This Review is part of a thematic series on Gene Expression in Hypertrophy and Stress, which includes the
following articles:
Gene Expression in Fibroblasts and Fibrosis: Involvement in Cardiac Hypertrophy
Molecular Mechanisms of Csx/Nkx2-5 Involvement in Cardiac Hypertrophy
Ras, Akt, and Mechanotransduction in the Cardiac Myocyte
G Protein–Coupled Signaling and Gene Expression
Genetic Models and Mechanisms of Transcription in Cardiac Hypertrophy
Calcineurin Pathway
Ryozo Nagai, Guest Editor
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Gene Expression in Fibroblasts and Fibrosis
Involvement in Cardiac Hypertrophy
Ichiro Manabe, Takayuki Shindo, Ryozo Nagai
Abstract—Structural remodeling of the ventricular wall is a key determinant of clinical outcome in heart disease. Such
remodeling involves the production and destruction of extracellular matrix proteins, cell proliferation and migration, and
apoptotic and necrotic cell death. Cardiac fibroblasts are crucially involved in these processes, producing growth factors
and cytokines that act as autocrine and paracrine factors, as well as extracellular matrix proteins and proteinases. Recent
studies have shown that the interactions between cardiac fibroblasts and cardiomyocytes are essential for the progression
of cardiac remodeling. This review addresses the functional role played by cardiac fibroblasts and the molecular
mechanisms that govern their activity during cardiac hypertrophy and remodeling. A particular focus is the recent
progress toward our understanding of the transcriptional regulatory mechanisms involved. (Circ Res. 2002;91:11031113.)
Key Words: transcription 䡲 fibrosis 䡲 myofibroblast 䡲 cardiac remodeling
R
diomyocyte hypertrophy, cardiac fibroblast proliferation, fibrosis, and cell death.3 Fibrosis, which is a disproportionate
accumulation of fibrillar collagen, is an integral feature of the
remodeling characteristic of the failing heart. Accumulation
of type I collagen, the main fibrillar collagen found in cardiac
fibrosis, stiffens the ventricles and impedes both contraction
and relaxation.4 Fibrosis can also impair the electrical coupling of cardiomyocytes by separating myocytes with extracellular matrix (ECM) proteins.3 Furthermore, fibrosis results
in reduced capillary density and an increased oxygen diffusion distance that can lead to hypoxia of myocytes.5 Thus,
fibrosis profoundly affects myocyte metabolism and performance and ultimately ventricular function.6
In the myocardium, ECM proteins are mainly produced by
fibroblasts that also produce matrix metalloproteinases
egardless of the origin, injury to the heart evokes a
diverse and complex array of cellular responses involving both cardiomyocytes and nonmuscle cells that initiate and
sustain a process of structural remodeling of the myocardium.1 The importance of the structural remodeling has been
addressed in randomized clinical trials.2 For example, angiotensin-converting enzyme (ACE) inhibitors, which may delay
and, in some cases, reverse cardiac remodeling, have proven
to have a beneficial effect on morbidity and mortality in heart
failure. Thus, remodeling is now generally accepted as a key
determinant of the clinical course of heart failure.2
Cardiac remodeling is manifested clinically as changes in
the size, shape, and function of the heart.2 Histopathologically, it is characterized by a structural rearrangement of
components of the normal chamber wall that involves car-
Original received August 19, 2002; revision received October 30, 2002; accepted October 30, 2002.
From the Departments of Cardiovascular Medicine (I.M., T.S., R.N.) and Clinical Bioinformatics (I.M., R.N.), University of Tokyo, Tokyo, Japan.
Correspondence to Ichiro Manabe, MD, PhD, Department of Cardiovascular Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655,
Japan. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000046452.67724.B8
1103
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(MMPs), growth factors, and cytokines, all of which are
involved in the maintenance of myocardial structure, and in
diseased hearts play pivotal roles in remodeling. In this
review, we present an overview of the mechanism that
controls the functions of cardiac fibroblasts in cardiac remodeling with a special emphasis on transcriptional regulation of
gene expression.
Cardiac Interstitium and ECM Regulation
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The cardiac interstitium, ie, the space between the cardiomyocytes, contains fibroblasts, blood vessels, lymphatic vessels, adrenergic nerve endings, and ECM.4 The myocardial
ECM is made up of a fibrillar collagen network, a basement
membrane, proteoglycans, and glycosaminoglycans and contains a diverse array of bioactive signaling molecules.4,7 The
major ECM proteins are type I and III collagens, although
type IV, V, and VI collagens, as well as elastin are also
present. The fibrillar collagen network ensures the structural
integrity of the adjoining myocytes,7 provides the means by
which myocyte shortening is translated into ventricular pump
function, and is essential for maintaining alignment of the
myofibrils within the myocytes through a collagen-integrincytoskeletal myofibril relation.8
The disproportionate increase in synthesis and/or inhibition
of degradation of ECM proteins would result in fibrosis.
