Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts.
 

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Vol. 280, Issue 1, C53-C60, January 2001
 

Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts

Deborah A. Siwik¹, Patrick J. Pagano², and Wilson S. Colucci¹

¹ Myocardial Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, and Cardiovascular Division, Boston Medical Center, Boston, Massachusetts 02118; and ² Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan 48202

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that oxidative stress can regulate extracellular matrix in cardiac fibroblasts. Neonatal and adult rat cardiac fibroblasts in vitro were exposed to H₂O₂ (0.05-5 µM) or the superoxide-generating system xanthine (500 µM) plus xanthine oxidase (0.001-0.1 mU/ml) (XXO) for 24 h. In-gel zymography demonstrated that H₂O₂ and XXO each increased gelatinase activity corresponding to matrix metalloproteinases (MMP) MMP-13, MMP-2, and MMP-9. H₂O₂ and XXO decreased collagen synthesis (collagenase-sensitive [³H]proline incorporation) without affecting total protein synthesis ([³H]leucine incorporation). H₂O₂ and XXO decreased the expression of procollagen ₁(I), ₂(I), and ₁(III) mRNA but increased the expression of fibronectin mRNA, suggesting a selective transcriptional effect on collagen synthesis. H₂O₂, but not XXO, also decreased the expression of nonfibrillar procollagen ₁(IV) and ₂(IV) mRNA. To determine the role of endogenous antioxidant systems, cells were treated with the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid (DDC, 100 µM) to increase intracellular superoxide or with the glucose-6-phosphate dehydrogenase inhibitor dehydroisoandrosterone 3-acetate (DHEA; 10 µM) to increase intracellular H₂O₂. DDC and DHEA decreased collagen synthesis and increased MMP activity, and both effects were inhibited by an SOD/catalase mimetic. Thus increased oxidative stress activates MMPs and decreases fibrillar collagen synthesis in cardiac fibroblasts. Oxidative stress may play a role in the pathogenesis of myocardial remodeling by regulating the quantity and quality of extracellular matrix.

reactive oxygen species; H₂O₂; superoxide; superoxide dismutase; glucose-6-phosphate dehydrogenase; in vitro

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

FIBRILLAR COLLAGEN PLAYS AN IMPORTANT role in determining the structural integrity of the myocardium (22). The quantity and quality of the extracellular collagen is determined by the balance between synthesis and degradation (27). Collagen synthesis is regulated transcriptionally and posttranslationally. Degradation is mediated by matrix metalloproteinases (MMPs) that are regulated transcriptionally, posttranslationally through activation of latent proenzymes (pMMPs), and by endogenous tissue inhibitors (TIMPs). Collagen synthesis, MMPs, and TIMPs are localized to the cardiac fibroblasts (8, 11, 12).

Recently it has been shown that MMP activity is increased in the failing myocardium of patients (9, 35) and animal models of myocardial remodeling and failure (32). It was further shown that inhibition of MMPs can decrease the severity of remodeling early post-myocardial infarction (28) and in chronic pacing-induced failure (31). The mechanisms responsible for these changes in collagen metabolism are not known. It has been shown that there is increased oxidative stress in the myocardium of patients with heart failure (21) and animal models of heart failure (10) and that antioxidants attenuate the development of myocardial failure (17). Reactive oxygen species (ROS) are known to regulate collagen metabolism in a variety of noncardiac cell types, including rat lung, dermal fibroblasts, and human venous endothelial cells (2, 19, 25). However, it is not known whether ROS can regulate collagen metabolism in cardiac fibroblasts, which are the major cell type responsible for collagen synthesis and degradation in the myocardium.

