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Cell Biology International (2006) 30, 21–30 (Printed in Great Britain)
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Purified human chondroitin-4-sulfate reduced MMP/TIMP imbalance induced by iron plus ascorbate in human fibroblast cultures
Giuseppe M. Campoa*, Angela Avenosoa, Salvatore Campoa, Angela D'Ascolaa, Alida M. Ferlazzob, Dario Samàc and Alberto Calatronia
aDepartment of Biochemical, Physiological and Nutritional Sciences, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy
bDepartment of Morphology, Biochemistry, Physiology and Animal Production, School of Veterinary Medicine, University of Messina, contrada Annunziata, 98168 Messina, Italy
cHaematologic Operative Unit, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy

Abstract

Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) is an important control point in tissue remodelling. Several findings have reported a marked MMP/TIMP imbalance in a variety of in vitro models in which oxidative stress was induced. Since previous studies showed that commercial hyaluronan and chondroitin-4-sulphate are able to limit lipid peroxidation during oxidative stress, we investigated the antioxidant capacity of purified human plasma chondroitin-4-sulfate in reducing MMP and TIMP imbalance in a model of ROS-induced oxidative injury in fibroblast cultures.

Purified human plasma chondroitin-4-sulfate was added to the fibroblast cultures exposed to FeSO4 plus ascorbate. We assayed cell death, MMP and TIMP mRNA expression and protein activities, DNA damage, membrane lipid peroxidation, and aconitase depletion. FeSO4 plus ascorbate produced severe death of cells and increased MMP-1, MMP-2 and MMP-9 expression and protein activities. It also caused DNA strand breaks, enhanced lipid peroxidation and decreased aconitase. TIMP-1 and TIMP-2 protein levels and mRNA expression remain unaltered.

Purified human plasma C4S, at three different doses, restored the MMP/TIMP homeostasis, increased cell survival, reduced DNA damage, inhibited lipid peroxidation and limited impairment of aconitase.

These results further support the hypothesis that these biomolecules possess antioxidant activity and by reducing ROS production C4S may limit cell injury produced by MMP/TIMP imbalance.

Keywords: Metalloproteinases, Antioxidants, Chondroitin-4-sulfate, Oxidative stress, Fibroblasts.

*Corresponding author. Tel.: +39 90 221 3334; fax: +39 90 221 3330.

1 Introduction

Oxidative damage is a consequence of the inefficient utilization of molecular oxygen (O2) by cells. The bulk of the O2 absorbed by cells is used for mitochondrial generation of energy in the form of ATP (Droge, 2002). A small percentage of the O2 taken into cells, however, escapes conventional metabolism and is reduced to radicals and non-radical products which, because of their high reactivity, are damaging to subcellular structures. Cells possess a number of defence mechanisms to protect themselves against the toxic effects of free radicals. When the rate of free radical generation exceeds the cell capacity for their removal, a number of alterations of cell constituents, including inactivation of enzymes, damage of nucleic acid bases and proteins, and peroxidation of membrane lipids occur. While the damage to lipids, proteins and DNA seems to be of greatest interest, the injury that occurs is not restricted to these large molecules. In fact, the reactive species generated by these mechanisms attack any molecule in the vicinity of where they are produced (Halliwell and Gutteridge, 1989).

Transition metals such as iron and copper have an incomplete outer shell of electrons and are thus able to undergo changes in oxidation states involving one electron. The easy access to two oxidation states allows iron and copper to participate in redox processes making them essential biological catalysts. The Fenton reaction utilizes this redox cycling ability of iron and copper to increase the rate of reactive oxygen species (ROS) production (Ercal et al., 2001).

The extracellular matrix (ECM), that is an essential component to many tissues of the body, contains proteoglycans (PGs), which are complex macromolecules of a core protein bounded with one or more glycosaminoglycan (GAG) chains. The GAGs are a family of acid polysaccharides that display a variety of fundamental biological roles (Prydz and Dalen, 2000). The typical GAG structure consists of alternating units of uronic acid and hexosamine. Except for hyaluronic acid (HA), GAGs also contain sulphate groups that allow different electrostatic interactions, with a number of biological constituents (Iozzo, 1998). There are two major classes of sulphated GAGs distinguished by the nature of hexosamine units, glucosamine or galactosamine. Chondroitin-4-sulfate (C4S) consists of an alternating polymer of sulphated N-acetylgalactosamine and uronic acid residues linked by glycosidic bonds (Prydz and Dalen, 2000).

