Published online before print November 14, 2002, doi: 10.1161/01.RES.0000047090.08299.D5
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© 2002 American Heart Association, Inc.
Molecular Medicine |
Tumor Necrosis Factor-
–Induced AT1 Receptor Upregulation Enhances Angiotensin II–Mediated Cardiac Fibroblast Responses That Favor Fibrosis
From the Department of Medicine, Division of Cardiology, University of California, San Diego, Calif.
Correspondence to Barry Greenberg, MD, Dept of Medicine/Cardiology, UCSD Medical Center, 200 W Arbor Dr, San Diego, CA 92103-8411. E-mail bgreenberg{at}ucsd.edu
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Abstract |
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Extracellular matrix (ECM) remodeling after myocardial infarction (MI) is an important determinant of cardiac function. Tumor necrosis factor-
(TNF-) and angiotensin (Ang) II levels increase after MI and both factors affect fibroblast functions. The type 1 (AT1) receptor that mediates most Ang II effects is upregulated after MI in cardiac fibroblasts, and there is evidence that this is caused by TNF-. We sought to determine if TNF-–induced AT1 receptor upregulation alters fibroblast responsiveness to Ang II and if this effect differs from direct TNF- effects on fibroblast functions. In cultured neonatal rat cardiac fibroblasts, TNF- reduced cellular [3H]-proline incorporation, increased matrix metalloproteinase-2 (MMP-2) activity and protein, and increased TIMP-1 protein levels. In cardiac fibroblasts with TNF-–induced AT1 receptor upregulation, Ang II–stimulated [3H]proline incorporation and TIMP-1 protein production was approximately 2-fold greater than in nonpretreated fibroblasts. Angiotensin II reduced MMP-2 activity and protein level only in TNF-–pretreated fibroblasts. Angiotensin II effects were inhibited by selective AT1 (but not AT2) receptor blockers. Thus, TNF-–induced AT1 receptor upregulation enhances Ang II–mediated functions that favor fibrosis. These effects are mostly directionally opposite of direct TNF- effects on cardiac fibroblasts. Recognition of multifaceted TNF- effects provides new insights into post-MI ECM remodeling.
Key Words: collagen synthesis • tumor necrosis factor- • matrix metalloproteinase • tissue inhibitor of matrix metalloproteinase • angiotensin II
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Post-myocardial infarction (MI) remodeling is a complex process involving biochemical, structural, and geometric changes in the heart. The resulting alterations in ventricular size and shape are an important cause of heart failure. Although alterations in myocyte structure and function clearly play a role in remodeling, fibroblast effects on the extracellular matrix (ECM) contribute significantly to the remodeling process.1 ECM remodeling involves tissue breakdown and formation of the replacement scar at the infarct site. Progressive accumulation of fibrous tissue including reactive perivascular/interstitial fibrosis and reparative fibrosis in response to myocyte loss also occurs in noninfarcted segments of myocardium.1–3 Both the adequacy of replacement scar formation and the extent of fibrosis in noninfarcted myocardium are determinants of systolic and diastolic function of the heart.4,5 Thus, better understanding of factors that influence fibroblast function during ECM remodeling should provide important insights into the pathogenesis of heart failure.
After MI there is initial tissue breakdown at the infarct site followed by ECM deposition in this region and in noninfarcted myocardium.1–6 Collagen breakdown involves the activation of degradative enzymes termed matrix metalloproteinases (MMPs).7 These endogenous zinc-dependent enzymes are regulated by proteins known as tissue inhibitors of metalloproteinases (TIMPs). Cardiac fibroblasts and phenotypically transformed myofibroblasts8,9 play a critical role in remodeling through ECM protein deposition and both MMP and TIMP production. Cytokines,10,11 mechanical stretch,12 and oxidative stress,13 all present in the post-MI heart, regulate ECM protein production and control the balance between MMPs and TIMPs in cultured cardiac fibroblasts. Angiotensin II (Ang II) binding to its type I receptor (AT1) stimulates fibroblasts and myofibroblasts to express ECM proteins14,15 and has multiple profibrotic effects16 that contribute to remodeling. Cytokines such as tumor necrosis factor- (TNF-) increase in the heart after MI17,18 where they also appear to influence post-MI remodeling.17,19,20 Studies implicating the renin-angiotensin system and TNF- in ECM remodeling, however, have mostly considered their effects independently and potential interactions have not been explored.
