Reduction In Proteoglycan Synthesis Research Articles

TWEAK/Fn14 signaling: a promising target in intervertebral disc degeneration.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a potent chemoattractant cytokine with various biological functions, such as stimulation of angiogenesis, induction of proinflammatory cytokines, regulation of cellular proliferation and apoptosis. Therefore, it has also been implicated in several pathological processes, from cancer to inflammatory diseases. Remarkably, TWEAK and its receptors, fibroblast growth factor inducible 14 (Fn14), are also present in intervertebral disc (IVD) tissue, where they play a role in the pathogenesis of IVD degeneration. The interaction of TWEAK with Fn14 is involved in physiological and pathological activities of IVD degeneration patients, which includes apoptosis of endplate chondrocytes, extracellular matrix degradation, reduction in proteoglycan synthesis and so on. The blockade of this interaction results in suppressing over-production of proinflammatory factors and cell death in in vivo or in vitro experiments, suggesting that TWEAK/Fn14 signaling may be therapeutically relevant in IVD degeneration, and the targeting of TWEAK or Fn14 has been proposed as a potential therapeutic approach for autoimmune diseases such as Rheumatoid arthritis (RA). In this article, we discuss the biological features of TWEAK/Fn14 signaling and summarize recent advances in our understanding of the role of TWEAK/Fn14 signaling in the pathogenesis and treatment of IVD degeneration. We think that the blockade of TWEAK/Fn14 signaling may be a promising therapeutic strategy for IVD degeneration in the near future.

In VitroandIn VivoModulation of Cartilage Degradation by a StandardizedCentella asiaticaFraction

Osteoarthritis (OA) is a degenerative joint disease in which focal cartilage destruction is one of the primary features. The present study aims to evaluate the effect of a Centella asiatica fraction on in vitro and in vivo cartilage degradation. Bovine cartilage explants and bovine chondrocytes cultured in alginate were stimulated with IL-1 beta in the presence or absence of different concentrations (2, 5 and 10 microg/ml) of a standardized Centella asiatica triterpenes (CAT) fraction. The CAT fraction inhibited the IL-1 beta-induced proteoglycan (PG) release and nitric oxide (NO) production by cartilage explants in a dose-dependent manner. The IL-1 beta-induced reduction in PG synthesis and proliferation of chondrocytes cultured in alginate were counteracted by the CAT fraction at a concentration of 10 microg/ml. In a zymosan-induced acute arthritis model, the CAT fraction inhibited PG depletion without modulating joint swelling and inflammatory cell infiltration. In conclusion, the present study demonstrated for the first time that the tested Centella asiatica fraction was able to inhibit the zymosan-induced cartilage degradation in vivo without affecting the zymosan-induced inflammatory cell infiltration and joint swelling. The in vitro data indicate that the cartilage protective activity might at least partially be induced by the inhibition of NO production. The overall results indicate a possible disease modifying osteoarthritic activity of the Centella asiatica fraction.

F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-beta activation.

In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (approximately 7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-beta1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-beta and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (approximately 20%; P<0.05), which were both TGF-beta and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-beta and induce cartilage degradation via pathways that involve production of PGE2.

Proteoglycan Synthesis in Porcine Nasal Cartilage Grafts Following Nd:YAG (λ= 1.32 μm) Laser-Mediated Reshaping

Mechanically deformed morphologic cartilage grafts undergo temperature-dependent stress relaxation during sustained laser irradiation resulting in stable shape changes. In this study, porcine nasal septal cartilage specimens were evaluated for viability by measuring the incorporation of Na235SO4 into proteoglycan (PTG) macromolecules in whole tissue culture following laser-mediated reshaping. Synthesis rates of PTG were determined by scintillation counting lyophilized specimens and normalizing these values by total protein content. Positive controls were established by inducing chondrocyte apoptosis using prolonged exposure to nitric oxide (NO). In chondrocytes, apoptosis induced using NO resulted in significantly lower PTG synthesis rates compared to untreated native specimens. Cartilage specimens were irradiated with light emitted from a Nd:YAG laser (25 W/cm2, λ= 1.32 μm) while recording simultaneously radiometric surface temperature, internal stress and back-scattered light intensity from a probe laser. Each specimen received one, two or three sequential laser exposures. The duration of each exposure was determined from real-time measurements of characteristic changes in back-scattered light intensity that correlate with accelerated stress relaxation. A 5 min time interval between each laser exposures allowed the cartilage specimen to return to thermal equilibrium. Average PTG synthesis rates decreased with successive laser exposures, though these were always higher than baseline rates established for NO-treated tissues, suggesting that laser-mediated cartilage reshaping acutely does not eliminate the entire population of viable chondrocytes. The reduction in PTG synthesis is correlated with the time–temperature-dependent heating profile created during laser irradiation, supporting our hypothesis that careful monitoring of laser dosimetry is required to ensure chondrocyte viability.

