Differential direct effects of cyclo-oxygenase-1/2 inhibition on proteoglycan turnover of human osteoarthritic cartilage: an <Emphasis Type=Italic>in vitro</Emphasis>study

Treatment of osteoarthritis (OA) with nonsteroidal anti-inflammatory drugs (NSAIDs) diminishes inflammation along with mediators of cartilage destruction. However, NSAIDs may exert adverse direct effects on cartilage, particularly if treatment is prolonged. We therefore compared the direct effects of indomethacin, naproxen, aceclofenac and celecoxib on matrix turnover in human OA cartilage tissue. Human clinically defined OA cartilage from five different donors was exposed for 7 days in culture to indomethacin, naproxen, aceclofenac and celecoxib – agents chosen based on their cyclo-oxygenase (COX)-2 selectivity. As a control, SC-560 (a selective COX-1 inhibitor) was used. Changes in cartilage proteoglycan turnover and prostaglandin E₂ production were determined. OA cartilage exhibited characteristic proteoglycan turnover. Indomethacin further inhibited proteoglycan synthesis; no significant effect of indomethacin on proteoglycan release was found, and proteoglycan content tended to decrease. Naproxen treatment was not associated with changes in any parameter. In contrast, aceclofenac and, prominently, celecoxib had beneficial effects on OA cartilage. Both were associated with increased proteoglycan synthesis and normalized release. Importantly, both NSAIDs improved proteoglycan content. Inhibition of prostaglandin E₂ production indirectly showed that all NSAIDs inhibited COX, with the more COX-2 specific agents having more pronounced effects. Selective COX-1 inhibition resulted in adverse effects on all parameters, and prostaglandin E₂ production was only mildly inhibited. NSAIDs with low COX-2/COX-1 selectivity exhibit adverse direct effects on OA cartilage, whereas high COX-2/COX-1 selective NSAIDs did not show such effects and might even have cartilage reparative properties.     

Biosynthesis of proteoglycan in vitro by cartilage from human osteochondrophytic spurs

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [(35)S]sulphate or [(14)C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [(35)S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [(35)S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60-80% of the [(35)S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (K(av.)) of the proteoglycan monomer on Sepharose CL-2B was 0.28-0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2-D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [(35)S]sulphate and [(14)C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60-70%), with 9-11% keratan sulphate in the monomer proteoglycan; (3) K(av.) values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH(4)-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.     

Salmon nasal cartilage proteoglycan enhances growth of normal human dermal fibroblast through Erk1/2 phosphorylation

Proteoglycan (PG) is a heavily glycosylated protein, localized to cell surface and extracellular matrix, and has various functions. Recently, it has been gradually revealed that PG interacts with various growth factors and morphogens and regulates cellular functions. Although salmon nasal cartilage PG (Salmon-PG) increases proliferation of immortalized cells, its mechanism remains unclear. In this study, we confirmed the effect of Salmon-PG on normal human dermal fibroblast (NHDF) and investigated the mechanism of PG action on NHDF. Salmon-PG dose- and time-dependently increased NHDF proliferation. Receptor tyrosine kinase array revealed that Salmon-PG increased only Erk1/2 signaling. Erk1/2 phosphorylation was significantly increased by Salmon-PG in a time-(10 min) and dose-(400 or 800 μg/mL) dependent manner. MEK inhibitor suppressed the enhancement of NHDF proliferation by Salmon-PG. The overall findings indicate that Salmon-PG plays a role as a growth factor in NHDF via Erk1/2 activation, suggesting that Salmon-PG contributes to the maintenance of skin homeostasis.     

The effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with [35S]sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of 35S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the 35S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with [35S]sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the 35S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible. Cartilage cultures maintained with retinoic acid for 1 day then switched to medium with 20% (v/v) fetal calf serum for the remainder of the culture period exhibited decreased rates of loss of 35S-labeled proteoglycans from the matrix and increased tissue hexuronate contents to levels near those observed in tissue maintained in medium with 20% (v/v) fetal calf serum throughout. Furthermore, following switching to 20% (v/v) fetal calf serum, the relative proportions of the 35S-labeled proteoglycan species remaining in the matrix of these cultures were similar to those of control cultures.     

