Paola Traina

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.

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.

Glycosaminoglycans reduced inflammatory response by modulating toll-like receptor-4 in LPS-stimulated chondrocytes

Lipopolysaccharide (LPS)-mediated activation of toll-like receptor-4 (TLR-4) complex induces specific signaling pathways, such as the myeloid differentiation primary response protein-88 (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), involving NF-kappaB activation. As previous data reported that hyaluronan (HA) and heparan sulfate (HS) may interact with TLR-4, the aim of this study was to investigate whether glycosaminoglycans (GAGs) may modulate the TLR-4 receptor in a model of LPS-induced inflammatory cytokines in mouse chondrocytes. LPS stimulation up-regulated all inflammation parameters. The GAG treatment produced various effects: HA reduced MyD88 and TRAF-6 levels and NF-kappaB activation at the higher dose only, and exerted a very low anti-inflammatory effect; chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate significantly inhibited MyD88, TRAF-6 and NF-kappaB activation, the inflammation cytokines, and inducible nitric oxide synthase; HS, like C4S, significantly reduced MyD88, TRAF-6, NF-kappaB and inflammation. Specific TLR-4 blocking antibody confirmed that TLR-4 was the target of GAG action.

The antioxidant activity of chondroitin-4-sulphate, in carbon tetrachloride-induced acute hepatitis in mice, involves NF-κB and caspase activation

Reactive oxygen species (ROC) are the main causes of carbon tetrachloride (CCl4)‐induced acute liver injury. Chondroitin‐4‐sulphate (C4S) is known to inhibit lipid peroxidation through antioxidant mechanisms. Activation of nuclear factor (NF)‐κB and caspases may strongly intensify inflammation and cell damage, in addition to that directly exerted by ROS. We investigated whether treatment with C4S, besides exerting antioxidant activity, was able to modulate NF‐κB and apoptosis activation in CCl4‐induced liver injury in mice.

Chondroitin-4-sulphate inhibits NF-kB translocation and caspase activation in collagen-induced arthritis in mice

OBJECTIVE Free radical damage, inflammation, and apoptosis play a critical role in the onset and progression of cartilage erosion in arthritis. Many studies have demonstrated that glycosaminoglycans (GAGs), and chondroitin-4-sulphate (C4S) in particular, possess antioxidant activity that is able to inhibit lipid peroxidation which is the main mechanism of free radical-mediated biological injury. In addition to the effect directly exerted by reactive oxygen species (ROS), the activation of nuclear factor kB (NF-kB) and caspases may contribute substantially to increase inflammation and cell damage. We studied whether the antioxidant action of chronic C4S treatment to reduce ROS injury involves NF-kB and caspases modulation using an experimental model of collagen-induced arthritis in mice. METHODS Arthritis was induced in mice via an intradermal injection at the base of the tail of 100 microl of emulsion containing bovine type II collagen in complete Freunds adjuvant. RESULTS Arthritis provoked the following: severe oedema and inflammation in the hind paws; lipid peroxidation in the joints [measured by 8-isoprostane (8-IPE) levels]; reduction of the endogenous antioxidants catalase (CAT) activity and reduced glutathione (GSH) levels; induction of NF-kB translocation; a loss of cytoplasmic NF-kB inhibitor alpha (IkBalpha); an increase in metalloproteinase-13 (MMP-13), caspase-3 and caspase-7 gene expression and their related protein; the induction of cartilage polymorphonuclear (PMN) activation and infiltration [evaluated by elastase (ELA) assay] and cartilage alterations evaluated by histological analysis. Intraperitoneal administration of different doses of C4S (for 25 days), ameliorated all the symptoms of inflammation in the articular knee and paw joints, limited lipid peroxidation, inhibited NF-kB activation and IkBalpha protein loss, decreased mRNA MMP-13 and caspases expression and their related protein, restored endogenous antioxidants, and reduced PMN accumulation in the damaged cartilage. CONCLUSION The evidence that C4S was able to inhibit NF-kB and apoptosis activation supports the hypothesis that the C4S effect depends on reduction of ROS production, although other direct effects cannot be excluded.

