Published online before print January 15, 2004, doi: 10.1161/01.RES.0000117070.86556.9F
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© 2004 American Heart Association, Inc.
Molecular Medicine |
B-Type Natriuretic Peptide Exerts Broad Functional Opposition to Transforming Growth Factor-ß in Primary Human Cardiac Fibroblasts
Fibrosis, Myofibroblast Conversion, Proliferation, and Inflammation
From Scios Inc, Fremont, Calif.
Correspondence to Andrew A. Protter, Scios Inc, 6500 Paseo Padre Pkwy, Fremont, CA 94555. E-mail protter{at}sciosinc.com
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Abstract |
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The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-ß (TGF-ß) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-ß–induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-ß–induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-ß and BNP for 24 and 48 hours. TGF-ß, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-ß effects; 88% and 85% of all TGF-ß–regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-ß–regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (
-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNF-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate–dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-ß–induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.
Key Words: B-type natriuretic peptide • transforming growth factor-ß • cardiac fibroblasts • fibrosis
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Introduction |
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Natriuretic peptides comprise a family of vasoactive hormones that play important roles in the regulation of cardiovascular and renal homeostasis.1–3 Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are predominantly produced in the heart and exert vasorelaxant, natriuretic, and antigrowth activities. Binding of ANP and BNP to type-A natriuretic peptide receptor (NPRA) leads to the generation of cyclic guanosine monophosphate (cGMP), which mediates most biological effects of the peptides. Mice lacking NPRA exhibit cardiac hypertrophy, fibrosis, hypertension, and increased expression of fibrotic genes, including TGFß1, TGFß3, and collagen 1.4–6 Furthermore, targeted disruption of the BNP gene in mice results in cardiac fibrosis and enhanced fibrotic response to ventricular pressure overload, suggesting that BNP is involved in cardiac remodeling.7
Cardiac remodeling is viewed as a key determinant of the clinical outcome in heart disease. It is characterized by a structural rearrangement of the cardiac chamber wall that involves cardiomyocyte hypertrophy, fibroblast proliferation, and increased deposition of extracellular matrix (ECM) proteins. Cardiac fibrosis is a major aspect of the remodeling process typically seen in the failing heart. The proliferation of interstitial fibroblasts and increased deposition of ECM components results in myocardial stiffness and diastolic dysfunction, which ultimately leads to heart failure. Several neurohumoral or growth factors have been implicated in the development of cardiac fibrosis. These include angiotensin II (Ang II), endothelin-1 (ET-1), cardiotrophin-1 (CT-1), norepinephrine (NE), aldosterone, fibroblast growth factor 2 (FGF2), platelet-derived growth factor (PDGF), and transforming growth factor-ß (TGF-ß). TGF-ß expression is also stimulated by Ang II and ET-1 in cardiac myocytes and fibroblasts,8 additionally supporting its involvement in cardiac fibrosis.
TGF-ß mediates fibrosis by modulating fibroblast proliferation and ECM production, particularly of collagen and fibronectin.9 TGF-ß also promotes the phenotypic transformation of fibroblasts into myofibroblasts characterized by expression of -smooth muscle actin.10 Several studies have demonstrated increased myocardial TGF-ß expression associated with cardiac hypertrophy and fibrosis.11–13 Moreover, functional blockade of TGF-ß prevents myocardial fibrosis and diastolic dysfunction in pressure-overloaded rats, indicating that TGF-ß has a crucial role in the process of myocardial remodeling, particularly in cardiac fibrosis.14
Although BNP has been implicated in cardiac fibrosis, it is unclear whether BNP has direct effects on cardiac fibroblasts to inhibit the fibrotic response. In the present study, we explore the effects of BNP on TGF-ß–induced fibrosis in cultured human cardiac fibroblasts.