Fibrosis has been classified into two groups: reparative and
reactive fibrosis.9,10 Reparative (replacement) fibrosis or scarring accompanies myocyte death. Reactive fibrosis appears as
“interstitial” or “perivascular” fibrosis and does not directly
associate with myocyte death. In interstitial fibrosis, fibrillar
collagen appears in intermuscular spaces. Perivascular fibrosis refers to the accumulation of collagen within the adventitia of intramyocardial coronary arteries and arterioles. “Focal” and “diffuse” are also used to describe the distribution of
fibrosis. Although there are a number of apparent differences
between reparative and reactive fibrosis (eg, cells involved
and the time course of fibrotic change), many factors likely
work in common to control fibroblast function (discussed
subsequently).
Collagens are degraded by a family of MMPs capable of
enzymatically digesting a wide variety of ECM proteins. The
activity of MMPs is controlled at the transcriptional level as
well as through activation and inhibition by other proteins
including tissue inhibitors of MMPs (TIMPs). Cardiac fibroblasts produce both ECM proteins and MMPs, thus playing a
central role in the regulation of ECM.
The expressions of both ECMs and MMPs change dynamically during the developmental process of heart failure. For
instance, loss of collagen and enhanced MMP activity begin
within minutes of the onset of myocardial ischemia.11 This
rapid initial loss of collagen is followed by a rapid and
progressive increase in collagen and fibronectin gene expression. MMPs seem to be involved in several aspects of infarct
healing processes, including early ECM degradation, cell
migration of inflammatory cells and fibroblasts, angiogenesis, remodeling of newly synthesized connective tissue, and
the regulation of growth factor activities.11 Progressive activation of MMPs has also been demonstrated to accompany
the progression of left ventricular (LV) dilation and dysfunc-
tion.8 Moreover, increased MMP activity was found in the
hearts of patients with ischemic and dilated cardiomyopathy.12
Recent studies using genetic and pharmacological manipulation of collagens and MMPs have further demonstrated
that the balance between matrix synthesis and degradation
plays an essential role in the maintenance of the integrity of
the myocardium. Disruption of this balance would lead to
cardiac remodeling and dysfunction.13 Cardiac-specific overexpression of MMP-1 (interstitial collagenase) resulted in
structural changes in ECM and marked deterioration of
systolic and diastolic function.13 Disruption of MMP inhibitory control by TIMP-1 gene knockout resulted in LV
dilation.14 MMP-9 (gelatinase B) gene knockout mice showed
attenuation of LV enlargement and collagen accumulation
after myocardial infarction.15 MMP inhibitor treatment of
pigs with chronic rapid pacing and spontaneously hypertensive heart failure rats attenuated the degree of LV dilation and
improved LV function.16,17
ECM remodeling in the processes of the progression and
repair of myocardial infarction has also been investigated
using mouse models. For instance, urokinase-type plasminogen activator (u-PA) deficiency protected against rupture at
the acute phase.18 However, the u-PA knockout mice showed
impaired scar formation. Reduced leukocyte infiltration and
angiogenesis were observed in the infarcted region. In a
separate study, the lack of wound healing and infiltration of
inflammatory cells were observed in plasminogen knockout
mice.19 Therefore, although suppression of ECM degradation
may inhibit acute myocardial rupture, it could impede the
normal healing processes later. Collectively, these studies
have clearly demonstrated the importance of dynamic regulation of the synthesis and degradation of ECM in the repair
of acute cardiac damage and subsequent development of
cardiac remodeling.
Cardiac Fibroblasts and Myofibroblasts
In the normal heart, two thirds of the cell population is
composed of nonmuscle cells, the majority of which are
fibroblasts.20 External stress causes fibroblasts to change their
phenotype.21,22 These altered cells are referred to as myofibroblasts because they express several smooth muscle (SM)
markers, including SM ␣-actin, SM22␣, SMemb/nonmuscle
myosin heavy chain-B, and tropomyosin.22–24 However, more
stringent SM markers (eg, SM myosin heavy chains) are not
expressed in myofibroblasts.25
With the exception of heart valve leaflets, myofibroblasts
are not found in normal cardiac tissue.26 Upon injury,
myofibroblasts appear in the myocardium and are generally
believed to arise from resident interstitial and/or adventitial
fibroblasts.21 Their origin is not yet completely clear; however, they might also originate from progenitor stem cells.
Given that recent reports suggest that stem cells in local
tissues and bone marrow may be involved in various diseases,27 the differentiation of cardiac myofibroblasts from stem
cells cannot be ruled out. In any case, a growing body of
evidence indicates that by producing growth factors, cytokines, chemokines, ECM proteins, and proteases,21,22 myofibroblasts play a pivotal role in inflammation, tissue repair,
fibrosis, and organogenesis.