Dysregulated metabolism of extracellular matrix, principally due to focal overexpression of matrix metalloproteinases (MMPs), may contribute to tissue damage (Nagase and Woessner, 1999). ROS are known to react with thiol groups, such as those involved in preserving MMP latency, in this way they could modulate the activity of MMPs (Rajagopalan et al., 1996). In addition oxidative stress may regulate MMP production by the regulation of their expression at mRNA level (Herrmann et al., 1993). MMP-1, MMP-2 and MMP-9 seem to be the molecules more involved during tissue disruption, while the respective inhibitors (TIMPs) remain almost unchanged (Herrmann et al., 1993; Wainwright, 2004).

Recently, several studies have shown antioxidant properties of GAGs, mainly for HA and C4S both in the in vitro and in vivo experimental models (Arai et al., 1999; Albertini et al., 1999; Campo et al., 2003a; Balogh et al., 2003; Campo et al., 2004a; Ha, 2004). This antioxidant activity is probably due to their capacity to chelate transition metals like Fe++ or Cu++ that are in turn responsible for the initiation of Fenton reaction (Albertini et al., 1999; Campo et al., 2004a; Scott, 1968). In these studies, GAGs of commercial origin were studied.

Normal human plasma contains low concentration of circulating GAGs (Calatroni et al., 1992; Campo et al., 2001). These levels are in part originated from connective tissue catabolism (Varma and Varma, 1983). At present the exact meaning of these molecules is unclear.

Starting by these previous data the aim of this study was to evaluate the effects of C4S, which have been purified from normal human plasma, on reducing MMP/TIMP imbalance and cell damage in a model of iron-induced oxidative injury in human skin fibroblast cultures.

2 Methods

2.1 Materials

Bio-Gel P-2 was obtained from Bio-Rad, Hercules, CA, USA. The ion exchangers Ecteola-cellulose and Dowex 1×2 were purchased from Fluka (Sigma–Aldrich). Dulbecco's minimal essential medium (DMEM), foetal bovine serum (FBS), l-glutamine, penicillin/streptomycin, trypsin–EDTA solution and phosphate buffered saline (PBS) were obtained from GibcoBRL (Grand Island, NY, USA). All cell culture plastics were obtained from Falcon (Oxnard, CA, USA). Ascorbic acid, iron (II) sulphate, sucrose, ethylenediaminetetracetic acid (EDTA), potassium phosphate, butylated hydroxytoluene (BHT), dichloromethane (DCM), trypan blue, RNase, proteinase K, protease inhibitor cocktail, sodium dodecylsulphate (SDS), and all other general laboratory chemicals were obtained from Sigma–Aldrich S.r.l. (Milan, Italy).

2.2 Isolation and purification of C4S from human plasma

Plasma samples were obtained from the Haematologic Operative Unit of the University of Messina, from both female and male healthy volunteers aged 23–54 years with informed consent to take part in the study. The GAG isolation from plasma and serum preparations was performed by using the technique previously published with some modification (Calatroni et al., 1992). The purity of preparation and the percentage of C4S isolated from the plasma samples were determined by the analysis of unsaturated disaccharides by using a capillary electrophoresis method after treatment with chondroitinase ABC/AC (Al-Hakim and Linhardt, 1991).

2.3 Cell culture

Normal human skin fibroblasts type CRL 2056 were obtained from American Type Culture Collection (Promochem, Teddington, UK). Fibroblasts were cultured in 75cm2 plastic flasks containing DMEM supplemented with 10% FBS, l-glutamine (2.0mM) and penicillin/streptomycin (100U/ml, 100μg/ml), and incubated in an incubator at 37°C in humidified air with 5% CO2.

2.4 Oxidative stress induction

Fibroblasts were cultured into six-well culture plates at a density of 1.3×105 cells/well. Twelve hours after plating (time 0), when cells were firmly attached to the substratum (about 1×105 cells/well), the culture medium was replaced by 2.0ml of fresh medium containing the purified human plasma C4S (P-HC4S) in concentrations of 0.5, 1.0 and 2.0mg/ml. After 4h of incubation, oxidative stress was induced in the cells in the following way: 10μl of 400μM FeSO4 was added in a series of wells (final concentration 2.0μM) pretreated with P-HC4S or the vehicle. Then, 15min after, 10μl of 200mM of ascorbic acid was added for free radical production (Collis et al., 1996). After 1.5h, in all experiments, the medium was discarded and replaced by 2.0ml of the same fresh medium. Twenty-four hours later cells were subjected to morphological and biochemical evaluation.

