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RECEPTORS AND SIGNAL TRANSDUCTION

Interleukin-1 increases expression and activity of matrix metalloproteinase-2 in cardiac microvascular endothelial cells: role of PKC/₁ and MAPKs

Department of Physiology, James H. Quillen College of Medicine, James H. Quillen Veterans Affairs Medical Center, East Tennessee State University, Johnson City, Tennessee

Submitted 7 April 2006 ; accepted in final form 18 September 2006

	ABSTRACT

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Matrix metalloproteinases (MMPs), a family of extracellular endopeptidases, are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Interleukin-1 (IL-1), increased in the heart post-myocardial infarction (post-MI), plays a protective role in the pathophysiology of left ventricular (LV) remodeling following MI. Here we studied expression of various angiogenic genes affected by IL-1 in cardiac microvascular endothelial cells (CMECs) and investigated the signaling pathways involved in the regulation of MMP-2. cDNA array analysis of 96 angiogenesis-related genes indicated that IL-1 modulates the expression of numerous genes, notably increasing the expression of MMP-2, not MMP-9. RT-PCR and Western blot analyses confirmed increased expression of MMP-2 in response to IL-1. Gelatin in-gel zymography and Biotrak activity assay demonstrated that IL-1 increases MMP-2 activity in the conditioned media. IL-1 activated ERK1/2, JNKs, and protein kinase C (PKC), specifically PKC/₁, and inhibition of these cascades partially inhibited IL-1-stimulated increases in MMP-2. Inhibition of PKC/₁ failed to inhibit ERK1/2. However, concurrent inhibition of PKC/₁ and ERK1/2 almost completely inhibited IL-1-mediated increases in MMP-2 expression. Inhibition of p38 kinase and nuclear factor-B (NF-B) had no effect. Pretreatment with superoxide dismutase (SOD) mimetic, MnTMPyP, increased MMP-2 protein levels, whereas pretreatment with SOD and catalase mimetic, EUK134, partially inhibited IL-1-stimulated increases in MMP-2 protein levels. Exogenous H₂O₂ significantly increased MMP-2 protein levels, whereas superoxide generation by xanthine/xanthine oxidase had no effect. This in vitro study suggests that IL-1 modulates expression and activity of MMP-2 in CMECs.

MMP-2; protein kinase C; ERK1/2; JNK

Our laboratory has shown that IL-1 activates protein kinase C (PKC), mitogen-activated protein kinases (MAPKs; ERK1/2 and JNKs), and nuclear factor-B (NF-B) in cardiac fibroblasts (56–58) and that these signaling pathways play a critical role in the regulation of MMP-2 and -9 (56). Reactive oxygen species (ROS) are recognized as signaling intermediates for cytokines, including IL-1 (6, 31, 43, 46), and are shown to increase the expression and activity of MMPs in various cell types, including cardiac fibroblasts and myocytes (4, 22, 35, 38, 41, 42). ROS play an important role in the regulation of signal transduction via MAPKs (28, 38, 48, 55, 58) and NF-B (25).

The present study was undertaken to examine IL-1-stimulated angiogenic gene regulation in cardiac microvascular endothelial cells (CMECs), the major players in the processes of angiogenesis. Here, using cDNA array analysis of angiogenic genes affected by IL-1, we identified that IL-1 increases expression of MMP-2 in CMECs. The data presented here suggest that IL-1 activates ERK1/2, JNKs, and PKC/₁. The molecular mechanisms involved in the regulation of MMP-2 expression appear distinct in CMECs compared with cardiac fibroblasts.

	MATERIALS AND METHODS

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Isolation and culture of CMECs. Adult rat CMECs were isolated as described (55), with minor modifications. Briefly, hearts from adult male Sprague-Dawley rats (200–225 g) were removed under sterile conditions and perfused with DMEM supplemented with 0.1% penicillin-streptomycin (PS). After removing atria, visible connective tissue, valvular tissue, and the right ventricle, the left ventricle was immersed in 70% ethanol for 10 s to devitalize epicardial mesothelial and endocardial endothelial cells. After peeling away the outer one-third of the ventricular wall, the remaining tissue was washed in Hanks’ balanced salt solution (HBSS). The tissue was finely minced and digested in 15 ml HBSS containing 30 mg collagenase at 37°C with gentle shaking for 20 min. After a second digestion under the same conditions with the addition of 3 mg trypsin, the solution was passed through an 80-µm nylon mesh to remove undigested tissue. The dissociated cells were pelleted at 1,050 rpm for 5 min, washed in HBSS, resuspended in DMEM supplemented with 0.2% PS and 20% heat-inactivated FBS, and plated on laminin-coated dishes. The culture medium was replaced after 1 h to remove nonadherent cells. All experiments were carried out using primary culture cells. Using Griffonia simplicifolia lectin-1 cytochemical staining, we found that the CMECs culture is 95% pure. Cells were grown to 80–90% confluence and serum-starved for 24 h before use. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animal protocol was approved by the University Committee on Animal Care.