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Materials and Methods |
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Cell Culture
Two lots of primary human cardiac fibroblasts, derived from an 18-year old white man (lot 1) and a 56-year old white man (lot 2), were provided by Cambrex Bio Science (Walkersville, Md). Cells stained positive for
-smooth muscle actin and vimentin antibodies, corroborating their identity as cardiac fibroblasts and myofibroblasts (data not shown). Both lots were used for the real-time reverse transcription–polymerase chain reaction (RT-PCR) studies; lot 1 was used for the microarray analysis. Cells at passages 3 through 5 were cultured in FGM containing 15% FBS. At confluence, cells were split and cultured in 6-well plates for 24 hours. Cells were changed to serum-free medium and treated with human BNP (American Peptide Company, Sunnyvale, Calif) in the presence or absence of 5 ng/mL of TGF-ß (R&D Systems) for 6, 24, and 48 hours. BNP- or TGF-ß-treated cells were also incubated in the presence of cGMP-dependent protein kinase (PKG) inhibitor KT5823 (1 µmol/L, Calbiochem), mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (0.1 to 10 µmol/L, Sigma), or PD98059 (10 µmol/L, Sigma) for 48 hours. BNP (100 nmol/L) was added into the medium 3 times a day, such that the total calculated concentrations of exogenous BNP were 200, 600, and 900 nmol/L at 6, 24, and 48 hours, respectively. This dosing protocol was necessary to maintain the levels of BNP in culture because two distinct clearance pathways are responsible for the rapid degradation of natriuretic peptides.15–17 Without this treatment regimen, we found that BNP was significantly degraded in the cardiac fibroblasts; 50% of added BNP was metabolized within 24 hours, as measured by immunoreactive assays and cGMP stimulation cell bioassays (data not shown).
Intracellular cGMP Assay
Cells were cultured in 6-well plates for 24 hours and then changed to serum-free medium and preincubated with 0.1 mmol/L of 3-isobutyl-1-methylxanthine for 1 hour before treatment with 10-9 to 10-6 mol/L of BNP for 10 minutes. The medium was aspirated, and 0.5 mL of cold PBS was added to each well. Cells were scraped and mixed with 2 volumes of cold ethanol by vortex. After 5 minutes of incubation at room temperature, the precipitate was removed by centrifugation at 1500g for 10 minutes. The supernatant was dried by vacuum centrifugation, and levels of cGMP were measured using the cyclic GMP EIA kit (Cayman Chemical).
BrdU Incorporation
Cells were placed in 96-well plates and cultured for 24 hours before changing to serum-free medium. Cells were treated with BNP (100 nmol/L, 3 times a day) in the presence or absence of 5 ng/mL of TGF-ß for 24 hours. Subsequently, 10 µmol/L of 5-bromo-2'-deoxyuridine (BrdU) was added to the cells, and they were cultured for an additional 24 hours. BrdU incorporation was detected using the Cell Proliferation ELISA kit (Roche). Data were analyzed by ANOVA using the Newman-Keuls test to assess significance.
cDNA Microarray
Gene expression profiles were determined from cDNA microarrays containing 8600 elements derived from clones isolated from normalized cDNA libraries or purchased from ResGen (Invitrogen Life Technologies). Differential expression values were expressed as the ratio of the median of background-subtracted fluorescent intensity of the experimental RNA to the median of background-subtracted fluorescent intensity of the control RNA. For ratios 
1.0, the ratio was expressed as a positive value. For ratios <1.0, the ratio was expressed as the negative reciprocal (ie, a ratio of 0.5=-2.0). An expanded discussion of the microarrays can be found in the online Materials and Methods section, available in the online data supplement at http://circres.ahajournals.org.
Real-Time RT-PCR
Real-time RT-PCR18 was performed in a two-step manner. cDNA synthesis and real-time detection were carried out in a PTC-100 Thermal Cycler (MJ Research) and an ABI Prism 7700 Sequence Detection System (Applied Biosystems), respectively. Random hexamers (Qiagen) were used to generate cDNA from 200 ng RNA, as described in Applied Biosystems User Bulletin No. 2. TaqMan PCR Core Reagent Kit or TaqMan Universal PCR Master Mix (Applied Biosystems) were used in subsequent PCR reactions according to the manufacturer’s protocols. Relative quantitation of gene expression was performed using the relative standard curve method. All real-time RT-PCR reactions were performed in triplicate.
Sequence-specific primers and probes were designed using Primer Express Version 2 software (Applied Biosystems). Sequences of primers and probes can be found in online Table 1 in the data supplement, available at http://circres.ahajournals.org. Expression levels were normalized to 18S rRNA. The selection of 18S rRNA as an endogenous control was based on an evaluation of the 
CT levels (Applied Biosystems document No. 4308134C) of the following 6 housekeeping genes: cyclophilin A, 18S, GAPDH, ß-actin, ß-glucuronidase, and hypoxanthine guanine phosphoribosyl transferase. The CT levels of 18S did not differ significantly between treatment conditions; thus, they were expressed at constant levels between samples (data not shown).
