Inhibition of cardiac myofibroblast formation and collagen synthesis by activation and overexpression of adenylyl cyclase

Transformation of fibroblasts to myofibroblasts, characterized by expression of α-smooth muscle actin (α-SMA) and production of extracellular matrix (ECM) components, is a key event in connective tissue remodeling. Approaches to inhibit this transformation are needed in tissues, such as the heart, where excessive ECM production by cardiac fibroblasts (CFs) causes fibrosis, myocardial stiffening, and cardiac dysfunction. We tested whether adenylyl cyclase (AC) activation (increased cAMP levels) modulates the transformation of adult rat CF to myofibroblasts, as assessed by immunofluorescent microscopy, immunoblotting, and collagen synthesis. A 24-h incubation of CF with TGF-β or angiotensin II increased α-SMA expression, which was inhibited by the AC agonist forskolin and a cAMP analog that activates protein kinase A. Treatment with forskolin blunted serum-, TGF-β-, and angiotensin II-stimulated collagen synthesis. CFs engineered to overexpress type 6 AC had enhanced forskolin-promoted cAMP formation, greater inhibition by forskolin of TGF-β-stimulated α-SMA expression, and a decrease in the EC₅₀ of forskolin to reduce serum-stimulated collagen synthesis. The AC stimulatory agonist adrenomedullin inhibited collagen synthesis in CF that overexpressed AC6 but not in controls. Thus, AC stimulation blunts collagen synthesis and, in parallel, the transformation of adult rat CF to myofibroblasts. AC overexpression enhances these effects, “uncovering” an inhibition by adrenomedullin. These findings implicate cAMP as an inhibitor of ECM formation by means of blockade of the transformation of CF to myofibroblasts and suggest that increasing AC expression, thereby enhancing cAMP generation through stimulation of receptors expressed on CF, could provide a means to attenuate and prevent cardiac fibrosis and its sequelae.     

Vinpocetine Attenuates Pathological Cardiac Remodeling by Inhibiting Cardiac Hypertrophy and Fibrosis

Purpose

Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II).

Methods

Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts.

Results

We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFβ-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1).

Conclusions

Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling.

     

The effects of cardiac myocytes on interstitial fibroblasts in toxic iron overload

Iron deposits preferentially in myocytes in mixed cultures of cardiac myocytes and nonmyocytic fibroblasts. In vivo, iron overload is associated with cardiac fibrosis. Therefore, we examined whether iron loading of cardiac myocytes in culture could trigger a response in nonmyocytes characteristic of a fibrogenic phenotype. We found that the nonmyocytes adopted a myofibroblast phenotype in culture. The rate of DNA synthesis (measured by [³H]thymidine incorporation) by the nonmyocytes was decreased by the myocyte-conditioned medium, compared to that of the unconditioned medium, and this activity was retained in >10-kDa fractions. The rate was partially restored when the medium was obtained from iron-loaded myocytes, and in this medium, the >10-kDa fraction was even more effective in reversing the suppression of proliferation. This suppression suggests a decreased secretion of a growth-inhibitory substance in the iron-loaded myocytes, and this effect was partially reversed when the iron-loaded cells were treated with the iron chelator, deferoxamine. This indicates that cardiac myocytes may play a paracrine role in suppressing the proliferation of myofibroblasts that is partially overcome when the myocytes are iron overloaded. The myocyte-conditioned medium also affects the myofibroblast phenntype, increasing the cells' fibronectin mRNA content and decreasing α-smooth-muscle actin mRNA. The myocyte-conditioned medium increases transforming growth factor-β (TGF-β) secretion by myofibroblasts, but the TGF-β content of the conditioned medium was found to play, at most, a minor role in determining the response of the myofibroblast.     

Tumor necrosis factor-alpha-induced AT1 receptor upregulation enhances angiotensin II-mediated cardiac fibroblast responses that favor fibrosis

Extracellular matrix (ECM) remodeling after myocardial infarction (MI) is an important determinant of cardiac function. Tumor necrosis factor-alpha (TNF-alpha) 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-alpha. We sought to determine if TNF-alpha-induced AT1 receptor upregulation alters fibroblast responsiveness to Ang II and if this effect differs from direct TNF-alpha effects on fibroblast functions. In cultured neonatal rat cardiac fibroblasts, TNF-alpha 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-alpha-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-alpha-pretreated fibroblasts. Angiotensin II effects were inhibited by selective AT1 (but not AT2) receptor blockers. Thus, TNF-alpha-induced AT1 receptor upregulation enhances Ang II-mediated functions that favor fibrosis. These effects are mostly directionally opposite of direct TNF-alpha effects on cardiac fibroblasts. Recognition of multifaceted TNF-alpha effects provides new insights into post-MI ECM remodeling.     

