Brief Reviews

Transforming Growth Factor ␤ and Atherosclerosis: So Far,

So Good for the Protective Cytokine Hypothesis
David J. Grainger

Abstract—The role of the anti-inflammatory cytokine transforming growth factor ␤ (TGF-␤) in atherosclerosis has been
the subject of considerable debate for a decade. In the early 1990s, we postulated that TGF-␤ played an important role
in maintaining normal vessel wall structure and that loss of this protective effect contributed to the development of
atherosclerosis. We termed this the protective cytokine hypothesis. This proposal was slow to gain broad acceptance,
however, because at that time there were little data available on the role of TGF-␤ during the development of
atherosclerosis but much information about its role during trauma-induced neointima formation. Because TGF-␤
apparently aggravates neointima formation, both by inhibiting endothelial regeneration and by promoting fibrosis, it was
difficult to accept that its presence might ameliorate the superficially similar atherogenesis process. But several recent
studies revealed beyond doubt the fact that TGF-␤ protects against lipid lesion formation, at least in mouse models of
atherosclerosis. Therefore, two important questions remain. First, is the role of TGF-␤ in vascular biology similar in
Downloaded from http://atvb.ahajournals.org/ by guest on April 30, 2018

humans and in mice? Secondly, how important, compared with defects in thrombosis or lipoprotein metabolism, is the
protective role of TGF-␤ during atherogenesis? (Arterioscler Thromb Vasc Biol. 2004;24:399-404.)
Key Words: TGF-␤ 䡲 myocardial infarction 䡲 inflammation

he transforming growth factor ␤ (TGF-␤) superfamily

T consists of a large number of structurally related cyto-
kines that participate in a vast range of biological processes
from axis specification and tissue differentiation in early
development through to regulation of a range of immune
functions in the adult organism. Members of the TGF-␤
superfamily are typically produced as dimeric latent precur-
sors with no biological activity, which are subsequently
activated in the extracellular environment to release the
receptor-binding ligands that share a common structural motif
known as the cysteine knot. Several members of the TGF-␤
superfamily are likely to play a role in vascular biology.
TGF-␤1 (the first member of the family to be discovered and
the best-studied member to date) is present at high levels in
the healthy blood vessel wall, whereas the closely related
TGF-␤2 and TGF-␤3 isoforms are either absent (␤2) or
present only at low levels (␤3). Of the remaining members of
the superfamily, activin1 and several of the bone morphoge-
netic proteins2,3 are known to be expressed in the blood vessel
wall and, although substantially less well-studied than TGF-
␤1, are thought to play broadly similar roles.
The protective cytokine hypothesis has its origins in cell
culture studies performed to examine the effects of TGF-␤ on
vascular smooth muscle cells (VSMCs).4,5 Although some of
the earliest studies suggested that TGF-␤ could promote
smooth muscle proliferation,6,7 it soon became clear that
under the majority of conditions, and particularly under any
conditions in which proliferation was stimulated by growth
factors, that TGF-␤ inhibits smooth muscle proliferation.8 –10
The small stimulatory effects seen in the early studies most
likely owe their existence to the exceptional ability of TGF-␤
to promote extracellular matrix (ECM) formation. Cells
placed into culture simply adhere to the culture plastic better
if TGF-␤ is present to stimulate ECM production. In addition
to its affect on VSMC proliferation, TGF-␤ also inhibited
VSMC migration11 and promoted the expression of an array
of proteins that make-up the contractile apparatus of the
cell.8 –10 In short, TGF-␤ possessed a portfolio of coordinated
effects in vitro that would contribute to the maintenance of
the normal blood vessel wall architecture if similar effects
occurred in vivo.5
Early in its existence, the protective cytokine idea was
expanded beyond the smooth muscle component of the vessel
wall. Gene knockout studies demonstrated that TGF-␤ ex-
erted a dramatic anti-inflammatory signal in constitutive
fashion. Loss of TGF-␤1 resulted in perinatal death after
unbridled leukocyte extravasation in almost every organ
system.12,13 Because accumulation of leukocytes, particularly
macrophages but also T cells, is a characteristic signature of
the change from normal vessel wall structure to early athero-
sclerotic lesion,14 we postulated that lowered TGF-␤ activity
in the vessel wall might lead to leukocyte accumulation, in

Received July 9, 2003; revision accepted November 26, 2003.