Effects of Electromagnetic Fields on Proteoglycan Metabolism of Bovine Articular Cartilage Explants

Electromagnetic field (EMF) exposure has been proposed for the treatment of osteoarthritis. In this study, we investigated the effects of EMF (75 Hz, 2,3 mT) on proteoglycan (PG) metabolism of bovine articular cartilage explants cultured in vitro, both under basal conditions and in the presence of interleukin-1 g (IL-1 g ) in the culture medium. Proteoglycan synthesis and the residual PG tissue content resulted significantly higher in EMF-exposed explants than in controls, whereas no effect was observed on PG release and nitric oxide (NO) production. IL-1 g induced both a reduction in PG synthesis and an increase in PG release, related to a strong stimulation of NO production, which resulted in a net loss of tissue PG content. In IL-1 g -treated explants, EMF increased PG synthesis, whereas in spite of a slight stimulation of NO production EMF did not modify PG release. This resulted in the residual PG tissue content being maintained at the control level. In both experimental conditions, the effects of EMF were associated with an increase in lactate production. The results of our study show that EMFs are able to promote anabolic activities and PG synthesis in bovine articular cartilage explants. This effect also is maintained in the presence of IL-1 g , thus counteracting the catabolic activity of the cytokine. Altogether, these data suggest that EMF exposure exerts a chondroprotective effect on articular cartilage in vitro.

Radiation-induced reduction of BMP-induced proteoglycan synthesis in an embryonal mesenchymal tissue equivalent using the chicken "limb bud" test.

Heterotopic ossification (HO) is a common complication following total hip replacement. Clinical studies showed the effectiveness of irradiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to find a suitable cell system, which can reproduce in-vitro data resulting from clinical in-vivo studies. The establishment of such a cell model allows detailed analyses of the mechanism of radiotherapy. The chicken limb bud test was used as an in-vitro model. The cells acquired by the limb bud test were irradiated with different doses (0 Gy, 3 Gy, 7 Gy, 10 Gy, 20 Gy). Irradiation was set either 1 hour before, or 1 or 3 days after BMP-2 incubation. The synthesis of proteoglycans (PGS) upon treatment with bone morphogenetic protein (BMP)-2 was measured in cells incubated with BMP-2 for 4 days followed by 35SO4(2-) labeling for 6 hours. Labeled proteoglycans were precipitated using Alcian blue and measured in a raytest radio-TLC analyzer. The incubation with BMP-2 was defined to correlate the in-vivo stimulus meaning the operation. The proteoglycan synthesis was significantly reduced by irradiation 1 hour before or 1 day after BMP-2 incubation, if the dosage was at least 7 Gy. Higher doses than 7 Gy did not lead to lower proteoglycan levels. There was only a trend for a reduction of proteoglycan synthesis by 3 Gy irradiation, but no significant difference compared to the non-irradiated control. An irradiation 3 days after BMP-2 incubation had no effect on proteoglycan. A dose and time dependent effect of radiation on BMP-2-induced proteoglycan synthesis was observed. Therefore the results of clinical in-vivo studies were reproduced exactly by the limb bud test. We established an in-vitro cell model to analyze the mechanism of the prevention of heterotopic ossification by radiotherapy on cellular or sub-cellular level.

Proteoglycan synthesis in porcine nasal cartilage grafts following Nd:YAG (lambda = 1.32 microns) laser-mediated reshaping.

Mechanically deformed morphologic cartilage grafts undergo temperature-dependent stress relaxation during sustained laser irradiation resulting in stable shape changes. In this study, porcine nasal septal cartilage specimens were evaluated for viability by measuring the incorporation of Na2(35)SO4 into proteoglycan (PTG) macromolecules in whole tissue culture following laser-mediated reshaping. Synthesis rates of PTG were determined by scintillation counting lyophilized specimens and normalizing these values by total protein content. Positive controls were established by inducing chondrocyte apoptosis using prolonged exposure to nitric oxide (NO). In chondrocytes, apoptosis induced using NO resulted in significantly lower PTG synthesis rates compared to untreated native specimens. Cartilage specimens were irradiated with light emitted from a Nd:YAG laser (25 W/cm2, lambda = 1.32 microns) while recording simultaneously radiometric surface temperature, internal stress and back-scattered light intensity from a probe laser. Each specimen received one, two or three sequential laser exposures. The duration of each exposure was determined from real-time measurements of characteristic changes in back-scattered light intensity that correlate with accelerated stress relaxation. A 5 min time interval between each laser exposures allowed the cartilage specimen to return to thermal equilibrium. Average PTG synthesis rates decreased with successive laser exposures, though these were always higher than baseline rates established for NO-treated tissues, suggesting that laser-mediated cartilage reshaping acutely does not eliminate the entire population of viable chondrocytes. The reduction in PTG synthesis is correlated with the time-temperature-dependent heating profile created during laser irradiation, supporting our hypothesis that careful monitoring of laser dosimetry is required to ensure chondrocyte viability.

