Salvatore Campo

Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats

To evaluate the antioxidant activity of the glycosaminoglycans hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S), we used a rat model of collagen-induced arthritis (CIA). Arthritis was induced in Lewis rats by multiple intradermal injections of 250 μl of emulsion containing bovine type II collagen in complete Freunds adjuvant at the base of the tail and into three to five other sites on the back. Rats were challenged again with the same antigen preparation 7 days later. Disease developed about 11 days after the second immunization. The effects of treatment in the rats were monitored by biochemical parameters and by macroscopic and histological evaluations in blood, synovial tissue and articular cartilage. Arthritis produced the following symptoms: severe periarticular erythema, edema and inflammation in the hindpaws; membrane peroxidation in the cartilage of the joints; endogenous antioxidant wasting; high tumour necrosis factor-α (TNF-α) plasma levels; and synovial neutrophil accumulation. Treatment with HYA and C4S, starting at the onset of arthritis for 10 days, limited the erosive action of the disease in the articular joints of knee and paw, reduced lipid peroxidation, restored the endogenous antioxidants reduced glutathione (GSH) and superoxide dismutase, decreased plasma TNF-α levels, and limited synovial neutrophil infiltration. These data confirm that erosive destruction of the joint cartilage in CIA is due at least in part to free radicals released by activated neutrophils and produced by other biochemical pathways. The beneficial effects obtained with the treatment suggest that HYA and C4S could be considered natural endogenous macromolecules to limit erosive damage in CIA or as a useful tool with which to study the involvement of free radicals in rheumatoid arthritis.

Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes

Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflammatory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappa B (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation. Lipopolysaccharide (LPS) -mediated activation of TLR-4 complex induces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. The aim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4 modulation in LPS-induced inflammatory response in mouse chondrocyte cultures. Messenger RNA and related protein levels were measured for TLR-4, MyD88, and TRAF-6 in both untreated and LPS-treated chondrocytes, with and without the addition of HA (two doses for each MW). NF-kB activation, TNF-alpha and IL-1beta levels, matrix metalloprotease-13 (MMP-13), and inducible nitric oxide synthase (iNOS) gene expression were also evaluated. LPS increased all the parameters studied as well as NF-kB activation. Low MW HA upregulated TLR-4 expression, increased MyD88 and TRAF-6 and the inflammation mediators in untreated chondrocytes, and it enhanced the LPS effect in LPS-treated cells. Medium and high MW HA exerted no activity in untreated cells and only the latter reduced the LPS effects. Specific TLR-4 blocking antibody was utilised to confirm TLR-4 as the target of HA action. These findings suggest that the regulatory effect exerted by HA (at any MW) on NF-kB activation may depend upon the interaction between HA and TLR-4 and HA may thereby modulate pro-inflammatory activity via its different state of aggregation.

Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes

Small degradation fragments of hyaluronan (HA) may stimulate an inflammatory response in a variety of tissues at the injury site. HA oligosaccharides are endogenous ligands for the cluster determinant 44 (CD44) receptor as well as for toll-like receptor 4 (TLR-4). Previous data have shown that HA fragments may induce pro-inflammatory cytokine expression by interacting with both the CD44 receptor and TLR-4. CD44 and TLR-4 stimulation activates different inflammatory pathways that culminate with the activation of the transcriptional nuclear factor kappaB (NF-kappaB) which is responsible for the expression of inflammation mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 beta (IL-1beta). The aim of this study was to investigate the inflammatory effects of very small HA oligosaccharides on both TLR-4 and CD44 involvement in normal human articular chondrocytes. Adding HA fragments to chondrocyte cultures up-regulated CD44 and TLR-4 expression, activated NF-kappaB translocation and increased the pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1beta. The addition of a specific CD44 blocking antibody reduced CD44 and all inflammatory cytokine expression as well as protein production. However, cytokine expression remained significantly higher than in untreated chondrocytes. TLR-4 expression was not affected. The treatment with TLR-4 blocking antibody decreased TLR-4 and inflammatory cytokine expression, although cytokine expression was significantly higher than in control cells. CD44 expression was unaffected. The addition of both CD44 and TLR-4 blocking antibodies significantly reduced CD44, TLR-4 and inflammatory cytokine expression.