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Genetically Engineered Mouse Models Reported to Have Cardiac Fibrosis
Receptor
Growth factor, cytokine
Signaling molecule
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Ion channel
Transcription factor
ECM protein
Contractile protein
Enzyme
Overexpression
Knockout/Knockdown
␤1-Adrenergic receptor*1
Angiotensin II type 1 receptor*1
Ro1 Gi-coupled receptor*3
Bradykinin B2 receptor
Mineralocorticoid receptor*3, *6
Natriuretic peptide receptor A
Transforming growth factor-␤
Monocyte chemoattractant protein-1*1
Tumor necrosis factor-␣*1
Fas ligand*1
Brain natriuretic peptide
G proteins (G␣s*1, G␣q*1, *4)
Gh (tissue-type transglutaminase)*1
Protein kinase C ␤2*1
RhoA*1
TGF-␤–activated kinase-1*1, *4
MAP kinase kinase 6bE*1
Ca2⫹/calmodulin-dependent protein kinase-IV*1
Calcineurin*1, *4
Calsequestrin*1
䡠䡠䡠
䡠䡠䡠
Nuclear factors of activated T cells*1,
Myf5
Retinoic acid receptor ␣*1, *4
Csx/Nkx2-5*2, *7
Serum response factor*1
Kv4.2 K⫹ channel*1, *6
*4
cAMP-responsive element binding protein*1,
*6
Collagen type I*5
␣ Myosin heavy chain*1, *7
␤ Myosin heavy chain*2, *7, *8
␣ Tropomyosin*1, *7
Cardiac troponin T*7
Muscle-specific LIM-only protein
Long-chain acyl-CoA synthetase*1
Cathepsin L
A complete list of references for this Table is available online in the data supplement (http://www.circresaha.org).
*1
and *2 indicate mouse models of cardiac muscle–specific overexpression of transgenes using mouse cardiac ␣*1 or ␤*2 myosin
heavy chain regulatory regions; *3, cardiac muscle–specific conditional overexpression model; *4, activated form mutants; *5, mutant
form resistant to collagenase; *6, overexpression of antisense mRNA or dominant-negative forms; *7, other mutant forms; and *8,
transgenic rabbits.
As summarized in the Table, a number of genetically
engineered mouse models have been reported to have cardiac
fibrosis. Of note, cardiac fibrosis developed in the myocardium of both hypertrophy and dilated cardiomyopathy models. Therefore, the mechanisms of fibrosis induction in these
animals appear to be divergent. In some cases, the alterations
in target gene function per se may have induced fibrosis. In
others, the deterioration of myocardial function due to the
genetic manipulation may have secondarily caused fibrosis.
In mice with cardiac-specific overexpression of Fas and
tumor necrosis factor-␣ (TNF-␣), inflammatory cells likely
activated fibroblasts. These studies indicate that even if the
dysfunction is initially confined within nonfibroblasts (eg,
cardiomyocytes), disturbances in myocardial function would
lead to fibroblast activation. Humoral factors, ECM proteins,
and adhesion molecules mediate the interaction between
cardiac fibroblasts and other cell types.
Autocrine and Paracrine Factors Controlling
Cardiac Fibroblasts
Humoral factors that affect the phenotype and function of
cardiac fibroblasts include angiotensin II (Ang II), basic
fibroblast growth factor (bFGF/FGF-2), transforming growth
factor-␤ (TGF-␤), catecholamines, and insulin-like growth
factor-1 (IGF-1).28,29 Among these factors, Ang II appears to
be one of the most important regulating cardiac fibrosis and
remodeling, although other factors are also important, as
clearly demonstrated in recent studies in which pressure
overload caused cardiac hypertrophy and fibrosis in type 1a
Ang II (AT1a) receptor knockout mice.30 Evidence suggests
that Ang II synthesis and degradation in the heart is compartmentalized and mediated by mechanisms different than those
in intravascular spaces.31 Furthermore, most components of
the renin-angiotensin-aldosterone (RAA) system are present
in the heart and are produced locally,3 although the extent of
local production of the RAA system components and how
they are produced still remains somewhat controversial.32,33
Regardless of the exact mechanisms, the RAA system appears to be upregulated by mechanical stretch and other
insults to the myocardium within which Ang II acts as an
autocrine/paracrine factor critically affecting the progression
of hypertrophy and remodeling.26
Recent studies have also demonstrated the local production
of aldosterone in the heart and the expression of mineralocorticoid receptors in cardiac cells, suggesting a paracrine
function for aldosterone in the heart.34 This is particularly
interesting, because a clinical study, Randomized Aldactone
Evaluation Study (RALES), showed that treatment of patients
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with spironolactone, an antagonist of the aldosterone receptor
(mineralocorticoid receptor), improved both morbidity and
mortality in heart failure patients.35 Subsequently, this beneficial effect of spironolactone was shown to be at least partly
a result of the reduced ECM turnover.36
Ang II acts via receptors that are members of the G
protein– coupled receptor (GPCR) superfamily.37 There are
two types of Ang II receptors: type 1 (AT1) and type 2 (AT2).
Most of the known physiological effects of Ang II are
mediated through the AT1 receptor. Binding of Ang II to the
AT1 receptor leads to activation of well-defined G protein–
linked pathways, such as activation of phospholipase C
(PLC), which causes the release of Ca2⫹ and subsequent
activation of calmodulin kinase and protein kinase C (PKC).
In addition to these classical pathways, AT1 receptors can
activate tyrosine kinases, such as Jak2, Tyk2, c-Src, and
Pyk2,38 – 40 and can transactivate cell surface receptors possessing an intrinsic tyrosine kinase domain (RTKs), in particular the EGF receptor.41 This effect is at least partly
mediated by heparin-binding EGF-like growth factor (HBEGF).42,43 Importantly, Ang II may activate different signaling pathways in cardiac fibroblasts than in cardiomyocytes.