Accordingly, this study had two goals. We first examined the ability of ROS to regulate collagen metabolism in cardiac fibroblasts by measuring the effect of two sources of ROS (H₂O₂ and the superoxide-generating system of xanthine plus xanthine oxidase) on collagen synthesis and MMP activity. Second, we tested the role of endogenous antioxidant systems in regulating collagen metabolism by 1) inhibiting cytosolic Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and extracellular SOD with diethyldithiocarbamic acid (DDC) (16) to increase intracellular superoxide levels or 2) inhibiting glucose-6-phosphate dehydrogenase (G6PD), which is essential for regeneration of reduced glutathione and catalase activity, with dehydroisoandrosterone 3-acetate (DHEA) (36, 37) to increase intracellular H₂O₂ levels.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Rat cardiac fibroblast cultures and treatments. Neonatal rat cardiac fibroblasts were prepared as previously described (5). Adult rat cardiac fibroblasts were prepared by plating the nonmyocyte fraction of adult rat hearts digested as described previously (33). First and second passage fibroblasts plated on 6- or 24-well plates, or 35- or 100-mm dishes (Falcon) were grown to confluence in DMEM (GIBCO) containing 7% (vol/vol) heat-inactivated fetal bovine serum (GIBCO) and 1% (vol/vol) penicillin-streptomycin (PS; GIBCO) and changed to serum-free DMEM containing PS for 48-72 h before exposure to experimental treatments.

Fibroblasts were treated in DMEM containing PS for 24 h with H₂O₂ (0.05-5 µM; stabilized; Sigma) or xanthine (500 µM; Sigma) plus xanthine oxidase (0.001-0.1 mU/ml; cow's milk; Boehringer Mannheim) (XXO). Control cells were treated with DMEM containing PS alone. To inhibit antioxidant systems, fibroblasts in DMEM containing PS were treated for 24 h with DDC (100 µM; Sigma) or DHEA (10 µM; Sigma) alone or in combination with the superoxide dismutase/catalase mimetic EUK-134 (50 µM; 30 min pretreatment; Eukarion) (1). Control cells were treated with DMEM containing PS alone for DDC groups or DMEM containing PS and 0.1% DMSO (vehicle) for DHEA groups.

 

G6PD activity. G6PD activity was measured in fibroblasts plated on 100-mm dishes treated for 24 h with 10 µM DHEA as described by Tian et al. (37). Briefly, cells are scraped into ice-cold homogenization buffer (in mM: 320 sucrose, 20 HEPES, and 0.5 EDTA, pH 7.2). Samples were homogenized and centrifuged at 2,000 g for 10 min. Protein concentration in the supernatant was determined by the Bradford assay (Bio-Rad Protein Dye Reagent; Bio-Rad) against a BSA standard. Equal amounts of protein were added to the total dehydrogenase assay buffer (in mM: 50 Tris base, 1 MgCl₂, 0.2 glucose 6-phosphate, 0.2 6-phosphogluconate, and 0.1 NADP⁺, pH 8.1; all reagents from Sigma) and the 6-phosphogluconate dehydrogenase (6PGD) assay buffer (in mM: 50 Tris base, 1 MgCl₂, 0.2 6-phosphogluconate, and 0.1 NADP⁺, pH 8.1). NADP⁺ reduction to NADPH was measured as the rate of change of the absorbance at 340 nm over 6 min. G6PD activity was calculated as the total dehydrogenase activity minus the 6PGD activity.

Dichlorofluoroscein fluorescence. The ability of DHEA treatment to increase H₂O₂ in cardiac fibroblasts was measured as the EUK-134-inhibitable 2,7-dichlorofluoroscein (DCF) fluorescence. Fibroblasts were plated on 24-well plates and were treated with 10 µM DHEA alone or in combination with 50 µM EUK-134 for 24 h. Cells were washed three times with phenol-free DMEM and were incubated for 1 h at 37°C with 10 µM DCF diacetate (Molecular Probes) in phenol-free DMEM. Cells were again washed three times with phenol-free DMEM, and fresh phenol-free DMEM was added. DCF fluorescence (485 nm excitation; 538 nm emission) was measured as the average of nine 100-ms readings at room temperature.

Superoxide dismutase activity. Superoxide dismutase (SOD) activity in fibroblasts plated on 100-mm dishes and treated for 24 h with 100 µM DDC was measured as inhibition of pyrogallol auto-oxidation as previously described (30).