Reduction of carbon tetrachloride-induced rat liver injury by IRFI 042, a novel dual vitamin E-like antioxidant

Carbon tetrachloride (CCl4)-induced hepatotoxicity is likely the result of a CCl4-induced free radical production which causes membrane lipid peroxidation and activation of transcription factors regulating both the TNF-α gene and the early-immediate genes involved in tissue regeneration. IRFI 042 is a novel vitamin E-like compound having a masked sulphydryl group in the aliphatic side chain. We studied the effect of IRFI 042 on CCl4-induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of CCl4 (1 ml/kg in vegetal oil). Serum alanine aminotransferase (ALT) activity, liver malondialdehyde (MAL), hydroxyl radical formation (OH·), calculated indirectly by a trapping agent, hepatic reduced glutathione (GSH) concentration, plasma TNF-α, liver histology and hepatic mRNA levels for TNF-α were evaluated 48 h after CCl4 administration. Hepatic vitamin E (VE) levels were evaluated, in a separate group of animals, 2 h after CCl4 injection. A control group with vitamin E (100 mg/kg) was also treated in order to evaluate the differences versus the analogue treated groups. Intraperitoneal injection of carbon tetrachloride produced a marked increase in serum ALT activity (CCl4= 404.61 ± 10.33 U/L; Controls= 28.54 ± 4.25 U/L), liver MAL (CCl4= 0.67 ± 0.16 nmol/mg protein; Controls= 0.13 ± 0.06 nmol/mg protein), OH· levels assayed as 2,3-DHBA (CCl4= 8.73 ± 1.46 μM; Controls= 0.45 ± 0.15 μM) and 2,5-DHBA (CCl4= 24.61 ± 3.32 μM; Controls= 2.75 ± 0.93 μM), induced a severe depletion of GSH (CCl4= 3.26 ± 1.85 μmol/g protein; Controls= 17.82 ± 3.13 μmol/g protein) and a marked decrease in VE levels (CCl4= 5.67 ± 1.22 nmol/g tissue; Controls= 13.47 ± 3.21 nmol/g tissue), caused liver necrosis, increased plasma TNF-α levels (CCl4= 57.36 ± 13.24 IU/ml; Controls= 7.26 ± 2.31 IU/ml) and enhanced hepatic mRNA for TNF-α (CCl4= 19.22 ± 4.38 a.u.; Controls= 0.76 ± 0.36 a.u.). IRFI 042 (100 mg/kg, 30 min after CCl4 injection) blunted liver MAL (0.32 ± 0.17 nmol/mg protein), decreased the serum levels of ALT (128.71 ± 13.23 U/L), and restored the hepatic concentrations of VE (9.52 ± 3.21 nmol/g tissue), inhibited OH· production (2,3-DHBA= 3.54 ± 1.31 μM; 2,5-DHBA= 7.37 ± 2.46 μM), restored the endogenous antioxidant GSH (12.77 ± 3.73 mmol/g protein) and improved histology. Furthermore IRFI 042 treatment suppressed plasma TNF-α concentrations (31.47 ± 18.25 IU/ml) and hepatic TNF-α mRNA levels (11.65 ± 3.21 a.u.). The acute treatment with vitamin E failed to exert any protective effect against CCl4-induced hepatotoxicity. These investigations suggest that IRFI 042 treatment may be of benefit during free radical-mediated liver injury.

Glycosaminoglycans modulate inflammation and apoptosis in LPS-treated chondrocytes

Previous studies reported that hyaluronic acid (HA), chondroitin sulphate (CS) and heparan sulphate (HS) were able to reduce the inflammatory process in a variety of cell types after lypopolysaccharide (LPS) stimulation. The aim of this study was to investigate the anti‐inflammatory effect of glycosaminoglycans (GAGs) in mouse articular chondrocytes stimulated with LPS. Chondrocyte treatment with LPS (50 µg/ml) generated high levels of TNF‐α, IL‐1β, IL‐6, IFN‐γ, MMP‐1, MMP‐13, iNOS gene expression and their related proteins, increased NO concentrations (evaluated in terms of nitrites formation), NF‐κB activation and IkBα degradation as well as apoptosis evaluated by the increase in caspase‐3 expression and the amount of its related protein. The treatment of chondrocytes using two different doses (0.5 and 1.0 mg/ml) of HA, chondroitin‐4‐sulphate (C4S), chondroitin‐6‐sulphate (C6S), HS, keratan sulphate (KS) and dermatan sulphate (DS) produced a number of effects. HA exerted a very small anti‐inflammatory and anti‐apoptotic effect while it significantly reduced NO levels, although the effect on iNOS expression and activity was extremely slight. C4S and C6S reduced inflammation mediators and the apoptotic process. C6S failed to decrease NO production, although iNOS expression and activity were significantly reduced. HS, like C4S, was able to reduce all the effects stimulated by LPS treatment. KS and DS produced no reduction in any of the parameters considered. These results give further support to the hypothesis that GAGs actively participate in the regulation of inflammatory and apoptotic processes. J. Cell. Biochem. 106: 83–92, 2009.