From the Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK.
Sources of Support: D.J.G. is supported by the British Heart Foundation.
Correspondence to David J. Grainger, Department of Medicine, University of Cambridge, Box 157, Addenbrooke’s Hospital, Cambridge, CB2 2QQ,
UK. E-mail djg15@cam.ac.uk
© 2004 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000114567.76772.33

399
 400 Arterioscler Thromb Vasc Biol. March 2004
parallel with increased VSMC proliferation, migration, and
dedifferentiation.5
Over a period of years, a range of other potentially
protective effects of TGF-␤ in cell culture were identified,
including suppression of proinflammatory adhesion molecule
expression by the vascular endothelium,15 and reduced foam
cell formation in cultured macrophages.16 Recently, Mallat
and Tedgui17 have provided an excellent overview of the
immunomodulatory role of TGF-␤ during the development of
atherosclerosis, in which they highlight the central role of
TGF-␤ in the regulation of T-cell biology and it implications
for the chronic inflammatory component of atherogenesis.
Recent evidence from Gojova et al18 underlines the role of
TGF-␤ as a deactivating cytokine for T cells, because
disrupting TGF-␤ signaling specifically in the T-cell popula-
tion increased the number of T cells, as well as MHC class
II-positive macrophages, in the lesions. These T-cell–rich
lesions had a characteristic phenotype of rupture-prone
plaques in humans: lowered collagen content and decreased
Downloaded from http://atvb.ahajournals.org/ by guest on April 30, 2018
representation of smooth muscle cells expressing smooth
muscle-specific actin isoforms. As Mallat and Tedgui con-
clude,17 the effect of TGF-␤ on T-cell differentiation is
undoubtedly an important component of TGF-␤ action during
atherogenesis.
However, TGF-␤ signaling in the inflammatory cell pop-
ulation cannot be the whole story: Gojova et al18 report a
small but significant decrease in lesion size in the aortic sinus
when TGF-␤ signaling is abrogated specifically in the T-cell
population, whereas generalized suppression of TGF-␤ levels
(using neutralizing antibodies,19 soluble receptor fragments,20
or heterozygous deletion of the tgfb1 gene21) led to either no
change in lesion size or an increase in lesion size. This is
likely to reflect the complexity of the role of TGF-␤ in the
vessel wall, with important effects not only on the leukocyte
populations but also on the endothelial and vascular smooth
muscle cell compartments. This review therefore comple-
ments the article by Mallat and Tedgui17 by focusing primar-
ily on the impact of TGF-␤ signaling on the vascular smooth
muscle cell population in the vessel wall.
This interplay between the leukocyte and smooth muscle
cell compartments is likely to be particularly important when
considering the impact of TGF-␤ signaling on ECM produc-
tion. Elaboration of ECM is one of the major functions of the
smooth muscle cell, and the balance between leukocytes and
smooth muscle cell populations in the developing atheroscle-
rotic plaque is therefore a major determinant of plaque
stability.22 Consequently, TGF-␤ is likely to play a central
role in regulating this “stability balance” in at least two
distinct ways: (1) through its immunoregulatory role it
suppresses leukocyte recruitment; and (2) it is one of the most
powerful fibrogenic cytokines described to date. TGF-␤
therefore not only protects against the initial formation of
fatty streak lesions by maintaining normal blood vessel wall
structure19,21 but also protects against the development of
unstable lesions by driving matrix production by the smooth
muscle cells of the fibrous cap.19,20
Potentially, suppression of TGF-␤ signaling is progres-
sively and doubly dangerous. To lose TGF-␤ activity tempo-
rarily is merely careless (leading to formation of stable
Figure 1. A model for the action of TGF-␤ on VSMCs during
atherogenesis. In the normal vessel wall (left cycle), latent
TGF-␤ is efficiently converted into active TGF-␤ (black line),
which signals to cells rich in type II TGF-␤ receptor (RII). This
maintains normal vessel wall structure and a high level of RII on
the VSMCs. If TGF-␤ activity is transiently diminished (red line;
the “first hit”), then RII levels decrease and the vessel wall shifts
to the right cycle. While TGF-␤ activity remains adequate, sig-
naling to cells rich in type I TGF-␤ receptor (RI) stimulates ECM
expression and a stable plaque phenotype results. Physiologi-
cally, this process is irreversible because TGF-␤ activity no
longer promotes RII expression. If TGF-␤ activity is again
reduced (thin red line; the “second hit”), inflammation and loss
of ECM results converting the lesion to an unstable phenotype
prone to rupture.
matrix-rich lesions), but to lose TGF-␤ activity repeatedly is
potentially fatal (leading to formation of leukocyte-rich,
matrix-poor lesions that are at high risk for rupture).
More recently, a plausible molecular basis for this double
role of TGF-␤ during atherogenesis has evolved: McCaffrey
et al demonstrated that VSMCs (the cells that make all the
ECM in the vessel wall) had different TGF-␤ receptor
expression profiles in atherosclerotic lesions compared with
normal vessel wall.23 In the diseased vessel, the cells domi-
nantly expressed the type I TGF-␤ receptor, whereas in
normal vessel wall the type II receptors dominated. This
change had functional consequences, at least in vitro. The
type I dominated VSMCs responded to TGF-␤ very differ-
ently from the type II dominated cells, by massively elevating
ECM production.23 In contrast, the type II dominated cells
barely increase ECM production at all in response to TGF-␤.
We confirmed and extended these studies, demonstrating that
when VSMCs from healthy artery are first dispersed into cell
culture, they have high levels of the type II receptor and
respond to TGF-␤ by increasing contractile protein expres-
sion, but not ECM production. In the absence of added
TGF-␤, over a number of cell cycles in culture, the ratio of
type II to type I receptor decreases, and the cells switch their
responses over. Now TGF-␤ stimulates ECM production but
not contractile protein expression. Most interestingly, if the
freshly dispersed cells were maintained continually in
TGF-␤, they maintained high levels of type II receptor
indefinitely in culture, and with it maintained a differentiated
phenotype with relatively low ECM production.
Taken together, these studies allow a plausible molecular
model for the role of TGF-␤ in the vessel wall to be
constructed (Figure 1). High levels of TGF-␤ maintain a