Cell treatment. Cells were treated with IL-1 (4 ng/ml; R&D Systems, Minneapolis, MN) for 24 h to measure differential expression of angiogenic genes and MMP-2 expression and activity, for 15 min to measure MAPKs activity, or for 5 min to measure PKC activity. UO126 (10 µM; MEK1/2-specific inhibitor), SP600125 (10 µM; inhibitor of JNKs), SB202190 and SB203580 (10 µM; inhibitors of p38), chelerythrine chloride (1 µM; PKC inhibitor), Gö6976 (100 nM; a PKC/₁ inhibitor), Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride [MnTMPyP; 10 µM; superoxide dismutase (SOD) mimetic], EUK134 (50 µM; SOD and catalase mimetic), and SN50 and IB kinase inhibitor peptide (10 µM and 50 µg/ml, respectively; NF-B inhibitors) were added 30 min prior to the addition of IL-1. EUK134 was a gift from Proteome Systems (Boston, MA). All other inhibitors were purchased from Calbiochem, La Jolla, CA. Treatment with xanthine (500 µM; Sigma-Aldrich, St. Louis, MO)/xanthine oxidase (0.15 mU/ml; Roche Applied Science, Indianapolis, IN) or H₂O₂ (5 µM; Sigma-Aldrich) for 24 h was also used to measure MMP-2 protein levels.

RNA isolation, GEArray analysis, and RT-PCR. Total RNA was extracted using the RNAqueous-4PCR kit (Ambion, Austin, TX) according to the manufacturer’s instructions. Two micrograms RNA was used as a template to generate [³²P]dCTP-labeled cDNA probes according to the manufacturer’s instructions (SuperArray Bioscience, Frederick, MD). The cDNA probes were denatured and hybridized at 60°C with a GEArray membrane, containing 96 angiogenesis-related genes. After washing, the membrane was incubated with a chemiluminescent substrate. The spots were digitized with ScanAlyze software, and signal intensities were normalized to GAPDH using the GEArray analyzer program (SuperArray Bioscience).

For RT-PCR, 2 µg RNA was reverse transcribed using the MMLV RT kit (Promega, Madison, WI) according to the manufacturer’s instructions. Primer sequences for RT-PCR amplification were as follows: MMP-2, 5'-CTGATAACCTGGATGCAGTCGT-3' and 5'-CCAGCCAGTCCGATTTGA-3', resulting in a 135-bp fragment; and GAPDH, 5'-CTCATGACCACAGTCCATGC-3' and 5'-TTCAGCTCTGGGATGACCTT-3', resulting in a 155-bp fragment. An initial denaturation was performed at 94°C for 2 min followed by a 20-cycle (MMP-2) or 30-cycle (GAPDH) amplification phase under the following conditions: 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s. A final extension was carried out at 72°C for 2 min. PCR products were analyzed by gel electrophoresis on a 2% agarose gel stained with ethidium bromide. Band intensities were quantified using Kodak photodocumentation system (Eastman Kodak, Rochester, NY).

Western blot analyses. Total cell lysates were prepared in lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, and 0.4 mM phenylmethylsulfonyl fluoride), and protein contents were measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Membrane fractions were prepared as described (1). Equal amounts of proteins (25 µg) were resolved by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes. After blocking, the membranes were probed with anti-MMP-2 (Chemicon International, Temecula, CA), anti-phospho-ERK1/2 (Cell Signaling Technology, Danvers, MA), anti-phospho-JNK (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-PKC(pan) or anti-phospho-PKC/_II (Cell Signaling Technology) antibodies. These anti-phospho-PKC/_II antibodies also recognize phosphorylated PKC₁. The membranes were then incubated with appropriate secondary antibodies, and immune complexes were detected using chemiluminescence and autoradiography. Band intensities were quantified using Kodak photodocumentation system (Eastman Kodak).