Western Blot Analysis
Cells were cultured in 6-well plates and treated with BNP (100 nmol/L, 3 times daily) in the presence or absence of 5 ng/mL TGF-ß for 48 hours. See the online data supplement for detailed methods. Briefly, 20 µg of protein from each sample was transferred to nitrocellulose membranes. The membranes were incubated with rabbit anti-human collagen 1 antibody (Cortex Biochem, San Leandro, Calif), HRP-conjugated anti-human fibronectin antibody, or goat anti-Actin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif). For extracellular signal-related kinase (ERK) phosphorylation, the membranes were incubated with rabbit anti-human phospho-ERK 1/2 antibody or rabbit anti-human ERK 1/2 antibody (Cell Signaling, Beverly, Mass). Subsequently, the membranes were incubated with HRP-conjugated anti-rabbit antibody or HRP-conjugated anti-goat antibody.
An expanded Materials and Methods section can be found in the online data supplement, available at http://circres.ahajournals.org
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cGMP Production in Cardiac Fibroblasts
To determine whether NPRA was expressed in the cultured fibroblast cells, we performed cGMP accumulation assays. BNP dose-dependently induced intracellular cyclic GMP production in cardiac fibroblasts with an EC50 of 50 nmol/L (data not shown). These results are consistent with the report of Cao and Gardner19 showing NPRA expression in cardiac fibroblasts.
Effects of BNP on TGF-ß–Induced Fibroblast Proliferation
To examine the effects of TGF-ß and BNP on cell proliferation, BrdU incorporation was measured in cardiac fibroblasts treated with TGF-ß in the presence or absence of BNP. TGF-ß modestly increased (
50%) cardiac fibroblast proliferation, and BNP inhibited TGF-ß–induced proliferation by 65% (online Figure 1, available in the online data supplement at http://circres.ahajournals.org).
Effects of BNP on TGF-ß–Induced Gene Expression
To determine the effects of BNP on gene expression profiles induced by TGF-ß in cardiac fibroblasts, we performed a microarray analysis. Fluorescently labeled cDNA probes were prepared from pooled mRNAs generated from duplicate wells of cells from 4 groups: unstimulated (control), TGF-ß–treated, BNP-treated, and cotreated with TGF-ß and BNP for 24 and 48 hours (as described in Materials and Methods). Arrays were probed in duplicate for a total of 12 hybridizations (6 at each time point), as follows: control compared with TGF-ß–treated, TGF-ß–treated compared with TGF-ß plus BNP-treated, and control compared with BNP-treated.
We observed that BNP had no significant effects on gene expression in unstimulated human cardiac fibroblasts (Figure 1). In contrast, TGF-ß induced 394 and 501 gene expression changes at 24 and 48 hours, respectively. These differentially expressed genes represent 7% to 8% of the target genes on the array. Interestingly, BNP had dramatic effects on the gene expression changes induced by TGF-ß (Figure 2). Approximately 88% and 85% of TGF-ß–regulated gene expression events were opposed by BNP at 24 and 48 hours, respectively. These results demonstrate that BNP has strikingly different effects on gene expression in TGF-ß–stimulated fibroblasts compared with unstimulated cells.
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Gene Expression Clustering
To identify different gene expression patterns after TGF-ß stimulation, we performed a hierarchical cluster analysis. A visualization of this analysis is shown in Figure 3. A complete listing of differentially expressed genes is available online (online Table 2). The clustered expression patterns showed temporal effects of TGF-ß–responsive genes (compare A with B). In addition, the dramatic effects of BNP in opposing TGF-ß–induced upregulated and downregulated gene changes were revealed in the clusters (compare A and B with C and D). The insignificant effects of BNP on gene expression in unstimulated cardiac fibroblast cells were evident in groups E and F.
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Genes were grouped according to functional categories by using a combination of gene expression clustering and functional annotations. A cluster of genes involved in fibrosis and ECM production was upregulated in cells stimulated with TGF-ß; these genes were downregulated when treated with BNP (Figure 4A). This cluster includes the following extracellular matrix components: collagen 1a2 (COL1A2), collagen 15A (COL15A), collagen 7A1 (COL7A1), microfibril-associated glycoprotein-2 (MAGP2), matrilin 3 (MATN3), fibrillin 1 (FBN1), and cartilage oligomeric matrix protein(COMP). Also included in the cluster are known markers of fibrosis, such as TIMP3,CTGF, IL11, and SERPINE1 (PAI-1). Furthermore, the cluster revealed that BNP opposed TGF-ß induction of myofibroblast markers, including -smooth muscle actin 2 (ACTA2) and nonmuscle myosin heavy chain (MYH9).