2.5 Cell viability assay

After 24h of oxidative stress, cell viability was determined under photozoom invert microscope (Optech GmbH, Munchen, Germany) connected with a digital camera (mod. Coolpix 4500, Tokyo, Japan). The number of viable cells was then quantitated by trypan blue dye exclusion test from several randomly chosen areas of each well (Krischel et al., 1998).

2.6 MMP and TIMP ELISA assay

The total MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 protein levels were determined by specific commercial ELISA assay kits (Biotrak cod. RPN2610, cod. RPN2617, cod. RPN2614, cod. RPN2611 and cod. RPN2618, Amersham Bioscences, Piscataway, USA) according to the protocols of the manufacturer. Briefly, anti-MMP and anti-TIMP antibodies were precoated onto microtiter wells. Culture media, 24h after oxidative stress induction, were added to each well, followed by incubation at 25°C for 2h. Then, after washing, a specific chromogenic peptide substrate was added and incubated at 25°C for 30min. Finally, the reaction was stopped with an acid solution, and the absorbance was measured at 450nm by using a microplate reader (DAS srl, Rome, Italy). The concentration of MMPs and TIMPs in each sample was determined by interpolation from a standard curve.

2.7 RNA isolation, cDNA synthesis and real-time quantitative PCR amplification

Total RNA for reverse-PCR real-time analysis of MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 was isolated from 4 to 5×106 cells by using the Omnizol Reagent Kit (Euroclone, West York, UK). Before cDNA synthesis, residual genomic DNA was digested with DNase I for 60min at 37°C. The first strand of cDNA was synthesized from 1.0μg total RNA using 200 units of Superscript II RnaseH− Reverse Transcriptase (Gibco, BRL) and random decamer primers (Ambion, Austin, USA). To allow the relative quantification of MMP and TIMP mRNAs, β-actin mRNA was used as an endogenous control (Bustin, 2000). Specific TaqMan primers and probes were designed with the “Primer Express” 1.0 software (Applied Biosystems). The internal fluorogenic probes were labelled at the 5′ end with the reporter dye FAM, at the 3′ end with the quencher dye TAMRA and phosphate-blocked at the 3′ end to prevent extension. The β-actin mRNA probe was labelled with the VIC reporter dye at its 5′ end and the TAMRA quencher dye at its 3′ end. The amplified PCR products were quantified by measuring the MMPs, TIMPs and β-actin mRNA thresholds cycle (CT). The CT values were plotted against log input RNA concentration in samples in serially diluted total RNA of fibroblasts and used to generate standard curves for all mRNAs analysed. The amount of specific mRNA in samples was calculated from the standard curve, and normalized with the β-actin mRNA.

2.8 8-Hydroxy-2′-deoxyguanosine (8-OHdG) assay

DNA extraction and digestion was performed from 4 to 5×106 cell samples obtained 24h after oxidative stress induction. The 8-OHdG levels were analysed as an index of DNA damage. The assay was carried out by using a specific EIA test kit (Bioxytech, cat no. 21026, OxisResearch, Portland, USA). Briefly, samples were added into a microtiter well together with a primary antibody and incubated a 37°C for 1h. After washing, a secondary antibody was added into the well and incubated at 37°C for 1h. Then, after the addition of a chromogen solution, and a further incubation, in the dark at room temperature, for 15min, the reaction was stopped and the absorbance was read at 450nm by using a microplate reader. The concentration of 8-OHdG in each sample was determined by interpolation from a standard curve.

2.9 Lipid peroxidation estimation

Measurement of hydroxyalkenals (HAE) in the cell lysate samples was performed to estimate the extension of lipid peroxidation in the fibroblast cultures. Cell samples of 4–5×106 obtained 24h after oxidative stress induction were collected in 500μl of PBS containing 200μM BHT and were stored at −80°C. Samples were first extracted with dichloromethane, centrifuged at 500×g for 5min at 4°C, and the pellet was resuspended and sonicated in 250μl of sterile H2O (Transsonic Model 420, Elma instrumentation, Germany). HAE evaluation was carried out according to the manufacturer's protocol of a colorimetric commercial kit (Bioxytech HAE-586 cat no. 21043, OxisResearch, Portland, USA). Finally absorbance was measured spectrophotometrically at 586nm. A calibration curve of an accurately prepared standard HAE solution (from 0 to 32nmol/ml) was also run for quantification. The concentration of HAE in cell samples was expressed as nmol/mg protein.