In-gel zymography. MMP activities were measured in conditioned media as previously described (40). Briefly, the conditioned media were lyophilized, and the pellets were resuspended in water. Equal amounts of protein (2 µg) were electrophoresed under nonreducing conditions into 10% SDS-PAGE gels polymerized with 1 mg/ml gelatin (type A from porcine skin). Following electrophoresis, the gels were washed in 2.5% Triton X-100 for 30 min with gentle shaking followed by a 30-min wash in distilled water. The gels were incubated overnight at 37°C in substrate buffer with gentle shaking (50 mM Tris·HCl, pH 8.0, 5 mM CaCl₂, and 0.02% NaN₃). The gels were then stained with Coomassie blue (R-250) and destained with a solution containing 7% acetic acid and 40% methanol. Unstained, digested regions represent areas of MMP activity. These translucent bands were quantified using Kodak photodocumentation system (Eastman Kodak).

MMP-2 Biotrak activity assay. The levels of active MMP-2 in the concentrated conditioned media containing 1 µg of total protein were measured using the MMP-2 activity assay kit (GE Healthcare Biosciences, Piscataway, NJ) according to the manufacturer’s instructions.

Statistical analysis. All data are reported as means ± SE. Statistical analyses were performed using Student’s t-test or one-way ANOVA and a post hoc Tukey test. Probability (P) values of <0.05 were considered to be significant.

	RESULTS

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IL-1 regulates angiogenic gene expression in CMECs, notably increasing MMP-2 expression. To investigate whether IL-1 modulates expression of angiogenic genes, CMECs were treated with IL-1 (4 ng/ml) for 24 h. Total RNAs were reverse transcribed and radiolabeled, and the resulting cDNAs were hybridized simultaneously with two GEArray membranes containing 96 angiogenesis-related genes. This analysis indicated that IL-1 modulates expression of angiogenic genes, notably increasing expression of MMP-2 (Fig. 1). There was no major effect on MMP-9 gene expression. IL-1-stimulated increases in MMP-2 mRNA levels were confirmed by RT-PCR. IL-1 increased MMP-2 mRNA levels by 3.8 ± 0.12-fold vs. control (P < 0.001; n = 5; Fig. 2A).

View larger version (75K): [in this window] [in a new window]   Fig. 1. Angiogenic SuperArray analysis. Confluent cultures of cardiac microvascular endothelial cells (CMECs) were treated with interleukin-1 (IL-1, 4 ng/ml) for 24 h. Total RNAs were reversed transcribed, and resulting cDNAs were hybridized to a GEArray membrane containing 96 angiogenesis-related genes. The solid arrow indicates the matrix metalloproteinase-2 (MMP-2) spot on the membrane. The dashed arrow indicates the MMP-9 spot on the membrane. Bottom: GAPDH spots used for normalization.

View larger version (15K): [in this window] [in a new window]   Fig. 2. IL-1 increases MMP-2 gene expression. Confluent cultures of CMECs were treated with IL-1 (4 ng/ml) for 24 h. A: RT-PCR analyses. Total RNAs were reverse transcribed, and resulting cDNAs were subjected to amplification of MMP-2 and GAPDH genes. Mean data normalized to GAPDH is in bar graph. *P < 0.001 vs. control (C); n = 5. B: Western blot analyses. Total cell lysates were analyzed by Western blot using anti-MMP-2 antibodies. Equal protein loading in each lane was verified using anti-actin antibodies. *P < 0.01 vs. control (C); n = 4.

IL-1 stimulation increases MMP-2 protein levels and activity in CMECs.

View larger version (16K): [in this window] [in a new window]   Fig. 3. IL-1 increases MMP-2 activity. Confluent cultures of CMECs were treated with IL-1 (4 ng/ml) for 24 h. Conditioned media were collected and lyophilized to dryness. Concentrated conditioned media were used to measure MMP-2 activity using gelatin in-gel zymography (A) or Biotrak activity assay kit (B). Bar graphs show the means ± SE. *P < 0.05 vs. control (C); n = 3–4.