Purified human plasma glycosaminoglycans reduced NF-κB activation, pro-inflammatory cytokine production and apoptosis in LPS-treated chondrocytes

Introduction: There have been several cases reporting a significant increase in chondroitin sulphate plasma levels in patients with different types of disease, such as systemic lupus erythematosus, rheumatoid arthritis, and liver disease. At present, the precise role of chondroitin sulphate molecules in blood is unclear. Previous investigations have shown that the addition of purified human plasma glycosaminoglycans (GAGs), containing a high percentage of chondroitin-4-sulphate (C4S) was able to inhibit lipid peroxidation and to protect cells from reactive oxygen species damage, suggesting antioxidant activity. Starting from these reports, the aim of this study was to evaluate the effectiveness of GAG structures purified from normal human plasma in reducing inflammation using a model of lipopolysaccharide (LPS)-induced increase of pro-inflammatory cytokines in mouse articular chondrocyte cultures. Results: Chondrocyte stimulation with LPS (50 μg/ml) for 24 h enhanced gene expression of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin 6 (IL-6), interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS) and increases in their related protein levels, as well as NF-κB activation, IκBα phosphorylation and apoptosis evaluated by the increase in caspase-3 expression and its related protein amount. LPS treatment also generated a high amount of nitric oxide (NO). The addition of different doses of purified human GAGs to LPSstimulated chondrocytes reduced inflammatory cytokines and iNOS both at mRNA and protein levels, blocked NF-κB activation and cytoplasmic IκBα phosphorylation, limited cell death by inhibiting apoptosis, and reduced NO concentrations. Conclusions: These results further support the hypothesis that plasma GAGs may function as immunomodulators and their increased release and degradation could be a biological response acting to modulate inflammation during disease.

Differential effect of molecular mass hyaluronan on lipopolysaccharide-induced damage in chondrocytes

Hyaluronan is a biological polysaccharide that may exist in different degrees of polymerization. Several investigations reported that low molecular mass hyaluronan may have pro-inflammatory activity, while high molecular mass hyaluronan can exert beneficial effects. Starting from these data, the aim of this study was to investigate the effect of hyaluronan of different molecular mass in mouse articular chondrocyte cultures stimulated with lipopolysaccharide (LPS). Inflammation was induced in chondrocytes by acute treatment with 2.0 µg/ml LPS. High levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ and iNOS gene expression and their related proteins were found in chondrocytes 24 h after treatment with LPS. High concentrations of NO, NF-κB activation, IκBα phosphorylation and apoptosis, evaluated by the increase in caspase-3 expression and its related protein amount were also produced by LPS stimulation. In contrast, LPS reduced aggrecan and collagen type II (Col2A) expression and their protein production. The treatment of chondrocytes with hyaluronan of different molecular mass produced the following effects: (i) low molecular mass hyaluronan exerted a slight inflammatory effect in untreated chondrocytes, while in LPS-treated chondrocytes it enhanced cytokine production and decreased aggrecan and Col2A compared with cells treated with LPS alone; (ii) no effect was exerted on LPS-induced apoptosis and NO production; (iii) medium molecular mass hyaluronan did not exert any inflammatory/anti-inflammatory activity in LPS-untreated/treated cells and failed to reduce apoptosis; and (iv) high molecular mass hyaluronan had no inflammatory effect in LPS-untreated cells while it was able to reduce all the detrimental effects stimulated by LPS treatment. These data confirm the multifactorial role played by hyaluronan and suggest, in particular, that hyaluronan may modulate inflammation during pathologies by its different degrees of polymerization.

