylene blue assay. Laser-induced temperature changes were measured during irradiation with a diode laser and a neodymium:yttrium aluminum garnet (Nd:YAG) laser, which produces radiation at a wavelength of 1,064 nm, using conditions that were reported in previous studies to increase explant metabolism. RESULTS: After incubation for 24 or 72 hours, rate of 33SO4 uptake or endogenous GAG, CS, or KS content in irradiated explants was not significantly different than in nonirradiated explants. Cartilage temperature increased < 4.75 C during diode laser application. Cartilage temperature increased 5 to 12 C during Nd:YAG laser application. CONCLUSIONS: Minimal thermal increases in cartilage explants with use of a low-intensity diode laser resulted in no change in proteoglycan metabolism of chondrocytes. An increase in tissue temperature over a narrow range with use of a Nd:YAG laser may have contributed to the metabolic alteration of chondrocytes reported in previous studies.     

Angiotensin-converting enzyme inhibition in experimental in-situ immune complex glomerulonephritis: influence on renal function, proteinuria, and morphology

OBJECTIVE: To identify antigen(s) among purified deglycosylated aggrecan peptides spanning the chondroitin sulphate domain that may be responsible for the initiation or perpetuation of the autoimmune responses in rheumatoid arthritis (RA). METHODS: Aggrecan was purified from human articular cartilage and deglycosylated with either bacterial glycosidases or trifluoromethanesulphonic acid (TFMS). Twelve overlapping peptides (15 residues) spanning the chondroitin sulphate domain with N-terminal residues offset by three amino acids were synthesised. T cell responses to these antigens in RA patients and age matched controls were assessed in vitro by antigen specific T cell proliferation assays. RESULTS: Enzymically deglycosylated aggrecan (EDA) stimulated proliferation of T cells isolated from the peripheral blood in a greater proportion of patients with RA than controls. In a subset (12.5%) of RA patients, the magnitude of stimulation lay outside the control range. T cell proliferative responses to TFMS treated aggrecan were greater than, but well correlated with, responses to EDA. T cells from 15 patients were also stimulated with the pooled synthetic peptides. Four of seven patients who demonstrated T cell reactivity to EDA (seven of 15) also showed enhanced T cell proliferation to synthetic peptides. CONCLUSION: These data suggest that an autoantigenic T cell epitope may lie within the chondroitin sulphate domain of aggrecan.     

Localization of glycosaminoglycans (GAGs) in pleomorphic adenoma (PA) of salivary glands: an immunohistochemical and histochemical evaluation

The tumor matrix of salivary pleomorphic adenoma (PA) is characteristically rich in glycosaminoglycans (GAGs), which contribute to its complex histoarchitecture. This study evaluated the microscopic localization of various GAGs in 17 PAs, using a panel of anti-GAG monoclonal antibodies and biotinylated hyaluronic acid (HA)-binding protein. Both epithelial and mesenchymal-like tissues were confirmed to contain GAGs. Luminal epithelial cells mostly lacked GAGs, whereas GAGs were seen both in the cytoplasm and cell membrane of non-luminal epithelial cells. In addition, small intercellular accumulations of GAGs were often present in solid epithelial areas, implying the epithelial origin of GAGs. GAGs did not appear to be a main component of the hyaline matrix. The myxoid region was consistently stained for both chondroitin 6-sulfate (CS-6) and HA but variably for chondroitin 4-sulfate (CS-4), dermatan sulfate (DS) and keratan sulfate (KS); heparan sulfate (HS) was not detected. The chondroid region showed increased staining for CS-6 but reduced staining for HA when compared with the myxoid region. In addition, CS-4, DS and KS were seen both in chondroid cells and the territorial matrix, whereas HS was present only in the cells. It is suggested that GAGs in PA are mainly produced by non-luminal cells and influence the proliferation, differentiation, secretory activity and shape of tumor cells, thus contributing to the morphological diversity of this tumor.     