2.10 Aconitase analysis

Aconitase activity was analysed as an index of oxidative damage. Because this enzyme is very sensitive to oxidant agents, such as the hydroxyl radical (OH) and the superoxide anion (O2), the measurement of active aconitase may be a good biomarker to assess oxidative damage in biological systems.

Cell samples (4–5×106) obtained 24h after oxidative stress induction were collected in 500μl of PBS containing 50μl of protease and phosphatase inhibitor cocktails and were stored at −80°C. Aconitase activity was assayed according to the manufacturer's protocol of a spectrophotometric commercial kit (Bioxytech Aconitase-340, cat no. 21041, OxisResearch, Portland, USA). Finally the rate of change of absorbance was measured spectrophotometrically at 340nm per minute (ΔA410). The concentration of aconitase in cell samples was expressed as mU/mg protein.

2.11 Protein analysis

The amount of protein was determined using the Bio-Rad protein assay system (Bio-Rad Lab., Richmond, CA, USA) and bovine serum albumin as a standard according to the published method (Bradford, 1976).

2.12 Statistical analysis

Data are expressed as means±S.D. of at least seven experiments for each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. The statistical significance of differences was set at p<0.05.

3 Results

3.1 Effects of purified human plasma C4S on fibroblast viability

The exposure of cells to FeSO4 plus ascorbate produced a large fibroblast death and growth inhibition as shown in Fig. 1. In particular, the percent of cell viability ranged about 10%. The treatment with human plasma C4S exerted a protective effect in a dose-dependent way. The maximum protection was exerted with the dose of 2.0mg/ml and viability of cells was about 63% while the doses of 0.5 and 1.0mg/ml protected about 30% and 48% of fibroblasts, respectively (Fig. 1).

Fig. 1

Effect of purified human plasma C4S on fibroblast viability (% of control) in the considered model of oxidative stress. Values are the mean±S.D. of seven experiments.

3.2 MMP and TIMP protein levels

The protein amount of MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 in fibroblasts exposed to FeSO4 plus ascorbate was assayed in order to evaluate the effect of purified human plasma C4S on the activity of these enzymes and their inhibitors during oxidative stress (Fig. 2A,B). In the CTRL group low levels of MMPs were measured, consistent with the physiological concentrations of these enzymes. The cells exposed to FeSO4 plus ascorbate only showed a marked increase in MMP-1, MMP-2 and MMP-9 concentrations (Fig. 2B), no effect was seen on TIMP activities (Fig. 2A). The treatment of stressed cells with human plasma C4S was able to reduce MMP activities with all used doses (Fig. 2B). Also in this case the treatment had no effect on TIMP levels (Fig. 2A).

Fig. 2

Effect of purified human plasma C4S on fibroblast TIMP-1, TIMP-2, MMP-1, MMP-2 and MMP-9 protein levels in the considered model of oxidative stress. Values are the mean±S.D. of seven experiments.

3.3 MMP and TIMP mRNA expression

The amount of mRNA of MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 in fibroblasts exposed to FeSO4 plus ascorbate was measured in order to assess the effect of purified human plasma C4S on gene expression in cells during oxidative stress (Fig. 3A,B). In the control group, a low expression of MMPs and TIMPs was observed. After FeSO4 plus ascorbate administration the mRNA expression of all MMPs was significantly increased (Fig. 3B), while no effect was found on TIMPs gene transcription. The addition of purified human plasma C4S to the injured fibroblasts exerted a significant effect on the inhibition of MMPs expression (Fig. 3B). No variations were observed on TIMPs mRNA levels (Fig. 3A).

Fig. 3

Effect of purified human plasma C4S on fibroblast TIMP-1, TIMP-2, MMP-1, MMP-2 and MMP-9 mRNA expressions in the considered model of oxidative stress. Values are the mean±S.D. of seven experiments.