Cytochrome c reduction. The ability of DDC treatment to increase superoxide in cardiac fibroblasts was measured as SOD-inhibitable cytochrome c reduction. Fibroblasts were plated on six-well plates and were treated with 100 µM DDC for 24 h. Cells were washed three times with phenol-free DMEM and were incubated for 1 h at 37°C with 15 U/ml acetylated cytochrome c (Sigma) and 1 mM diethylenetriaminepentaacetic acid with or without 600 U/ml bovine erythrocyte SOD (Sigma) in phenol-free DMEM. Reduction of cytochrome c was measured as the absorbance of the media at 550 nm.

Collagen synthesis. Collagenase-sensitive [³H]proline incorporation was determined by a modification of the technique described by Botstein et al. (3). Briefly, confluent fibroblasts in 35-mm dishes were treated with H₂O₂, XXO, DHEA, or DDC alone or in combination with EUK-134 for 24 h, and 10 µCi/ml [³H]proline (NEN) and 50 µg/ml ascorbate (Sigma) were added for the final 4 h of treatment. Cells and media were collected by scraping and proteins were precipitated overnight in 20% (wt/vol) trichloroacetic acid at 4°C. Precipitated proteins were washed and digested with chromatographically purified collagenase (0.5 mg/ml; Worthington Biochemical) as described by Guarda et al. (14). The percent of total protein synthesis sensitive to collagenase was calculated as described by Guarda et al. (15).

Total protein synthesis. Fibroblasts were plated on 24-well plates and treated with H₂O₂, XXO, DHEA, or DDC alone or in combination with EUK-134 for 24 h in the presence of 1 µCi/ml [³H]leucine (NEN) as previously described (34). To account for any changes in cell number with experimental treatments, cell number was determined in parallel plates by trypsinization (GIBCO) and counting with a hemacytometer (Hausser). [³H]leucine incorporation was calculated as disintegrations per minute (dpm)/1,000 cells.

In-gel zymography. MMP activity was determined in conditioned media from fibroblasts treated with H₂O₂, XXO, DHEA, or DDC alone or in combination with EUK-134 for 24 h in 100-mm dishes. The media were collected, centrifuged for 5 min at 500 g to remove cells and debris, lyophilized to dryness, resuspended in 1/20 volume of water, and protein was determined by the Bradford assay. MMP activity per 500 ng protein was measured by in-gel zymography with gelatin (Type A from porcine skin; Sigma) as the substrate. Samples were loaded under nonreducing conditions onto 4% stacking/10% separating SDS-polyacrylamide gels with 1 mg/ml gelatin polymerized in the separating gel and were electrophoresed at 15 mA while stacking and 20 mA while separating. After separation, gels were washed in 2.5% Triton X-100 for 30 min with gentle shaking and then were rinsed with water for an additional 30 min. MMP identity was confirmed by an additional 30-min incubation of selected gels with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF; 5 mM), or the metal chelators EDTA (10 mM), or 1,10-phenanthroline (1 mM). All gels were incubated overnight at 37°C in substrate buffer (50 mM Tris · HCl, pH 8, 5 mM CaCl₂, and 0.02% NaN₃), stained in Coomassie blue R-250 in 7% acetic acid and 40% methanol, and then destained in 7% acetic acid and 40% methanol. Clear, digested regions representing MMP activity were quantified using an imaging densitometer (GS700; Bio-Rad), and molecular weights were estimated using prestained molecular weight markers.