Simvastatin reduces human atrial myofibroblast proliferation independently of cholesterol lowering via inhibition of RhoA

OBJECTIVE: Adverse atrial and ventricular myocardial remodeling is characterized by fibrosis, myocyte death or hypertrophy and fibroblast proliferation. HMG-CoA reductase inhibitors (statins) are widely prescribed cholesterol-lowering drugs that also appear to have beneficial effects on myocardial remodeling. Although statins are known to reduce myocyte hypertrophy, their effect on cardiac fibroblast proliferation is unknown. The purpose of this study was to investigate the effects of simvastatin on human atrial myofibroblast proliferation. METHODS: Cardiac myofibroblasts were cultured from biopsies of human right atrial appendage. Proliferation was quantified by cell counting and cell cycle progression determined by immunoblotting for Cyclin A. The expression, activation and intracellular localization of RhoA were investigated using immunoblotting and immunocytochemistry. RESULTS: Simvastatin (0.1-1.0 micromol/l) inhibited serum-induced myofibroblast proliferation in a concentration-dependent manner at a point upstream of Cyclin A expression. These effects were reversed by mevalonate or geranylgeranyl pyrophosphate (GGPP), but not squalene or farnesyl pyrophosphate (FPP), indicating a mechanism involving inhibition of Rho-family GTPases and independent of cholesterol synthesis. The effects of simvastatin were mimicked by inhibiting Rho geranylgeranylation or Rho-kinase activation. Furthermore, we demonstrated that simvastatin inhibited RhoA function by preventing its association with the plasma membrane and hence, its interaction with downstream effectors required for cell proliferation. CONCLUSIONS: Simvastatin reduced proliferation of cultured human atrial myofibroblasts independently of cholesterol synthesis via a mechanism involving inhibition of RhoA geranylgeranylation. Statins may therefore have an important role in preventing adverse myocardial remodeling associated with cardiac myofibroblast proliferation.     

Manipulation of collagen synthesis influences progenitor cell engraftment and angiomyogenesis

Myocardial infarction(MI)results in loss of cardiomyocytes(CM) in the ischemic area of the heart followed by an inflammatory response and replacement of contractile CM with fibrosis.Myocardial fibrosis,a key contributor to cardiac dysfunction after MI,presents as a secondary response to the pathophysiological remodeling of long-standing disease including ischemia,obstruction,and microvascular abnormalities.Cardiac fibroblasts and myofibroblasts are responsible for post-MI remodeling which occurs via regulation of extracellular matrix (ECM),presenting as increased collagenⅠandⅢinto the interstitial and perivascular space.In addition to the pluripotency of stem cells following stem/ progenitor cell transplantation,decreased apoptosis, hypertrophy,and fibrosis in the infarcted heart have been demonstrated.This has made transplantation of progenitor/stem cells a primary research focus in the field of tissue regeneration.Unfortunately,the accumulation of ECM and myofibroblasts in areas of tissue injury presents a barrier that can impair penetration of reparative stem/progenitor cells mobilized from peripheral reservoirs.Therefore,cardiac fibroblast production and degradation of ECM are critical in regulating cardiac remodeling and stem/progenitor cell mobilization.This study used transgenic mice overexpressing adenylyl cyclaseⅥ(AC6) in which collagen synthesis was decreased to determine the role of collagen deposition on the engraftment of iPSC from a tri-cell patch applied to infarcted area after MI.     