Zou et al44 recently reported that in cardiac fibroblasts, Ang
II activated extracellular signal-regulated kinases (ERKs)
through a pathway including the G␤␥ subunit of Gi and
tyrosine kinases (eg, Src, Ras, and Raf), whereas Gq and PKC
are important in cardiomyocytes. These differences in signal
activation may lead to differential activation of genes in these
two cell types.
In certain cases, the AT2 receptor has been shown to
counterregulate the AT1 receptor effects. AT2 receptor stimulation suppresses the growth of cardiomyocytes and cardiac
fibroblasts.45 AT2 receptor expression is upregulated in human failing hearts mainly in cardiac fibroblasts.46 Therefore,
it has been suggested that selective block of the AT1 signal
might have an additional beneficial effect in heart failure via
AT2 receptors. However, previous studies have also provided
conflicting results regarding the role of the AT2 receptor.47
AT2-deficient mice showed reduced cardiac fibrosis in pressure overload and chronic Ang II infusion.48,49 Therefore, the
role of AT2 may depend on other signals, including that via
AT1.50
In vitro studies of cultured cardiac fibroblasts have shown
that Ang II directly stimulates fibroblast proliferation, collagen synthesis, and the expression of ECM proteins (collagen,
fibronectin, and laminin) via AT1 receptors.29 Ang II also
regulates cardiac fibroblast function indirectly through induction of TGF-␤, endothelin-1 (ET-1), IL-6, and osteopontin,
which function in autocrine and paracrine manners.29,51–54
Recent studies suggest that this indirect effect may be the
major mechanism by which Ang II controls cardiac fibroblasts.55 This mechanism has been directly addressed in
recent studies using knockout mice. TGF-␤1– deficient mice
subjected to chronic subpressor doses of Ang II showed no
significant LV hypertrophy or fibrosis.56 In another study, an
Ang II– dependent renovascular hypertension model was
applied to FGF-2– (bFGF) deficient mice.57 In these mice,
compensatory hypertrophy and fibrosis were abolished, at
least partly because of the deficiency in the paracrine function
of cardiac fibroblasts. That is, FGF-2⫺/⫺ cardiac fibroblasts
had a defective capacity for releasing growth factors, including FGF-2, to induce hypertrophic responses in cardiomyocytes. These studies clearly demonstrate the importance of
cell-cell communication in cardiac remodeling.
Cell-Cell Communication in
Cardiac Remodeling
As discussed above, acute injury to the myocardium activates
the RAA system. For example, mechanical stretch of cardiomyocytes induces the release of Ang II,58,59 which in turn
stimulates the release of multiple growth factors and cytokines from cardiac fibroblasts that affect both the fibroblasts
themselves and other cell types, including cardiomyocytes
(Figure). Although the paracrine factors released by cardiac
fibroblasts are required for induction of hypertrophic responses in cardiomyocytes, the paracrine activities of cardiomyocytes are also crucially important for regulating the
function of cardiac fibroblasts. For example, Matsusaka et
al60 generated chimeric mice that expressed both AT1a receptor–intact and AT1a receptor–null cells. Infusion of Ang II
caused mild cardiac hypertrophy and fibrosis in the chimeric
mice, and importantly most of the proliferating fibroblasts
were found to be surrounding cardiomyocytes carrying the
intact AT1a gene. Fibroblasts adjacent to AT1a-null myocytes
showed a lesser degree of cell proliferation. Consistent with
these findings, chimeric mice carrying cells overexpressing
Gs␣ protein and wild-type cells showed clustered fibrosis and
hypertrophy in loci predominantly containing myocytes overexpressing Gs␣,61 again indicating the importance of local
communication between fibroblasts and myocytes, presumably via a paracrine mechanism. Finally, cardiac fibrosis is
observed in several transgenic mouse lines specifically overexpressing genes in cardiomyocytes (Table).
Other cell types, including endothelial cells, pericytes/
smooth muscle cells (SMCs), and immune cells are also
involved in the cell-cell communication mediating the pathogenesis of cardiac remodeling. The myocardium contains a
dense network of blood vessels, and all of the muscle fibers
are situated in close proximity to the vessels.7 As evident in
the pathogenesis of atherosclerosis and other chronic inflammatory conditions, endothelial cells act as a mediator of
inflammatory responses by promoting the infiltration of
inflammatory cells and interacting with the surrounding
SMCs and pericytes. It is therefore likely that cells comprising the blood vessels and blood cells are also involved in the
pathogenesis of cardiac remodeling.
The role of inflammatory cells has been extensively studied in myocardial infarction. In reperfused myocardial infarction, inflammatory cells are involved in both myocardial
injury and healing. Ischemic myocardial injury activates the
complement cascade, production of reactive oxygen species,
and cytokine cascade triggered by mast cell– derived mediators, all of which directly affect the function and fate of
cardiac cells as well as recruit inflammatory cells.62 The
major cell type first recruited to the reperfused area is the
neutrophil. Subsequently, monocytes and lymphocytes infiltrate the infarcted myocardium in the first few hours of
reperfusion. Histamine and TNF-␣ released from mast cells
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Effects of Ang II on cardiac fibroblasts.
