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Cell Biology International (2006) 30, 2130 (Printed in Great Britain)
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
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 FeSO
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.
Oxidative damage is a consequence of the inefficient utilization of molecular oxygen (O
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.
Bio-Gel P-2 was obtained from Bio-Rad, Hercules, CA, USA. The ion exchangers Ecteola-cellulose and Dowex 1
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 75
2.4 Oxidative stress induction
Fibroblasts were cultured into six-well culture plates at a density of 1.3
2.5 Cell viability assay
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
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
2.8 8-Hydroxy-2′-deoxyguanosine (8-OHdG) assay
DNA extraction and digestion was performed from 4 to 5
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
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
Cell samples (4–5
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
3.1 Effects of purified human plasma C4S on fibroblast viability
The exposure of cells to FeSO
Effect of purified human plasma C4S on fibroblast viability (% of control) in the considered model of oxidative stress. Values are the mean
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 FeSO
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
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 FeSO
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
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 FeSO
Effect of purified human plasma C4S on fibroblast 8-OHdG concentrations in the considered model of oxidative stress. Values are the mean
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 FeSO
Effect of purified human plasma C4S on fibroblast HAE content in the considered model of oxidative stress. Values are the mean
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
Effect of purified human plasma C4S on fibroblast aconitase activity in the considered model of oxidative stress. Values are the mean
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 FeSO
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 FeSO
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 FeSO
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 FeSO
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.
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.
Albertini R, Ramos, P, Giessauf, A, Passi, A, De Luca, G, Esterbauer, H. Chondroitin 4-sulphate exhibits inhibitory effect during Cu2+-mediated LDL oxidation. FEBS Lett 1997:403:154-8
Albertini R, De Luca, G, Passi, A, Moratti, R, Abuja, PM. Chondroitin-4-sulfate protects high-density lipoprotein against copper-dependent oxidation. Arch Biochem Biophys 1999:5:143-9
Arai H, Kashiwagi, S, Nagasaka, Y, Uchida, K, Hoshii, Y, Nakamura, K. Oxidative modification of apoliprotein E in human very-low-density lipoprotein and its inhibition by glycosaminoglycans. Arch Biochem Biophys 1999:367:1-8
Balogh GT, Illes, J, Szekely, Z, Forrai, E, Gere, A. Effect of different metal ions on the oxidative damage and antioxidant capacity of hyaluronic acid. Arch Biochem Biophys 2003:410:76-82
Beaudeux JL, Giral, P, Bruckert, E, Foglietti, MJ, Chapman, MJ. Matrix metalloproteinases, inflammation and atherosclerosis: therapeutic perspectives. Clin Chem Lab Med 2004:42:121-31
Bode W, Maskos, K. Structural basis of the matrix metalloproteinases and their physiological inhibitors, the tissue inhibitors of metalloproteinases. Biol Chem 2003:384:863-72
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976:72:248-54
Calabrò L, Musolino, C, Spatari, G, Vinci, R, Calatroni, A. Increased concentration of circulating acid glycosaminoglycans in chronic lymphocytic leukaemia and essential thrombocythaemia. Clin Chim Acta 1998:269:185-99
Calatroni A. Extraction and purification of glycosaminoglycans (GAGs) from biological fluids. Analytical techniques to evaluate the structure and function of natural polysaccharides, glycosaminoglycans 2002:15-22
Calatroni A, Vinci, R, Ferlazzo, AM. Characteristics of the interactions between acid glycosaminoglycans and proteins in normal human plasma as revealed by the behaviour of the protein–polysaccharide complexes in ultrafiltration and chromatographic procedures. Clin Chim Acta 1992:206:167-80
Campo GM, Campo, S, Ferlazzo, AM, Vinci, R, Calatroni, A. Improved high-performance liquid chromatographic method to estimate aminosugars and its application to glycosaminoglycan determination in plasma and serum. J Chromatogr B 2001:765:151-60
Campo GM, Avenoso, A, Campo, S, Ferlazzo, AM, Altavilla, D, Micali, C. Aromatic trap analysis of free radicals production in experimental collagen-induced arthritis in the rat: protective effect of glycosaminoglycans treatment. Free Radic Res 2003:37:257-68
