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Cardiovascular Research 2004 64(3):507-515; doi:10.1016/j.cardiores.2004.07.020
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Tumor necrosis factor {alpha} induces human atrial myofibroblast proliferation, invasion and MMP-9 secretion: inhibition by simvastatin

Karen E. Portera,*, Neil A. Turnera, David J. O'Reganb and Stephen G. Balla

aInstitute for Cardiovascular Research, Academic Unit for Cardiovascular Medicine, Worsley Building, University of Leeds, Leeds LS2 9JT, UK
bDepartment of Cardiac Surgery, The Yorkshire Heart Centre, Leeds General Infirmary, Leeds LS1 3EX, UK

* Corresponding author. Tel.: +44 113 3434807; fax: +44 113 3434803. Email address: medkep{at}leeds.ac.uk

Received 22 April 2004; revised 20 July 2004; accepted 26 July 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Tumor necrosis factor {alpha} (TNF{alpha}) is implicated in myocardial remodeling, a process in which activated cardiac fibroblasts (myofibroblasts) secrete matrix-degrading metalloproteinases (MMPs) and undergo increased proliferation and invasion. Statins are cholesterol-lowering drugs that also have direct cellular effects, which may underlie their ability to reduce myocardial remodeling. This study investigated the effect of TNF{alpha} on human cardiac myofibroblast proliferation, invasion and MMP-9 secretion, and determined whether these properties were modulated by simvastatin.

Methods: Human cardiac myofibroblasts were cultured from right atrial appendage. TNF receptor expression was quantified by immunoblotting. Cell proliferation, invasion, MMP-9 secretion and MMP-9 mRNA expression were determined by cell counting, Matrigel-coated modified Boyden chamber assays, gelatin zymography and RT-PCR, respectively.

Results: Human atrial myofibroblasts expressed the TNF-RI and TNF-RII receptor subtypes. TNF{alpha} (1 ng/ml) induced a 23.1±3.9% increase in cell number after 4 days (P<0.001). Additionally, TNF{alpha} (1–10 ng/ml) significantly (P<0.01) increased myofibroblast invasion, with a concomitant increase in MMP-9 secretion, that was due to increased MMP-9 mRNA levels. Using TNF-R-specific neutralizing antibodies, we determined that these cellular effects of TNF{alpha} were predominantly TNF-RI-mediated. Simvastatin (0.1–10 µmol/l) concentration dependently inhibited TNF{alpha}-induced myofibroblast proliferation, invasion and MMP-9 secretion.

Conclusions: TNF{alpha}, acting predominantly via the TNF-R1 receptor, increased human atrial myofibroblast proliferation, invasion and MMP-9 secretion, all of which were inhibited by simvastatin. Inhibition of cytokine-induced cardiac myofibroblast activation by statins provides a rationale for their use in patients with cardiac pathologies characterized by adverse myocardial remodeling.

KEYWORDS Tumor necrosis factor {alpha}; Human atrial myofibroblast; MMP-9


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Structural remodeling of the left ventricle (LV) is a key feature in the development of heart failure (HF) [1]. In addition, atrial remodeling not only plays an important role in HF progression [2], but also in the development of atrial fibrillation (AF) [3], one of the commonest arrhythmias in man. Adverse myocardial remodeling is characterised by fibrosis, myocyte death, hypertrophy of surviving myocytes and proliferation of cardiac fibroblasts [1]. Fibroblasts account for up to two-thirds of the total cells in the normal heart and are responsible for maintaining its structural integrity through controlled proliferation and extracellular matrix turnover. During the remodeling process, ordinarily quiescent cardiac fibroblasts transform into a proliferative and invasive myofibroblast phenotype [4] and initiate structural changes to the cardiac interstitium mediated in part by increased expression of matrix-degrading metalloproteinases (MMPs) [5,6]. A key event in the remodeling process is activation of MMP-2 and MMP-9, gelatinases that are necessary for degrading the basement membrane matrix [7], a pre-requisite for both cell proliferation and invasion in vivo. Both ventricular [8,9] and atrial [2] MMP-9 activity is increased in animal models of cardiac injury, and targeted deletion of MMP-9 attenuates myocardial remodeling in mice [10]. In HF patients, both plasma and LV MMP-9 activity is markedly increased [11,12]. Moreover, it has recently been shown that atrial remodeling in patients with AF is associated with significant increases in the expression and activity of atrial MMP-9 [13,14].

