© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Proinflammatory cytokines regulate tissue inhibitors of metalloproteinases and disintegrin metalloproteinase in cardiac cells
Research Laboratories, 538 Scaife Hall, Division of Cardiology, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15213, USA
* Corresponding author. Tel.: +1-412-647-1666; fax: +1-412-383-8857. E-mail address: feldmanam@msx.upmc.edu (A.M. Feldman)
Received 2 June 1998; accepted 14 September 1998
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: Tissue inhibitors of metalloproteinases (TIMPs) are downregulated in the failing human heart. The objective of the present study was to test the hypothesis that cytokines might be involved in the regulation of TIMPs in cardiac cells. Methods: Neonatal Sprague–Dawley rat ventricular cells were exposed to 100 units/ml tumor necrosis factor-
and/or 5 ng/ml interleukin-1β. The mRNA and protein expression of TIMPs-1–4 and disintegrin metalloproteinase was analyzed using Northern blot, ELISA and/or Western blot, respectively. Proteolytic activity and extracellular matrix degradation and turnover were determined using gelatin zymography and pulse-chase experiments. Results: The TIMP-1 mRNA was upregulated in cardiac cells, while TIMP-1 protein levels were unchanged in myocytes but downregulated in non-myocytes. The TIMP-2 expression did not change with the cytokine treatment. TIMP-3 was downregulated at both the mRNA and protein levels in cardiac cells. TIMP-4 protein was transiently increased and then returned to control level. In contrast, disintegrin metalloproteinase mRNA and protein were significantly upregulated in those cells. The gelatinolytic activity and extracellular matrix protein degradation were significantly increased. Conclusions: Tumor necrosis factor- and interleukin-1β regulate the expression of TIMPs and disintegrin metalloproteinase, which may in turn contribute to the increased matrix degradation in cardiac cells. Since heart failure in humans is characterized by both re-expression of myocardial cytokines and remodeling of the extracellular matrix, those in vitro results suggest a potential role for those cytokines in the regulation of extracellular matrix remodeling and therefore in the transition to the end-stage heart failure phenotype.
KEYWORDS Cytokines; Extracellular matrix; Gene expression; Myocytes; Metalloproteinases
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The integrity of the cardiac extracellular matrix is important in maintaining hemodynamic stability. This integrity is maintained by a balance between matrix degradation and production, degradation occurring in large part through the action of a family of metalloproteinases [1]. During hemodynamic stress as seen in the failing and hypertrophied heart, the initiating event in the transition to severe congestive heart failure is the myocardial remodeling which occurs as a result of an imbalance between the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) [2, 3]. It has been well documented that the activity of matrix metalloproteinases increases during the progression of ventricular dilatation and dysfunction [4–6]. Four TIMPs have been cloned and purified, the most recent being TIMP-4 [7–10]. Each TIMP is encoded by a unique gene, yet all share both structural and functional similarities with conservation of 12 cysteine residues important for tertiary structure (Table 1). Although each TIMP displays a unique profile of tissue expression, all are expressed in the heart with TIMP-4 being largely restricted to the heart and brain [10, 11].
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The disintegrin metalloproteinases (ADAMs) are an emerging new family of transmembrane metalloproteinases containing a mosaic structure with pro-, metalloproteinase-like, disintegrin-like, cysteine rich, epidermal growth factor-like, transmembrane and cytoplasmic domains [12, 13]. These multiple domains can be proteolytically released and it has been suggested that they play a variety of roles in development and potentially in the pathophysiology of disease. ADAM10 has unique properties by virtue of its multiple functional domains. Furthermore, the cell adhesion and proteolytic domains can process or remove the extracellular domains of cell surface proteins, including tumor necrosis factor-alpha (TNF-
) [14–17].