Differential allelic expression of the type II collagen gene (COL2A1) in osteoarthritic cartilage.

Osteoarthritis (OA) is a common debilitating disease resulting from the degeneration of articular cartilage. The major protein of cartilage is type II collagen, which is encoded by the COL2A1 gene. Mutations at this locus have been discovered in several individuals with inherited disorders of cartilage. We have identified 27 primary OA patients who are heterozygous for sequence dimorphisms located in the coding region of COL2A1. These dimorphisms were used to distinguish the mRNA output from each of the two COL2A1 alleles in articular cartilage obtained from each patient. Three patients demonstrated differential allelic expression and produced < 12% of the normal level of mRNA from one of their COL2A1 alleles. The same allele shows reduced expression in all three patients, and this allele is more frequent in a well-defined OA population than in a control group, suggesting the possible existence of a rare COL2A1 allele that predisposes to OA.     

In vitro inhibition of IL-1beta catabolic effects on cartilage: a mechanism involved on diacerein anti-OA properties

Osteoarthritis (OA) is a progressive joint disease which represents a combination of several disorders leading to cartilage degradation. The main characteristic of OA is an imbalance between chondrocyte anabolic and catabolic activities. Cytokines produced by the synovium and chondrocytes, especially interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), play a significant role in the degradation of cartilage. They stimulate the production of nitric oxide (NO), which is involved in cartilage catabolism and also may induce the apoptosis of chondrocytes. The IL-1beta produced in activated chondrocytes or synovium may modulate disease progression in OA and should therefore be considered a potential target for therapeutic interventions. Drug and non-drug treatments are used to relieve pain and/or swelling in OA. Diacerein is a slow-acting drug that may slow down the breakdown of cartilage and relieve pain and swelling. It is not clear whether diacerein works but it has been proposed that diacerein acts as a symptom-modifying and perhaps disease-structure modifying drug.     

Chondroptosis: An immunohistochemical study of apoptosis and Golgi complex in chondrocytes from human osteoarthritic cartilage

The Golgi complex is thought to play an important role in the apoptotic process of osteoarthritic (OA) chondrocytes. However, the exact relationship between modifications of the Golgi complex and apoptosis in human OA cartilage requires to be established. We compared the patterns and immunolabeling intensities for anti-Golgi 58 K protein with apoptosis markers such as TUNEL and caspase-2L in OA cartilage removed from patients during knee total replacement surgery. We observed important modifications in labeling of the Golgi 58 K protein in OA chondrocytes compared with normal cell. Immunohistochemical analysis revealed co-localization between 58 K protein and caspase-2L, suggesting that this enzyme was localized in Golgi complex of OA chondrocytes. In addition, these cells labeled positive with the TUNEL technique, but in different proportions to caspase-2L. Our results support the concept, previously reported, that apoptosis in OA cartilage (chondroptosis) might be a variant of the classical apoptosis.     

Age-related effects of TGF-beta on proteoglycan synthesis in equine articular cartilage

The synthesis of proteoglycans was measured in normal equine articular cartilage of ages 9 months to 20 years and the effect of TGF-beta1 on this activity was investigated. The rate of incorporation of [(35)S]Na(2)SO(4) decreased with age as did the responsiveness of the tissue to the growth factor. The enhanced synthesis of proteoglycan induced at all ages by TGF-beta1 was down-regulated by IL-1 beta and retinoic acid. The expression of mRNA for TGF-beta1, 2, and 3 was also measured, and although the level of TGF-beta1 was highest at all ages, the expression of each growth factor decreased with age.     