The density of AT1 receptors on fibroblast-like cells and myofibroblasts increases substantially after MI.21,22 We have previously reported that TNF- increases functional AT1 receptors on cardiac fibroblasts.23 These findings suggest that TNF-–induced AT1 receptor upregulation might influence cardiac fibroblast functions related to ECM remodeling. In the present study we examined the consequences of this interaction on processes involved in ECM remodeling. The hypotheses tested were that TNF-–induced AT1 receptor upregulation on cardiac fibroblasts enhances Ang II–mediated functions in favor of ECM deposition and that these effects differ from direct TNF- effects.
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Materials and Methods |
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Cell Cultures
Neonatal rat cardiac fibroblasts were prepared from hearts of 1- to 2-day-old Sprague Dawley rats (Harlan, Ramona, Calif) as described.23 Cells were plated from frozen stock (passage 1) in medium (DMEM high glucose, Gibco-BRL) containing 10% FBS. At confluence, cells were placed in serum-free media for 24 hours and treated as described below. The protocol was approved by the UCSD Animal Subject Committee for the preparation of cells. UCSD is accredited by AAALAC International.
Experimental Design
The following experimental design was used to distinguish direct effects of TNF- on neonatal rat cardiac fibroblasts from indirect effects mediated by induction of AT1 receptors. 
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To determine direct effects of TNF-, confluent cells were incubated for 24 hours in serum-free media with or without TNF- (0.01 to 1000 ng/mL). The effects of TNF- on fibroblast functions due to increased AT1 receptor density were determined in cells that had been exposed to TNF- (10 ng/mL) in serum-free media for 48 hours followed by 24 hours TNF- removal. Preliminary studies demonstrated that AT1 receptor density was increased 2- to 3-fold above control, whereas the effects of TNF- on fibroblast functions related to ECM remodeling were attenuated at this time. Control fibroblasts (not exposed to TNF-) were cultured under identical conditions. Fibroblasts were then exposed to serum-free media with Ang II (10-10 to 10-6 mol/L). The differences in Ang II effects between TNF- pretreated and control (nonpretreated) fibroblasts were used to measure TNF- effects due to increased AT1 receptor density. Angiotensin II has been shown to induce TNF- production in some settings.24,25 However, TNF- levels measured by ELISA (Biosource International) in the culture media from neonatal rat cardiac fibroblasts after exposure to Ang II according to the conditions described above were <15 pg/mL, a concentration well below the threshold for affecting fibroblast functions that we evaluated. To determine which angiotensin receptor subtype was involved, both TNF- pretreated and control fibroblasts were incubated with losartan (Merck Pharmaceuticals, Inc) or PD123319 (RBI Signaling Innovation, Sigma), both at 10-6 mol/L for 45 minutes before and during exposure to Ang II.
[3H]Proline Incorporation Assay
Collagen synthesis was assessed by measurement of cellular [3H]proline uptake as described previously.26 For all experiments, [3H]proline (L-[4-3H(N)]-Proline, NEN) was added to each well at a final concentration of 1 µCi/mL for the last 24 hours. After incubation, the media was removed and ice-cold 10% trichloroacetic acid (TCA) was added for 30 minutes at 4°C. After 2 rinses with cold 10% TCA, the acid-precipitable material was solubilized overnight in 0.5 mL of 1N NaOH at 37°C and neutralized with 0.46 mL 1N HCl per well. Total protein was determined from an aliquot of each well by Bradford assay (Sigma). Incorporated radioactivity (dpm) from the remaining cell lysate was measured in a liquid scintillation counter and was expressed as percent of control.