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Many genes involved in cell proliferation were also regulated by TGF-ß and were opposed by BNP (Figure 4B). For example, TGF-ß induced the expression of positive regulators of cell proliferation, including PDGFA, IGFBP10, IGF1, and parathyroid hormone-like hormone (PTHLH). We also found that TGF-ß downregulated both positive and negative regulators of proliferation, such as CDC25B and cullin 5 (CUL5), respectively. All of these TGF-ß–regulated gene events were opposed by BNP.
BNP affected TGF-ß–induced genes involved in inflammation (Figure 4C). For example, BNP reversed TGF-ß induction of PTGS2 (COX2), TNF-induced protein 6 (TNFAIP6), and TNF superfamily, member 4 (TNFSF4) (Figure 4C and data not shown). TNFAIP6 and TNFSF4 were not included in Figure 4C, because some of the data points at 48 hours did not meet acceptable criteria (see the online Materials and Methods section); at 24 hours, both genes were elevated 3-fold by TGF-ß and opposed by BNP. TGF-ß also downregulated many proinflammatory genes, including IL1B, CCR2 (MCP1-R), CXCL1 (GRO1), CXCL3 (GRO3), and CCL13 (MCP4), which were reversed by BNP. The significance of these inflammatory changes is discussed below.
Validation of Microarray by Real-Time RT-PCR and Western Blot Analyses
Representative microarray data were validated using real-time RT-PCR and Western analyses. TGF-ß induced collagen 1 mRNA levels in human cardiac fibroblasts at 6, 24, and 48 hours; this induction was blocked by BNP at all three time points (Figure 5A). Collagen 1 protein synthesis was also induced (3-fold) at 48 hours, and BNP inhibited this stimulation by 75% (Figure 5B). BNP also inhibited TGF-ß–induced fibronectin mRNA and protein expression at 48 hours (Figures 5C and 5D). These data corroborate the microarray results, with the exception of fibronectin, which did not exceed the array differential expression threshold value, most likely because of the lower sensitivity of the microarray compared with real-time RT-PCR. The effects of BNP on TGF-ß stimulation of profibrotic genes CTGF, PAI-1, TIMP3, IL11, and ACTA2 were also confirmed by real-time RT-PCR (Figure 6). Additional verification was obtained for the proinflammatory genes COX2 and IL6 at 6, 24, and 48 hours (Figure 6). Again, most likely because of sensitivity issues, IL6 was not included in Figure 4C, because it did not exceed the array differential expression threshold value.
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In addition, real-time RT-PCR assays were performed for 9 genes on primary cultures of human cardiac fibroblasts from a second independent donor lot of fibroblasts (online Table 3). The effects of BNP on TGF-ß–induced gene expression in both donors were similar, although donor lot 2 was slightly less responsive to TGF-ß.
Taken together, these results confirm our microarray data using independent assay methods as well as multiple human cardiac fibroblast donors.
MEK/ERK Pathway Involved in the Antifibrotic Role of BNP
Natriuretic peptides were previously shown to stimulate ERK activity in cardiac myocytes and vascular endothelial cells.20,21 The MEK/ERK pathway has been linked to the repression of TGF-ß/Smad signaling.22 To determine whether PKG or ERK signaling is involved in BNP-dependent attenuation of TGF-ß signaling, we treated cultured cells with BNP or TGF-ß in the presence of a PKG inhibitor (KT5823) or two different MEK inhibitors (U0126 and PD98059). BNP-induced ERK phosphorylation was completely blocked by KT5823 and U0126, indicating that BNP activates ERK via PKG and MEK signaling cascades (Figure 7A). Both MEK inhibitors (U0126 and PD98059) reversed BNP inhibition of TGF-ß–induced collagen-1 expression analyzed by Western blot (Figure 7B) and real-time RT-PCR (Figure 7C). A similar result was demonstrated for PAI-1 using real-time RT-PCR (data not shown). These findings suggest that the ERK pathway plays an important role in BNP-dependent inhibition of the fibrotic response induced by TGF-ß in human cardiac fibroblasts.