Recently, we have found that TIMP-1 and -3 were downregulated at both the mRNA and protein levels in the failing human heart, while TIMP-4 protein was selectively downregulated in ischemic cardiomyopathy [18]. These studies suggest that the downregulation of TIMPs might be involved in the pathophysiology of congestive heart failure in humans. However, the mechanisms responsible for the regulation of myocardial TIMPs are not understood. A recent study suggests that proinflammatory cytokine can induce cardiac interstitial fibrosis in transgenic mice overexpressing TNF- [19]. Furthermore, we recently utilized mRNA differential display to identify novel genes that were differentially expressed in neonatal myocytes exposed to TNF- and interleukin-1-beta (IL-1β) [20]. These studies identified a group of differentially expressed genes including rat Eph (erythropoietin producing hepatoma cell line) receptor tyrosine kinase type A3 (r-EphA3), TIMP-3 and ADAM10. We demonstrated that the proinflammatory cytokine IL-1β attenuates r-EphA3 expression; however, the effects of these cytokines on ADAM10 and TIMPs in cardiac myocytes remain undefined. Therefore, the present study was performed to examine the role of proinflammatory cytokines in regulating the expression of TIMPs and ADAM10 in cardiac cells, which may reflect the role that these cytokines play in the downregulation of TIMPs in the failing human heart.
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2 Methods |
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2.1 Cell culture and cytokine treatment
Cardiac myocytes were isolated from ventricles of 1-day-old Sprague–Dawley rat hearts and cultured exactly as previously described by us [21]. After plating on Pronectin (Promega, Madison, WI, USA) coated tissue culture plates, myocytes were grown at 37°C in 5% CO2 for 24 h. The media was changed and the cells were allowed to grow for an additional 48 h before media containing 100 units/ml TNF-
and/or 5 ng/ml IL-1β (both from Biosource, Camarillo, CA, USA) was added to the cultures and incubated for 18 h, one more dose of cytokines was added and incubated for an additional 18 h. For time course studies at 1, 15 and 36 h, the initiation of treatment was staggered so that collection of the cells occurred at the same time. After the media was removed and rinsed once with phosphate buffered saline (PBS), pH 7.4, the cells were snap frozen by floating on liquid nitrogen and kept at –80°C until use. The cytokine concentrations were chosen based on previous studies in our laboratory assessing the effects of these cytokines on cardiac myocyte gene expression, contractility and Ca2+ homeostasis [20, 21]. As determined by immunostaining with anti-sarcomeric-myosin antibody, the cardiac myocyte preparations routinely contained >95% sarcomeric-myosin positive cells [21]. Cardiac non-myocytes were recovered from the adherent pre-plated cells prepared as described above, and cultured in the same media as for the cardiac myocytes but lacking bromodeoxyuridine. After two passages, cells were allowed to grow to subconfluency and cytokine treatment was initiated as described for the cardiac myocytes. For RNA or protein preparations from cardiac myocytes or non-myocytes, at least four independent cytokine treatment experiments were performed.
All experimental procedures were carried out under sterile conditions, and conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).
2.2 Total RNA and poly A+ mRNA isolation
Total RNA was isolated from cultured cells via acid phenol extraction [22]. RNA yields were assessed by spectrophotometric absorbance at 260 nm. Due to the low absolute transcript levels of TIMPs and ADAM10 in cardiac cells, RNA samples were enriched for polyadenylated species by oligo(dT)/magnetic bead capture with a PolyATtract mRNA isolation system (Promega).
2.3 Northern blot analysis
The cDNA probes for TIMPs, ADAM10 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were prepared as previously reported [18]. The nucleotide sequences of all cDNA probes were determined to confirm their identities. Poly A+ mRNA enriched samples isolated from 150 µg of total RNA were resolved in formaldehyde-agarose gels, transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA) by pressure blotting, and fixed by ultraviolet crosslinking. Prehybridization, hybridization and quantification of the signals were performed as previously reported [21]. The membranes were stripped after each hybridization with 2 mM Tris–HCl, pH 8.0, 0.2 mM EDTA, 0.1% SDS at 75°C for 1 h, and re-hybridized sequentially with radiolabeled TIMPs-1–4, ADAM10 and GAPDH probes, respectively. Membranes were then re-exposed and quantified with IMAGEQUANT software (Molecular Dynamics, Sunnyvale, CA, USA). The hybridization results of TIMPs and ADAM10 were normalized to that of the GAPDH probe from the same lane to correct for differences in mRNA mass and efficiency of transfer, and in turn normalized to the mean of the controls that was arbitrarily set as 100%.
