doi: 10.1161/01.RES.0000190403.87462.bf
© 2005 American Heart Association, Inc.
Editorials |
NAD(P)H Oxidases and TGF-ß–Induced Cardiac Fibroblast Differentiation
Nox-4 Gets Smad
From the Departments of Physiology (P.R.) and Pharmacology (P.A.L.), Louisiana State University Health Sciences Center, New Orleans, La.
Correspondence to Pamela A. Lucchesi, Department of Comparative Biomedical Sciences, Louisiana State University School of Veterinary Medicine, Skip Bertman Dr, Rm 2530, Baton Rouge, LA 70803. E-mail plucch{at}lsuhsc.edu
See related article, pages 900–907
Key Words: NAD(P)H oxidase • cardiac fibrosis • myofibroblasts • collagen
Under normal conditions, cardiac fibroblasts play a pivotal role in the maintenance of the structural integrity of the myocardium by regulating extracellular matrix (ECM) production. In response to myocardial infarction and pressure overload, paracrine and autocrine signals in the interstitial milieu stimulate fibroblast proliferation and differentiation into myofibroblasts. The phenotype of these cells is characterized by an upregulation of smooth muscle 
-actin (SM -actin), collagen gel contraction in vitro, increased proliferation, decreased secretion of matrix degrading enzymes (matrix metalloproteinases), and enhanced production of extracellular matrix proteins, plasminogen activator inhibitor-1, and tissue inhibitors of matrix metalloproteineases. Thus, fibroblast transformation into myofibroblasts is associated with enhanced matrix production and decreased matrix turnover, resulting in interstitial fibrosis. This adverse ECM structural remodeling facilitates abnormalities of both systolic and diastolic cardiac function, because fibrosis leads to increase in chamber stiffness.1
The renin–angiotensin system and transforming growth factor-ß1 (TGF-ß1) play a critical role in the development of cardiac fibrosis. Angiotensin II (Ang II)–dependent upregulation of TGF-ß1 expression in cardiac fibroblasts is absolutely required for Ang II–induced fibrosis.2 TGF-ß1 induces the proliferation of cardiac fibroblasts, their phenotypic conversion to myofibroblasts, deposition of ECM proteins such as collagen, fibronectin, and proteoglycans, and hypertrophic growth of cardiomyocytes, thereby mediating Ang II–induced structural remodeling of the ventricular wall in an autocrine/paracrine manner.2 The profibrotic effects of TGF-ß1 are primarily attributable to the differentiation of fibroblasts to myofibroblasts, because acute exposure (<24 hours) of primary rat cardiac fibroblasts fails to increase collagen production.3 On the other hand, chronic exposure to TGF-ß1 caused maximal collagen production that was accompanied by an increase in SM -actin and the phenotypic conversion of fibroblasts to myofibroblasts.
The signaling cascades that control cardiac fibroblast differentiation into myofibroblasts have not been fully elucidated. TGF-ß1 elicits profibrotic responses by binding to a heteromeric complex of TGF-ß1 receptor types I and II on cardiac fibroblasts.2 The conventional TGF-ß1 signaling cascade involves phosphorylation of R-type Smad proteins (Smad2/3) in response to receptor activation. Phosphorylated Smad2/3 associate with a co-Smad (eg, Smad 4) and translocate to the nucleus where they act as transcription factors (reviewed by Massague4). TGF-ß1–activated kinase (TAK1) is also involved in TGF-ß1 signaling and, together with Smads, has been shown to regulate GATA-4 and ATF-2.5
More recently, it has been shown that reactive oxygen species (ROS) are released during the phenotypic transformation of fibroblasts to myofibroblasts and are important in the control of collagen deposition. Both cardiac myocytes and cardiac fibroblasts express the NAD(P)H oxidase that is a major source of superoxide (O2·–) production within the myocardium. This is particularly relevant to cardiovascular disease because increasing evidence points to the pivotal role of oxidative stress in left ventricular remodeling associated with the development and progression of heart failure. Indeed, activation of the NAD(P)H oxidases and the subsequent production of ROS have been reported in human and animal models of heart failure and myocardial infarction.
Ang II, a potent profibrotic agent, activates NAD(P)H oxidase in many cardiovascular cells, including cardiac fibroblasts and vascular smooth muscle cells by regulation of Rac1 and p47phox6 phosphorylation, which are critical in the regulation of the assembly of the functional oxidase. Moreover, pharmacological blockade or genetic depletion of the oxidase blunts fibrosis in response to Ang II. The central question that remains is whether other profibrotic also signal through the NAD(P)H oxidase to control the phenotype and function of cardiac fibroblasts. A recent report suggest that ROS scavenger superoxide dismutase decreased TGF-ß1–induced cardiac fibroblast differentiation, hinting at a role for O2·– in TGF-ß1 signaling.7
In this issue of Circulation Research, Cucoranu et al8 report a new role for the NAD(P)H oxidase in TGF-ß–induced Smad2/3 phosphorylation, SM -actin expression, and cardiac fibroblast differentiation to myofibroblasts (Figure). Although it has been previously shown that Smad2/3 mediate TGFß1 induction of SM -actin in human lung fibroblasts,9 this is the first study to link NAD(P)H-dependent ROS production to Smad signaling.