Tumor necrosis factor {alpha} (TNF{alpha}) is a proinflammatory cytokine that acts at the cellular level by binding to two distinct cell surface receptors, TNF-R1 and TNF-RII, both of which are expressed in the adult human myocardium [15]. A large number of studies, both clinical [16,17] and experimental [18,19], have demonstrated a strong association between circulating levels of TNF{alpha} and/or its receptors and the progression of LV remodeling and HF. At the cellular level, TNF{alpha} induces apoptosis [20] or hypertrophy [21] of cardiac myocytes, and stimulates proliferation of rat cardiac fibroblasts [22]. Transgenic mice with cardiac-specific overexpression of TNF{alpha} develop progressive LV remodeling and HF with an accompanying increase in gelatinase activity [23,24]. Additionally, anti-TNF{alpha} treatment attenuates gelatinase activity and improves cardiac function in this model [23]. Taken together, these data suggest that TNF{alpha} may directly stimulate synthesis of MMP-9, a theory supported by in vitro studies in a variety of cell types [25–27].

Several large clinical trials have concluded that administration of HMG-CoA reductase inhibitors (statins), commonly prescribed cholesterol-lowering drugs, significantly improves the morbidity and mortality associated with atherosclerosis [28]. In addition, statins exhibit beneficial effects unrelated to lipid-lowering. For example, there is strong evidence from animal studies that statins have pleiotropic effects on adverse myocardial remodeling by reducing LV hypertrophy, improving LV function and increasing survival, independently of cholesterol-lowering [29–31]. In clinical studies, a retrospective analysis of the 4S study demonstrated that long-term simvastatin treatment reduced the incidence of HF [32]. More recently, statins were shown to reduce LV mass in hypertensive patients [33], protect against AF in patients with coronary artery disease [34] and improve cardiac function and survival in HF patients [35,36]. In all of these studies, the beneficial effects of statin therapy occurred irrespective of changes in serum cholesterol levels.

Given the benefits conferred by statins in the control of adverse myocardial remodeling, and the importance of TNF{alpha} in its genesis and progression, we sought to determine whether TNF{alpha} could induce human atrial myofibroblast proliferation, invasion and MMP-9 secretion, and whether these effects could be directly inhibited by the commonly prescribed statin, simvastatin.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Reagents
All cell culture reagents were purchased from Invitrogen (Paisley, UK), with the exception of fetal calf serum (FCS) that was from Biowest (Ringmer, East Sussex, UK). Human recombinant TNF{alpha}, gelatin and monoclonal vimentin antibody were obtained from Sigma (Poole, Dorset, UK). Simvastatin was a gift from Merck, Sharpe and Dohme (Hoddesdon, Herts., UK). Monoclonal TNF-RI and TNF-RII neutralizing antibodies were from R&D Systems (Abingdon, Oxon., UK). Phospho-specific (Thr180/Tyr182) p38 mitogen-activated protein kinase (MAPK) antibody was from Cell Signaling Technology (Hitchin, Herts., UK).

2.2 Culture and characterization of human cardiac myofibroblasts
Biopsies of right atrial appendage were obtained from patients without LV dysfunction (ejection fraction normal by cardiac ultrasound and/or LV angiography) undergoing elective coronary artery bypass surgery. Local ethical committee approval and written informed patient consent were obtained. Primary cultures of cardiac fibroblasts were harvested, characterized as myofibroblasts ({alpha}-smooth muscle actin and vimentin positive) and cultured as we have described previously [37,38]. Experiments were performed on cells from passages 2–4. The investigations conformed to the principles outlined in the Declaration of Helsinki, 1997.