MMP Substrate Zymography
MMP activity was measured by zymography in cell culture media as described previously.27 In each experiment, equivalent numbers of cells were plated and treated as per the experimental design. Cell culture media were collected and equal volumes were electrophoresed in a SDS-polyacrylamide gel-containing gelatin (1 mg/mL, Sigma Chemical Co). The gels were washed in 2.5% Triton X-100 for 30 minutes, incubated for 24 hours in a substrate buffer (50 mmol/L Tris-Cl pH 8, 5 mmol/L CaCl2, 0.5% Brij-35, and 0.02% sodium azide) at 37°C, stained using 0.1% Coomassie blue R-250 in acetic acid:methanol:water (1:4:5), and then destained in the same solvent. Protease activity indicated by removal of substrate protein was determined using a flatbed scanner (Hewlett Packard ScanJet 6300C) and analyzing with NIH image software. Identification of MMP-2 band was based on estimated molecular weight.12,13
Immunoblotting for MMPs and TIMPs
The relative abundance of secreted MMPs and TIMPs in cell culture media was determined by immunoblotting as described previously.28 Briefly, culture media was collected and concentrated 40:1 using an Amicon B-15 concentrator (Amicon Inc). Aliquots of concentrated media were separated on 12% SDS-PAGE and electroblotted onto Hybond PVDF membrane (Amersham Pharmacia Biotech). MMP-2 and -9 and TIMP-1, -2, and -3 proteins were probed with goat polyclonal antibodies (1:500 for TIMP-1, 1:250 for MMP-2, TIMP-2 and TIMP-3, Santa Cruz Biotechnology, Inc.). MMP-13 protein was probed with either a monoclonal antibody (1:500, Oncogene Research Products) or goat anti–MMP-13 polyclonal antibody (1:100, Chemicon International). TIMP-4 protein was detected with sheep anti–TIMP-4 polyclonal antibody (1:500 dilution, Chemicon International). Horseradish peroxidase–conjugated donkey anti-goat or anti-sheep IgG (1:5000, Santa Cruz Biotechnology, Inc) or anti-mouse IgG (1:5000, GIBCO BRL) was used as the secondary antibody. The reactions were developed with enhanced chemiluminescence reagents (ECL plus, Amersham Pharmacia Biotech), and the images were obtained by exposure to x-ray films. The films were digitized and quantified with NIH Image. The results are presented as a percentage of untreated controls with the control means arbitrarily set as 100%. Prestained molecular weight makers (Amersham Pharmacia Biotech) were used to determine protein molecular weight and to ensure adequate protein separation and transfer.
Statistical Analysis
Data were collected for each assay from 3 to 7 independent experiments performed in either duplicate or triplicate. Data were analyzed by a one-way ANOVA using the Dunnett’s nonparametric post test to determine significant differences between untreated cells and cells treated with TNF- and/or Ang II according to the experimental protocol. A value of P<0.05 was considered significant.
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Results |
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Direct Effects of TNF-
[3H]Proline Incorporation
The effects of TNF-
on collagen synthesis as assessed by [3H]proline incorporation in neonatal rat cardiac fibroblasts are depicted in Figure 1. Overall, there was a significant reduction in [3H]proline incorporation (P<0.007). Whereas lower concentrations of TNF- did not alter [3H]proline incorporation, concentrations of 50, 100, and 1000 ng/mL of TNF- reduced [3H]proline incorporation by 18±4%, 15±4%, and 13±5%, respectively. Although none of these individual changes reached statistical significance on post-ANOVA analysis, the reduction in [3H]proline incorporation at the higher TNF- concentrations was similar to that previously reported.10
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MMP Activity and Protein Levels
As shown in Figure 2A, TNF- (0.01 to 1000 ng/mL) increased MMP-2 activity in the culture media in a dose-dependent manner (P<0.0005) with a maximum 2.6-fold increase at a TNF- concentration of 1000 ng/mL. Most of the MMP-2 secreted was in the active form.13 As shown in Figure 2B, TNF- produced similar dose-dependent increases in MMP-2 protein levels as in MMP-2 proteolytic activity. Although increases in MMP-9 and MMP-13 activities were seen, the very low levels of MMP-9 and MMP-13 in culture media precluded accurate quantification.
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TIMP Protein Levels
As shown in Figure 3, TNF- increased TIMP-1 protein production in a dose-dependent fashion with a maximal 3.7-fold increase (P<0.0001). The EC50 of the TNF- effect was 35 ng/mL (2 nmol/L). TNF did not significantly alter TIMP-2 protein level (data not shown). TIMP-3 and TIMP-4 protein levels were undetectable in the culture media.
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TNF- Effects Related to Increased AT1 Receptor Density
Ang II Induced [3H]Proline Incorporation
The effects of Ang II (10-10 to 10-6 mol/L) on [3H]proline incorporation was assessed in nonpretreated control fibroblasts and in fibroblasts pretreated with TNF- (10 ng/mL, Figure 4A). Angiotensin II significantly increased [3H]proline incorporation in a dose-dependent fashion (P<0.0001). In nonpretreated fibroblasts, the maximal increase of 31±7% occurs at a concentration of 10-8 mol/L Ang II. In TNF-–pretreated fibroblasts, Ang II had a greater effect on [3H]proline incorporation as evidenced by 44±11%, 67±6%, and 54±6% increases at 10-9, 10-8, and 10-7 mol/L Ang II, respectively, relative to nontreated cells. As shown in Figure 4B, AT1 receptor blockade with losartan inhibited Ang II effects in both untreated and TNF-–pretreated cells, whereas AT2 receptor antagonism with PD123319 (10-6 mol/L) did not significantly affect [3H]proline incorporation.