View larger version (72K): [in this window] [in a new window]   Fig. 4. IL-1 activates ERK1/2 and JNKs. Confluent cultures of CMECs were pretreated with UO126 (10 µM; inhibitor of ERK1/2 pathway) or SP600125 (10 µM; inhibitor of JNKs) for 30 min followed by treatment with IL-1 for 15 min. Total cell lysates were analyzed by Western blot using phospho-specific antibodies for ERK1/2 (A) and JNKs (B). Blots are representative of 3 independent experiments. Equal protein loading in each lane was verified using anti-actin antibodies.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Inhibition of ERK1/2 and JNKs inhibits IL-1-stimulated increase in MMP-2 protein levels and activity. Confluent cultures of CMECs were pretreated with UO126 (10 µM; inhibitor of ERK1/2 pathway) or SP600125 (10 µM; inhibitor of JNKs) for 30 min then stimulated with IL-1 for 24 h. A: Western blot analyses. Total cell lysates were analyzed by Western blot using anti-MMP-2 antibodies. Anti-actin antibodies were used as a protein loading control. Bar graph shows means ± SE. *P < 0.01 vs. IL-1; n = 4. B: in-gel zymography. Conditioned media from the samples in A were subjected to gelatin in-gel zymography to measure MMP-2 activity. Bar graph shows the means ± SE. *P < 0.01 vs. IL-1; n = 4.

View larger version (21K): [in this window] [in a new window]   Fig. 6. IL-1 activates protein kinase C (PKC), specifically PKC/1. Confluent cultures of CMECs were pretreated with chelerythrine chloride (1 µM; non-isoform-specific PKC inhibitor) for 30 min then stimulated with IL-1 for 5 min. Membrane fractions were collected and subjected to Western blot analyses using phospho-specific PKC(pan) (A) or phospho-specific PKC/1 antibodies (B). Equal protein loading in each lane was verified using anti-actin antibodies. Blots are representative of 3 independent experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 7. A: inhibition of PKC/1 partially inhibits IL-1-stimulated increase in MMP-2 protein levels. Confluent cultures of CMECs were pretreated with Gö6976 (100 nM; PKC/1 inhibitor) alone or in combination with UO126 (10 µM; inhibitor of ERK1/2 pathway) for 30 min, then stimulated with IL-1 for 24 h. Total cell lysates were analyzed by Western blot using anti-MMP-2 antibodies. Bar graph indicates MMP-2 protein levels normalized with actin as a loading control. *P < 0.001 vs. IL-1; n = 3; #P < 0.001 vs. Gö6976+IL-1; n = 3. B and C: inhibition of PKC/1 does not affect IL-1-stimulated activation of ERK1/2 or JNKs. Confluent cultures of CMECs were pretreated with Gö6976 (100 nM; PKC/1 inhibitor) for 30 min, then stimulated with IL-1 for 15 min. Total cell lysates were analyzed by Western blot using phospho-specific antibodies for ERK1/2 (B) and JNKs (C). Equal protein loading in each lane was verified using anti-actin antibodies. Blots are representative of 3 independent experiments.

Role of PKC/₁ in the activation of MAPKs.

Role of ROS in MMP-2 regulation. Inflammatory cytokines, including IL-1, are known to increase intracellular ROS (6, 31, 43, 46). MAPKs are suggested to be redox sensitive in certain cell types, including cardiac fibroblasts and myocytes (28, 38, 42, 48, 55, 58). To study whether IL-1-stimulated activation of ERK1/2 and JNKs could be ROS sensitive, CMECs were pretreated with the SOD mimetic, MnTMPyP (10 µM), for 30 min followed by 15-min treatment with IL-1. Pretreatment with MnTMPyP had no effect on IL-1-stimulated phosphorylation of ERK1/2 or JNKs (Fig. 8, A and B). Interestingly, pretreatment with MnTMPyP potentiated IL-1-stimulated increases in MMP-2 protein levels by 37 ± 5% (P < 0.001; n = 4; Fig. 8C). Pretreatment with EUK134 (an SOD and catalase mimetic, 50 µg/ml) for 30 min partially inhibited (24 ± 8%; P < 0.05; n = 4; Fig. 8D) IL-1-stimulated increases in MMP-2 protein levels. Treatment with xanthine/xanthine oxidase, an extracellular source of superoxide (54), had no significant effect on MMP-2 protein levels (n = 3; Fig. 9). However, exogenous H₂O₂ increased MMP-2 protein levels (61 ± 11%; P < 0.001; n = 3; Fig. 9) with no effect on ERK1/2 or JNKs phosphorylation (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 8. A and B: pretreatment with MnTMPyP does not affect IL-1-stimulated activation of ERK1/2 or JNKs. Confluent cultures of CMECs were pretreated with MnTMPyP (10 µM; SOD mimetic) for 30 min followed by treatment with IL-1 for 15 min. Total cell lysates were analyzed by Western blot analyses using phospho-specific ERK1/2 (A) or JNKs (B) antibodies. Equal protein loading in each lane was verified using anti-actin antibodies. Blots are representative of 3 independent experiments. C: pretreatment with MnTMPyP potentiates IL-1-stimulated increases in MMP-2 protein levels. Confluent cultures of CMECs were pretreated with MnTMPyP (10 µM; SOD mimetic) for 30 min followed by treatment with IL-1 for 24 h. Total cell lysates were analyzed by Western blot using anti-MMP-2 antibodies. Bar graph shows the mean data normalized to actin. *P < 0.001 vs. IL-1; n = 4. D: pretreatment with EUK134 inhibits IL-1-stimulated increases in MMP-2 protein levels. Confluent cultures of CMECs were pretreated with EUK134 (50 µM; SOD and catalase mimetic) for 30 min followed by treatment with IL-1 for 24 h. Total cell lysates were analyzed by Western blot using anti-MMP-2 antibodies. Bar graph shows the mean data normalized to actin. *P < 0.05 vs. IL-1; n = 4.