Assessment of mRNA levels. Fibroblasts plated on 100-mm dishes were treated with H₂O₂ or XXO for 24 h. Total RNA was collected as previously described (34). Northern blots and hybridizations were performed as previously described (34) except for the hybridization buffer [100 µg/ml herring sperm DNA, 20% (vol/vol) dextran sulfate, 1% (wt/vol) SDS, 50% (vol/vol) formamide, and 15 mM NaCl]. cDNAs for procollagen ₁(I), ₂(I), ₁(III), ₁(IV), ₂(IV), and fibronectin (American Type Culture Collection) were labeled with [³²P]dCTP (NEN) as previously described (34). Blots were exposed to storage phosphor screens (Molecular Imaging Screen BI; Bio-Rad) for 2-3 h and quantified with a storage phosphor imager (GS-363; Bio-Rad), or exposed to XOMAT-AR film (Kodak) overnight and quantified with an imaging densitometer (GS-700; Bio-Rad). Images were analyzed with Molecular Analyst Software (Bio-Rad). The size of the hybridized messages was estimated by using 18S and 28S rRNA bands as standards. To normalize for potential variations in the amount of RNA loaded or transferred, all blots were reprobed with a ³²P-labeled oligonucleotide complimentary to 18S rRNA.

Statistical methods. All data are presented as means ± SE. Statistical analysis was performed using the Student's t-test or a one-way analysis of variance followed by the Student's-Newman-Keuls test for multiple comparisons, as appropriate. A value of P  0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of oxidative stress on collagen synthesis. Exposure of neonatal rat cardiac fibroblasts to H₂O₂ or XXO for 24 h had concentration-dependent effects on cell number. At the highest concentrations used, H₂O₂ (5 µM) and XXO (xanthine, 500 µM; xanthine oxidase, 0.1 mU/ml) decreased cell number by 18 ± 10% [n = 4; P = not significant (NS)] and 38 ± 7% (n = 12, P < 0.001), respectively. Lower concentrations of H₂O₂ (0.05 and 0.5 µM) or XXO (xanthine oxidase, 0.001-0.1 mU/ml) had no effect on cell number (data not shown). After correction for cell loss, neither H₂O₂ nor XXO had an effect on total protein synthesis as reflected by [³H]leucine incorporation (Fig. 1A). However, both H₂O₂ and XXO caused a concentration-dependent decrease in collagen synthesis measured as collagenase-sensitive [³H]proline incorporation normalized to total protein synthesis (Fig. 1A).

 

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Reactive oxygen species (ROS) specifically affect collagen synthesis in neonatal rat cardiac fibroblasts. Neonatal (A) and adult (B) cardiac fibroblasts were treated for 24 h with DMEM alone, H2O2 (0.05-5 µM), or xanthine plus xanthine oxide (XXO; 500 µM + 0.001-0.1 mU/ml). Fibroblasts were treated in the presence of [3H]leucine (total protein synthesis) or [3H]proline (last 4 h; collagen synthesis), and [3H]leucine incorporation and collagenase-sensitive [3H]proline incorporation were determined. Data for [3H]leucine are calculated as dpm/1,000 cells. Data for collagenase-sensitive [3H]proline incorporation are calculated as the percent of total protein synthesis. Means ± SE from 6-12 experiments. Significance was determined by Student's t-test; *P  0.05.

 

H₂O₂ and XXO had similar effects in adult rat cardiac fibroblasts. H₂O₂ (5 µM) and XXO (500 µM + 0.1 mU/ml) decreased cell number by 26 ± 2% and 43 ± 17% (n = 3; P < 0.05), respectively. Neither XXO nor H₂O₂ had an effect on total protein synthesis (n = 3; P = NS; Fig. 1B). However, XXO and H₂O₂ decreased collagen synthesis (normalized to total protein synthesis) by 24 ± 7% and 19 ± 6%, respectively (n = 3; P < 0.05; Fig. 1B).

 

Effect of oxidative stress on collagen mRNA expression. The effects of H₂O₂ (5 µM) and XXO (500 µM + 0.1 mU/ml) on collagen synthesis were further examined by Northern analysis. Treatment of neonatal fibroblasts with H₂O₂ or XXO for 24 h decreased the expression of mRNA for procollagens ₁(I), ₂(I), and ₁(III), the major fibrillar collagen forms in the rat heart (Fig. 2). In contrast, H₂O₂ and XXO tended to increase the expression of fibronectin (Fig. 2). H₂O₂ also decreased the expression of mRNA for the nonfibrillar procollagens ₁(IV) and ₂(IV), whereas XXO had no effect on the mRNA levels of ₁(IV) and ₂(IV) (Fig. 2).