Ang II stimulation evokes multiple signaling pathways in cardiac fibroblasts.
Upon binding of Ang II, the AT1 receptor
activates G proteins, which triggers multiple pathways including PKC, MAPK,
PI3K/Akt, and Jak/Stat. Ang II stimulation
also transactivates RTKs, such as the
EGF receptor. Reactive oxygen species
(ROS) may be involved in Ang II signaling
in cardiac fibroblasts. Activation of these
pathways results in changes in gene
expression. Growth factors and cytokines are induced by Ang II and act as
autocrine and paracrine factors that
affect both cardiac fibroblasts and myocytes. Changes in ECM also affect signaling and gene expression in cardiac
fibroblasts and myocytes.
induce IL-6 in myocytes and mononuclear cells. IL-6, in turn,
induces ICAM-1 expression in myocytes, leading to ligandspecific adhesion of neutrophils that injure myocytes. During
the healing phase, the infarcted tissue is repaired and remodeled by degradation and production of matrix, angiogenesis,
and cell apoptosis and proliferation. Macrophages and mast
cells accumulate in the healing scar and secrete a variety of
growth factors and cytokines, inducing fibroblast proliferation. Fibroblasts become myofibroblasts that produce ECM
proteins and growth factors.63 In the healing phase, angiogenesis is also induced by a variety of angiogenic factors, such as
VEGF, bFGF, and IL-8. As such, the complex processes of
tissue repair and remodeling in myocardial infarction are
mediated by a network of molecules, such as cytokines and
growth factors.62 In the network, these factors interact synergistically and antagonistically with each other and have
pleiotropic effects depending on cellular, spatial, and temporal variables. Therefore, cytokines and other inflammatory
mediators that play an injurious role in the early stages of the
inflammatory response may also be necessary as regulators of
cardiac repair.62
Taken together, the networks of various molecules and
cells control the complex processes of tissue injury, repair,
and remodeling in cardiac diseases. The functional role of
each player in the networks is highly dependent on environmental variables. Therefore, it would be important to address
the function of each molecule in the context of networks for
understanding of the molecular mechanisms of cardiac remodeling as well as the development of novel therapeutics.
Transcriptional Control of
Cardiac Remodeling
Injury to the myocardium evokes multiple intracellular signaling pathways that ultimately lead to changes in gene
expression, which are largely conducted by transcription
factors and cofactors. The first group of transcription factors
activated by external stress (eg, growth factors, cytokines,
hypoxia, and mechanical stretch) includes products of immediate early response genes, such as AP-1, NF-␬B, Egr-1, and
Stat3. Activation of these transcription factors leads to
sequential transcriptional control events that coordinately
change gene expression and control the function of cells
responding to environmental cues. In the next section, we
present an overview of the transcription factors activated at
the immediate early stage and later stages. After this overview, we discuss specific gene regulation by the cooperative
interaction between transcription factors.
Immediate Early Response Genes
Activating Protein-1 (AP-1)
AP-1 is activated by diverse environmental cues and is
involved in numerous cellular functions. It often functions
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primarily to control cell proliferation and apoptosis.64 AP-1
proteins are homodimers and heterodimers composed of basic
region-leucine zipper (bZIP) proteins, which include Jun,
Fos, and Jun dimerization partners and activating transcription factors.64 External stimuli can differentially activate
stimulus- and cell type–specific sets of AP-1 proteins. It is
likely that the different AP-1 dimers execute specific cellular
programs.64,65
In cardiac fibroblasts, mechanical stretch, Ang II, and
hypoxia have all been shown to activate AP-1.66,67 Ang II
directly induces c-fos, c-jun, and JunB expression in cardiac
fibroblasts.67 Potential AP-1 binding sites have been identified in the transcriptional regulatory regions of numerous
genes, including transcription factors, ECM proteins, MMPs,
cell adhesion molecules, growth factors, cytokines, and cyclins.65 In cardiac fibroblasts, the potential target genes identified so far include collagen,68 fibronectin,69 ICAM, and
VCAM.70 It is very likely that AP-1 also controls genes
involved in the cell cycle and apoptosis in cardiac fibroblasts.
Nuclear Factor-␬B (NF-␬B)
NF-␬B is a redox-sensitive transcription factor that controls a
number of genes involved in inflammation and apoptosis; it
mediates cell survival or apoptosis depending on the cell type
and environment.71 NF-␬B consists of homodimers or heterodimers of NF-␬B/Rel family proteins.71 In unstimulated
cells, NF-␬B is bound by an inhibitory I␬B family protein and
is retained within the cytoplasm, where it can be activated by
a wide range of stimuli, including reactive oxygen species
(ROS), hypoxia, hyperoxia, cytokines, growth factors, bacterial and viral products such as lipopolysaccharide (LPS), and
UV radiation.72 Cell stimulation leads to degradation of I␬B.
Free NF-␬B translocates into the nucleus and works as a
transcription factor.