2.3 Expression of TNF receptor subtypes
Early passage cardiac myofibroblasts were homogenized using a Polytron homogenizer in phosphate buffered saline containing 1% Igepal CA-630, 0.1% SDS and 0.1 mg/ml phenylmethylsulfonyl fluoride. After incubation of homogenates on ice for 30 min, cellular debris was removed by centrifugation and supernatants mixed with 2x non-reducing SDS-PAGE sample (62.5 mmol/l Tris, pH 6.8, 40% glycerol, 4% SDS). Samples were boiled and protein-standardized before resolving 20 µg of each sample by 10% SDS-PAGE. Immunoblotting was performed by simultaneous probing with TNF-RI and TNF-RII antibodies (each diluted 1:250) as described previously [39]. Blots were then reprobed with vimentin antibody (1:1000) to confirm equal loading of samples.

2.4 Proliferation assays
Cardiac myofibroblasts were plated into 24-well tissue culture plates at a density of 2x104 cells/well in growth medium comprising Dulbecco's Modified Eagle Medium supplemented with 10% FCS. After incubation overnight, cells were rendered quiescent in serum-free medium (SFM) for 48 h before addition of medium containing 2.5% FCS (minimal growth medium, MGM) supplemented with TNF{alpha} alone and/or appropriate additional supplements. Medium and drugs were replaced after 2 days and cell number was determined after 4 days in triplicate using a hemocytometer.

2.5 Invasion assays
Invasion assays were performed essentially as described previously [40] using a modified Boyden chamber technique with Matrigel basement membrane matrix coated membranes (BD Biosciences, Oxford, UK). Assays were performed in medium containing 0.4% FCS, a concentration that supports cell invasion, but has no intrinsic gelatinase activity (data not shown). TNF{alpha} was loaded into the lower chamber and cardiac myofibroblasts loaded into the upper chamber (1x105 cells). Simvastatin was added to both the upper and lower chambers as appropriate. After a 24-h incubation period, processing and evaluation were performed as described previously [40]. Cell supernatants were collected from the upper chambers, any detached cells removed by centrifugation, and supernatants analyzed by gelatin zymography to quantify MMP-9 secretion (Section 2.6).

2.6 Gelatin zymography
Invasion assay supernatants were mixed with 2x non-reducing SDS-PAGE sample buffer and resolved through an 8.5% polyacrylamide gel impregnated with 1.5 mg/ml gelatin. As a positive control, conditioned medium from HT-1080 cells (a human fibrosarcoma cell line that constitutively secretes MMP-2 and MMP-9) was also analyzed. After electrophoresis, gels were washed, incubated and stained as described previously [40]. The relative density of gelatinolytic bands was determined from negative scanned images of gels using ImageQuant software (Amersham Life Science, Amersham, Bucks., UK).

2.7 Semi-quantitative RT-PCR
Serum-starved cells were treated with fresh 0.4% FCS-containing medium supplemented with 0.1–10 ng/ml TNF{alpha} for 24 h. RNA was extracted and RT-PCR performed as we have described previously [37]. MMP-9 forward (5'-TTCATCTTCCAAGGCCAATC-3') and reverse (5'-CTTGTCGCTGTCAAAGTTCG-3') primers and GAPDH primers [37] were synthesized by Invitrogen. PCR conditions were 25 (GAPDH) or 30 (MMP-9) cycles of 94 °C (30 s), 60 °C (1 min) and 72 °C (1 min). PCR products were resolved by 2% agarose gel electrophoresis and the relative intensity of MMP-9 (287 bp) and GAPDH (240 bp) bands determined using a Typhoon 9410 Imager and ImageQuant software (Amersham Life Science). Reactions were optimised for the linear phase of PCR by serial dilution of RNA to ensure that band intensity was proportional to the amount of RNA template. Samples were then standardized for equal expression of GAPDH.

2.8 Neutralizing antibody experiments
For p38 MAPK experiments, quiescent cardiac myofibroblasts were exposed to TNF-RI or TNF-RII neutralizing antibody for 30 min prior to addition of TNF{alpha} for 20 min. Whole cell homogenates were prepared, protein-standardized and immunoblotted for phospho-p38 MAPK as described previously [39]. In subsequent neutralizing antibody experiments, cells were pretreated with 10 µg/ml TNF-RI or TNF-RII antibody for 30 min before performing proliferation (Section 2.4) or invasion (Section 2.5) assays.