 

View larger version (45K): [in this window] [in a new window]   Fig. 2.   ROS alter expression of extracellular matrix mRNAs. A: representative Northern hybridization showing decreased expression of procollagen 1(I), 2(I), and 1(III) mRNA and differential expression of procollagen 1(IV) and 2(IV) and fibronectin mRNA with 24-h treatment of neonatal cardiac fibroblasts with H2O2 (5 µM) or XXO (500 µM + 0.1 mU/ml). The level of 18S mRNA was used to control for differences in loading and transfer. B: quantification of the procollagen 1(I), 2(I), 1(III), 1(IV), 2(IV) and fibronectin mRNA level changes in fibroblasts exposed to H2O2 or XXO. Data are presented as the percent change from control; means ± SE of 8-15 experiments. *P  0.05 vs. control.

 

Effect of oxidative stress on MMP activities. MMP activity in the media of cultures treated with H₂O₂ or XXO was determined by in-gel zymography using gelatin as the substrate. H₂O₂ and XXO each increased total MMP activity in neonatal and adult cardiac fibroblasts (Fig. 3). These effects were concentration dependent in neonatal fibroblasts (Fig. 3B). Specific bands corresponding to the molecular masses of MMP13 (57-55/48-45 kDa; type I collagenase), MMP2 (72/66 kDa; gelatinase A), and MMP 9 (95/88 kDa; gelatinase B) were increased by treatment with H₂O₂ and XXO in both neonatal and adult fibroblasts (Fig. 4). Notably, H₂O₂ and XXO increased both the proenzyme and active enzyme bands for MMP13, MMP2, and MMP9. All MMP activities were inhibited by the metal chelators EDTA and 1,10 phenanthroline, but not the serine protease inhibitor PMSF (data not shown), confirming their identity as MMPs.

 

View larger version (33K): [in this window] [in a new window]   Fig. 3.   ROS increase matrix metalloproteinase (MMP) activities in conditioned media. A: a representative zymograph depicting the effects of H2O2 (0.05-5 µM) or XXO (500 µM + 0.001-0.1 mU/ml) on the activities of MMPs in the media of cultured neonatal cardiac fibroblasts. B and C: summary data of the effects of H2O2 or XXO on the activities of MMPs in the media of cultured neonatal (B) or adult (C) cardiac fibroblasts. Values are the percentage change vs. control; means ± SE of 7-13 experiments. *P  0.05 vs. control.

 

View larger version (27K): [in this window] [in a new window]   Fig. 4.   ROS increases MMP activities in conditioned media. Summary data of the effects of H2O2 (5 µM) or XXO (500 µM + 0.1 mU/ml) on the activity of individual MMPs in the media of cultured neonatal cardiac fibroblasts. Values are the percentage change vs. control; means ± SE of 7-13 experiments. *P  0.05 vs. control.

 

Inhibition of SOD and G6PD increases oxidative stress in cardiac fibroblasts. Treatment of neonatal fibroblasts with 100 µM DDC for 24 h inhibited total SOD activity 54 ± 19% (n = 3; P < 0.05) and increased superoxide production 87 ± 5% (n = 3; P = 0.013; Fig. 5A). Treatment of neonatal fibroblasts with 10 µM DHEA for 24 h inhibited G6PD activity by 23 ± 6% (n = 5; P = 0.015) and increased H₂O₂ production by 20 ± 4% (n = 4; P = 0.001; Fig. 5B). Neither DDC (4.1 ± 2.0%; P = NS; n = 4) nor DHEA (+0.4 ± 3.0%; P = NS; n = 4) affected cell number.