2.4 Lysis of cells, isolation of glycoprotein, Western blot analyses
The total protein of cultured cells was obtained through lysis of the cells into RIPA buffer (1xPBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail, Sigma cat. No. P2714, St. Louis, MO, USA). Glycoprotein was isolated from the above cell lysates using lentil lectin-Sepharose affinity chromatography as described in [23]. For the detection of TIMP-3 protein, the extracellular matrix was collected as described previously [24]. Protein concentration was determined with the Bradford protein assay using bovine IgG as a standard (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of either total protein (60 µg) or glycoprotein (32 µg) were separated on standard SDS-polyacrylamide gel (12% for TIMPs and 7% for ADAM10) and electroblotted onto nitrocellulose membrane (Micron Separations, Westborough, MA, USA). TIMP-1 and -3 positive controls (Chemicon, Temecula, CA, USA) were included and electrophoresed in parallel with the cellular protein samples. TIMP-1 protein was detected using a monoclonal antibody at 1 µg/ml (Oncogene Research Products, Cambridge, MA, USA). TIMP-3 or -4 proteins were detected with rabbit polyclonal antibodies (1:600 for TIMP-3, 1:3000 for TIMP-4) raised against the respective TIMPs (Chemicon). The TIMP-3 protein was also probed with a monoclonal antibody at 5 µg/ml (Oncogene Research Products). ADAM10 protein was detected with antiserum against the synthetic peptide FDANQPEGKKC corresponding to amino acids 485–495 of the deduced human polypeptide sequences. Horseradish peroxidase conjugated goat anti-rabbit IgG (1:7500, Schleicher & Schuell) or anti-mouse IgG (1:20 000, Pierce, Rockford, IL, USA) was used as secondary antibodies for the polyclonal or monoclonal primary antibodies, respectively. The reactions were developed with enhanced chemiluminescence reagents (Pierce) and the images were obtained by exposing to X-ray films. The films were digitized with a ScanJet 4C scanner (Hewlett-Packard, Englewood, CO, USA) and quantified with IMAGEQUANT software. The results were presented as percent changes compared with untreated controls, the means of which were arbitrarily set as 100%.
2.5 Enzyme linked immunosorbent assay (ELISA)
TIMP-2 protein in cardiac cells was measured using a commercial ELISA kit that detects both free and metalloproteinases-complexed TIMP-2 (Amersham Life Science, Arlington Heights, IL, USA). Recombinant human TIMP-2 provided by the manufacturer was used as a standard. All samples and the standard were assayed in duplicates. The assay is based on a two-site ELISA sandwich format. A peroxidase labelled Fab antibody to TIMP-2 was added to samples, and the mixture was incubated in microtitre wells precoated with anti-TIMP-2 antibody. The amount of peroxidase bound to each well was determined by the addition of tetramethylbenzidine–hydrogen peroxide. The absorbance at 630 nm was read spectrophotometrically using a microtiter plate reader. The detection limits of this assay are 5–128 ng/ml.
2.6 Gelatin zymography
Gelatin zymography of cardiac cell lysates was performed as described with modifications [25]. To detect the gelatinolytic activity, the samples were mixed with Laemmeli sample loading buffer containing 2.5% SDS without β-mercaptoethanol or boiling and electrophoresed in 10% SDS-polyacrylamide gels impregnated with 1.5 mg/ml type I gelatin from porcine skin, constant voltage 110 V. After electrophoresis, gels were washed in 2.5% Triton X-100 for 30 min to allow proteins to renature. Gels were then incubated at 37°C overnight in substrate buffer (50 mM Tris–HCl, pH 8.0, 10 mM CaCl2, 1 µM ZnCl2), and were stained with Coomassie Blue R250 to reveal zones of lysis. Zones of lysis resulting from gelatinolytic activity appear as white bands on the Coomassie Blue R250 stained background.