Critical roles of glycosaminoglycan side chains of cartilage proteoglycan (aggrecan) in antigen recognition and presentation

Systemic immunization of BALB/c mice with proteoglycan (aggrecan) from fetal human cartilage induces progressive polyarthritis, an experimental disease similar to human rheumatoid arthritis. The development of the disease in this genetically susceptible murine strain is based on cross-reactive immune responses between the immunizing fetal human and mouse self-proteoglycans. One of the cross-reactive and arthritogenic T cell epitopes (92GR/QVRVNSA/IY) is localized in the G1 domain of human/murine proteoglycan. Susceptible BALB/c mice, however, develop arthritis only if both the chondroitin sulfate (CS) and keratan sulfate (KS) side chains of the arthritogenic human proteoglycans are removed. The function of these two glycosaminoglycan side chains is opposite. The presence of a KS side chain in adult proteoglycan inhibits the recognition of arthritogenic T cell epitopes, prevents the development of T cell response, and protects animals from autoimmune arthritis. In contrast, the depletion of the CS side chain generates clusters of CS stubs and provokes a strong B cell response. These carbohydrate-specific B cells are the most important proteoglycan APC. Taken together, proteoglycan-induced progressive polyarthritis is dictated by three major components: genetic background of the BALB/c strain, highly specific T cell response to epitope(s) masked by a KS chain in aging tissue, and the presence of proteoglycan (CS stub)-specific B cells required for sufficient Ag presentation.     

Polarized infrared spectra of crystalline glycosaminoglycans

Polarized infrared spectra have been recorded for oriented, crystalline specimens of hyaluronates, chondroitin 4-sulfate and 6-sulfate, dermatan sulfate, and a cartilage proteoglycan, having different known chain conformations as determined by X-ray diffraction. The dichroism data for the vibrational modes of the amide and carboxyl groups have been interpreted with respect to the particular molecular structures.     

Extracellular matrix of opacified anterior capsule after endocapsular cataract surgery

BACKGROUND: We determined the types and distribution of glycosaminoglycans (GAGs) and collagens, in anterior capsular opacification after endocapsular phacoemulsification and aspiration (ECPEA) and intraocular lens implantation. METHODS: Opacified anterior capsules were removed from human eyes after ECPEA. Immunohistochemical staining was performed to determine GAGs with monoclonal antibodies to chondroitin, chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS), and keratan sulfate (KS); collagens with monoclonal antibodies to types I, II, and III collagens; and cellular characteristics with monoclonal antibodies to vimentin, desmin, alpha-smooth muscle actin, and cytokeratin. Decorin mRNA and type I collagen mRNA were detected by in situ hybridization. RESULTS: In the capsules, the C6S, DS, KS, and types I and III collagens were similar to the chemical components found at the adhesion site of the anterior and posterior capsules after extracapsular cataract extraction, and cellular components contained vimentin, desmin, alpha-smooth muscle actin, cytokeratin, decorin mRNA, and type I collagen mRNA. CONCLUSIONS: The GAGs and collagens in opacified anterior capsule after ECPEA were similar to those found during wound healing, although KS is present in normal anterior segment tissue during development and only in the cornea postnatally. These chemical components may be produced by myofibroblast-like cells presumably transformed from lens epithelial cells.     

Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val6, Ala7]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Association of proteoglycan degradation with catabolic cytokine and stromelysin release from cartilage cultured with fibronectin fragments

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.     