2.9 Statistical analysis
All results are expressed as mean±S.E.M. with n representing the number of different patients. Differences between treatment groups were analyzed using paired t-tests and P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Expression of TNF receptor subtypes in human cardiac myofibroblasts
Immunoblotting studies demonstrated that human cardiac myofibroblasts expressed both the TNF-RI (~55 kDa) and TNF-RII (~75 kDa) subtypes (Fig. 1). The absolute levels of each receptor subtype varied between cells from different patients, as did the relative levels of TNF-RI and TNF-RII.


Figure 1
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  		Fig. 1 TNF-R subtype expression in cultured human cardiac myofibroblasts. (A) Immunoblotting with TNF-RI and TNF-RII antibodies. Positions of molecular weight markers are indicated to the left (in kDa). (B) Blots were reprobed with vimentin antibody to confirm equal loading.

 
3.2 Effect of TNF{alpha} on cardiac myofibroblast proliferation
Myofibroblasts grown in MGM (2.5% FCS) alone exhibited a low level of proliferation, resulting in a 27.4±6.1% increase in cell number after 4 days, consistent with our previous report [37]. Supplementation of MGM with 1 ng/ml TNF{alpha} induced a further mean increase in cell number of 23.1±3.9% (P<0.001, n=41) (Fig. 2A). Interestingly, we observed that TNF{alpha} did not induce proliferation in all myofibroblast populations, whereas large increases (up to 80% over MGM alone) were observed in others (Fig. 2A).


Figure 2
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  		Fig. 2 TNF{alpha} stimulates myofibroblast proliferation. (A) Cells from 41 different patients were treated with 1.0 ng/ml TNF{alpha} in MGM for 4 days. Data are expressed as % cell number observed in MGM alone. (B) Dose response of TNF{alpha} on responsive cell populations. Data are expressed as % cell number observed in MGM alone. *P<0.05, **P<0.01 vs. MGM alone (n=4).

 
To determine the concentration of TNF{alpha} that induced maximal proliferation, we exposed TNF-responsive cell populations to 0.1–10 ng/ml TNF{alpha} and observed a significant increase in cell number at all concentrations, with maximal effect at 1 ng/ml (Fig. 2B).

3.3 Effects of TNF{alpha} on cardiac myofibroblast invasion and MMP-9 secretion
In our modified Boyden chamber assay, degradation of basement membrane matrix is a prerequisite for migration towards a chemotactic stimulus, a scenario that mimics the in vivo situation. Using TNF-responsive cell populations, we determined that TNF{alpha} (0.1–10 ng/ml) promoted cardiac myofibroblast invasion in a concentration-dependent manner, with a maximal effect at 10 ng/ml (Fig. 3).


Figure 3
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  		Fig. 3 TNF{alpha} stimulates myofibroblast invasion. Invasion assays were performed on TNF-responsive cell populations. (A) Representative images of myofibroblasts migrating to underside of membrane in response to TNF{alpha}. Pores in the membrane are visible as dark circles. Scale bar=30 µm. (B) Pooled data expressed as % invasion observed with 10 ng/ml TNF{alpha}. NS not significant, **P<0.01, ***P<0.001 vs. vehicle (0.4% FCS) (n=4).

 
To determine whether the cells in the invasion assay were secreting gelatinases in response to TNF{alpha}, gelatin zymography was performed on invasion assay supernatants (Fig. 4A). MMP-9 (92 kDa) secretion was markedly induced by TNF{alpha} in a concentration-dependent manner, with maximum secretion observed at 10 ng/ml. In contrast, both the pro- (72 kDa) and active (66 kDa) forms of MMP-2 were consistently detected and were unaffected by TNF{alpha} treatment (Fig. 4A).


Figure 4
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  		Fig. 4 TNF{alpha} stimulates MMP-9 secretion and increases MMP-9 mRNA levels. (A) Gelatinase activity was determined on supernatants from the invasion assays in Fig. 3. Representative gelatin zymogram is shown. HT-1080 conditioned medium was included as a positive control. Bar chart of pooled data depicts densitometric analysis of MMP-9 band intensity. NS not significant, ***P<0.001 vs. vehicle (0.4% FCS) (n=4). (B) Representative agarose gel of MMP-9 and GAPDH RT-PCR products following stimulation with TNF{alpha}.