NF-␬B target genes include proinflammatory cytokines,
chemokines, leukocyte adhesion molecules, MMPs, NO synthase, and antiapoptotic factors.72 In cultured cardiac fibroblasts, NF-␬B has been shown to be activated by hypoxia,
TNF-␣, and IL-1␤,70,73 and its potential target genes include
ICAM, VCAM, and monocyte chemoattractant protein-1
(MCP-1). It may also induce expression of angiotensinogen,
AT1 receptor, and IL-6.70,73–75
Early Growth Response Factor-1 (Egr-1)
Egr-1 is a zinc-finger transcription factor that is rapidly and
transiently activated by growth factors and other injurious
stimuli.76 In cardiac fibroblasts, Egr-1 is induced by Ang II,
EGF, and IGF-1. Once expressed, Egr-1 has been shown to
control a battery of genes important for cell growth and
inflammatory responses. In cardiac fibroblasts, Egr-1 has
been shown to induce PDGF-A and KLF5.77 It is also known
to control PDGF-B, FGF-2, TNF-␣, IL-2, tissue factor,
plasminogen activator, macrophage colony stimulating factor
(M-CSF), apolipoprotein A1, ICAM-1, and NF-␬B in other
cells.76
Signal Transducers and Activators of Transcription (Stat)
Janus kinase (Jak) and Stat proteins are found mainly coupled
with cytokine receptors. However, recent studies have shown
Jak/Stat to be involved in other receptors, including receptor
tyrosine kinases (eg, PDGF receptor) and GPCRs (eg, AT1
receptor).78 Upon ligand binding, Jaks phosphorylate Stat
proteins, resulting in their rapid translocation to the nucleus,
where they function as specific transcription factors.79
There are seven Stat proteins, many of which can be
activated in cardiac cells.80 Stat3 is unique among Stats in that
it appears to be involved in multiple signaling pathways and
biological activities.80,81 Stat3 is activated by cytokines that
signal through gp130 (eg, IL-6, LIF, and CT-1). The importance of the gp130/Stat3 pathway in heart disease was clearly
demonstrated by recent studies with transgenic and knockout
mice.82– 84 Continuous activation of gp130 signaling resulted
in cardiac hypertrophy,82 whereas cardiac-specific ablation of
gp130 caused massive apoptosis of cardiomyocytes and
dilated cardiomyopathy.85 Activation of Stat3 and other Stats
in the heart has been documented in acute infarction, ischemic preconditioning, and pressure overload.86 – 88 In both
cultured cardiac myocytes89 and fibroblasts,90 the Jak/Stat
pathway is activated by Ang II. On the other hand, Stat3
activates transcription of angiotensinogen via direct binding
to its promoter,89 suggesting a close interaction between Ang
II and the Jak/Stat pathways. Ang II also induces expression
of IL-6, CT-1, and LIF in cardiac fibroblasts, which in turn
activate Jak/Stat in adjacent cardiomyocytes.53 In ischemia
preconditioning of the myocardium in vivo, Stat1 and Stat3
activate expression of inducible NO synthase (iNOS).91
Furthermore, Stat1 is also involved in apoptosis.92 All of
these results indicate that the Jak/Stat pathway is crucially
involved in the pathogenesis of cardiac remodeling.
Transcription Factors Activated at the
Later Stages
Smad
TGF-␤ participates in the regulation of cell proliferation,
differentiation and migration, apoptosis, and ECM production. Moreover, TGF-␤ plays a central role in the development of fibrosis in a variety of chronic inflammatory conditions,93,94 for instance, it stimulates fibroblast chemotaxis and
the production of collagen and fibronectin while inhibiting
collagen degradation. It can also induce expression of SM
␣-actin in fibroblasts and is thus considered to be one of the
factors responsible for myofibroblast formation.22,25 In the
heart, TGF-␤ is induced by pressure overload, infarction, and
Ang II infusion,26,95–98 and this upregulation persists for at
least 4 to 8 weeks, during which concomitant expression of
fibronectin and collagen is observed.96,98 Cardiac specific
overexpression of TGF-␤1 resulted in cardiac hypertrophy
and fibrosis.99 Neutralization of TGF-␤ by anti–TGF-␤ antibody resulted in reduced fibrosis in pressure-overloaded
rats.96 Furthermore, TGF-␤1 knockout mice showed no hypertrophic responses to subpressor doses of Ang II.56
TGF-␤ can activate multiple signaling pathways, including
mitogen-activated protein kinase (MAPK) pathways, but the
Smad pathway is thought to be the predominant one.100 After
ligand activation, activated TGF-␤ receptor phosphorylates
R-Smads (Smad2 and Smad3), which in turn associate with a
Co-Smad (Smad4) and are translocated into the nucleus
where they act as transcription factors.101
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Transcriptional control by Smads involves interactions
with other transcription factors.101 For instance, analysis of
COL1A2 demonstrated that responsiveness to TGF-␤ is
governed by the overlapping potential cis-elements for Smad,
AP-1, and NF-␬B.102 Association with coactivators and
corepressors is also critically important for the function of
Smads. Indeed, Smads can either activate or repress target
genes depending on the binding partners.103
Changes in Smad expression have been associated with
heart diseases, for instance, the levels of Smad2 and Smad4
are upregulated in scar tissue after myocardial infarction.104
This effect is attenuated by the AT1 receptor blocker losartan,
suggesting the presence of crosstalk between Ang II and
Smad signaling. Conversely, Smad7, an I-Smad that inhibits
phosphorylation of R-Smads, is downregulated in scar tissues
after myocardial infarction.105 Interestingly, Smad7 expression is induced by the proinflammatory cytokines
interferon-␥ (IFN-␥) and TNF-␣ via NF-␬B and Stat in at
least some cell types, suggesting a crosstalk between signaling pathways important for chronic inflammation.106
(glucocorticoids and estrogens), nonsteroidal ligands such as
retinoids, and various lipid metabolites.117 A number of NRs
are expressed in the cardiovascular system, and their respective functions in cardiovascular disease have been extensively
studied.118
In addition to activation and repression via direct binding
to target sites, NRs can affect the activity of other classes of
transcription factors. For example, ligand-coupled glucocorticoid receptors can inhibit the transcriptional activities of
AP-1 and NF-␬B without directly binding to the DNA.117
Such transrepression is considered to be the major mechanism by which glucocorticoids inhibit inflammatory gene
expression.119 Transrepression is not limited to glucocorticoid
receptors; however, the activity of KLF5 is transrepressed by
retinoic acid receptors,111 and those of AP-1, STAT, and
NF-␬B are transrepressed by peroxisome proliferator-activated receptors (PPAR-␥).120 Thus, NRs appear to be critically involved in chronic inflammatory responses by modifying the functions of other transcription factors.