The role of Interleukin Receptor Associated Kinase (IRAK)-M in regulation of myofibroblast phenotype in vitro,and in an experimental model of non-reperfused myocardial infarction

In the infarcted myocardium, necrotic cardiomyocytes activate innate immune pathways, stimulating pro-inflammatory signaling cascades. Although inflammation plays an important role in clearance of the infarct from dead cells and matrix debris, repair of the infarcted heart requires timely activation of signals that negatively regulate the innate immune response, limiting inflammatory injury. We have previously demonstrated that Interleukin receptor-associated kinase (IRAK)-M, a member of the IRAK family that suppresses toll-like receptor/interleukin-1 signaling, is upregulated in the infarcted heart in both macrophages and fibroblasts, and restrains pro-inflammatory activation attenuating adverse remodeling. Although IRAK-M is known to suppress inflammatory activation of macrophages, its role in fibroblasts remains unknown. Our current investigation examines the effects of IRAK-M on fibroblast phenotype and function. In vitro, IRAK-M null cardiac fibroblasts have impaired capacity to contract free-floating collagen pads. IRAK-M loss reduces transforming growth factor (TGF)-β-mediated α-smooth muscle actin (α-SMA) expression. IRAK-M deficient cardiac fibroblasts exhibit a modest reduction in TGF-β-stimulated Smad activation and increased expression of the α-SMA repressor, Y-box binding protein (YB)-1. In a model of non-reperfused myocardial infarction, IRAK-M absence does not affect collagen content and myofibroblast density in the infarcted and remodeling myocardium, but increases YB-1 levels and is associated with attenuated α-SMA expression in isolated infarct myofibroblasts. Our findings suggest that, in addition to its role in restraining inflammation following reperfused infarction, IRAK-M may also contribute to myofibroblast conversion.     

Deficiency in TIMP-3 increases cardiac rupture and mortality post-myocardial infarction via EGFR signaling: beneficial effects of cetuximab

Cardiac rupture is a fatal complication of myocardial infarction (MI); however, its underlying molecular mechanisms are not fully understood. This study investigated the role of tissue inhibitor of metalloproteinase-3 (TIMP-3)/matrix metalloproteinase (MMP)/epidermal growth factor (EGF)/transforming growth factor (TGF)-β1 pathway in infarct healing and effects of cetuximab on cardiac rupture after MI. Induction of MI was achieved by left coronary artery ligation in wild-type (WT) and TIMP-3^−/− mice. TIMP-3 deficiency resulted in a fourfold increase in cardiac rupture and 50% decrease in survival after MI. Hydroxyproline content, collagen synthesis and myofibroblast cell number in the infarct region, and the force required to induce rupture of the infarct scar were significantly decreased, while MMP activity was increased in TIMP-3^−/− mice. EGF proteins were increased by threefold in TIMP-3^−/− mice following MI, while TGF-β1 mRNA levels were decreased by 68%. Cell proliferation of cultured adult cardiac myofibroblasts was significantly decreased in TIMP-3^−/− compared to WT myofibroblasts. EGF treatment significantly decreased collagen synthesis and TGF-β1 expression. Conversely, TGF-β1 treatment increased collagen synthesis in cardiac myofibroblasts. Treatment with cetuximab significantly decreased the incidence of cardiac rupture and improved survival post-MI in TIMP-3^−/− mice. We conclude that deficiency in TIMP-3 increases cardiac rupture post-MI via EGF/epidermal growth factor receptor (EGFR) signaling which downregulates TGF-β1 expression and collagen synthesis. Inhibition of EGFR by cetuximab protects against cardiac rupture and improves survival post-MI.     

The interaction of coronary tone and cardiac fibrosis

Regulation of coronary vascular tone is critical for proper perfusion and function of the myocardium. Many disease processes result in compromised regulation of coronary vascular tone and impaired myocardial perfusion. A common result of coronary vascular dysfunction is the development of areas of replacement fibrosis within the myocardium and surrounding the vasculature. Both intravascular processes, such as coronary atherosclerosis and endothelial dysfunction, and extravascular processes, including compromised myocardial metabolism, hormone excesses, and altered local signaling, may result in coronary vascular dysregulation. Coronary occlusion events, in turn, lead to myocardial damage and the activation of inflammatory cells and fibroblasts. The role of fibroblasts in regulating myocardial fibrosis and the contribution of myofibroblasts, cells that have limited contractile potential while retaining many of the extracellular matrix regulating processes of the fibroblast, may also contribute to the development of myocardial disease. In this review we examine the recent literature on myocardial fibrosis and myofibroblast activity, highlighting the effects of several classes of cardiovascular agents on the remodeling process.     