 
We then investigated the effects of TNF{alpha} on myofibroblast MMP-9 mRNA expression and observed a concentration-dependent increase in steady-state mRNA levels (Fig 4B). These data indicate that the TNF{alpha}-induced increase in MMP-9 secretion is the result of enhanced MMP-9 mRNA expression.

3.4 The effects of TNF{alpha} on myofibroblast function are predominantly TNF-RI-mediated
To identify the TNF-R subtypes that mediate the effects of TNF{alpha}, we used neutralizing antibodies to TNF-RI and TNF-RII. We firstly determined that the TNF-RI antibody inhibited TNF{alpha}-induced p38 MAPK activation, with maximal effect at 10 µg/ml (Fig. 5). The TNF-RII antibody did not inhibit p38 MAPK activation (Fig. 5). This was anticipated since the TNF-RII is not coupled to the p38 MAPK signaling pathway [41], but in addition confirmed that there was no cross-reactivity between neutralizing antibodies.


Figure 5
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  		Fig. 5 Effect of TNF-R neutralizing antibodies on TNF{alpha}-induced p38 MAPK activation. Cells were pre-treated with 2.5–10 µg/ml TNF-RI or TNF-RII antibody prior to addition of 10 ng/ml TNF{alpha} for 20 min. Figure shows representative immunoblot of phosphorylated p38 MAPK. Bar chart depicts pooled densitometric data as a percentage of that observed with TNF{alpha} alone (n=4). Closed bars=TNF-RI neutralizing antibody, open bars=TNF-RII neutralizing antibody. NS not significant, *P<0.05, **P<0.01, ***P<0.001 for the effect of TNF-R antibody (n=4).

 
We then used these neutralizing antibodies (10 µg/ml) to determine the relative contribution of TNF-R subtypes to TNF{alpha}-induced myofibroblast proliferation, invasion and MMP-9 secretion. Proliferation was fully inhibited by the TNF-RI neutralizing antibody, but the TNF-RII antibody had no effect (Fig. 6A). In contrast, invasion was inhibited 67% by the TNF-RI antibody and 37% by the TNF-RII antibody (Fig. 6B). Analysis of invasion assay supernatants showed that TNF{alpha}-induced MMP-9 secretion was almost fully inhibited by the TNF-RI neutralizing antibody (Fig. 6C). The TNF-RII antibody attenuated MMP-9 secretion by 35%, although this did not achieve statistical significance (Fig. 6C).


Figure 6
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  		Fig. 6 Effect of TNF-R neutralizing antibodies on proliferation, invasion and MMP-9 secretion. (A) Proliferation. Cells were treated with 1 ng/ml TNF{alpha} alone or in combination with either 10 µg/ml TNF-RI (RI) or TNF-RII (RII) neutralizing antibody for 4 days. Data are expressed as % cell number observed in MGM alone. NS not significant, *P<0.05 for the effect of TNF-R antibody; {dagger}{dagger}P<0.01 for the effect of TNF{alpha} vs. MGM alone (n=4). (B) Invasion towards a 10 ng/ml TNF{alpha} stimulus was determined in cells pretreated with either 10 µg/ml TNF-RI (RI) or TNF-RII (RII) neutralizing antibody. Data are expressed as % invasion observed with TNF{alpha} alone. *P<0.05, ***P<0.001 for the effect of TNF-R antibody; {dagger}{dagger}{dagger}P<0.001 for the effect of TNF{alpha} vs. vehicle control (n=5). (C) Gelatinase activity of supernatants from the invasion assays in (B). Bar chart depicts densitometric analysis of MMP-9 band intensity expressed as a percentage of that observed with 10 ng/ml TNF{alpha} alone. NS not significant, ***P<0.001 for the effect of TNF-R antibody; {dagger}{dagger}{dagger}P<0.001 for the effect of TNF{alpha} vs. vehicle control (n=5).