feedback loop in which type II receptor remains high and the
vessel wall retains its differentiated phenotype with lots of
contractile proteins and relatively little ECM. If some exter-
nal factor intervenes to reduce TGF-␤ activity sufficiently in
a particular region, the VSMCs respond not only by dedif-
ferentiating, proliferating, and migrating into the vessel in-
tima but also by reducing type II receptor expression. This is
the “first hit” of reduced TGF-␤ activity that promotes early
fatty streak lesion formation.
However, the changes in TGF-␤ receptor expression pat-
terns has the effect of locking in the vessel wall changes that
have occurred, because even if TGF-␤ activity returns to
normal, those dedifferentiated intimal VSMCs respond by
producing ECM rather than by returning to the contractile
state. The result would be a stable matrix-rich atherosclerotic
plaque. However, if TGF-␤ activity remained suppressed or
was lowered still further, then the “second hit” of reduced
TGF-␤ activity occurs. Enhanced recruitment of leukocytes
Downloaded from http://atvb.ahajournals.org/ by guest on April 30, 2018
to the plaque coupled with reduced ECM production by the
remaining VSMCs results in formation of a rupture-prone
plaque typical of those that go on to have clinical sequelae,
including myocardial infarction and death.
Such a model provides one possible explanation for a range
of features of the atherogenic process, but is it correct? And
what might trigger localized suppression of TGF-␤ activity?
Direct Evidence Supports the Protective
Cytokine Hypothesis in Mice
Genetic manipulation has become the standard method for
determining the importance of gene products in disease
processes. The gene of interest is knocked out, and the impact
on disease progression monitored. Unfortunately, the tgfb1
null mouse dies soon after birth from severe multifocal
inflammation,12,13 preventing us from studying vessel wall
architecture in the absence of TGF-␤1. Two approaches have
been taken to get around this problem: tgfb1 null mice can be
bred on a SCID background, allowing them to survive into
adulthood;24 alternatively, mice heterozygous for the tgfb1
deletion are studied.21,25,26 These tgfb1⫹/⫺ mice have lower
levels of TGF-␤1 in a number of tissues,25 including the
blood vessel wall,21,26 and provide a direct method to study
the impact of lowered TGF-␤1 levels on vessel wall
architecture.
Adult tgfb1⫹/⫺ mice have reduced levels of smooth muscle
differentiation markers, including smooth muscle specific-␣-
actin and smooth muscle-specific myosin heavy chain in the
aortic wall,21,26 consistent with the expected effect of reduced
levels of TGF-␤1. However, neither tgfb1⫺/⫺ mice on a SCID
background24 nor tgfb1⫹/⫺ mice21 showed signs of endothelial
activation (marked by VCAM-1 and ICAM-1 expression).
Interestingly, when the mice were fed a high-fat diet, endo-
thelial activation and lipid deposition was observed in the
tgfb1⫹/⫺ mice but not in their wild-type littermate controls.21
These observations demonstrate that reduced TGF-␤1 pro-
duction renders the vessel wall susceptible to endothelial
activation and development of early stage lipid lesions when
subjected to a proatherogenic challenge. This study, however,
provides no information as to when or where the level of