Endogenous thrombospondin 1 protects the pressure-overloaded myocardium by modulating fibroblast phenotype and matrix metabolism

The matricellular protein thrombospondin (TSP) 1 is induced after tissue injury and may regulate reparative responses by activating transforming growth factor-β, by suppressing angiogenesis and by modulating inflammation and matrix metabolism. We hypothesized that endogenous TSP-1 may be involved in the pathogenesis of cardiac remodeling in the pressure-overloaded heart. Myocardial TSP-1 expression was increased in a mouse model of pressure overload because of transverse aortic constriction. TSP-1(-/-) mice exhibited increased early hypertrophy and enhanced late dilation in response to pressure overload. Pressure-overloaded TSP-1 null mice had intense degenerative cardiomyocyte changes, exhibiting more extensive sarcomeric loss and sarcolemmal disruption when compared with wild-type hearts. Accentuated hypertrophy and cardiomyocyte injury in TSP-1(-/-) hearts was accompanied by increased myofibroblast density. However, despite a 2-fold higher infiltration of the cardiac interstitium with myofibroblasts, pressure-overloaded TSP-1 null hearts did not exhibit significantly increased collagen content when compared with wild-type hearts. The disproportionately low collagen content in TSP-1 null hearts was attributed to infiltration with abundant, but functionally defective, fibroblasts that exhibited impaired myofibroblast differentiation and reduced collagen expression in comparison with wild-type fibroblasts. Impaired myofibroblast activation in TSP-1 null hearts was associated with reduced Smad2 phosphorylation reflecting defective transforming growth factor-β signaling. Moreover, TSP-1 null hearts had increased myocardial matrix metalloproteinase 3 expression and enhanced matrix metalloproteinase 9 activation after pressure overload. TSP-1 upregulation in the pressure-overloaded heart critically regulates fibroblast phenotype and matrix remodeling by activating transforming growth factor-β signaling and by promoting matrix preservation, thus preventing chamber dilation.     

TGF-beta1 and angiotensin networking in cardiac remodeling

The renin-angiotensin system (RAS) and transforming growth factor-beta1 (TGF-beta1) play a pivotal role in the development of cardiac hypertrophy and heart failure. Recent studies indicate that angiotensin II (Ang II) and TGF-beta1 do not act independently from one another but rather act as part of a signalling network in order to promote cardiac remodeling, which is a key determinant of clinical outcome in heart disease. This review focuses on recent advances in the understanding, how Ang II and TGF-beta1 are connected in the pathogenesis of cardiac hypertrophy and dysfunction. Increasing evidence suggests that at least some of the Ang II-induced effects on cardiac structure are mediated via indirect actions. Ang II upregulates TGF-beta1 expression via activation of the angiotensin type 1 (AT1) receptor in cardiac myocytes and fibroblasts, and induction of this cytokine is absolutely required for Ang II-induced cardiac hypertrophy in vivo. TGF-beta induces the proliferation of cardiac fibroblasts and their phenotypic conversion to myofibroblasts, the deposition of extracellular matrix (ECM) proteins such as collagen, fibronectin, and proteoglycans, and hypertrophic growth of cardiomyocytes, and thereby mediates Ang II-induced structural remodeling of the ventricular wall in an auto-/paracrine manner. Downstream mediators of cardiac Ang II/TGF-beta1 networking include Smad proteins, TGFbeta-activated kinase-1 (TAK1), and induction of hypertrophic responsiveness to beta-adrenergic stimulation in cardiac myocytes.     

Actions of Angiotensin II on Isolated Cardiac Fibroblasts

Angiotensin (Ang) II contributes to myocardial hypertrophy by modulating fibroblast function. In the nonhypertrophied adult rat, rabbit, monkey and human heart, both angiotensin receptor subtypes, AT1 and AT2, are expressed. AT1 seems to be present on almost all myocardial cells, whereas AT2 has so far been localized to coronary endothelial cells and to fibroblasts.AT1 signaling in fibroblasts resembles in many aspects growth factor and cytokine signaling. It includes Ca2+ influx, protein kinase C activation, tyrosine phosphorylation of a number of proteins, activation of the JAK–STAT pathway, and protooncogene induction. Effects of Ang II on the cellular level include proliferation, migration, activation of paracrine–autocrine loops via TGF-1 and other mediators, stimulation of cell–cell interaction, and synthesis of matrix proteins, i.e., collagens I and III and fibronectin. Ang II stimulation induces an increase in osteopontin, which then engages to the v3 integrin receptor on the cell surface of the fibroblasts. These events appear necessary for increased DNA synthesis and collagen gel contractions.Several modulators of fibroblast function interfere with the effects of Ang II, including prostaglandins, which interact with matrix synthesis; nitric oxide, which modulates proliferation at the level of cell cycle regulation; and endothelin, which transmits Ang II-induced proliferation signals. Mechanical loading of intact hearts also affects isolated fibroblast function, and therefore, isolated fibroblasts from pressure- and volume-loaded animals exhibit specific features that interfere with matrix synthesis and calcium handling.Fibroblasts from explanted human hearts respond to Ang II and to Ang (1–7) by DNA and protein synthesis. So far, binding assays to identify angiotensin receptor subtypes have been inconclusive. PCR has consistently revealed the presence of AT1. It is possible that AT2 receptors are also present on human cardiac fibroblasts in vivo, but their number is downregulated by growth factors in cell culture. The question concerning the conditions under which Ang II stimulates collagen formation in human cardiac fibroblasts cannot yet be conclusively answered.     