Effects of glucose and TGF-beta 1 on the proliferation of cultured human peritoneal mesothelial cells]

OBJECTIVE: The aggrecan proteoglycan is a major component of articular cartilage and supports the biomechanical function of this tissue. A variable number tandem repeat (VNTR) polymorphism has been discovered recently in a region of the human aggrecan gene that codes for the chondroitin sulfate attachment sites. We examined whether alleles of this polymorphism displayed a non-random association with bilateral hand or knee osteoarthritis (OA) in men from the Baltimore Longitudinal Study of Aging (BLSA). DESIGN: DNA was obtained from 93 Caucasian men, aged 60 and above, who had bilateral hand and standing knee radiographs read for changes of OA. The DNA was analyzed by polymerase chain reaction (PCR) and/or Southern blotting for the presence of the VNTR alleles. RESULTS: Bilateral hand OA and knee OA were present in 46 and 30% of the men respectively. The following distribution of alleles was observed: allele 33 (0.5%), 29 (2.2%), 28 (31.7%), 27 (43.0%), 26 (16.7%), 25 (3.2%), 22 (2.2%) and 19 (0.5%). This distribution was similar to that detected in a random population of individuals from a separate study. In multiple logistic regression analysis, adjusting for age and body mass index, the presence of allele 27 was associated with bilateral hand OA with an odds ratio (OR) = 3.23 (95% confidence intervals (CI): 1.24-8.41). No other alleles showed an association with bilateral hand OA and the association between allele 27 and bilateral knee OA was not statistically significant (OR = 1.14; 95% CI: 0.45-2.88). CONCLUSIONS: These data demonstrate the first association between a human aggrecan gene polymorphic allele and hand OA. This finding supports the concept that genetic factors may play a role in the development and/or progression of some forms of age-onset OA.     

Differential release of proteoglycans during human B lymphocyte maturation

Proteoglycans interact with soluble proteins such as growth factors and thereby regulate extracellular signals. During B lymphocyte maturation, secretion of proteoglycans may be functionally related to the different requirements of the respective maturation stage. In order to address this question we compared structures of proteoglycans released by three B lymphocyte lines which correspond to different maturation stages. Plasma-cell type U266 cells secreted the largest proteoglycans (150 kDa), followed by mature B cells JOK-1 (130 kDa) and pre-B cells Nalm 6 (90 kDa). On average, secreted proteoglycans carried four glycosaminoglycan chains with molecular masses ranging each from 32 kDa (U266) to 23 kDa (Nalm 6). All three cell lines secreted more than 90% of their proteoglycans possessing chondroitin sulfate chains having chondroitin-4-sulfate (delta Di-4S) as the prevalent disaccharide unit. In these proteochondroitin sulfates, unsulfated chondroitin (delta Di-0S) was present in smaller quantities and chondroitin-6-sulfate (delta Di-6S)-containing proteoglycan was released only by Nalm 6 and U266 cells. Cell line Nalm 6 exclusively produced proteochondroitin sulfate, whereas in culture medium of JOK-1 and U266 a small amount of proteoheparan sulfate was found also. In all three cell lines, treatment with chondroitinase ABC released a protein of 30 kDa and chemical deglycosylation resulted in a core protein of 21 kDa. In addition to pure proteochondroitin sulfate, a small portion of proteoheparan sulfate with a protein moiety of 30 kDa was detected after heparitinase treatment in supernatants of JOK-1 and U266. Thus, our results indicate that released proteoglycans may undergo modulations in their glycosaminoglycan moieties during B-cell differentiation. This may have functional consequences at the level of growth factor regulation.     

Liquid ventilation for treatment of meconium aspiration syndrome in a piglet model

OBJECTIVES: Osteoarthritis (OA) is a common joint deterioration initiated by multiple factors. To better understand related factors in the development of this disease, we focused on the mechanical stress loaded on articular cartilage. MATERIALS AND METHODS: The anterior cruciate ligaments of rabbit knee joints were transected, and expression of protein kinase C (PKC) examined immunohistochemically. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was then administered intraarticularly. To determine the involvement of gas mediators, a cartilage defect was made on the medical femoral condyle of rabbit knee joints. Hydrostatic pressure was loaded on the cartilage taken from the surrounding defects, and levels of superoxide anion and nitric oxide (NO) were measured. Bovine chondrocytes were subjected to cyclic mechanical stretch using a Flexercell Strain Instrument. Proteoglycan synthesis and PKC activity were measured. Expression of matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 in articular cartilages obtained from OA patients were examined using Northern blots. RESULTS: Chondrocytes from experimentally induced OA were stained positively with anti-alpha-PKC antibody. Intraarticular administration of TPA prevented the development of OA changes. Cyclic tensile stretch loaded on chondrocytes decreased proteoglycan synthesis and PKC activity. Thus, PKC is involved in the stress-mediated degradation of articular cartilage. Cartilage defects led to degradation of surrounding cartilage and to enhanced superoxide anion and NO synthesis. We also noted increased and decreased expressions of MMP-3 and TIMP-1 mRNA in human OA cartilage, respectively. CONCLUSION: PKC, gas mediators (superoxide anion, NO), and proteinases are all involved in OA.     