 
3.5 Simvastatin reduces TNF{alpha}-induced proliferation, invasion and MMP-9 secretion
We next investigated the effects of simvastatin on TNF{alpha}-induced myofibroblast proliferation, invasion and MMP-9 secretion. Proliferation induced by either MGM alone or MGM supplemented with TNF{alpha} was inhibited by simvastatin in a concentration-dependent (0.1–1.0 µmol/l) manner (Fig. 7A). Simvastatin (1 µmol/l) inhibited MGM-induced proliferation by 39.9±5.1% (P<0.001, n=6) and also inhibited the increase in proliferation specific to TNF{alpha} by 42.7±3.4% (P<0.001, n=6), indicating an effect on both components (FCS and TNF{alpha}) of the proliferative response.


Figure 7
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  		Fig. 7 Effect of simvastatin on TNF{alpha}-induced proliferation, invasion and MMP-9 secretion. (A) Proliferation. Cells were treated with MGM alone (open bars) or MGM plus 1 ng/ml TNF{alpha} (closed bars), together with 0–1.0 µmol/l simvastatin for 4 days. Data are expressed as % cell number observed in MGM alone. *P<0.05, **P<0.01, ***P<0.001 for the effect of simvastatin vs. MGM plus TNF{alpha}; {dagger}{dagger}P<0.01, {dagger}{dagger}{dagger}P<0.001 for the effect of simvastatin vs. MGM alone; {ddagger}P<0.05 for the effect of MGM plus TNF{alpha} vs. MGM alone (n=6). (B) Invasion towards a 10 ng/ml TNF{alpha} stimulus was determined in myofibroblasts treated with 0.5–10 µmol/l simvastatin. Data are expressed as % invasion observed with TNF{alpha} alone. *P<0.05, **P<0.01 for the effect of simvastatin; {dagger}{dagger}{dagger}P<0.001 for the effect of TNF{alpha} vs. vehicle control (n=4). (C) MMP-9 secretion. Gelatinase activity was determined on supernatants from the invasion assays in (B). Bar chart depicts densitometric analysis of MMP-9 band intensity expressed as a percentage of that observed with TNF{alpha} alone. NS not significant, *P<0.05, **P<0.01 for the effect of simvastatin; {dagger}{dagger}{dagger}P<0.001 for the effect of TNF{alpha} vs. vehicle control (n=4).

 
Simvastatin inhibited TNF{alpha}-induced invasion in a concentration-dependent manner, with maximum inhibition (88%) observed at 10 µmol/l (Fig. 7B). Furthermore, MMP-9 secretion was inhibited ~50 % by 5–10 µmol/l simvastatin (Fig. 7C).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In the present study, we demonstrate that TNF{alpha} induces proliferation, invasion and MMP-9 secretion in cultured human cardiac myofibroblasts predominantly via activation of the TNF-RI receptor. These observations are of significant clinical interest in view of the pivotal role of myofibroblasts in the remodeling process during the development of HF, and the role of TNF{alpha} in its genesis and progression. We also establish that simvastatin directly inhibits TNF{alpha}-induced myofibroblast functions, effects that could underlie the ability of statins to reduce adverse myocardial remodeling in HF patients.

Expression of both the TNF-RI and TNF-RII receptor subtypes has been demonstrated in the adult human myocardium on cardiac myocytes, vascular smooth muscle cells and vascular endothelial cells [15]. Herein we demonstrate that human cardiac myofibroblasts also express both TNF-RI and TNF-RII, the relative levels of which varied between different cell populations. The effects of TNF{alpha} on myofibroblast proliferation were mediated specifically via activation of TNF-RI. Although TNF{alpha}-induced invasion and MMP-9 secretion appeared to require both TNF-RI and TNF-RII, the predominant effect was again via TNF-RI. Our findings are of particular relevance to a recent transgenic mouse study in which TNF{alpha}-induced myocardial remodeling and HF mortality was shown to be mediated via activation of TNF-RI, but not TNF-RII [42].