Hyaluronan reduces inflammation in experimental arthritis by modulating TLR-2 and TLR-4 cartilage expression.

Previous studies have reported that low molecular mass HA and highly polymerized HA respectively elicited pro- and anti-inflammatory responses by modulating the toll-like receptor 4 (TLR-4) and the TLR-2. The activation of TLR-4 and TLR-2 mediated by collagen-induced arthritis (CIA) induces the myeloid differentiation primary response protein (MyD88) and the tumor necrosis factor receptor-associated factor 6 (TRAF6), and ends with the liberation of NF-kB which, in turn, stimulates pro-inflammatory cytokine production. The aim of this study was to investigate the influence of high molecular weight HA at different concentrations on TLR-4 and TLR-2 modulation in CIA in mice. Arthritis was induced in mice via intradermal injection of an emulsion containing bovine type II collagen in complete Freunds adjuvant. Mice were treated with HA intraperitoneally daily for 30days. CIA increased TLR-4, TLR-2, MyD88 and TRAF6 mRNA expression and the related protein in the cartilage of arthritic joints. High levels of both mRNA and related protein were also detected for tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL-1-β), interleukin-17 (IL-17), matrix metalloprotease-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in the joint of arthritic mice. HA treatment significantly limited CIA incidence and decreased all the parameters up-regulated by CIA. The improvement of biochemical parameters was also supported by histological analysis, plasma and synovial fluid HA levels. These results suggest that the TLR-4 and TLR-2 play an important role in the arthritis mechanism and the interaction/block of HA at high molecular mass may reduce inflammation and cartilage injury.

Hyaluronan differently modulates TLR‐4 and the inflammatory response in mouse chondrocytes

Hyaluronic acid (HA) may exert different action depending on its degree of polymerization. Small HA fragments induce proinflammatory responses, while highly polymerized HA exerts a protective effect in inflammatory pathologies such as rheumatoid arthritis. In both cases the toll-like receptor 4 (TLR-4) seems to be involved in the modulation of the inflammation process. The aim of this study was to investigate the influence of short HA oligosaccharides (HA 4-mers) and high molecular weight HA (HMWHA) in the inflammatory response in normal mouse chondrocytes. Messenger RNA and related protein levels were measured for TLR-4, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and interleukin-18 (IL-18) in cells with and without the addition of HA. NF-kB activation was also evaluated. 4-mer HA treatment produced a significant up-regulation of all parameters considered while HMWHA did not exert any activity in untreated cells although it was able to reduce the effects of 4- mers HA significantly. Specific TLR-4 small interference RNA (siRNA) was used to confirm TLR-4 as the target of HA action. This study suggests that HA may modulate proinflammatory cytokines via its different degree of polymerization and inflammatory action may be modulated as a result of the interaction between HA and TLR-4.

The inhibition of hyaluronan degradation reduced pro‐inflammatory cytokines in mouse synovial fibroblasts subjected to collagen‐induced arthritis

Hyaluronan (HA) degradation produces small oligosaccharides that are able to increase pro‐inflammatory cytokines in rheumatoid arthritis synovial fibroblasts (RASF) by activating both CD44 and the toll‐like receptor 4 (TLR‐4). CD44 and TLR‐4 stimulation in turn activate the NF‐kB that induces the production of pro‐inflammatory cytokines. Degradation of HA occurs via two mechanisms: one exerted by reactive oxygen species (ROS) and one controlled by different enzymes in particular hyaluronidases (HYALs). We aimed to investigate the effects of inhibiting HA degradation (which prevents the formation of small HA fragments) on synovial fibroblasts obtained from normal DBA/J1 mice (NSF) and on synovial fibroblasts (RASF) obtained from mice subjected to collagen induced arthritis (CIA), both fibroblast types stimulated with tumor necrosis factor alpha (TNF‐α). TNF‐α stimulation produced high mRNA expression and the related protein production of CD44 and TLR‐4 in both NSF and RASF, and activation of NF‐kB was also found in all fibroblasts. TNF‐α also up‐regulated the inflammatory cytokines, interleukin‐1beta (IL‐1beta) and interleukin‐6 (IL‐6), and other pro‐inflammatory mediators, such as matrix metalloprotease‐13 (MMP‐13), inducible nitric oxide synthase (iNOS), as well as HA levels and small HA fragment production. Treatment of RASF with antioxidants and specific HYAL1, HYAL2, and HYAL3 small interference RNA (siRNAs) significantly reduced TLR‐4 and CD44 increase in the mRNA expression and the related protein synthesis, as well as the release of inflammatory mediators up‐regulated by TNF‐α. These data suggest that the inhibition of HA degradation during arthritis may contribute to reducing TLR‐4 and CD44 activation and the inflammatory mediators response. J. Cell. Biochem. 113: 1852–1867, 2012.