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Discussion |
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In the present study, we demonstrate the antifibrotic activity of BNP on human cardiac fibroblasts treated with TGF-ß. BNP suppressed TGF-ß–stimulated cardiac fibroblast proliferation and TGF-ß–induced expression of profibrotic genes. These findings provide the first demonstration that BNP has a direct effect on cardiac fibroblasts to oppose a TGF-ß–induced fibrotic response, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.
Fibrosis and ECM
One of the key features of cardiac fibrosis is the increased deposition of the ECM. The dynamic turnover of ECM proteins is controlled by several regulatory mechanisms, including de novo biosynthesis of ECM components, proteolytic degradation of ECMs by matrix metalloproteinases (MMPs), and inhibition of MMP activities by endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs). All of these processes have been shown to be profoundly affected by TGF-ß.23,24 The results from this study suggest that TGF-ß–induced ECM deposition in human cardiac fibroblasts occurs largely by increasing ECM gene expression, including fibronectin, COL1A2, COL15A, COL7A1, MAGP2, MATN3, FBN1, and COMP. Fibronectin and collagen expression in cardiac fibroblasts has been well-established in the fibrotic response; however, this is the first report of TGF-ß induction of other ECM genes, including MAGP2, MATN3, FBN1, and COMP, additionally corroborating the role of TGF-ß in ECM induction. Interestingly, COMP, which is a member of the thrombospondin family, has been shown to have a direct interaction with fibronectin,25 supporting its role in fibrotic processes. We also found thrombospondin 2, which is involved in the activation of latent TGF-ß,26 regulated by TGF-ß in our studies and opposed by BNP (online Table 2). Also sharing close identity with the latent TGF-ß family of binding proteins is FBN1, a component of extracellular microfibrils.27 The opposing effects of BNP on these gene regulatory events suggests that BNP modulates cardiac fibrosis.
In addition to the suppression of TGF-ß–induced ECM biosynthesis, BNP may also modulate the degradation of ECM proteins by opposing elevated TIMP3 levels in TGF-ß–stimulated cells. The TIMP family of proteins is believed to play significant roles in controlling extracellular matrix remodeling. Elevation of TIMP3 expression has been observed in animal models of myocardial infarction, suggesting that it may be a contributor to matrix remodeling in the failing heart.28
Another hallmark of the fibrotic process is the transformation of cardiac fibroblasts to myofibroblasts and the induction of profibrotic mediators. Myofibroblasts acquire contractile properties similar to smooth muscle cells. In this study, we demonstrate that BNP inhibited TGF-ß induction of several myofibroblast markers, including ACTA2 and MYH9. BNP also inhibited TGF-ß profibrotic mediators, such as CTGF, PAI-1, and IL11. CTGF and PAI-1 are well-established downstream signaling genes of the TGF-ß pathway, and IL11 has been associated with tissue remodeling and fibrosis.29,30 Our results raise the possibility that increased IL11 expression in cardiac fibroblasts may contribute to TGF-ß–mediated fibrosis, and the suppression of this response by BNP may have a protective effect.
Collectively, these effects of BNP on gene expression in TGF-ß–stimulated cells support a role for BNP in antifibrotic processes in cardiac fibroblasts. In striking contrast to TGF-ß–treated cells, BNP had no significant effects in unstimulated fibroblasts. This is consistent with the physiological actions of BNP, working only in opposition to other hormonal systems, such as the renin-angiotensin-aldosterone system.3
Changes in Cell Proliferation
The effects of TGF-ß on cell growth is cell-type dependent.31–33 In this study, we demonstrate that TGF-ß stimulated cardiac fibroblast proliferation. Whether TGF-ß has a direct effect on cell cycle or an indirect effect through other mechanisms is still unclear. Our cDNA microarray revealed that BNP markedly inhibits the expression of several TGF-ß–induced growth factors or growth factor–like genes, including PDGFA, IGF1, FGF18, and IGFBP10 (CYR61). The upregulation of these genes by TGF-ß could partially explain the induction of cell proliferation, suggesting that it may be mediated indirectly through the stimulation of growth factor productions. TGF-ß also induced the expression of PTHLH (PTHrP), which has known chronotropic and vasodilatory effects. In osteoblast-like cells, PTHrP can induce cell proliferation.34 Interestingly, in the myocardium, PTHrP levels are increased in congestive heart failure.35
The growth-inhibitory effects of natriuretic peptides have previously been reported. Cao and Gardner19 first demonstrated that natriuretic peptides inhibit PDGF, FGF2, and mechanical stretch-induced DNA synthesis in neonatal rat cardiac fibroblasts. Consistent with these findings, natriuretic peptides and cyclic GMP have been reported to inhibit cell proliferation induced by Ang II, ET-1, and norepinephrine in many cell types, including cardiac fibroblasts, vascular smooth muscle cells, endothelial cells, and mesangial cells.36–39 Taken together, our study and others suggest an important role for BNP in regulating fibroblast growth during cardiac remodeling.