Scleroderma,fibroblasts, signaling,and excessive extracellular matrix

Excessive extracellular matrix (ECM) deposition in the skin, lung, and other organs is a hallmark of systemic sclerosis (SSc). The pathogenesis of SSc is still poorly understood, but increasing evidence suggests that various cytokines such as transforming growth factor (TGF)-β and their signaling pathways are key mediators of tissue fibrosis as a consequence of ECM accumulation in the pathogenesis of fibrosis such as SSc. TGF-β regulates diverse biologic activities including cell growth, cell death or apoptosis, cell differentiation, and ECM synthesis. TGF-β is known to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. This paper focuses on the possible role of ECM, various cytokines, especially TGF-β signal transduction pathways in the pathogenesis of fibrosis in SSc.     

Reduced scar maturation and contractility lead to exaggerated left ventricular dilation after myocardial infarction in mice lacking AMPKα1

Cardiac fibroblasts (CF) are crucial in left ventricular (LV) healing and remodeling after myocardial infarction (MI). They are typically activated into myofibroblasts that express alpha-smooth muscle actin (α-SMA) microfilaments and contribute to the formation of contractile and mature collagen scars that minimize the adverse dilatation of infarcted areas. CF predominantly express the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1), while AMPKα2 is the major catalytic isoform in cardiomyocytes. AMPKα2 is known to protect the heart by preserving the energy charge of cardiac myocytes during injury, but whether AMPKα1 interferes with maladaptative heart responses remains unexplored. In this study, we investigated the role of AMPKα1 in modulating LV dilatation and CF fibrosis during post-MI remodeling. AMPKα1 knockout (KO) and wild type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery. The absence of AMPKα1 was associated with increased CF proliferation in infarcted areas, while expression of the myodifferentiation marker α-SMA was decreased. Faulty maturation of myofibroblasts might derive from severe down-regulation of the non-canonical transforming growth factor-beta1/p38 mitogen-activated protein kinase (TGF-β1/p38 MAPK) pathway in KO infarcts. In addition, lysyl oxidase (LOX) protein expression was dramatically reduced in the scar of KO hearts. Although infarct size was similar in AMPK-KO and WT hearts subjected to MI, these changes resulted in compromised scar contractility, defective scar collagen maturation, and exacerbated adverse remodeling, as indicated by increased LV diastolic dimension 30 days after MI. Our data genetically demonstrate the centrality of AMPKα1 in post-MI scar formation and highlight the specificity of this catalytic isoform in cardiac fibroblast/myofibroblast biology.     

TGF-β1 Induces Human Bronchial Epithelial Cell-to-Mesenchymal Transition in Vitro

