Cell Biology International 30 (2006) 21e30
www.elsevier.com/locate/cellbi
Purified human chondroitin-4-sulfate reduced MMP/TIMP imbalance
induced by iron plus ascorbate in human fibroblast cultures
Giuseppe M. Campo a,*, Angela Avenoso a, Salvatore Campo a, Angela D’Ascola a,
Alida M. Ferlazzo b, Dario Samà c, Alberto Calatroni a
a
Department of Biochemical, Physiological and Nutritional Sciences, School of Medicine, University of Messina,
Policlinico Universitario, Torre Biologica, 5 piano, Via C. Valeria, 98125 Messina, Italy
b
Department of Morphology, Biochemistry, Physiology and Animal Production, School of Veterinary Medicine,
University of Messina, contrada Annunziata, 98168 Messina, Italy
c
Haematologic Operative Unit, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica,
5 piano, Via C. Valeria, 98125 Messina, Italy
Received 17 May 2005; revised 11 June 2005; accepted 20 August 2005
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.
Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
Keywords: Metalloproteinases; Antioxidants; Chondroitin-4-sulfate; Oxidative stress; Fibroblasts
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
* Corresponding author. Tel.: C39 90 221 3334; fax: C39 90 221 3330.
E-mail address: [email protected] (G.M. Campo).
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
1065-6995/$ - see front matter Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.cellbi.2005.08.009
G.M. Campo et al. / Cell Biology International 30 (2006) 21e30
22
Nomenclature
ATP
BHT
C4S
C6S
DCM
DMEM
ECM
EDTA
GAGs
HA
HAE
MMPs
8-OHdG
OHc
PBS
PGs
P-HC4S
ROS
SDS
TIMPs
adenosine triphosphate
butylated hydroxytoluene
chondroitin-4-sulphate
chondroitin-6-sulphate
dichloromethane
Dulbecco’s minimal essential medium
extracellular matrix
ethylenediaminetetracetic acid
glycosaminoglycans
hyaluronic acid
hydroxyalchenals
matrix metalloproteinases
8-hydroxy-2-deoxyguanosine
hydroxyl radical
phosphate buffered saline
proteoglycans
purified human chondroitin-4-sulphate
reactive oxygen species
sodium dodecylsulphate
tissue inhibitor metalloproteinases
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 FeCC or CuCC
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
(SigmaeAldrich). Dulbecco’s minimal essential medium (DMEM), foetal
bovine serum (FBS), L-glutamine, penicillin/streptomycin, trypsineEDTA
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 SigmaeAldrich 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 23e54 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 75 cm2 plastic flasks containing DMEM supplemented with 10%
FBS, L-glutamine (2.0 mM) and penicillin/streptomycin (100 U/ml, 100 mg/
ml), and incubated in an incubator at 37 C in humidified air with 5% CO2.
G.M. Campo et al. / Cell Biology International 30 (2006) 21e30
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 2.0 ml of fresh medium containing the purified human
plasma C4S (P-HC4S) in concentrations of 0.5, 1.0 and 2.0 mg/ml. After
4 h of incubation, oxidative stress was induced in the cells in the following
way: 10 ml of 400 mM FeSO4 was added in a series of wells (final concentration 2.0 mM) pretreated with P-HC4S or the vehicle. Then, 15 min after, 10 ml
of 200 mM of ascorbic acid was added for free radical production (Collis
et al., 1996). After 1.5 h, in all experiments, the medium was discarded and
replaced by 2.0 ml of the same fresh medium. Twenty-four hours later cells
were subjected to morphological and biochemical evaluation.
2.5. Cell viability assay
After 24 h 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, 24 h after oxidative stress induction, were added to each well, followed by incubation at 25 C for 2 h.
Then, after washing, a specific chromogenic peptide substrate was added
and incubated at 25 C for 30 min. Finally, the reaction was stopped with
an acid solution, and the absorbance was measured at 450 nm 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, MMP9, 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 60 min at 37 C. The
first strand of cDNA was synthesized from 1.0 mg total RNA using 200 units
of Superscript II RnaseHÿ Reverse Transcriptase (Gibco, BRL) and random
decamer primers (Ambion, Austin, USA). To allow the relative quantification
of MMP and TIMP mRNAs, b-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 b-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 b-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 b-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 24 h 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
23
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 1 h. After washing, a secondary antibody was added
into the well and incubated at 37 C for 1 h. Then, after the addition of a chromogen solution, and a further incubation, in the dark at room temperature, for
15 min, the reaction was stopped and the absorbance was read at 450 nm 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 4e5 ! 106 obtained 24 h after oxidative stress induction were collected in 500 ml of PBS containing 200 mM BHT and were
stored at ÿ80 C. Samples were first extracted with dichloromethane, centrifuged at 500 ! g for 5 min at 4 C, and the pellet was resuspended and sonicated in 250 ml 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 586 nm. A calibration curve of an accurately prepared
standard HAE solution (from 0 to 32 nmol/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
(OHc) and the superoxide anion (O2c), the measurement of active aconitase
may be a good biomarker to assess oxidative damage in biological systems.