Hemoglobin system of Sparus aurata: changes in fishes farmed under extreme conditions

In order to gain more knowledge on the stress responses of gilhead seabream (Sparus aurata) under extreme conditions, this study investigated the functional properties of the hemoglobin system and globin gene expression under hypoxia and low salinity. The oxygen affinity for the two hemoglobin components present inside the S. aurata erythrocyte was practically identical as was the influence of protons and organic phosphates (Root effect). The quantification of S. aurata hemoglobin fractions performed by HPLC and the data on gene expression of globin chains assayed by PCR indicate that under hypoxia and low salinity there is a change in the ratio between the two different hemoglobin components. The result indicating that the distinct hemoglobins present in S. aurata erythrocyte have almost identical functional properties, does not explain the adaptive response (expression change) following exposure of the animal to hypoxia or low salinity on the basis of their function as oxygen transporter. We hypothesize that other parallel biological functions that the hemoglobin molecule is known to display within the erythrocyte are involved in adaptive molecular mechanisms. The autoxidation-reduction cycle of hemoglobin could be involved in the response to particular living conditions.

Chondroitin-4-Sulphate Reduced Oxidative Injury in Caerulein-Induced Pancreatitis in Mice: The Involvement of NF-κB Translocation and Apoptosis Activation

Activation of nuclear factor κB (NF-κB) and caspases may greatly amplify inflammation and cell damage in addition to that directly exerted by free radicals. Since reactive oxygen species (ROS) are involved in acute pancreatitis, we studied whether the administration of chondroitin-4-sulphate (C4S), in addition to its antioxidant activity, was able to modulate NF-κB and caspase activation in an experimental model of caerulein-induced acute pancreatitis in mice. Hyperstimulating doses of caerulein (50 μg/ kg), five injections per mouse given at hourly intervals produced the following: high serum lipase and amylase activity; lipid peroxidation, evaluated by 8-isoprostane concentrations; loss of antioxidant defenses such as glutathione reductase (GR) activity; NF-κB activation and loss of cytoplasmic IκBα protein; increases in tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), caspase-3, and caspase-7 gene expression and their related protein; accumulation and activation of neutrophils in the damaged tissue, evaluated by elastase (ELA) determination; and pancreatic injury, evaluated by histologic analysis. Pretreatment of mice with different doses of C4S, given 1 hr before caerulein injections and 1 and 2 hrs after the last caerulein injection, reduced lipid peroxidation, inhibited NF-κB translocation and cytoplasmic IκBα protein loss, decreased TNF-α, IL-6, and caspase gene expression and their related protein levels, limited endogenous antioxidant depletion, and reduced tissue neutrophils accumulation and tissue damage. Since molecules with antioxidant activity can block NF-κB and apoptosis activation, we suggest that C4S administration is able to block NF-κB and caspase activation by reducing the oxidative burst.

Effect of cytokines on hyaluronan synthase activity and response to oxidative stress by fibroblasts

Cytokines such as tumour necrosis factor-α (TNFα), interferon-γ (IFNγ), and transforming growth factor-β (TGF1β) modulate hyaluronan synthase (HAS) gene expression and protein activity. The aim of this research is to evaluate the response of HAS gene expression and the related protein synthesis in fibroblasts after treatment with TNFα, IFNγ and TGF1β and to assess the potential protective effect of increased hyaluronan (HA) synthesis during oxidative stress. In this study, gene expression, protein synthesis, hyaluronan content, cell death, lactate dehydrogenase (LDH) activity, membrane lipid peroxidation and endogenous antioxidant depletion are determined for HAS1, HAS2 and HAS3. Messenger RNA (mRNA) expression and protein formation of the three HAS genes is modulated using different cytokines and various doses and correlated with increased HA synthesis. Protection of fibroblasts from injury induced by exposure to reactive oxygen species was significantly increased by TGF1β and was associated with increased gene expression and protein formation of HAS1 and HAS2 enzymes synthesising high-molecular-weight HA. It is proposed that specific HAS enzyme activity and HA molecular weight specificity is involved in the protective mechanism.