Krüppel-Like Factor (KLF)
Krüppel-like factors are a family of transcription factors
that contain three Krüppel-like C2H2-type zinc finger
domains107–109 and have diverse functions in normal development and disease. Several KLFs are crucially involved
in the function of cells of mesenchymal origin: KLF2/
LKLF is required for the investment of SMCs in the
vascular wall110; KLF5/BTEB2/IKLF is required for cardiovascular remodeling111; and KLF6/zf9/GBF is required
for stellate cell activation in liver cirrhosis.112 In addition,
KLF10/TIEG1 and KLF11/TIEG2 are both induced by
TGF-␤ and could play a role in tissue remodeling.113,114
We recently identified KLF5 as a transcription factor that
binds to the promoter of SMemb, a marker of phenotypic
modulation of SMCs.115 KLF5 is abundantly expressed in
embryonic vascular SMCs, is downregulated in mature cells,
and is reexpressed in phenotypically modulated cells present
in neointimal lesions in rats and in atherosclerotic lesions in
human coronary arteries.115,116 We also noted that KLF5 was
expressed in activated fibroblasts (myofibroblasts).
We generated lines of KLF5 knockout mice.111 Homozygous mice died in utero at a very early stage of embryonic
development. KLF5⫹/⫺ mice are viable and apparently normal. The cardiovascular system showed relatively minor
abnormalities. However, KLF5⫹/⫺ mice demonstrated attenuated responses to cardiovascular injuries. Vascular injury
resulted in much less neointimal formation and reduced
reactions in the adventitia of KLF5⫹/⫺ mice. KLF5⫹/⫺ mice
also showed reduced cardiac hypertrophy and fibrosis by Ang
II infusion, indicating its important role in cardiac fibroblasts.
These results clearly demonstrate an important role played by
KLF5 in the activation of SMCs and fibroblasts. Furthermore,
KLF5 is induced by Ang II via the MEK pathway.77 Once
expressed, KLF5 directly controls expression of PDGF-A and
TGF-␤.111 As such, KLF5 appears to be a key element linking
external stress and cardiovascular remodeling.
Context-Dependent Combinatorial Transcriptional
Regulation in Cardiac Remodeling
Nuclear Receptor (NR)
Nuclear receptors are a group of ligand-inducible transcription factors that include receptors for steroid hormones
Injury to the myocardium activates a number of transcription
factors reviewed so far. These transcription factors, particularly immediate early factors, are activated in many cell types
and induce expression of genes generally required for stress
responses. Still, activation of these transcription factors also
leads to the coordinate control of genes specifically required
for the function of a given cell type. Thus, a key question is
how do these transcription factors control specific sets of
genes in a specific cell type in a context-dependent manner?
An interesting feature of the promoters of stress-responsive
genes is that they often contain binding sites for multiple
immediate early factors. For example, the c-fos promoter is
controlled by both AP-1 and Stat, IL-8 by AP-1 and NF-␬B,
and angiotensinogen by NF-␬B and Stat.121 Although AP-1
per se may not have specificity for a particular target gene,
interactions at composite regulatory elements likely produce
protein complexes with a high degree of sequence and
regulatory selectivity.122 Recent studies have demonstrated
that combinatorial transcriptional control of such genes as
IFN-␤ and TNF-␣ involves formation of higher-order nucleoprotein complexes called enhanceosomes.123–125 Enhanceosomes have important features that enable transcription factors to control context-dependent transcription. First, the
binding of all required transcription factors is necessary for
enhanceosome activation. Second, the activity of enhanceosomes is not determined by the simple linear sum of that of
individual transcription factors, but rather by the highly
synergistic interaction between transcription factors and cofactors involved.123 As a result, the activation of enhanceosomes requires inputs from multiple signaling pathways, each
of which leads to activation of a different set of transcription
factors. For instance, stimulation that activates an individual
subset of transcription factors does not turn on IFN-␤123;
activation of all relevant signaling pathways is required
before IFN-␤ is transcribed. Although enhanceosomes have
been formally analyzed for only a limited number of genes,
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they are almost certainly responsible for transcriptional control of many others.