This is the first report of a mitogenic effect of TNF{alpha} on cultured human cardiac myofibroblasts, consistent with two previous studies using rat cardiac fibroblasts [22,43]. In addition we show that TNF{alpha} induces cardiac myofibroblast invasion, a process that requires cells to degrade a basement membrane matrix before migrating towards a chemotactic stimulus. Invasion was associated with a specific increase in MMP-9 secretion due to increased MMP-9 mRNA expression. In contrast, MMP-2 levels remained unchanged. TNF{alpha} has been reported to increase MMP-9 secretion in other human cardiovascular cell types, including vascular smooth muscle cells [25,27] and vascular endothelial cells [26]. In agreement with our findings, these studies reported that TNF{alpha} stimulated MMP-9 secretion via increased MMP-9 mRNA expression, without affecting MMP-2 secretion [25–27]. Only one previous study using cardiac fibroblasts (derived from rat ventricle) has reported an increase in gelatinase secretion following treatment with TNF{alpha} [44]. However, it appeared that the major effects were on MMP-2 and -13 rather than on MMP-9. Although these apparent differences might be explained by inter-species variation, another important factor may be that these authors measured gelatinase secretion from fibroblasts in static monolayer culture [44]. This contrasts with our own assay in which we measured gelatinase secretion from invasive cells that were actively degrading a matrix barrier. Hence, the presence of matrix in our study may augment TNF{alpha}-induced MMP-9 secretion, as has been previously observed in collagen gel cultures of human dermal fibroblasts [45].

In our study, TNF{alpha} evoked a measurable proliferative response in only half of the cell populations studied. These differences did not appear to be related to passage number or TNF-R expression levels (data not shown). However, these observations were based upon a single concentration of TNF{alpha} (1 ng/ml) and therefore the possibility that less responsive cell populations might respond to higher TNF{alpha} concentrations cannot be excluded.

The ability to regulate myofibroblast proliferation and invasion represents a potentially important therapeutic strategy for the control of adverse myocardial remodeling. In view of the emerging potential of statins for the control of adverse myocardial remodeling, we hypothesized that these drugs would reduce the stimulatory effects of TNF{alpha} on human atrial myofibroblast function. Simvastatin not only reduced TNF{alpha}-induced proliferation, but also invasion and MMP-9 secretion. The inhibitory effect of simvastatin on TNF{alpha}-induced proliferation is in agreement with our recent report in FCS-stimulated myofibroblasts in which we demonstrated a role for inhibition of RhoA prenylation, Rho kinase and cell cycle progression [38]. The effect of simvastatin was not attributable to increased cell death [38].

In contrast to the effect of simvastatin on proliferation, the mechanisms underlying its inhibitory effect on cardiac myofibroblast invasion and MMP-9 secretion have yet to be elucidated. Studies in other cell types suggest roles for inhibition of small GTPases of both the Ras and Rho families [26,46,47]. Furthermore, from our own recently reported work, simvastatin inhibited the Rho/Rho kinase pathway, but not the Ras-mediated signaling pathways in human cardiac myofibroblasts [38]. By implication, therefore, the inhibitory effects of simvastatin on atrial myofibroblast invasion and MMP-9 secretion are also likely to be the result of Rho inhibition.

A major strength of our study in understanding the effects of statins in man was the use of adult human cardiac myofibroblasts, rather than the more commonly studied animal (often neonatal) cardiac fibroblasts. The cells utilized in the present study were cultured from right atrium. Whether atrial and ventricular myofibroblasts respond similarly to TNF{alpha} is an issue that requires further study. Whilst TNF{alpha} plays an important role in LV remodeling, its role in atrial remodeling, such as that observed during progression of AF, is unknown. Interestingly, TNF{alpha} is highly expressed in the human right atrium [48]. This observation, combined with our present findings, indicates a potential role for TNF{alpha} in atrial remodeling and impaired atrial function.

In conclusion, TNF{alpha} increases human cardiac myofibroblast proliferation, invasion and MMP-9 secretion. These effects are mediated predominantly via activation of TNF-RI and are inhibited by simvastatin. Recent clinical trials of anti-TNF{alpha} therapy in HF patients have proved disappointing with no demonstrable benefit [49]. Inhibition of cytokine-induced cardiac myofibroblast activation by statins may provide an alternative strategy for the treatment of cardiac pathologies characterized by adverse myocardial remodeling.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by a Project Grant from the British Heart Foundation (PG/03/128). We are grateful to Amy Siddall, Jean Kaye, Ebru Burton and Lynne Midgley for excellent technical assistance.


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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