View larger version (25K): [in this window] [in a new window]   Fig. 9. H2O2 increases MMP-2 levels, whereas generation of O2–· has no effect. Confluent cultures of CMECs were treated with H2O2 (5 µM) or xanthine (500 µM)/xanthine oxidase (0.15 mU/ml; XXO) for 24 h. Total cell lysates were analyzed by Western blot using anti-MMP-2 antibodies. Bar graph shows the mean data with actin as a loading control. *P < 0.001 vs. control (C); n = 3; NS, not significant.

Role of NF-B in MMP-2 regulation.

View larger version (25K): [in this window] [in a new window]   Fig. 10. Inhibition of nuclear factor-B (NF-B) does not affect IL-1-stimulated increase in MMP-2 protein levels. Confluent cultures of CMECs were pretreated with SN50 (10 µM, NF-B nuclear translocation inhibitor) or IB kinase inhibitor peptide (50 µg/ml; IBIP, inhibitor of NF-B activation) for 30 min followed by treatment with IL-1 for 24 h. Total cell lysates were subjected to Western blot analyses, and anti-actin antibodies were used as a loading control. P = NS vs. IL-1; n = 3.

	DISCUSSION

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The human MMP-2 promoter contains AP-2 and SP-1 transcription factor binding sites but does not seem to have an AP-1 transcription factor binding site (53). However, a functional AP-1 binding site in the promoter region of MMP-2 has been identified in rat cardiac cells (5). The transcription factor AP-1 is formed by dimerization of different members of Fos and Jun protein family members. The expression of Fos and Jun is mainly regulated via the activation of ERK1/2 and JNKs, respectively. In cardiac fibroblasts IL-1-mediated activation of JNKs and ERK1/2 plays an important role in the regulation of MMP-9, not MMP-2, expression and activity (56). Activation of ERK1/2 pathway is shown to play a critical role in the regulation of MMP-2 activity in transformed cells (24). The data presented here demonstrates that IL-1 increases expression and activity of MMP-2, not MMP-9, and IL-1-mediated activation of ERK1/2 and JNKs plays an important role in the regulation of MMP-2 expression in CMECs. Interestingly, concurrent inhibition of ERK1/2 and JNKs had no additive effect on IL-1-stimulated increases in MMP-2 expression and activity. These data suggest differential regulation of MMP-2 expression and activity in response to IL-1 in two cell types of the heart: cardiac fibroblasts and CMECs. The partial inhibition of MMP-2 following inhibition of ERK1/2 and JNKs suggests involvement of ERK1/2- and JNK-independent mechanism(s) in the regulation of MMP-2 expression.

PKC comprises a large 12-member family of serine-threonine kinases that play an important role in cardiovascular functions. PKC activation is suggested to be necessary for the induction of MMP gene expression in response to proinflammatory cytokines, such as IL-1 and TNF- in noncardiac and tumor cell lines (19, 29). In hepatoma cells, activation of PKC and PKA activated AP-2 (21) and therefore may play a role in MMP-2 gene expression (53). In cardiac fibroblasts, IL-1-stimulated activation of PKC/₁ plays a key role in the regulation of MMP-9, not MMP-2, expression and activity (56). Here we demonstrate that, similar to cardiac fibroblasts, IL-1 activates PKC in CMECs. However, activation of PKC/₁ plays an important role in the regulation of MMP-2 in CMECs.