Another important feature of the transcriptional control
mechanism that has been revealed recently is that gene
transcription is controlled by multiple cis-regulatory modules,
each executing one of the functions of the entire regulatory
program.126 One example involving fibroblasts is the controlling expression of type I collagen.127 Type I collagen is
synthesized by multiple cell types, including fibroblasts,
osteoblasts, and odontoblasts. Analysis in transgenic mice
showed that type I collagen genes were controlled by multiple
regulatory modules that were spread over large genomic
regions (more than several kilobases) and differentially required in the different types of cells. In other words, different
programs control the same type I collagen genes in different
cell types. Likewise, the SM ␣-actin gene is controlled by
multiple transcriptional regulatory modules, which appear to
differentially control expression of the gene in different cells,
such as SMCs and myofibroblasts.128 As such, the modularity
of the transcriptional regulatory system likely enables the
system to respond to divergent environmental cues.129,130
A view on transcriptional regulation in vivo emerging from
enhanceosomes and the modular control system is that genes
are controlled by the network rather than by individual
transcription factors. The network of factors participating in
the transcriptional control is linked with the network of
signaling molecules. In this way, cells control gene expression in response to changing environmental cues. Analysis of
these networks likely requires new technologies that enable
us to determine pathways linking factors and to evaluate the
network experimentally and computationally. In this regard,
global mRNA and protein expression analysis and various
bioinformatics techniques will become increasingly
important.
Microarray analysis has identified transcriptional changes
associated with cardiac hypertrophy, myocardial infarction,
cardiomyopathy, and heart failure in human and animal
models.131 Genome wide expression analysis has also been
applied to map the effects of upregulation or downregulation
of genes in mice.132,133 Redfern et al133 generated a conditional expression of a Gi-coupled receptor Ro1 mouse model,
in which Ro1 expression can be regulated by drug administration in a cardiomyocyte-specific manner. In this study, the
changes in gene expression caused by Ro1 expression were
mapped to the G protein signaling cascade and other functionally grouped molecules. This analysis revealed a complex
feedback regulation of each G protein signaling pathway and
interactions between the pathways. A number of genes
involved in fibrosis were also reported to be modulated in the
study. When linked with other databases, such as those of
promoter structures and protein interactions, expression profiling would potentially provide us valuable data with which
to analyze the network of signaling and transcriptional
regulation.
We did not discuss the involvement of chromatin structure
in transcriptional regulation in this review, although clearly
chromatin remodeling profoundly affects gene transcription.
For instance, chromatin remodeling is essential for transcrip-
tion of SM ␣-actin.129,134 Chromatin remodeling is also
crucially important for the activation of enhanceosomes.
Analysis of the complex interaction between protein,
DNA, and chromatin is clearly important for elucidation of
the molecular mechanism that regulates dynamic gene expression during the processes of cardiac remodeling and heart
failure. It would also provide us novel therapeutic targets for
heart diseases. For instance, if the interaction but not the
expression of transcription factors is specific to control a
gene, it might be possible to develop a drug targeting the
interaction.111,135
Conclusions
Cardiac fibroblasts, along with cardiomyocytes, play an
essential role in the progression of cardiac remodeling.
Injurious stimuli evoke multiple signaling pathways in cardiac cells that lead to coordinate and sequential gene regulation. Initial transcriptional events lead to the activation of
cardiac fibroblasts. Activated fibroblasts (myofibroblasts)
produce ECM proteins and proteinases, as well as autocrine/
paracrine factors, and mediate tissue remodeling processes.
Recent studies have demonstrated that specific transcriptional control is governed by the complex interaction between
factors participating in transcriptional control. The activity at
transcriptional regulatory regions (eg, enhancers and promoters) is not a collection of activities of individual transcription
factors, but instead emerges as a network of synergistic
interactions between cis-elements within the region and their
cognate transcription factors.125 Signaling molecules also
form networks. Therefore, to fully understand the mechanism
of cardiac remodeling, we will need to investigate the
molecular networks that control the activities of cardiac
fibroblasts and cardiomyocytes.
Acknowledgments
This work was supported in part by a Grant-in-Aid for Millennium
Projects from the Ministry of Health, Labor and Welfare, Japan, a
Grant-in-Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan (to R.N.), and grants
from the Takeda Medical Foundation and the Sankyo Foundation of
Life Science (to I.M.).
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Gene Expression in Fibroblasts and Fibrosis: Involvement in Cardiac Hypertrophy
Ichiro Manabe, Takayuki Shindo and Ryozo Nagai
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Circ Res. 2002;91:1103-1113
doi: 10.1161/01.RES.0000046452.67724.B8
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Online data supplement
for MS # 4683-R1
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