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The NAD(P)H oxidase is a membrane-bound oxidase that is found in a variety of cell types, including neutrophils, vascular smooth muscle cells, endothelial cells, cardiac myocytes, and both vascular and cardiac fibroblasts. The enzyme consists of two membrane-spanning subunits: p22phox, which is thought to serve as a stabilizing and regulatory subunit for the O2·– producing subunit called gp91phox (in myocytes, fibroblasts, and endothelial cells) and its homologues Nox1 and Nox4 (see Hanna et al10 for a comprehensive review). The cytoplasmic components of the oxidase, p47phox and p67phox, as well as Rac are thought to regulate the assembly of the functional oxidase, and thus, also its activity.
Unlike the neutrophil oxidases that release large bursts of superoxide (O2·–) extracellularly, the cardiovascular oxidases are thought to release lower levels of O2·– intracellularly. These lower amounts of ROS initiate cellular signaling events that control a variety of cellular functions, including proliferation, hypertrophy, apoptosis, cytokine production, and the expression of matrix regulatory proteins. Of the cardiovascular oxidases, the Nox1 and gp91phox-containing oxidases have been suggested to be inducible, whereas the Nox4-containing oxidases are thought to be constitutively active and may contribute to the regulation of the overall redox state of the cell.11 However, Cucoranu et al8 provide compelling data that Nox4 and Nox5 are the primary Nox subunits in cardiac fibroblasts and only Nox4 is upregulated by TGF-ß1.
The authors use four experimental strategies (real time RT-PCR, immunohistochemistry, and siRNA to conclusively demonstrate that the effects of TGF-ß are mediated through upregulation of the Nox-4 subunit of the oxidase. These studies clearly demonstrate the requirement of the oxidase subunit in myofibroblast differentiation because si-Nox4 blocked SM -actin, connective tissue growth factor, and fibronectin expression. Nox4 siRNA also partially prevented chronic TGF-ß1–induced Smad2/3 expression suggesting that oxidase-dependent ROS production controls fibroblast differentiation. Although TGF-ß1 has been shown to regulate the oxidase in human fetal lung fibroblasts12 and rat hepatocytes,13 this is the first report that the oxidase-derived ROS are involved in cardiac fibroblast differentiation in response to TGF-ß. These earlier studies suggested that upregulation of oxidase activity required new protein synthesis, but expression of specific subunits was not examined.
There are several caveats to this study that caution the in vivo significance of the findings. The experiments were performed in higher passages of human cardiac fibroblasts, which contained a significant amount of myofibroblasts under basal conditions. Because these cell types can release autocrine growth factors, including TGF-ß1,14 it is unclear how increased basal secretion of these factors would impact the results. Future studies with primary cultures of human cardiac fibroblasts will resolve this issue. Secondly, a more rigorous assessment of myofibroblast function (collagen secretion, collagen gel contraction) would conclusively indicate the myofibroblast phenotype
Several questions remain unanswered. The mechanisms by which TGF-ß1 upregulates Nox4 expression in cardiac fibroblasts are not known. In vascular smooth muscle, 7-ketocholesterol activated a Jun-NH(2)-terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression.15 Whether TGF-ß1 activates a similar pathway in cardiac fibroblasts is unclear. NAD(P)H oxidase–derived ROS have been shown to activate a variety of other transcription factors in cardiac fibroblasts including NFAT6 and CREB. It is unclear whether these transcription factors are involved in TGF-ß1–dependent gene transcription that controls fibroblast differentiation. Finally, increased ROS is also associated with increased ECM turnover. For example Siwik et al16 demonstrated that hydrogen peroxide and O2·– increased MMP activity and fibronectin expression but decreased collagen synthesis in low-passage neonatal and adult cardiac fibroblasts. Therefore, the consequence of NAD(P)H oxidase–derived ROS on left ventricular (LV) ECM remodeling may depend on the subcellular localization of the oxidase subunit isoforms, interaction with ROS produced by other cell types, as well as the duration and amount of ROS production.
The contribution of the myofibroblast Nox4-containing oxidase under pathological conditions that are characterized by fibroblast to myofibroblast conversion and increased interstitial fibrosis in vivo remains unclear. Ang II–induced cardiac fibrosis is attenuated in gp91phox-deficient mice despite normal levels of Nox1 and Nox4.17,18 On the other hand, pressure overload–induced LV hypertrophy and remodeling is not dependent on gp91phox and instead may rely on Nox4.19 Therefore, in vivo, the regulation of cardiac fibrosis by the NAD(P)H oxidase may require coordinate regulation of multiple NAD(P)H oxidase subunits.
In conclusion, Cucoranu et al8 provide novel evidence for a role of Nox4-Smad3/3 signaling in cardiac fibroblast differentiation to myofibroblasts and ECM production in response to TGF-ß1. Future studies in primary human cardiac fibroblasts from normal and diseased LV will provide definitive proof of the role of Nox4 in cardiac fibrosis.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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