 

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Diethyldithiocarbamic acid (DDC) and dehydroisoandrosterone-3-acetate (DHEA) increase oxidative stress in cardiac fibroblasts. A: neonatal cardiac fibroblasts were treated with DDC (100 µM) for 24 h. The ability of DDC to inhibit superoxide dismutase (SOD) activity and increase oxidative stress was measured as inhibition of pyrogallol autooxidation and quenchable cytochrome c reduction, respectively. B: neonatal cardiac fibroblasts were treated with DHEA (10 µM) for 24 h. The ability of DHEA to inhibit glucose-6-phosphate dehydrogenase (G6PD) activity and increase oxidative stress measured as NADP+ reduction and quenchable 2,7-dichlorofluoroscein (DCF) fluorescence, respectively. Data are reported as the percentage change vs. control; means ± SE from 3-5 experiments. *P  0.05 vs. control.

 

Effects of SOD and G6PD inhibition of collagen synthesis and MMP activity. Treatment (24 h) of neonatal cardiac fibroblasts with 100 µM DDC or 10 µM DHEA inhibited collagen synthesis by 24 ± 4% (n = 8; P < 0.001; Fig. 6A) and 12 ± 2% (n = 5; P = 0.001; Fig. 6B), respectively.

 

View larger version (37K): [in this window] [in a new window]   Fig. 6.   DHEA and DDC decrease collagen synthesis and increase MMP activities. Neonatal fibroblasts were treated with DDC (100 µM; A and C) or DHEA (10 µM; B and D) alone, or in combination with the SOD/catalase mimetic EUK-134 (EUK; 50 µM; 30 min pretreatment) and collagen synthesis (A and B) and total MMP activity (C and D) were determined. Data are presented as the percent change vs. control; means ± SE from 5-8 experiments. *P  0.05 vs. vehicle/control. #P  0.05 vs. DHEA/DDC alone.

 

Treatment (24 h) of neonatal cardiac fibroblasts with 100 µM DDC or 10 µM DHEA increased total MMP activity by 33 ± 4% (n = 11; P < 0.001; Fig. 6C) and 64 ± 20% (n = 7; P = 0.001; Fig. 6D), respectively. The effects of DDC and DHEA on collagen synthesis and MMP activation were inhibited by the SOD/catalase mimetic EUK-134 (Fig. 6, A-D).

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The major new finding of this study is that ROS may have profound effects on collagen metabolism in cardiac fibroblasts by affecting both synthesis and the activity of degradative enzymes. H₂O₂ and XXO each decreased collagen synthesis (as measured by collagenase-sensitive [³H]proline incorporation) and decreased the abundance of mRNAs for procollagens ₁(I), ₂(I), and ₁(III). Likewise, H₂O₂ and XXO caused an increase in MMP activity as measured by in-gel zymography. These effects were mimicked by inhibition of endogenous antioxidant systems and were reversed by an SOD/catalase mimetic.

ROS inhibit collagen synthesis. H₂O₂ and XXO each decreased collagenase-sensitive [³H]proline incorporation. This effect was not due to a generalized depression in protein synthesis since it occurred without a decrease in overall protein synthesis as reflected by [³H]leucine incorporation. The synthesis of collagen is regulated at the transcriptional and posttranslational levels (20). H₂O₂ and XXO decreased the levels of mRNA for procollagens ₁(I), ₂(I), and ₁(III), indicating that the ROS-stimulated decrease in collagen synthesis was due, at least in part, to a decrease in mRNA transcription and/or stability.

In cardiac fibroblasts, prostaglandin E₂ (4) and phorbol esters are known to decrease collagen synthesis (13). On the other hand, several other stimuli are known to increase collagen synthesis in cardiac fibroblasts, including endothelin (14), aldosterone (4), mechanical stretch (6), transforming growth factor-₁ (TGF-₁) (13), and angiotensin (4). TGF-₁ and angiotensin also stimulate the proliferation of cardiac fibroblasts.