3.4 DNA damage

8-OHdG was evaluated as an indicative marker of DNA strand breaking induced by oxidative stress. As shown in Fig. 4, high levels of this adduct, generated during DNA repair, were observed in fibroblasts after exposure to FeSO4 plus ascorbate in comparison with normal cells (151.2±22.3ng/106 cells and 1.76±0.38ng/106 cells, respectively). In contrast, the DNA damage appeared to be reduced in fibroblasts after exposure to the oxidant and when treated with purified human plasma C4S in a dose-dependent manner (120.6±17.3, 87.4±16.5 and 55.3±14.2ng/106 cells with the doses of 0.5, 1.0 and 2.0, respectively) (Fig. 4). No significant effect was seen in cells treated with human plasma C4S only.

Fig. 4

Effect of purified human plasma C4S on fibroblast 8-OHdG concentrations in the considered model of oxidative stress. Values are the mean±S.D. of seven experiments.

3.5 HAE assay

Evaluation of HAE levels was performed to estimate the degree of membrane lipid peroxidation on cell culture produced by oxidative injury (Fig. 5). A significant increase in HAE production was found in cells exposed to FeSO4 plus ascorbate, while low levels of HAE were found in the untreated fibroblasts. Purified human plasma C4S was able to reduce lipid peroxidation with all used doses. Fibroblasts treated with the lowest concentration were slightly protected, while the maximum effect was achieved with the dose of 2.0mg/ml (Fig. 5). No changes were observed after the only addition of the purified human C4S.

Fig. 5

Effect of purified human plasma C4S on fibroblast HAE content in the considered model of oxidative stress. Values are the mean±S.D. of seven experiments.

3.6 Aconitase levels

Aconitase activity was analysed in order to evaluate the degree of damage after free radical production (Fig. 6). In the untreated cells, aconitase concentrations ranged between 0.7 and 0.9nmol/mg protein. A significant reduction in this enzyme levels was observed in fibroblasts treated with FeSO4 plus ascorbate only (0.12±0.04nmol/mg protein). Also in this case, all used doses of purified human plasma C4S significantly restored the levels of aconitase and limited cellular energy depletion. The maximum effect was produced with the dose of 2.0mg/ml, while the lowest was exerted with the dose of 0.5mg/ml (Fig. 6). The levels of aconitase detected in fibroblasts treated only with purified human C4S were similar to those revealed in control cells.

Fig. 6

Effect of purified human plasma C4S on fibroblast aconitase activity in the considered model of oxidative stress. Values are the mean±S.D. of seven experiments.

4 Discussion

ECM components modulate cellular behaviour by creating influential cellular environments. Thus, the turnover of ECM is an integral part of development, morphogenesis, and tissue remodelling. While various types of proteinases participate in matrix turnover, the MMPs are the principal matrix-degrading proteinases (Nagase and Woessner, 1999).

The MMPs are a family of calcium-dependent zinc-containing endopeptidases, which are capable of degrading a wide variety of ECM components (Bode and Maskos, 2003). They are secreted in an inactive form, which is called a pro-MMP. These inactive MMPs require an activation step before they are able to cleave ECM components (Nagase and Woessner, 1999). MMPs are known to play important roles in tissue remodelling during physiological processes, including tissue repair. The activity of MMPs is regulated by several types of inhibitors, of which the TIMPs are the most important (Nagase and Woessner, 1999). The TIMPs are also secreted proteins, but they may be located at the cell surface in association with membrane-bound MMPs (Baker et al., 2002). The balance between MMPs and TIMPs regulates tissue remodelling under normal conditions. A deregulation of this balance is a characteristic of pathological conditions involving extensive tissue degradation and destruction, such as arthritis, diabetes, skin aging, liver injury, atherosclerosis, cardiac and pulmonary diseases, tumor invasion and metastasis (Murphy et al., 2002; Zaoui et al., 2000; Herouy, 2001; Arthur, 2000; Beaudeux et al., 2004; Lindsey et al., 2003; Suzuki et al., 2004; Polette et al., 2004).