2.7 Quantification of matrix protein degradation
The pulse-chase experiments were performed as described [26]. Medium without leucine and proline (MEM Select-Amine, Gibco-BRL, Gaithersburg, MD, USA) containing 3 µCi/ml [3,4,5-3H]-leucine (159 Ci/mmol, NEN Life Science, Boston, MA, USA) and 3 µCi/ml [3,4-3H]-proline (48 Ci/mmol, NEN) mixed in a single batch was used to pulse-label cellular and matrix proteins. Cells were incubated in the above medium for 24 h, the unincorporated radioisotopes were chased out by incubating with media containing 40 mM L-leucine and 17.4 mM L-proline for 1 h. The chasing medium was then replaced and the cells were washed twice with Hanks balanced salt solution. Cytokine treatment was initiated by changing to fresh media containing TNF- and/or IL-1β as described in Section 2.1.
The cellular and matrix proteins were extracted as follows: after cytokine treatment, the media were removed from the wells and a 500-µl aliquot taken for measurement of soluble radioactivity. The cells were washed with cold PBS containing 1 mM EDTA, the wash solution was discarded. The cells were then incubated for 30 min at 4°C with 0.25 M NH4OH, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM EDTA; washed once with PBS containing 1 mM EDTA; and incubated for 15 min at 4°C in 50 mM Tris–HCl, pH 7.4, 1 M NaCl, 1 mM PMSF and 1 mM EDTA. The culture wells were again washed with PBS containing 1 mM EDTA, and all the above solutions were combined as cell fraction, which was precipitated by adding 0.1 volume of 100% trichloroacetic acid (TCA). The TCA precipitates were washed twice with 10% TCA containing 1 mg/ml of L-proline and 1 mg/ml L-leucine, solubilized in 1 M NaOH, and neutralized with 1 M acetic acid. The solution was mixed with Ultra-Gold LSC-cocktail (Packard, Groningen, Netherlands), and the radioactivity was measured by liquid scintillation counting using a Wallac 1409 liquid scintillation counter (Gaithersburg, MD, USA). The results were presented as percent changes compared with untreated controls, the means of which were arbitrarily set as 100%.
The matrix fraction that remained on the well surface was solubilized in 1 M NaOH, and neutralized with an equal volume of 1 M acetic acid, and precipitated by the addition of 0.1 volume of 100% TCA in the presence of 1.5 mg bovine serum albumin. The precipitates were treated further as described for cell extracts.
2.8 Statistical analysis
One way analysis of variance was applied to compare the changes in TIMPs and ADAM10 expression and matrix degradation in different experimental groups. When a significant F value was obtained, comparison among the means was performed with the post-hoc Student–Newman–Kuels analysis test using a statistical analysis software (SPSS) [27]. The quantitative values were presented as mean±S.E.M. Statistical significance was considered at P<0.05.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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TNF-
and IL-1β have synergistic effects on the expression of various genes. We examined the combined as well as individual effects of TNF- or IL-1β on the expression of TIMPs and ADAM10 in cardiac myocytes and non-myocytes. The changes in gene expression of TIMPs and ADAM10 in these cells at both the mRNA and protein levels; and the effect of the altered gene expression on proteolysis and extracellular matrix turnover were quantitatively studied.