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Ang II Reduces MMP Activity and Protein Levels in TNF-–Pretreated Cells
As shown in Figure 5A, Ang II had no significant effect on MMP-2 activity in nonpretreated fibroblasts. However, Ang II significantly reduced MMP activity (P<0.0001) in fibroblasts pretreated with TNF- with the greatest reduction seen at concentrations of 10-9 and 10-8 mol/L. MMP-2 protein levels in response to Ang II followed the same pattern as MMP-2 activities (data not shown). As seen in Figure 5B (lanes 5 to 8), the effect of Ang II on MMP-2 protein levels is blocked by losartan but is unaffected by PD123319. Ang II also diminished zymographic activity and protein levels for MMP-9 and MMP-13 in fibroblasts pretreated with TNF-. However, the very low abundance of these MMPs in the culture media precluded reliable quantification of these changes.
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Ang II Induced TIMP Protein Production
As shown in Figure 6A, Ang II increased TIMP-1 production (P<0.04). In nonpretreated fibroblasts, Ang II stimulated TIMP-1 in a dose-dependent fashion with an approximately 2-fold increase at 10-8 and 10-7 mol/L concentrations. Pretreatment of the cells with TNF-, however, enhanced the profile of TIMP-1 protein production (lanes 5 to 8 in Figure 6A) with a maximal increase of approximately 4-fold at 10-8 mol/L. As shown in Figure 6B, losartan, but not PD123319 (both at 10-6 mol/L), prevented increases in TIMP-1 protein in response to Ang II in both nonpretreated and TNF- pretreated cells (P<0.0001). Fibroblasts pretreated with TNF- did not significantly enhance TIMP-2 protein production in response to Ang II stimulation (data not shown). TIMP-3 and TIMP-4 protein were not detectable in neonatal cardiac fibroblast culture media.
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Discussion |
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Post-MI remodeling of the ECM of the heart plays an important role in determining cardiac function. Although studies in experimental animal models and human patients indicate that both Ang II and TNF-
are involved, possible interactions between these factors have not been extensively explored. Evidence that TNF- increased functional AT1 receptors on cardiac fibroblasts23 suggested that, in addition to its direct cellular effects, TNF- might also enhance responsiveness to Ang II and thus alter ECM remodeling in ways not previously recognized. The major new findings of this study are that TNF-–induced AT1 receptor upregulation enhances Ang II–mediated proline incorporation and TIMP-1 protein expression and reduces MMP-2 activity in cardiac fibroblasts. In contrast to direct effects of TNF- on fibroblasts that mostly favor ECM degradation, these indirect effects would strongly favor ECM deposition when Ang II is present as in the post-MI heart.
Direct Effects of TNF- on Collagen Metabolism
In concentrations ranging from 50 to 1000 ng/mL, TNF- reduced [3H]proline incorporation. This result is similar to that reported previously10 and it suggests that when present at relatively high concentrations, TNF- impairs cardiac fibroblast collagen production. Because cell numbers are not significantly affected by TNF- under these conditions,23 decreased [3H]proline incorporation appears to be due to reduced collagen synthesis.
Matrix metalloproteinases are an endogenous family of enzymes responsible for ECM degradation.7 Increased MMP activity occurs in heart failure28,29 and in the post-MI heart.30 Cytokines and bioactive peptides can influence MMP activity.31 In the present experiments, TNF- produced dose-dependent increases in MMP-2 activity. These results are similar to those previously reported.10 The effects of TNF- on the protein level of MMP-2 followed the same pattern, suggesting that changes in MMP-2 activity involved increased protein synthesis and secretion.