Differential effects of 1,25-dihydroxyvitamin D3 on human lymphocytes and monocyte/macrophages: inhibition of interleukin-2 and augmentation of interleukin-1 production

Human peripheral blood monocytes and activated, but not resting, lymphocytes possess specific intracellular receptors for the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The effects of 1,25-(OH)2D3 on the function of these cells was therefore examined. The addition of physiologic concentrations of the hormone (0.001-0.1 nM) to lectin- or antigen-activated lymphocytes resulted in inhibition of lymphocyte proliferation. Supernatants from lectin-activated lymphocytes incubated with 1,25-(OH)2D3 had reduced interleukin-2 (IL-2) activity. The immediate biological precursor of 1,25-(OH)2D3, 25-hydroxyvitamin D3, did not affect function of lymphocytes or monocytes. The ability of exogenous recombinant IL-2 to reverse the inhibitory effects of the hormone on lymphocyte proliferation suggest that 1,25-(OH)2D3 does not alter the generation of IL-2 receptors. In contrast to its effects on IL-2 production, 1,25-(OH)2D3 caused a dose-dependent increase in the production of interleukin-1 (IL-1) by monocyte/macrophages. These results suggest that immune cells and their products can be regulated in a specific but diverse fashion by the vitamin D3-endocrine system.     

In vitro effects of ozone on human erythrocyte membranes: an EPR study

The effects of ozone at different concentrations (10, 30, 45 g/m3) on fluidity and thermotropic properties of erythrocyte membranes were investigated by EPR using two spin probes: 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA). The effect of ozone on the erythrocyte membrane fluidity was a dose-dependent process. The ozone at concentration of 10 g/m3 caused rigidization of the membrane while at concentration of 45 g/m3 increased fluidity both on the surface and in the deeper hydrocarbon region of the membrane. Temperature transitions close to the polar heads region (monitored by 5-DSA) were not sensitive to an increase in ozone concentration. In the case of 16-DSA, low temperature thermotropic transition (around 20 degrees C) gradually decreased with the increase of ozone concentration. High temperature transition (around 40 degrees C) significantly differed at the ozone concentration of 10 g/m3 and 45 g/m3, being higher and lower, respectively, as compared to untreated cells. For the ozone concentration of 45 g/m3 the disappearance of the low temperature break and the appearance of two breaks at 37 degrees C and 16 degrees C were observed.     

Differential regulation of cyclooxygenase-2 and inducible nitric oxide synthase by 4-hydroxynonenal in human osteoarthritic chondrocytes through ATF-2/CREB-1 transactivation and concomitant inhibition of NF-kappaB signaling cascade

4-hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues and was recently identified as a potent catabolic factor in OA cartilage. In this study, we provide additional evidence that HNE acts as an inflammatory mediator by elucidating the signaling cascades targeted in OA chondrocytes leading to cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression. HNE induced COX-2 protein and mRNA levels with accompanying increases in prostaglandin E2 (PGE(2)) production. In contrast, HNE had no effect on basal iNOS expression or nitric oxide (NO) release. However, HNE strongly inhibited IL-1beta-induced iNOS or NO production. Transient transfection experiments revealed that the ATF/CRE site (-58/-53) is essential for HNE-induced COX-2 promoter activation and indeed HNE induced ATF-2 and CREB-1 phosphorylation as well as ATF/CRE binding activity. Overexpression of p38 MAPK enhanced the HNE-induced ATF/CRE luciferase reporter plasmid activation, COX-2 synthesis and promoter activity. HNE abrogated IL-1beta-induced iNOS expression and promoter activity mainly through NF-kappaB site (-5,817/-5,808) possibly via suppression of IKKalpha-induced IkappaBalpha phosphorylation and NF-kappaB/p65 nuclear translocation. Upon examination of upstream signaling components, we found that IKKalpha was inactivated through HNE/IKKalpha adduct formation. Taken together, these findings illustrate the central role played by HNE in the regulation of COX-2 and iNOS in OA. The aldehyde induced selectively COX-2 expression via ATF/CRE activation and inhibited iNOS via IKKalpha inactivation.     

Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.     

Regulation of proteoglycan synthesis rate in cartilage in vitro: influence of extracellular ionic composition

Load-bearing cartilages regularly experience changes in fluid content as the result of changing load. It has been found that these changes in fluid content influence proteoglycan synthesis. The mechanism for this effect is not known. We have measured the influence of changes in cartilage hydration on the [35S]sulphate incorporation rate in both bovine nasal and human articular cartilage in medium whose concentration varied over the range 0.2-2-times physiological strength. In physiological medium the incorporation rate fell in proportion to fluid loss with a 10% fall in cartilage hydration resulting in a 30-50% decrease in 35S-incorporation rates. However, in medium of 0.5-times physiological strength, where the incorporation rate was only 40% of control values, the incorporation rate increased initially rather than falling as the cartilage lost fluid. These changes in hydration and hence proteoglycan content resulted in changes in the extracellular ionic composition of cartilage. When this was monitored in terms of [Na+]c, the internal sodium concentration, as a marker for changes in cartilage ionic composition, we found that incorporation rate varied with [Na+]c rather than directly with hydration.     