 

ROS stimulate collagen degradation. Collagen degradation is regulated by the activity of extracellular MMPs, which is determined by both transcriptional and posttranslational mechanisms. Posttranscriptional regulation occurs through the activation of latent proenzymes (pMMPs) by factors such as serum (39), heparin (40), and prostaglandin E₂ (4). Conversely, the activation of MMPs is opposed by the endogenous tissue inhibitors of metalloproteinase (TIMPs) (22).

H₂O₂ and XXO each increased total MMP activity as measured by in-gel zymography in both neonatal and adult fibroblasts. H₂O₂ and XXO increased the bands corresponding to MMP13, MMP2, and MMP9. The increases were due to both pMMPs and active MMPs, suggesting that the effects of ROS were mediated at both the transcriptional and posttranscriptional levels. ROS have previously been shown to cause direct activation of latent pMMPs in conditioned media from cardiac fibroblasts in vitro (38, 41). In this study, H₂O₂ and XXO were added to fresh medium in which pMMPs had not had time to accumulate. Therefore the increase in pMMPs and the activation of pMMPs in our experiments was likely not due to the oxidative burst of the stimuli, which lasted 1-2 h (data not shown).

 

Regulation of MMPs by oxidative stress has been shown in noncardiac cells. Hyperoxia increases expression of pMMP2 and pMMP9 mRNA in rat lung (25). XXO increases expression of pMMP2 and decreases expression of TIMP2 in dermal fibroblasts (19). H₂O₂ increases pMMP2, pMMP9, and MMP14 (which is responsible for pMMP2 activation) proteins and activates pMMP2 in human venous endothelial cells (2).

 

Inhibition of endogenous antioxidant systems. Antioxidant enzymes including SODs, catalase, and peroxidases regulate ROS by maintaining superoxide and H₂O₂ at low levels. DDC and DHEA each decreased collagen synthesis and increased MMP activity. DDC is a Cu chelator that inhibits CuZn-SOD and extracellular SOD (16). DHEA inhibits G6PD, which catalyzes the formation of NADPH during the conversion of glucose 6-phosphate to 6-phosphogluconate. NADPH is needed for the activity of both glutathione peroxidase and catalase, which are important enzymes in the conversion of H₂O₂ to water. The role of G6PD as an antioxidant enzyme has been shown by targeted disruption and overexpression experiments (24, 26, 29).

DDC and DHEA caused modest increases in ROS, and their effects on collagen synthesis and MMP activity were prevented by EUK-134, an antioxidant SOD/catalase mimetic, suggesting that their effects were mediated by ROS (1). The ability of DDC and DHEA to mimic the effects of H₂O₂ and XXO further indicate that the effects of ROS demonstrated here can occur at levels of ROS that can be made by the cardiac fibroblast.

 

Implications. ROS are increased in failing myocardium (21). There may be increased production of ROS due to mechanical strain (7), stimulated by substances such as angiotensin (23) or inflammatory cytokines (23), decreased activity of mitochondrial electron transport (18), and/or decreases in antioxidant systems (e.g., SOD and glutathione peroxidase) (17). The demonstration that ROS can cause both a decrease in fibrillar collagen synthesis and an increase in MMP activity suggests that ROS could play an important role in the pathophysiology of myocardial remodeling.

 

	ACKNOWLEDGEMENTS

EUK-134 was a generous gift of Eukarion. This work was supported by National Heart, Lung, and Blood Institute Grants HL-07224 (to D. A. Siwik), HL-42539 and HL-52320 (to W. S. Colucci), and HL-55425 (to P. J. Pagano), a Beginning Grant-in-Aid from the American Heart Association, Massachusetts Affiliate (to D. A. Siwik), and a Grant-in-Aid from the American Heart Association (to P. J. Pagano).

 

	FOOTNOTES

Address for reprint requests and other correspondence: W. S. Colucci, Cardiovascular Division Boston Univ. Medical Center, 88 East Newton St., Boston, MA 02118 (E-mail: wilson.colucci@bmc.org).

 

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 April 2000; accepted in final form 21 August 2000.

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Am J Physiol Cell Physiol 280(1):C53-C60
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