Effect of diacetyl rhein on the development of experimental osteoarthritis. A biochemical investigation

To investigate the effect of diacetyl rhein (DAR) on the synthesis, turnover and composition of cartilage in an experimental model of osteoarthritis in beagle bitches. Osteoarthritis was induced in mature beagle bitches by the transection of the cranial cruciate ligament. Six animals received DAR 20 mg/kg daily for 11 weeks. A matched group received empty capsules daily for the same period. At 11 weeks, articular cartilage was examined for the ratio of the 6:4-sulfated disaccharides of chondroitin and the tissue concentration of hydroxyproline and glycosaminoglycan. In addition, labeling studies were performed to estimate the effect of DAR on proteoglycan synthesis and turnover. DAR had no effect on body weight or food consumption but induced a mild diarrhea and slightly increased the incidence of vomiting. DAR tended to reduce proteoglycan synthesis, however, DAR did reduce proteoglycan turnover in the femoral cartilage. DAR produced changes in the composition of the osteoarthritic cartilage that could only partly be accounted for by changes in hydration and/or swelling. In addition, it was noted that induction of osteoarthritis increased the ratio of chondroitin 6-sulfated to chondroitin 4-sulfated disaccharides; DAR reduced the ratio in tibial plateau cartilage from osteoarthritic joints compared with untreated tissue from osteoarthritic joints. DAR showed moderate reduction on the biosynthesis of proteoglycans. DAR also produced a reduction in proteoglycan turnover from all anatomical areas compared with non-treated controls in both the lateral and medial femoral condyles. DAR was well tolerated by the experimental animals, but did not produce significant changes in the synthesis or turnover of proteoglycans. The slight reduction in proteoglycan synthesis may prove to be biologically significant after chronic dosing. DAR's effects on the hydroxyproline and glycosaminoglycan content suggest, however, that it must influence the swelling of cartilage and loss of glycosaminoglycan. This indicates that small changes can translate, to significant differences in cartilage composition over an 11-week time period.

Fibronectin attachment is permissive for IL-1 mediated gene regulation

This study examines the effect of cell-matrix interaction on IL-1 induced gene regulation. In fibroblasts and smooth muscle cells attached to fibronectin, IL-1 caused a pronounced reduction in proteoglycan synthesis, while no reduction occurred in cells plated on bare plastic. Further, fibronectin attachment was permissive for IL-1 mediated suppression of both versican and collagen mRNA levels, initially noted after 4–6 h of IL-1 incubation. Attachment to vitronectin was less potent in influencing regulation, and collagen had no effect, suggesting specificity of the matrix modulation of the IL-1 induced response. Similar fibronectin induced dependence was demonstrated for IL-1 regulation of IL-6 gene expression, supporting the notion of a general effect of fibronectin receptor engagement on IL-1 induced signal transduction.

The relationship between proteoglycan synthesis in Swarm chondrocytes and pathways of cellular energy and UDP-sugar metabolism