The course of age-related macular degeneration following bilateral cataract surgery

OBJECTIVE: The aim of this study was to define the relative regulation of matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinases-1 (TIMP-1), in chondrocytes and synovium in experimental osteoarthritis (EOA). METHODS: Partial-meniscectomized (PM) rabbits, surgical sham controls (SH), and normal non-surgical controls (N) were killed at times corresponding to early degenerative lesions (4 weeks) and increasingly progressive stages of EOA at 8 and 12 weeks post-PM. MMP-3 activity was measured in conditioned media from chondrocytes and synovium using a peptide cleavage assay with substance P (SP) as the substrate. TIMP-1 was quantitated using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Early degenerative lesions (4 weeks post-PM) were characterized by inflammatory responses in the synovium accompanied by a significant rise of MMP-3 activity in synovial cultures (P < 0.05). At 8 weeks there was no discernible inflammation, and MMP-3 activity in EOA synovial cultures was comparable to that in the controls; this was followed by a second increase in MMP-3 activity in EOA samples at 12 weeks. MMP-3 activity was significantly elevated in EOA chondrocyte cultures at 8 weeks post-PM relative to N controls, corresponding to the most destructive phase of EOA, but not in the early phase (4 weeks) or 'late' degenerative phase (12 weeks). Medium derived from chondrocytes contained little or no TIMP-1. Synovia secreted relatively higher amounts of TIMP-1, and this was elevated at 8 weeks post-PM relative to the SH controls. The majority (approximately 90%) of MMP-3 activity could be inhibited using recombinant TIMP-1 or a hydroxamate MMP inhibitor. Complete inhibition was achieved with EDTA or 1,10 phenanthroline. CONCLUSION: Together, these data indicate that in EOA, MMP-3 is initially upregulated in the synovium which may play a pivotal role in the pathogenesis of cartilage lesions. In contrast, chondrocyte-derived MMP-3 is upregulated in the later phases of EOA, contributing further to progression of cartilage lesions.     

Purification and properties of human N-acetylgalactosamine-6-sulfate sulfatase

1. Human N-acetylgalactosamine-6-sulfate sulfatase (EC 3.1.6.-) from human placenta has been purified more than 3000-fold by gel filtration, ion-exchange and substrate affinity chromatography. The enzyme has a molecular weight of 90 000 by gel filtration chromatography and 85 000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Enzyme purified from cultured human skin fibroblasts has similar properties. 2. The tritium-labeled chrondroitin 6-sulfate trisaccharide N-acetylgalactosamine 6-sulfate-(beta, 1-4)-glucuronic acid-(beta, 1-3(-N-acetyl[1-3H]galactosaminitol 6-sulfate as substrate demonstrated a Km of 0.12 mM at pH 4.5. Sulfate was hydrolyzed only from the non-reducing terminal of this disulfated trisaccharide. Hyaluronic acid, dermatan sulfate, chondroitin 4-sulfate, heparin and chondroitin 6-sulfate tetrasaccharide were slightly inhibitory, whereas 6-sulfated pentasaccharides and heptasaccharides were strongly inhibitory. The enzyme dose not hydrolyze sulfate from N-acetylglucosamine 6-sulfate.