In cardiac myocytes, TIMP-1 mRNA expression was significantly upregulated within 3 h of exposure to TNF- and IL-1β. The expression of TIMP-1 transcripts continued to increase until 36 h. An additive effect was observed in the induction of TIMP-1 mRNA, although IL-1β was more potent than TNF-. Northern blot analysis detected two TIMP-2 transcripts. The levels of these two transcripts did not change with cytokine treatment. The TIMP-3 cDNA probe detected three transcripts of 4.5, 2.3 and 0.9 kb. The expression of the 4.5 and 2.3 kb transcripts of TIMP-3 was downregulated in cardiac myocytes after 15 h stimulation with TNF- and IL-1β. Separate treatment with TNF- or IL-1β suggested that the suppression of TIMP-3 expression is caused by IL-1β, as TNF- seemed to have an opposite (though not significant) effect on the expression of TIMP-3. Similar to TIMP-2, TIMP-4 mRNA expression remained unchanged after stimulation with either TNF- or IL-1β (Figs. 1 and 2
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ADAM10 mRNA expression was significantly induced in cardiac myocytes exposed to either TNF-
or IL-1β for 3 to 36 h. No synergism was observed when cardiac myocytes were exposed simultaneously to TNF- and IL-1β (Figs. 1 and 2
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At the protein level, the amount of TIMP-1 in cardiac myocytes was not associated with changes in the amount of TIMP-1 mRNA, as cytokine treatment did not change the level of TIMP-1 protein. However, in contrast to TIMP-1, results of mRNA and protein level studies were consistent for TIMP-2 and -3. The TIMP-2 protein remained stable in myocytes exposed to cytokines, which was consistent with results at the mRNA level (Fig. 3). In the extracellular matrix where TIMP-3 is found, Western blot using both polyclonal antiserum and a monoclonal antibody against the synthetic peptide corresponding to the carboxyl terminus of TIMP-3 detected a 23 000 Da band that co-migrated with the TIMP-3 positive control. The TIMP-3 protein content was downregulated in the presence of IL-1β (Fig. 3C). A robust 24 000 Da TIMP-4 band was detected in myocyte lysates but barely detectable in non-myocyte lysates using Western blot analysis. TNF- and IL-1β both induced a transient increase and then a decrease in TIMP-4 protein level in myocytes (Fig. 3D).
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Western blot with antiserum directed against a peptide corresponding to residues in the disintegrin domain of ADAM10 detected a single 62 000 Da band in glycoprotein from cardiac myocytes. Stimulation of cardiac myocytes with TNF-
and/or IL-1β for 15 h significantly increased ADAM10 protein expression (Fig. 3E).
In cardiac non-myocytes, the continuous induction of TIMP-1 transcripts was more obvious than that seen in myocytes. Even after 36 h of administration of TNF- and IL-1β, the TIMP-1 transcripts continued to increase in these cells (Fig. 4). This effect was largely induced by IL-1β, though there was a marked additive effect of TNF- and IL-1β (Fig. 5A and B). The TIMP-2 transcripts in non-myocytes did not change with cytokine treatment. By contrast, the expression of the major TIMP-3 transcript (4.5 kb) was downregulated in cells exposed to TNF- and IL-1β, or IL-1β only (Fig. 5E and F). The TIMP-4 transcript was barely detectable in cardiac non-myocytes using Northern blot.
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The expression of ADAM10 was augmented by TNF-
and IL-1β co-treatment in non-myocytes and this was consistent to the results in myocytes. Furthermore, TNF- and IL-1β appeared to have an additive effect on the induction of ADAM10 gene expression in non-myocytes (Figs. 4 and 5).
In contrast to the results seen in cardiac myocytes, the expression of TIMP-1 protein in non-myocytes was downregulated by TNF- and IL-1β. Both TNF- and IL-1β had similar inhibitory effect on TIMP-1 protein expression (Fig. 6A). TIMP-2 protein levels as measured by ELISA did not change with the treatment. The downregulation of TIMP-3 protein by TNF- and/or IL-1β in extracellular matrix of non-myocytes was similar to that seen in cardiac myocytes (Fig. 6C). We were unable to detect measurable levels of TIMP-4 protein in cardiac non-myocytes (Fig. 3D), presumably due to the very low levels of its expression. The regulation of ADAM10 protein expression in non-myocytes by TNF- and IL-1β was similar to that seen in myocytes. Stimulation with TNF- and IL-1β for 15 h increased ADAM10 protein expression (Fig. 6D).
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Table 2 summarizes the effects of TNF-
and IL-1β on the expression of TIMPs and ADAM10 at both the mRNA and protein levels in cardiac myocytes and non-myocytes.