TIMPs provide posttranslational inhibition of MMP activity.7 Four different TIMPs, each encoded by a unique gene, have been identified. TIMP-1 is the most completely characterized and alterations in its expression have been identified during tissue remodeling.32 We found that TNF- produced dose-dependent increases in TIMP-1 protein content in media from cultured cardiac fibroblasts. These results differ from a study in which TNF- increased TIMP-1 mRNA but decreased TIMP-1 protein levels in neonatal rat cardiac fibroblast cell lysates.11 These disparate results could, however, be consistent if cellular TIMP-1 were depleted by increased secretion of the protein to the extracellular milieu. As noted previously, TIMP-2 was not affected by TNF-.11
These results suggest that direct TNF- effects on fibroblast functions are more complicated than previously recognized. Reduced [3H]proline incorporation and increased MMP-2 activity would result in a net reduction in ECM tissue. The increase in TIMP-1, however, would limit ECM breakdown because TIMPs deactivate MMPs in a 1:1 stoichiometric manner. Because TNF- induced substantial increases in TIMP-1 at relatively low concentrations (compared with that required to increase MMP-2), it is possible that TNF- coinduction of TIMP-1 reduced MMP-2 activity in these experiments and that it might also do so within the heart.
Indirect Effects of TNF-–Induced AT1 Receptor Upregulation on Collagen Metabolism
To determine if TNF-–induced AT1 receptor upregulation enhanced responsiveness to Ang II, we compared the effects of Ang II between TNF- pretreated and nonpretreated control cardiac fibroblasts. Previous studies from our laboratory show that 48-hour TNF- exposure increases AT1 receptor density 2- to 3-fold.23 The fact that increased receptor density is then maintained over 24 hours while TNF- direct effects on fibroblast functions are largely attenuated allowed us to isolate TNF- effects caused by AT1 receptor upregulation.
In nonpretreated fibroblasts, Ang II increased proline incorporation in a dose-dependent fashion. Similar findings have been reported on isolated human26 and rat cardiac fibroblasts.33 Ang II also modestly increased TIMP-1 protein production but had no significant effect on MMP-2 activity or protein levels. In fibroblasts pretreated with TNF- to increase AT1 receptor density, however, Ang II stimulated [3H]proline incorporation and TIMP-1 production were both 2-fold greater than in nonpretreated cells. Angiotensin II also significantly decreased MMP-2 activity and protein levels only in TNF-–pretreated cells. Application of specific Ang II receptor blockers demonstrated that these effects were mediated through the AT1 receptor. However, because Ang II had little effect on MMP-2 in nonpretreated cells its effect in TNF- pretreated cells might not be due to AT1 receptor upregulation but rather to an alteration in fibroblast phenotype so that AT1 activation reduced MMP-2 activity and protein. Overall, the effects of TNF- mediated AT1 receptor upregulation favor ECM deposition and, if present in vivo, would tend to enhance fibrosis in settings where Ang II is present.
Our previous work demonstrated that TNF-–induced AT1 receptor upregulation increased production of second messenger inositol-phosphates in response to Ang II.23 The present study demonstrates the substantial impact of increased AT1 receptor density on the responsiveness to Ang II for fibroblast functions related to ECM remodeling. These results indicate that enhanced Ang II responsiveness is not squelched by limiting numbers of signaling molecules within the fibroblasts or by activation of counter-regulatory mechanisms. In contrast to AT1 receptor upregulation in the post-MI heart,22,23 there is evidence that this receptor is downregulated in a rat model of sepsis and that inflammatory cytokines decrease AT1 receptor expression in rat mesangial cells.34 These differences are likely related to release of additional factors during sepsis and to the differing properties of cardiac fibroblasts and renal mesangial cells.
Potential Significance of Ang II and TNF- Interaction on Cardiac Fibroblasts
Fibroblasts and myofibroblasts play an essential role in ECM remodeling.1,6,9,35 In the post-MI rat heart, appearance of myofibroblasts at the infarct site on day 2 is associated with increased collagenase and gelatinase activities. These activities increase through day 7 as tissue breakdown and clearance progress, and they decline later as the replacement scar is formed. Fibroblasts and myofibroblasts are also associated with collagen deposition at the infarct site and in noninfarcted myocardium.6
TNF- is detected in rat heart within minutes of coronary artery ligation,36 and persistent increases are noted as late as 20 weeks after MI.17–19 Although the initial increase probably represents release of previously synthesized protein, later sustained increases are related to increased mRNA expression in leukocytes, macrophages, and fibroblasts within the periinfarction zone and myocytes in noninfarcted myocardium. Our findings that TNF- increases MMP-2 activity and decreases [3H]proline incorporation in cardiac fibroblasts suggest that this cytokine functions in an autocrine/paracrine manner to degrade ECM tissue at the scar site. It may also be involved in the transition to heart failure because in other experimental models, increased MMP activity is associated with degradation of the fibrous infrastructure of the heart, ventricular cavity enlargement, and reduced contractile performance.29 However, we also found that TNF- increased TIMP-1 production. Increased TIMP transcription occurs within the first week in the post-MI rat heart35 in fibroblast-like cells in regions around the infarct. Because increases in TNF- are noted at the periinfarction zone at this time,36 it is possible that TNF- induction of TIMPs restrains further fibrous tissue breakdown and mediates the transition to replacement scar formation.