The effect of anaerobic culture conditions and various metabolic inhibitors on 35S-proteoglycan synthesis, UDP-sugar pools, and the ATP pool were investigated in confluent, primary, day 1 cultures of Swarm chondrosarcoma chondrocytes. ( i)_Incubation under a nitrogen atmosphere for 6 h did not affect 35S-proteoglycan synthesis or the pool size for UDP-glucuronate, other UDP-sugars, or ATP. Incubation with 5 mM sodium azide brought about a 40% reduction of proteoglycan synthesis in the first 30 min but no further change over the subsequent 90 min. UDP-Glucuronate, other UDP-sugar pools, and the ATP level were not affected by azide treatment. The results indicate that proteoglycan synthesis and its energy requirements can be supported entirely by anaerobic metabolism in these cells. ( ii) 35S-Proteoglycan synthesis, UDP-sugar production, and nucleotide triphosphate pools were inhibited in a concentration-dependent fashion with sodium iodoacetate. A > 70% reduction of the ATP pool after 30 min treatment suggests that glycolysis is a major target for iodoacetate. Lactate production was inhibited by 40% after 3 h treatment with 10 −4 M iodoacetate. ( iii) Glutamine deprivation resulted in a 60% contraction in the UDP- N-acetylhexosamine pool and markedly inhibited 35S-proteoglycan and 3H-protein synthesis. At the same time the UDP-glucose pool expanded to 200%, but the UDP-glucuronate pool was unchanged. The sum of the UDP- N-acetylhexosamine and UDP-hexose pools remained constant. Restoration of glutamine to previously depleted cultures resulted in excessive expansion of the UDP- N-acetylhexosamine pool and excessive contraction of the UDP-hexose pool before both adjusted to normal levels. The UDP-xylose pool was very small. No increases were observed during inhibition of proteoglycan synthesis induced by glutamine deprivation. ( iv) 6-Diazo-5-oxo- l-norleucine (DON), a glutamine analogue and amino transferase inhibitor, induced a further contraction of the UDP- N-acetylhexosamine pool and a further decrease in proteoglycan synthesis in glutamine-deprived cultures. Thus cultures use endogenous glutamine during exogenous glutamine deprivation. DON unaccountably stimulated expansion of the UDP-glucuronate pool by 180%, irrespective of whether glutamine was present or not.

Antisense S-oligonucleotide against transforming growth factor-beta 1 inhibits proteoglycan synthesis in arterial wall.

Transforming growth factor-beta 1 (TGF-beta 1) is a potent stimulator of proteoglycan (PG) synthesis by cultured smooth muscle cells. To test the hypothesis that TGF-beta 1 stimulates PG synthesis in whole artery wall we have investigated the effect of blocking endogenous TGF-beta 1 using an antisense S-oligonucleotide (ASO) directed against the first 7 codons for the N-terminal region of active TGF-beta 1. This sequence reduced TGF-beta 1 secretion by cultured endothelial cells by 40-55%. To determine the effect of the ASO on PG synthesis in whole vessel we chose the rat carotid artery (RCA) maintained in organ culture, a model in which we previously documented endothelial-dependent increases in PG synthesis over time in culture. We report here that the increases in PG in the inner layers of the vessel wall are matched by similar increases in TGF-beta 1 mRNA. To test for the effect of ASO on PG synthesis, small segments of RCA, maintained in organ culture (Medium 199, supplemented with 1% FCS), were exposed on day 6 to either control media, antisense TGF-beta 1, sense TGF-beta 1, or a non-sense sequence at 5, 10 and 20 microM concentrations. Following 24 h exposure to the oligonucleotides, cultures were labelled for a further 24 h with [3H]glucosamine and processed for autoradiography. Antisense TGF-beta 1 at 10 and 20 microM produced a 60% reduction in PG synthesis in the endothelium and adjacent first layer of the media.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of reactive oxygen species in kidney damage.

There is considerable evidence suggesting that reactive oxygen species (ROS) are implicated in the pathogenesis of ischemic, toxic, and immunologically-mediated renal injury. In experimental renal ischemia, ROS sources include the electron transport chain, oxidant enzymes (xanthine oxidase), phagocytes, and auto-oxidation of epinephrine. ROS cause lipid peroxidation of cell and organelle membranes and, hence, disruption of the structural integrity and capacity for cell transport and energy production, especially in the proximal tubule segment. In experimental immune glomerulonephritis, ROS are generated by both infiltrating blood-borne cells (polymorphonuclear leukocytes and monocytes) and resident glomerular cells, mainly mesangial cells. Their formation results in morphologic lesions and in modifications of glomerular permeability to proteins through activation of proteases and reduction of proteoglycan synthesis. Additionally, they promote a reduction in glomerular blood flow and glomerular filtration rate through liberation of vasoconstrictory bioactive lipids (prostaglandins, thromboxane, and platelet activating factor) and, possibly, inactivation of relaxing nitric oxide. Further studies are needed to address the role of ROS in human glomerular diseases.

Development and Reversal of a Proteoglycan Aggregation Defect In Normal Canine Knee Cartilage After Immobilization

Healthy adult dogs were studied for a defect in proteoglycan aggregation by immobilizing one limb for varying periods of time. Immobilization for 6 days resulted in a 41% reduction in proteoglycan synthesis by articular cartilage from the restrained knee compared with the contralateral control knee. After 3 weeks of immobilization, proteoglycan aggregation was no longer demonstrable in cartilage from the constrained limb. The aggregation defect was rapidly reversible and aggregates were again normal size 2 weeks after removal of a cast that had been worn for 6 weeks.

Extracellular matrix metabolism by chondrocytes III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. Benzyl-β- D- xyloside , an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.