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To determine whether the changed expression of TIMPs indeed influences extracellular matrix protein turnover, gelatin zymography and pulse-chase experiments were performed. As shown in Fig. 7, the gelatinolytic activity of total lysates of both cardiac myocytes and non-myocytes were markedly increased by cytokine treatment. This increased enzymatic activity was also demonstrated in pulse-chase experiments, where [3H]-proline and [3H]-leucine pulse labelled matrix proteins were significantly reduced in TNF-
and IL-1β treated cells (Fig. 8). No changes in protein degradation were observed in cellular fractions of TNF- and IL-1β treated cells.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The present study characterized the regulation of TIMPs and ADAM10 by TNF-
and IL-1β in neonatal cardiac cells. TIMP-1 mRNA expression was upregulated in both myocytes and non-myocytes, however, TIMP-1 protein levels were not changed in cytokine treated myocytes and were downregulated in non-myocytes exposed to TNF- and/or IL-1β. The mechanism for the dissociation of TIMP-1 transcript and protein expression remains unclear. By Western blot analysis, the TIMP-1 levels in five-fold concentrated culture media were undetectable either before or after cytokine treatment of the cells. Post-transcriptional regulation, including altered efficiency of translation, decreased mRNA half-life or increased protein degradation could account for the discordance. TIMP-2 expression was not affected by proinflammatory cytokines, which is consistent with a previous report demonstrating that TIMP-2 is constitutively expressed [28], and suggests that basal TIMP-2 levels may be required for the integrity of the extracellular matrix. TIMP-3 was downregulated by IL-1β at both the mRNA and protein levels. While the most robust effect of cytokines on TIMPs was a downregulation of TIMP-3 mRNA and protein, both TNF- and IL-1β markedly upregulated the expression of ADAM10 mRNA and protein. Although no changes for TIMP-4 were observed at the mRNA level, TNF- and IL-1β induced a transient increase in TIMP-4 protein in cardiac myocytes. This may result from altered efficiency of translation, increased half-life of mRNA or decreased degradation of the protein.
The increased gelatinolytic activity and extracellular matrix degradation induced by proinflammatory cytokines may be a result of altered expression of TIMPs as well as matrix metalloproteinases (Figs. 7 and 8). The unchanged labelled protein levels in cellular fraction in pulse-chase experiments suggest that TIMPs and metalloproteinases function extracellularly and target to the matrix proteins. However, these studies could not differentiate the individual contribution of each TIMP or matrix metalloproteinase to the total gelatinolytic activity or matrix degradation.
The present results suggest that TNF- and IL-1β play a significant role in the regulation of TIMPs and ADAM10 in both myocytes and non-myocytes. Furthermore, the cytokine-induced changes in the present study are largely consistent with the downregulation of TIMPs and upregulation of metalloproteinases in the failing human heart [18]. Although some discrepancies exist, such as the down-regulation of TIMP-1 mRNA in the failing human heart and down-regulation of TIMP-4 protein in the ischemic failing human heart, the present experiments in cultured myocytes could not clarify this disparity. However, it should be noted that studies of the failing human heart are complex: (1) patients are at the end stage of the disease; (2) hemodynamic variations can affect study results; (3) both donors and recipients are receiving a variety of pharmacological agents; (4) the cellular effects of proinflammatory cytokines are pleiotropic and (5) the isolated myocytes may not fully represent the human condition. However, cultured myocytes have been used extensively over the past two decades to further understand the molecular mechanisms of various agents on the myocardium. To test whether the cytokine effects on the expression and activities of TIMPs are specific, we treated cardiac cells with other potent modulators of cardiac growth using either L-phenylephrine (12.5 to 200 µM) or angiotensin II (0.5 to 8 µM). No significant modulation in gelatinolytic activities was observed in either cardiac myocytes or non-myocytes (data not shown).