The subepithelial fibrosis component of airway remodeling in asthma is mediated through induction of transforming growth factor-β1 (TGF-β1) expression with consequent activation of myofibroblasts to produce extracellular matrix proteins. The number of myofibroblasts is increased in the asthmatic airway and is significantly correlated with the thickness of lamina reticularis. However, much is still unknown regarding the origin of bronchial myofibroblasts. Emerging evidence suggests that myofibroblasts can derive from epithelial cells by an epithelial-to-mesenchymal transition (EMT). In this study we investigated whether TGF-β1 could induce bronchial epithelial EMT in the human bronchial epithelial cell. Cultured human bronchial epithelial cells, 16HBE-14o, were stimulated with 10 ng/ml TGF-β1. Morphologic changes were observed and stress fiber by actin reorganization was detected by indirect immunostaining. The expression of α-SMA (α-smooth muscle actin) and the epithelial cell marker E-cadherin were detected in those 16HBE-14o cells after TGF-β1 stimulation for 72 h, using immunostaining and RT-PCR. The contents of collagen I were determined by radioimmunoassay, and the levels of endogenous TGF-β1 were measured with ELISA. Human bronchial epithelial cells stimulated with TGF-β1 were converted from a “cobblestone” epithelial structure into an elongated fibroblast-like shape. Incubation of human bronchial epithelial cells with TGF-β1 induced de novo expression of α-SMA, increased formation of stress fiber by F-actin reorganization, and loss of epithelial marker E-cadherin. Moreover, a significant increase in the levels of collagen I and endogenous TGF-β1 released from bronchial epithelial cells stimulated with TGF-β1 were observed. These results suggested that human bronchial epithelial cells, under stimulation of TGF-β1, underwent transdifferentiation into myofibroblasts.     

Involvement of reactive oxygen species in angiotensin II-induced endothelin-1 gene expression in rat cardiac fibroblasts

OBJECTIVES: The aim of this study was to investigate the effects of angiotensin II (Ang II) on fibroblast proliferation and endothelin-1 (ET-1) gene induction, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. BACKGROUND: Angiotensin II increases ET-1 expression, which plays an important role in Ang II-induced fibroblast proliferation. Angiotensin II also stimulates ROS generation in cardiac fibroblasts. However, whether ROS are involved in Ang II-induced proliferation and ET-1 expression remains unknown. METHODS: Cultured neonatal rat cardiac fibroblasts were stimulated with Ang II, and then [(3)H]thymidine incorporation and the ET-1 gene expression were examined. We also examined the effects of antioxidants on Ang II-induced proliferation and mitogen-activated protein kinase (MAPK) phosphorylation to elucidate the redox-sensitive pathway in fibroblast proliferation and ET-1 gene expression. RESULTS: Both AT(1) receptor antagonist (losartan) and ET(A) receptor antagonist (BQ485) inhibited Ang II-increased DNA synthesis. Endothelin-1 gene was induced with Ang II as revealed by Northern blotting and promoter activity assay. Angiotensin II increased intracellular ROS levels, which were inhibited with losartan and antioxidants. Antioxidants further suppressed Ang II-induced ET-1 gene expression, DNA synthesis, and MAPK phosphorylation. PD98059, but not SB203580, fully inhibited Ang II-induced ET-1 expression. Truncation and mutational analysis of the ET-1 gene promoter showed that AP-1 binding site was an important cis-element in Ang II-induced ET-1 gene expression. CONCLUSIONS: Our data suggest that ROS are involved in Ang II-induced proliferation and ET-1 gene expression. Our findings imply that the combination of AT(I) and ET(A) receptor antagonists plus antioxidants may be beneficial in preventing the formation of excessive cardiac fibrosis.     

Role of Extracellular Signal-Regulated Kinase, p38 Kinase, and Activator Protein-1 in Transforming Growth Factor-β1–Induced Alpha Smooth Muscle Actin Expression in Human Fetal Lung Fibroblasts In Vitro

Myofibroblasts characterized by alpha smooth muscle actin(α-SMA) expression play a key role in pulmonary fibrosis. Transforming growth factor-beta1 (TGF-β1) is likely to be involved in the emergence of myofibroblasts, but the intracellular signal pathways for this process have not been well determined. The aim of the present study was to investigate the role of mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) signaling pathways in TGF-β1–induced α-SMA expression in human fetal lung fibroblasts (HLF-02). We found that TGF-β1 treatment activated p38 kinase and extracellular signal-regulated kinase (Erk) in HLF-02 cells. The induction of α-SMA by TGF-β1 was suppressed by p38 kinase inhibitor (SB203580) and Erk inhibitor (PD98059). AP-1 inhibitor curcumin also inhibited TGF-β1–induced α-SMA expression. In addition, dominant negative mutant c-Jun (TAM67) downregulated TGF-β1–induced AP-1 transactivation and α-SMA expression. In additional, PD98059 but not SB203580 inhibited the AP-1 DNA binding activity induced by TGF-β1. Based on these findings, we conclude that p38 kinase, Erk, and AP-1 are responsible for the α-SMA expression induced by TGF-β1 in human fetal lung fibroblasts. Erk is involved in inducing α-SMA expression via AP-1 activation.