There is convincing evidence that Ang II plays an important role in ECM remodeling. Angiotensin II stimulation of fibroblasts and myofibroblasts has profibrotic effects including increased fibronectin and collagen gene transcription and protein synthesis, reduced collagenase activity, and increased expression of transforming growth factor (TGF)-ß1.15,37,38 Angiotensin II levels are increased in the post-MI rat heart39 likely due to local peptide synthesis because circulating levels are not elevated in this model.40 Increased expression of components of the renin-angiotensin system (RAS) are detected in the post-MI rat heart6,41 and myofibroblasts from the infarct scar can generate Ang II de novo.42 Upregulation of angiotensin converting enzyme (ACE) in fibroblasts and fibroblast-like cells is associated with increased collagen I mRNA expression and collagen deposition in the infarct zone and in remote segments of myocardium.6 The most compelling evidence, however, of Ang II involvement in ECM remodeling comes from studies in which ACE inhibitors and AT1 receptor blockers significantly attenuate the development of fibrosis in the post-MI heart.2,43–45 This effect appears to be AT1 specific because AT2 receptor blockade does not inhibit myofibroblast generated collagen accumulation during wound healing.46
Angiotensin II–stimulated cardiac fibrosis is due predominantly to activation of the AT1 receptor.15,37,38,46 We have previously reported that TNF- (and IL-1ß) was unique among a variety of growth factors found in the post-MI heart in its ability to increase AT1 mRNA and protein in cultured neonatal rat cardiac fibroblasts.23 In the post-MI rat heart, TNF- is increased in the infarct zone over the first several days.36 Increased AT1 receptor density is seen in this region by day 3 after MI,21 and it is then sustained over several weeks.22 Increased TNF- is also detected in noninfarcted myocardium17–19 where increased AT1 mRNA transcription has been noted.47 These temporal and spatial associations between appearance of TNF- and AT1 upregulation are consistent with the possibility that TNF- induces AT1 receptor expression in the post-MI heart. The present studies indicate that AT1 upregulation results in enhanced responsiveness to Ang II, and they suggest that this phenotypic change would increase fibrous tissue deposition in the post-MI heart.
Study Limitations
Neonatal cardiac fibroblasts were used in these experiments, and it is possible that different results might have been obtained had adult cardiac fibroblasts or myofibroblasts been used. However, we have previously shown that the phenomenon of AT1 receptor upregulation that occurs on myofibroblasts in the post-MI rat heart can be replicated in vitro by exposing either neonatal23 or adult rat cardiac fibroblasts (unpublished observations, 2000) to TNF-. Furthermore, neonatal fibroblasts demonstrate properties (ie, [3H]proline incorporation and production of both MMPs and TIMPs) relevant to ECM turnover in the post-MI heart.
Although these experiments were intended to isolate and compare the direct effects of TNF- to those due to AT1 receptor upregulation other factors present within the complex post-MI milieu (eg, TGF-ß) may greatly affect the fibroblasts functions that we studied. However, previous work demonstrating that AT1 receptor upregulation in cardiac fibroblasts was unique with TNF-23 (and IL-1ß) leads us to study only that cytokine in these experiments.
Summary and Conclusions
Our findings suggest that the role of TNF- in ECM remodeling may be more complex than previously considered. This cytokine has both direct effects on cardiac fibroblasts that predominantly favor ECM degradation and effects related to AT1 receptor upregulation that favor fibrous tissue deposition. Although enhanced responsiveness to Ang II due to TNF-–mediated AT1 receptor upregulation would promote wound healing, it would also have the unfavorable consequence of enhancing ECM deposition in noninfarcted myocardium. These results suggest that altering TNF- concentrations in the post-MI heart could be either beneficial or detrimental depending on both timing and location.
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Acknowledgments |
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The study was supported by NIH grant No. RO1 HL63909 (B.H.G.). We wish to thank Drs Jason Yuan, Francisco Villarreal, and Kirk Knowlton for their comments.
Received June 4, 2002; revision received October 15, 2002; accepted November 4, 2002.
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