Various isoforms of PKC are known to modulate various MAPK signaling pathways (10). In cardiac myocytes activation of PKC has been shown to selectively mediate ERK1/2, whereas PKC preferentially activates JNKs and p38 kinase (17). In cardiac fibroblasts PKC/₁ act upstream of JNKs, while PKC/ act upstream of both ERK1/2 and JNKs (56). In this study we show that inhibition of PKC/₁ with Gö6976 at 100 nM has no effect on ERK1/2 or JNKs activation. Concurrent inhibition of PKC/₁ and ERK1/2 results in almost complete inhibition of IL-1-stimulated increases in MMP-2 protein levels, supporting the possibility that ERK1/2 and PKC/₁ act via independent mechanisms to increase MMP-2 expression. At higher concentrations Gö6976 inhibited phosphorylation of JNKs. This, in addition to the lack of synergistic effect on MMP-2 regulation upon combined inhibition of PKC/₁ and JNKs, suggests the possibility of PKC/₁-dependent activation of JNKs. The differential effects of PKC isoforms in the activation of MAPKs and the regulation of MMP-2 expression among cardiac cells point toward cell-type specificity for downstream coupling. Of note, IL-1 has been shown to activate MAPKs (ERK1/2, p38, and JNKs) via PKC-independent pathways in noncardiac cell types (32).

Kinugawa et al. (23) have shown that ROS production is increased post-MI in mice and that chronic inhibition of hydroxyl radical (^·OH) production post-MI decreases LV remodeling. IL-1, one of the key players in inflammatory response, stimulates the production of ROS, and its signaling can be influenced by ROS (6, 43). ROS have been shown to increase the expression and activation of MMPs in various cell types (4, 22, 35), specifically MMP-13, -9, and -2 in cardiac fibroblasts (41). Oxidative stress also plays a role in the activation of MAPKs in a variety of cell types (28, 48, 55, 58). EUK134 is an SOD and catalase mimetic (3). These enzymes catalyze the dismutation of superoxide (O₂^–·) to O₂ and H₂O₂ and the decomposition of H₂O₂ to O₂ and H₂O, respectively. MnTMPyP, an SOD mimetic (13, 14), dimutates O₂^–·, resulting in the accumulation of H₂O₂. We found that H₂O₂ alone increases MMP-2 expression, and pretreatment with MnTMPyP potentiates IL-1-stimulated increases in MMP-2. This modulation of MMP-2 by ROS seems to be independent of MAPKs. On the other hand, generation of O₂^–· using xanthine/xanthine oxidase had no effect on MMP-2 expression, and pretreatment with EUK134 partially inhibited IL-1-stimulated increases in MMP-2. These data suggest that H₂O₂ may play a regulatory role in IL-1-stimulated increases in MMP-2 in CMECs. Of note, IL-1 is shown to increase MnSOD levels in a variety of cell types, including rat pulmonary microvascular endothelial cells (12, 49–51).

The human MMP-2 promoter does not contain an NF-B transcription factor-binding site (53). NF-B is suggested to be redox sensitive and activated by IL-1 (25, 26). NF-B is shown to regulate MMPs in a redox-sensitive manner in both cardiac and noncardiac cell types (7, 9, 36). In cardiac fibroblasts NF-B plays a key role in the regulation of IL-1-stimulated increases in MMP-2 and MMP-9 expression and activity (56). In CMECs activation of NF-B does not seem to play a role in IL-1-stimulated increases in MMP-2 expression. Of note, a concentration of 18 µM for SN50 is shown to maximally inhibit NF-B nuclear translocation in murine endothelial LE-II cells (27). The concentration of SN50 used in this study is 10 µM, 3.5 times lower than the concentration used for the treatment of cardiac fibroblasts. Higher concentrations (>10 µM) of SN50 were found cytotoxic for CMECs.

The signaling cascade leading to increased expression of MMP-2 appears distinct compared with cardiac fibroblasts. The differential regulation of MMPs among various cardiac cell types may be an indicator for cell-type-specific roles in angiogenesis. A better understanding of the differences in regulation of MMPs among cell types is essential for the development of pharmacological strategies targeting endothelial cell activities to maximize functional remodeling and angiogenesis of the myocardium post-MI.

	GRANTS

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This work is supported by National Heart, Lung, and Blood Institute Grant HL-071519 (to K. Singh) and by a Department of Veterans Affairs Merit Review Grant (to K. Singh).

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. Singh, Dept. of Physiology, James H. Quillen College of Medicine, East Tennessee State Univ., PO Box 70576, Johnson City, TN 37614 (e-mail: singhk{at}etsu.edu)

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.

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