Cell samples (4e5 ! 106) obtained 24 h after oxidative stress induction
were collected in 500 ml of PBS containing 50 ml of protease and phosphatase
inhibitor cocktails and were stored at ÿ80 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 340 nm per minute (DA410). 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 G 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 StudenteNewmaneKeuls 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
G.M. Campo et al. / Cell Biology International 30 (2006) 21e30
24
100
90
of control)
80
Control
P-HC4S (2.0 mg/ml)
FeSO4 + Asc
FeSO4 + Asc + P-HC4S (0.5 mg/ml)
FeSO4 + Asc + P-HC4S (1.0 mg/ml)
FeSO4 + Asc + P-HC4S (2.0 mg/ml)
*p<0.005 and **p<0.001 vs FeSO4 + Asc
°p<0.001 vs CTRL
**
70
60
**
Viability (
50
40
*
30
20
°
10
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 G 22.3
ng/106 cells and 1.76 G 0.38 ng/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 G
17.3, 87.4 G 16.5 and 55.3 G 14.2 ng/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.
0
Fig. 1. Effect of purified human plasma C4S on fibroblast viability (% of control) in the considered model of oxidative stress. Values are the mean G S.D.
of seven experiments.
was exerted with the dose of 2.0 mg/ml and viability of cells
was about 63% while the doses of 0.5 and 1.0 mg/ml protected
about 30% and 48% of fibroblasts, respectively (Fig. 1).
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).
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).
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 2.0 mg/
ml (Fig. 5). No changes were observed after the only addition
of the purified human C4S.
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 0.9 nmol/mg protein. A significant reduction in this
enzyme levels was observed in fibroblasts treated with FeSO4
plus ascorbate only (0.12 G 0.04 nmol/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 2.0 mg/ml, while the lowest was exerted with the
dose of 0.5 mg/ml (Fig. 6). The levels of aconitase detected
in fibroblasts treated only with purified human C4S were
similar to those revealed in control cells.
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
G.M. Campo et al. / Cell Biology International 30 (2006) 21e30
A
30
Control
P-HC4S (2.0 mg/ml)
FeSO4 + Asc
FeSO4 + Asc + P-HC4S (0.5 mg/ml)
FeSO4 + Asc + P-HC4S (1.0 mg/ml)
FeSO4 + Asc + P-HC4S (2.0 mg/ml)
°p<0.001 vs CTRL;
*p<0.05 and **p<0.001 vs FeSO4 + Asc
25
20
ng/mg protein
25
15
10
5
0
TIMP-1
TIMP-2
B
70
°
ng/mg protein
60
*
50
°
40
**
30
*
**
**
20
°
**
10
*
**
**
0
MMP-1
MMP-2
MMP-9
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 G S.D. of seven experiments.
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
G.M. Campo et al. / Cell Biology International 30 (2006) 21e30
A
TIMPs mRNA/ -Actin mRNA ratio (arbitrary units)
26
0,3
Control
P-HC4S (2.0 mg/ml)
FeSO4 + Asc
FeSO4 + Asc + P-HC4S (0.5 mg/ml)
FeSO4 + Asc + P-HC4S (1.0 mg/ml)
FeSO4 + Asc + P-HC4S (2.0 mg/ml)
°p<0.001 vs Control;
*p<0.05, **p<0.005 and ***p<0.001 vs FeSO4 + Asc
0,2
0,1
0
MMPs mRNA/ -Actin mRNA ratio (arbitrary units)
TIMP-1
B
TIMP-2
3,5
°
3
*
2,5
**
2
***
1,5
1
°
*
0,5
***
°
* ** ***
***
0
MMP-1
MMP-2
MMP-9
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 G S.D. of seven experiments.
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 highdensity 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,
G.M. Campo et al. / Cell Biology International 30 (2006) 21e30
°
150
8-OHdG (ng/106 cells)
*
125
**
100
75
**
Control
P-HC4S (2.0 mg/ml)
FeSO4 + Asc
FeSO4 + Asc + P-HC4S (0.5 mg/ml)
FeSO4 + Asc + P-HC4S (1.0 mg/ml)
FeSO4 + Asc + P-HC4S (2.0 mg/ml)
°p<0.001 vs Control;
*p<0.05, **p<0.005 and ***p<0.001 vs FeSO4 + Asc
0,5
***
**
*
°
50
0
25
Fig. 6. Effect of purified human plasma C4S on fibroblast aconitase activity in
the considered model of oxidative stress. Values are the mean G S.D. of seven
experiments.
0
Fig. 4. Effect of purified human plasma C4S on fibroblast 8-OHdG concentrations in the considered model of oxidative stress. Values are the mean G S.D.
of seven experiments.
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 Fe2C ions that are implicated in the initiation of
HabereWeis 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
3,5
°
Hydroxyalkenals (nmol/mg protein)
1
aconitase (mU/mg protein)
175
°p<0.001 vs Control;
*p<0.05 and **p<0.001 vs FeSO4 + Asc
Control
P-HC4S (2.0 mg/ml)
FeSO4 + Asc
FeSO4 + Asc + P-HC4S (0.5 mg/ml)
FeSO4 + Asc + P-HC4S (1.0 mg/ml)
FeSO4 + Asc + P-HC4S (2.0 mg/ml)
3
27
°p<0.001 vs Control;
*p<0.05, **p<0.005 and ***p<0.001 vs FeSO4 + Asc
Control
P-HC4S (2.0 mg/ml)
FeSO4 + Asc
FeSO4 + Asc + P-HC4S (0.5 mg/ml)
FeSO4 + Asc + P-HC4S (1.0 mg/ml)
FeSO4 + Asc + P-HC4S (2.0 mg/ml)
*
2,5
**
2
***
1,5
1
0,5
0
Fig. 5. Effect of purified human plasma C4S on fibroblast HAE content in the
considered model of oxidative stress. Values are the mean G S.D. of seven
experiments.
significant effects in all considered parameters and in a dosedependent 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 dosedependent 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
28
G.M. Campo et al. / Cell Biology International 30 (2006) 21e30
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 CuCC or FeCC
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 CuCC 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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