MMP activity is regulated at multiple levels, such as at the level of gene transcription and the synthesis of pro-MMPs. Furthermore, the activation of proenzymes and the inhibition of MMPs by TIMPs are important regulatory processes. MMPs are secreted in a latent form, which require activation. The expression and production of most MMPs is regulated at the transcriptional level by a number of factors, including cytokines, growth factors, mechanical force and several other mechanisms involved in pathological conditions, thereby disturbing the tenuous balance between them and TIMPs (Murphy et al., 2002; Zaoui et al., 2000; Herouy, 2001; Arthur, 2000; Beaudeux et al., 2004; Lindsey et al., 2003; Suzuki et al., 2004; Polette et al., 2004). Two members of the MMP family, MMP-2 and MMP-9, especially degrade type IV collagen and one is thought to specifically regulate basement membrane remodelling. Furthermore they can degrade gelatin after the cleavage of collagen molecules by interstitial collagenases, such as MMP-1 (Woessner, 1994).

ROS are known to react with thiol groups, such as those involved in preserving MMP latency, so they could modulate the activity of MMPs (Wainwright, 2004; Siwik and Colucci, 2004; Deem and Cook-Mills, 2004; Uemura et al., 2001). In particular both gelatinases MMP-2 and MMP-9, and the collagenase MMP-1 are activated by ROS and their expression seems to be regulated by oxidative stress (Uemura et al., 2001; Hemmerlein et al., 2004; Polte and Tyrrell, 2004), whereas TIMP synthesis remains unaltered (Herrmann et al., 1993; Hemmerlein et al., 2004; Wlaschek et al., 1995). One of the several approaches to reduce oxidative stress-induced MMP/TIMP imbalance is the use of antioxidant compounds as therapeutic agents (Orbe et al., 2003; Song et al., 2004; Tosetti et al., 2002).

Chondroitin sulphates (CS) are the more abundant GAGs in humans, and they are localized in connective tissues. Moreover, CS are the more representative components of circulating GAGs, and they also are constituents of normal urine, and are present in granulocytes, platelets and kurloff cells. These molecules may be distinguished by means of their sulfation. They may be sulphated at the C-4 position of galactosamine giving C4S or at the C-6 position of galactosamine giving C6S. In the last years, several findings reported an antioxidant activity of C4S capable of inhibiting lipid peroxidation and protecting cells from ROS damage (Albertini et al., 1999; Campo et al., 2003a; Campo et al., 2004a; Campo et al., 2003b; Campo et al., 2004b; Campo et al., 2004c; Albertini et al., 1997; Albertini et al., 2000). On the contrary, C6S showed no antioxidant properties in a model of high-density lipoprotein (HDL) peroxidation induced by transition metals, a difference with C4S probably due to the different position of the sulphate group (Albertini et al., 1999; Albertini et al., 1997; Albertini et al., 2000).

Acid GAGs are present in blood, usually in proteoglycan form. As stated before, C4S in low sulphate form is the main GAG of normal human plasma. KS, HS and HA are the other GAG structures commonly detected in human plasma (Calatroni, 2002). In animals, the total amounts of GAGs in plasma (Ferlazzo et al., 1997) are similar to those measured in humans (Calatroni et al., 1992). Nevertheless, a marked increase in plasma GAG levels was observed in a wide number of diseases, especially those involving free radical damage (Friman et al., 1987; Laurent et al., 1996; Radhakrishnamurthy et al., 1998; Calabrò et al., 1998; Roughley, 2001; Plevris et al., 2000). This increase in native plasma GAGs during diseases could be a biological response in an attempt to reduce the damage produced by oxidative stress. Nevertheless this increase in antioxidant activity exerted by GAGs is probably insufficient to neutralize the massive amount of ROS released, and the consequent cell injury. However, the exact meaning of their rise is at the moment unclear.

The toxic action of FeSO4 plus ascorbate produced high amount of Fe2+ ions that are implicated in the initiation of Haber–Weis and Fenton reaction. The use of metal chelating agents may then have therapeutic effect by reducing the oxidative burst and the consequent cell damage (Halliwell and Gutteridge, 1984; Gutteridge, 1998).

In the present study we investigated the protective effects of purified human plasma C4S obtained from human plasma in a simple culture system of fibroblasts following exposure to the prooxidant FeSO4 plus ascorbate. The data obtained by treating fibroblasts with this natural compound showed significant effects in all considered parameters and in a dose-dependent way.