Aromatic trap analysis of free radicals production in experimental collagen-induced arthritis in the rat: protective effect of glycosaminoglycans treatment.

Many findings demonstrated that Glycosaminoglycans (GAGs) and Proteoglycans (PGs) possess antioxidant activity. Collagen-induced arthritis (CIA) is an experimental animal model similar to human rheumatoid arthritis (RA) in which free radicals are involved. Sodium salicylate can be used as a chemical trap for hydroxyl radicals (OH ” ), the most damaging reactive oxygen species (ROS), yielding 2,5-dihydroxybenzoic acid), (2,5-DHBA) and 2,3-dihydroxybenzoic acid (2,3-DHBA). The measurement of these two acids in the plasma allows to indirectly assess the production of OH ” radicals. The aim of the study was to investigate the effect of hyaluronic acid (HYA) (30 mg/kg i.p.) or chondroitin-4-sulphate (C4S) (30 mg/kg i.p.), on free radical production in Lewis rats subjected to CIA. After the immunization with bovine collagen type II in complete Freunds adjuvant, rats developed an erosive hind paw arthritis, that produced high plasma OH ” levels assayed as 2,3-DHBA and 2,5-DHBA, primed lipid peroxidation, evaluated by analyzing conjugated dienes (CD) in the articular cartilage; decreased the concentration of endogenous vitamin E (VE) and catalase (CA) in the joint cartilage; enhanced macrophage inflammatory protein-2 (MIP-2) serum levels and increased elastase (ELA) evaluated as an index of activated leukocyte polymophonuclear (PMNs) accumulation in the articular joints. The administration of HYA and C4S starting at the onset of arthritis (day 11) for 20 days, limited inflammation and the clinical signs in the knee and paw, reduced OH ” production, decreased CD levels, partially restored the endogenous antioxidants VE and CA, reduced MIP-2 serum levels and limited PMNs infiltration. The results indicate that the GAGs HYA and C4S significantly reduce free radical production in CIA and could be used as a tool to investigate the role of antioxidants in RA.

Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA

BACKGROUND Hyaluronan (HA) fragments elicit the expression of inflammatory mediators through a mechanism involving the CD44 receptor. This study investigated the effects of HA at different molecular weights on PMA-induced inflammation in mouse chondrocytes. METHODS mRNA and related protein levels were measured for CD44, PKCdelta, PKCepsilon, TNF-alpha, IL-1beta, MMP-13, and iNOS in chondrocytes, untreated or PMA treated, with and without the addition of HA. The level of NF-kB activation was also assayed. RESULTS CD44, PKCdelta, and PKCepsilon mRNA expression resulted higher than controls in chondrocytes treated with PMA. PMA also induced NF-kB up-regulation and increased TNF-alpha, IL-1beta, MMP-13, and iNOS expression. HA treatment produced different effects: low MW HA up-regulated CD44 expression, increased PKCdelta and PKCepsilon levels, and enhanced inflammation in untreated chondrocytes; while in PMA-treated cells it increased CD44, PKCdelta, PKCepsilon, NF-kB, TNF-alpha, IL-1beta, MMP-13, and iNOS expression and enhanced the effects of PMA; medium MW HA did not exert action; high MW HA had no effect on untreated chondrocytes; however, it reduced PKCdelta, PKCepsilon, NF-kB activation and inflammation in PMA-stimulated cells. Specific CD44 blocking antibody was utilised to confirm CD44 as the target of HA modulation. GENERAL SIGNIFICANCE These data suggest that HA via CD44 may modulate inflammation via its different molecular mass.