Campo GM, Avenoso, A, Campo, S, Ferlazzo, AM, Altavilla, D, Calatroni, A. Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats. Arthritis Res Ther 2003:5:R122-31
Campo GM, Avenoso, A, Campo, S, D'Ascola, A, Ferlazzo, AM, Calatroni, A. Reduction of DNA fragmentation and hydroxyl radical production by hyaluronic acid and chondroitin-4-sulphate in iron plus ascorbate-induced oxidative stress in fibroblast cultures. Free Radic Res 2004:38:601-11
Campo GM, Avenoso, A, Campo, S, Ferlazzo, AM, Micali, C, Zanghì, L. Hyaluronic acid and chondroitin-4-sulphate treatment reduces damage in carbon tetrachloride-induced acute rat liver injury. Life Sci 2004:74:1289-305
Campo GM, D'Ascola, A, Avenoso, A, Campo, S, Ferlazzo, AM, Micali, C. Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H
Collis CS, Yang, M, Peach, SJ, Diplock, AT, Rice-Evans, C. The effects of ascorbic acid and iron co-supplementation on the proliferation of 3T3 fibroblasts. Free Radic Res 1996:25:87-93
Deem TL, Cook-Mills, JM. Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species. Blood 2004:104:2385-93
Ferlazzo AM, Campo, S, Vinci, R, Ferlazzo, A, Calatroni, A. Concentration and composition of serum and plasma glycosaminoglycans in domestic animal species. Comp Biochem Physiol 1997:118B:935-42
Gutteridge JMC. Iron in free radical reactions and antioxidant protection. Free radicals, oxidative stress, and antioxidants. Pathological and physiological significance 1998:1-14
Hemmerlein B, Johanns, U, Halbfass, J, Bottcher, T, Heuser, M, Radzun, HJ. The balance between MMP-2/9 and TIMP-1/2 is shifted towards MMP in renal cell carcinomas and can be further disturbed by hydrogen peroxide. Int J Oncol 2004:24:1069-76
Herrmann G, Wlaschek, M, Lange, TS, Prenzel, K, Goerz, G, Scharffetter-Kochanek, K. UVA radiation stimulates the synthesis of various matrix metalloproteinases (MMPs) in cultured human fibroblasts. Exp Dermatol 1993:2:92-7
Krischel V, Bruch-Gerharz, D, Suschek, C, Kroncke, KD, Ruzicka, T, Kolb-Bachofen, V. Biphasic effect of exogenous nitric oxide on proliferation and differentiation in skin derived keratinocytes but not fibroblasts. J Invest Dermatol 1998:111:286-91
Merce ALR, Carrera, LCM, Romanholi, LKS, Recio, MAL. Aqueous and solid complexes of iron(III) with hyaluronic acid potentiometric titrations and infrared spectroscopy studies. J Inorg Biochem 2002:89:212-8
Nieduszynski IA. Connective tissue polysaccharides. Polysaccharides. Topics in structure and morphology 1985:120-50
Orbe J, Rodriguez, JA, Arias, R, Belzunce, M, Nespereira, B, Perez-Ilzarbe, M. Antioxidant vitamins increase the collagen content and reduce MMP-1 in a porcine model of atherosclerosis: implications for plaque stabilization. Atherosclerosis 2003:167:45-53
Plevris JN, Haydon, GH, Simpson, KJ, Dawkes, R, Ludlum, CA, Harrison, DJ. Serum hyaluronan: a non invasive test for diagnosing liver cirrhosis. Eur J Gastroenterol Hepatol 2000:12:1121-7
Rajagopalan S, Meng, XP, Ramasamy, S, Harrison, DG, Galis, ZS. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability. J Clin Invest 1996:98:2572-9
Song XZ, Xia, JP, Bi, ZG. Effects of (−)-epigallocatechin-3-gallate on expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in fibroblasts irradiated with ultraviolet A. Chin Med J 2004:117:1838-41
Suzuki R, Miyazaki, Y, Takagi, K, Torii, K, Taniguchi, H. Matrix metalloproteinases in the pathogenesis of asthma and COPD: implications for therapy. Treat Resp Med 2004:3:17-27
Uemura S, Matsushita, H, Li, W, Glassford, AJ, Asagami, T, Lee, KH. Diabetes mellitus enhances vascular matrix metalloproteinase activity: role of oxidative stress. Circ Res 2001:88:1291-8
Varma R, Varma, RS. . Mucopolysaccharides (glycosaminoglycans) of body fluids in health and disease 1983:
Wlaschek M, Bolsen, K, Herrmann, G, Schwarz, A, Wilmroth, F, Heinrich, PC. UVA-induced autocrine stimulation of fibroblast-derived collagenase by Il-6. A possible mechanism in dermal photodamage? J Invest Dermatol 1993:101:164-8
Wlaschek M, Briviba, K, Stricklin, GP, Sies, H, Scharffetter-Kochanek, K. Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase. J Invest Dermatol 1995:104:194-8
Wlaschek M, Heinen, G, Poswing, A, Schwarz, A, Krieg, T, Scharffetter-Kochanek, K. UVA-induced autocrine stimulation of fibroblast derived collagenase/MMP-1 by interrelated loops of interleukin-1 and interleukin-6. Photochem Photobiol 1994:59:550-6
Zaoui P, Cantin, JF, Alimardani-Bessette, M, Monier, F, Halimi, S, Morel, F. Role of metalloproteases and inhibitors in the occurrence and progression of diabetic renal lesions. Diabetes Metab 2000:26:Suppl. 4:25-9
Received 17 May 2005/11 June 2005; accepted 20 August 2005doi:10.1016/j.cellbi.2005.08.009