The main findings in the present study were that the ratio between MMP-1/2/9 and TIMP-1/2 is shifted towards MMPs in fibroblasts exposed to FeSO4 plus ascorbate in comparison with unexposed cells, and that purified human plasma C4S reduced significantly this shift. This reduction in MMP/TIMP imbalance was revealed both at transcriptional level and also at post-transcriptional level. In fact, the fibroblasts treated with purified human plasma C4S showed a dose-dependent reduction in TIMP gene expression and protein synthesis, while TIMP mRNA levels and protein production remain unaffected in all experiments. In previous reports UVA irradiation elicited an increase in MMP-1 mRNA and protein levels (Wlaschek et al., 1995; Scharffetter-Kochanek et al., 1993). Similar studies indicated that ROS preceded and induced the synthesis and release of signalling peptides such as cytokines that mediated the induction of MMP mRNA (Wlaschek et al., 1995; Wlaschek et al., 1993; Wlaschek et al., 1994). In another study, it has been indicated that activation of cell membrane-associated Src tyrosine kinases and HaRas small guanosine-binding proteins occurs within minutes after UV irradiation-induced oxidative stress, indicating that the cell response to the ROS generation initiated at or near the plasma membrane (Devary et al., 1992). This leads to the activation of nuclear transcription factors, among them activator-protein 1, which in turn enhances MMP gene transcription. However, how ROS initiated the sequence of these events remains to be elucidated.

Interaction of ROS with DNA can induce a multiplicity of products of varying structures and with differing biological impacts. The antioxidant cell defence system intercepts ROS and normally inhibits cellular and nuclear damage. When the amount of ROS produced overwhelms these endogenous defences, an increase in oxidative DNA injury occurs (Marnett, 2002). We have shown that the high 8-OHdG levels generated by the fragmentation of DNA strands observed in the fibroblasts exposed to FeSO4 plus ascorbate was significantly reduced by purified C4S treatment.

Lipid peroxidation is considered a critical mechanism of injury occurring in cells during oxidative stress (Halliwell and Gutteridge, 1989). The evidence supporting these biochemical changes is based on analysis of a wide number of intermediate products (Esterbauer et al., 1991). An indicative method extensively used in evaluating lipid peroxidation is HAE analysis (Esterbauer et al., 1991). The increment of HAE concentrations found in the fibroblasts exposed to the oxidant agent is consistent with the occurrence of free-radical-mediated cell damage. The treatment with purified human plasma C4S limited membrane lipid peroxidation and consequently cell death as reported by cell viability data.

Protein impairment is one of the deleterious effects exerted by ROS to the cell structures. The function of aconitase is to isomerize citrate to isocitrate, a key intermediate of the citric acid cycle. Because of its role in cellular energy production, aconitase enzyme function is well positioned as an important marker relative to biological decline. The decrease in aconitase enzyme activity is used as a sensitive and specific indicator of oxidative damage during oxidative stress (Gardner and Fridovich, 1992). In our findings, the treatment of cells with the purified human plasma C4S limited aconitase inactivation by the reduction of free radical generation.

The antioxidant mechanism of C4S molecules is due to their particular chemical structure with the sulphated group in position 4 of the hexosamine at the opposite side of carboxylic group of uronic acid. These charged groups are supposed to interact with the transition metal ions like Cu++ or Fe++ that are in turn responsible for the initiation of Fenton's reaction. The ability of C4S to chelate different ions and transition metals was extensively reported by several authors (Albertini et al., 1999; Balogh et al., 2003; Albertini et al., 1997; Albertini et al., 2000; Merce et al., 2002; Nagy et al., 1998).

Cation positions have been elucidated for structures containing calcium ions. The co-ordination of the calcium ion, which bridges carboxylate groups in separate chains and also bridges the carboxylate and sulphate group within a single chain of chondroitin-4-sulphate, was shown (Nieduszynski, 1985). Moreover, C4S binds Cu++ ions more strongly than it binds calcium ions (Scott, 1968).

Taken together, these data strongly suggest that C4S is able to bind iron and copper cations in solution decreasing their availability for oxidation processes.

In conclusion, the results obtained from this study confirm the antioxidant activity of C4S and this could be useful knowledge in understanding the exact role played by GAGs in living organisms. Moreover, the data suggest that a physiological increase in GAG production following oxidative stress may be a natural defence in limiting cell damage and MMP/TIMP imbalance.

Acknowledgements

This study was supported in part by a grant ex 40% (COFIN 2002) of the MIUR, Italy and in part by a grant PRA (Research Athenaeum Project 2003) of the University of Messina, Italy.

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Received 17 May 2005/11 June 2005; accepted 20 August 2005

doi:10.1016/j.cellbi.2005.08.009
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