Changes in Inflammatory Genes
Cardiac expression of cytokines is thought to contribute to a decrease in left ventricle contractile performance and deleterious remodeling.40,41 We report for the first time that BNP blocks TGF-ß stimulation of several proinflammatory genes, including COX2, IL6, TNFAIP6, and TNFSF4. Our results are consistent with the observations that another natriuretic peptide, ANP, has antiinflammatory effects in cultured cells. ANP inhibits LPS-induced tumor necrosis factor- production and COX2 expression in murine macrophages.42,43 Additionally, ANP reduces tumor necrosis factor-–induced actin polymerization and cell permeability via inhibition of p38 mitogen-activated protein kinase activation in HUVECs.44
TGF-ß has a dual effect in the regulation of inflammatory processes. For example, it increases COX2 expression and prostaglandin E2 release in pulmonary artery smooth muscle cells, airway smooth muscle cells, and intestinal epithelial cells.45,46 On the other hand, TGF-ß downregulates the production of MCP-1 and complement components (C3 and C4) in human proximal tubular epithelial cells and macrophages.47,48 The present study corroborates the dual effect of TGF-ß in the modulation of inflammatory gene expression in cardiac fibroblasts. We found that whereas TGF-ß induced some inflammatory genes, it downregulated others, such as IL1ß, MCP1-R, GRO1, GRO3, and MCP4. Both effects are reversed by BNP. However, in the absence of TGF-ß stimulation, BNP had no significant effect on the expression of inflammatory genes. It is likely that a balance of proinflammatory and antiinflammatory stimuli is important in the process of cardiac remodeling.
Signaling Mechanism Underlying the Antifibrotic Role of BNP
Studies aimed at elucidating the mechanism of inhibition of a fibrotic response by BNP suggest that the ERK signaling pathway plays an important role. We demonstrate that BNP phosphorylates ERK via PKG-dependent signaling in primary human cardiac fibroblasts. Moreover, this activation attenuates the TGF-ß–induced fibrotic response as measured by collagen 1 expression. This is consistent with previous studies showing that ERK activation is required for both the antihypertrophic effect of ANP in cardiac myocytes20 and the inhibition of TGF-ß signaling in mammary and lung epithelial cells.22
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Conclusions |
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Along with the endothelin pathway and the renin-angiotensin and aldosterone system, the fibrosis-promoting TGF-ß pathway is important in the pathophysiology of heart failure.11–13,49 Our study demonstrates that BNP opposed TGF-ß–regulated gene expression related to fibrosis and myofibroblast conversion. Furthermore, we show that the opposition of BNP to the TGF-ß–stimulated fibrotic response is dependent on the PKG and the MEK/ERK pathways. These results are consistent with the observation that BNP-deficient mice show increased fibrosis and collagen 1 expression.7 In addition to the global effects of BNP on fibrosis, our study suggests that it also may have effects on other processes, such as inflammation and proliferation (Figure 8). Together these data suggest a beneficial role for BNP in the prevention of cardiac fibrosis and the treatment of cardiac diseases. Additional studies aimed at understanding the physiological and the pharmacological activities of BNP in the myocardium may allow for improved strategies in the treatment of cardiac diseases, especially heart failure.
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Acknowledgments |
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This work was supported by Scios Inc. We wish to thank Lisa Garrard, Kip Madden, Jie Hu, and Estelle Tham for their informatics expertise and support, all of which made this work possible. In addition, we are grateful to Evelyn Lawani and Carl Dowds for their assistance in clone annotations.
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Footnotes |
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*Both authors contributed equally to this study.
Original received August 27, 2003; resubmission received December 18, 2003; accepted December 22, 2003.
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