Effect of calcipenia on proteoglycan metabolism and aggregation in normal articular cartilage in vitro.

Glycosaminoglycan synthesis in normal adult dog knee cartilage cultured in medium containing 0, 0.3 MM- and 0.9 mM-Ca2+ was 52, 67 and 78%, respectively, of that in cartilage from the same joints cultured in a normal concentration of Ca2+, i.e. 1.8 mM. Pulse-chase experiments indicated that the rate of degradiation of glycosaminoglycans in cartilage cultured in the absence of Ca2+ was similar to that of glycosaminoglycans in cartilage cultured in 1.8 mM-Ca2+. Although [35S]sulphate incorporation into glycosaminoglycans was decreased in the presence of calcipenia, [3H]leucine incorporation into protein was unaffected. The average hydrodynamic size of newly synthesized proteoglycan aggregates and purified disaggregated proteoglycans from cartilage cultured in the absence of Ca2+ was similar to that of aggregates and disaggregated proteoglycans from cartilage cultured in 1.8 mM-Ca2+.     

Differential effects of human immunodeficiency virus type 1 Vpu on the stability of BST-2/tetherin

BST-2/CD317/tetherin is a host factor that inhibits the release of HIV-1 and other unrelated viruses. A current model proposes that BST-2 physically tethers virions to the surface of virus-producing cells. The HIV-1-encoded Vpu protein effectively antagonizes the activity of BST-2. How Vpu accomplishes this task remains unclear; however, it is known that Vpu has the ability to down-modulate BST-2 from the cell surface. Here we analyzed the effects of Vpu on BST-2 by performing a series of kinetic studies with HeLa, 293T, and CEMx174 cells. Our results indicate that the surface downregulation of BST-2 is not due to an accelerated internalization or reduced recycling of internalized BST-2 but instead is caused by interference with the resupply of newly synthesized BST-2 from within the cell. While our data confirm previous reports that the high-level expression of Vpu can cause the endoplasmic reticulum (ER)-associated degradation of BST-2, we found no evidence that Vpu targets endogenous BST-2 in the ER in the course of a viral infection. Instead, we found that Vpu acts in a post-ER compartment and increases the turnover of newly synthesized mature BST-2. Our observation that Vpu does not affect the recycling of BST-2 suggests that Vpu does not act directly at the cell surface but may interfere with the trafficking of newly synthesized BST-2 to the cell surface, resulting in the accelerated targeting of BST-2 to the lysosomal compartment for degradation.     

Nonenzymatic glycation of cartilage proteoglycans: an in vivo and in vitro study

In this study we have investigated whether proteoglycans (aggrecan) are modified by nonenzymatic glycation as in collagen. Purified human aggrecan from osteoarthritic and normal human knee articular cartilage was assayed for pentosidine, a cross-link formed by nonenzymatic glycation, using reverse-phase HPLC. In addition, an in vitro study was done by incubation of purified bovine nasal cartilage aggrecan with ribose. Pentosidine was found in all the purified human aggrecan samples. 2-3% of the total articular cartilage pentosidine was found in aggrecan. Purified link protein also contained penosidine. The in vitro study led to pentosidine formation, but did not appear to increase the molecular size of the aggrecan suggesting that pentosidine was creating intramolecular cross-links. Similar amounts of glycation were found in osteoarthritic and normal cartilage. Like collagen, aggrecan and link proteins are crosslinked by nonenzymatic glycation in normal and osteoarthritic cartilage. Crosslinking could be reproduced, in vitro, by incubating aggrecan with ribose. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of intermittent compressive force on proteoglycan content in calcifying growth plate cartilage in vitro

We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.