Increased activities of matrix metalloproteinases are believed to be a contributory mechanism for the initiation and the excessive extracellular matrix remodeling in the failing heart [6]. Since the levels of circulating proinflammatory cytokines are increased in patients with heart failure [29–31], and the failing human heart re-expresses TNF- [32], a pathophysiologic link between cytokine re-expression and extracellular matrix degradation may exist. The hypothesis is further supported by the fact that the TIMPs function as an important regulatory control on the activity of metalloproteinases by stabilizing the proenzymes and by inhibiting the active enzymes. Downregulation of TIMPs alters the extracellular matrix equilibrium towards matrix degradation and leads to an increase in tissue turnover and accelerated remodeling. Therefore, agents that restore the TIMP–metalloproteinase equilibrium in cardiac tissues might have clinical relevance. Indeed, the results of recent studies suggest that administration of synthetic metalloproteinase inhibitors improves left ventricular geometry and pump function, and blocks the progression of congestive heart failure [33, 34].
Alternatively, the expression of ADAM10 may also have potential pathological relevance via its ability to augment the production of its regulator, TNF-. The production of circulating TNF- is controlled by TNF- converting enzymes, such as ADAM10 and TACE [14–17]. ADAM10 actively participates in the processing of proTNF-, and may also regulate the interaction amongst matrix components. The disintegrin domain of ADAM10 may help to target the metalloproteinase by facilitating interaction with integrin in the matrix, or might mediate matrix–cell interactions and tissue remodeling. Therefore, any change in ADAM10 expression might have multiple cellular effects.
Several potential limitations exist in the use of cultured cells to examine physiological consequences of cytokine exposure in the intact adult animal. Neonatal myocytes used in the present study may not be representative of the adult myocardium. It has been noticed by both others [35]and ourselves [36]that the enzymes used to prepare adult cardiac myocytes contain significant amount of endotoxin and in fact lead to elevated TNF- expression by these cells. However, we have found that changes in the myocardial transcript levels of TIMP-1–4 in adult rats treated with lipopolysaccharide were virtually identical to the results seen in neonatal cardiac cells treated with proinflammatory cytokines (data not shown). The concentrations of TNF- (100 units/ml or 588 pM) and IL-1β (5 ng/ml or 294 pM) used in the present study were based on our previous work assessing both gene expression and cell function in cultured neonatal myocytes and represent those concentrations affecting cellular responses [20, 21]. However, it should be noted that the TNF- concentration is higher than that found in the plasma of patients with heart failure (
370 pM) [30], but substantially lower than that used in in vivo studies (100 µg/kg or 5882 pmol/kg) [37], and that used for inducing myocyte apoptosis (4000 pM) [38]. Finally, the variety of serum proteins such as cytokine antagonists or soluble receptors which are capable of modifying the biologic activity of IL-1β and TNF- complicate the determination of "physiological" concentrations of these cytokines. We could quantitatively detect the cytokines added to this media using anti-cytokine ELISA (data not shown), hence the presence of 5% serum does not alter the mass of detectable cytokine. However, we cannot rule out that other factors contributed from the serum, albeit at 1/20th the normal physiological concentration, could modify the biologic response to the cytokines added to the culture media.
In conclusion, the present study demonstrated that TNF- and IL-1β maladaptively regulate the expression of TIMPs and ADAM10, which may in turn contribute to the increased matrix degradation and turnover in both cardiac myocytes and non-myocytes. These regulations of TIMPs were similar to that seen in the failing human heart. Since heart failure in humans is characterized by both re-expression of myocardial TNF- and remodeling of the extracellular matrix, those in vitro results suggest a potential role for the proinflammatory cytokines in the regulation of extracellular matrix remodeling and therefore in the transition to the end-stage heart failure phenotype.
Time for primary review 31 days.
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Acknowledgements |
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This work was supported in part by NIH grant HL60032-01 (AMF). Part of the work was carried out during the tenure of a fellowship from the American Heart Association, Pennsylvania Affiliate (F98258P to YYL). We thank George S. Bounoutas and Bonnie Lemster for preparation of the cells, and Dr. Paul Glynn (Medical Research Council Toxicology Unit, UK) for kindly providing anti-ADAM10 antiserum. AMF is an established investigator of the American Heart Association.
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