Grainger Protective Cytokine Hypothesis 401
TGF-␤1 must be reduced to promote susceptibility to
atherogenesis.
More recently, several other groups have used different
methodological approaches to draw similar conclusions. Both
Mallat et al19 and Lutgens et al20 used in vivo neutralization
approaches to show that depletion of TGF-␤1 in the blood
vessel wall of adult apoE-deficient mice was sufficient to
exacerbate lipid lesion development. The amount of lipid
deposited was increased in both studies, as was the number of
macrophages and other inflammatory cells accumulating in
the developing lesion. Lutgens et al20 went on to demonstrate
that neutralizing TGF-␤ decreased ECM deposition, confirm-
ing the role of this cytokine in regulating the balance between
an unstable, proinflammatory lesion phenotype and a stable,
matrix-rich phenotype.
Taken together, these studies provide strong evidence that
TGF-␤ protects against unstable plaque development in at
least two ways. TGF-␤ acts constitutively to suppress the
changes in vessel wall architecture that are associated with
lipid lesion development in mice19,21 (the “first hit” of
reduced TGF-␤). Decreased TGF-␤ activity later during the
atherogenic process results in a switch to an unstable inflam-
matory lesion phenotype19,20 (the “second hit” of reduced
TGF-␤).
What Factors Might Trigger Suppression of
TGF-␤ Activity in Humans?
One of the best understood local triggers for suppression of
TGF-␤ activity in the blood vessel wall is the variant
lipoprotein particle Lp(a). Lp(a) consists of an additional
protein component, apo(a), covalently bonded to the apoB of
an LDL particle.27 The distinguishing apo(a) protein has
sequence homology to plasminogen but has been inactivated
in the catalytic triad, rendering it incapable of yielding an
enzymatically active form equivalent to plasmin. Rifkin et al
first demonstrated that Lp(a) could inhibit TGF-␤ activation
in cell culture,11 an observation that has been repeated many
times.28,29 We extended these studies and demonstrated that
transgenic expression of apo(a) in mice, which have no
equivalent endogenous gene, results in impaired TGF-␤
activation in the blood vessel wall.30 In a further study, we
demonstrated that apo(a) accumulates preferentially at sites
where TGF-␤ activation is already suppressed, leading to a
positive feedback loop that explains the highly focal suppres-
sion of TGF-␤ activity in apo(a) and Lp(a) transgenic mice.31
Feedback loops such as these may contribute to the focal
nature of atherosclerotic lesions, which affect some regions of
the vasculature but not others within the same individual. To
date, however, there are no data to determine whether the
presence of Lp(a) lowers TGF-␤ activation in humans.32
A range of other factors have also been postulated to
regulate TGF-␤ activation (Figure 2), including proteases that
are thought to cleave the LAP thereby promoting release of
mature TGF-␤, binding proteins that alter the conformation of
the complex promoting its dissociation, and physicochemical
factors (such as acidity and radiation) that also favor disso-
ciation of protein complexes. Recently, Rifkin et al33 have
proposed a unifying scheme, suggesting that the LAP dimer
acts as a “sensor” component of the latent TGF-␤ complex,
 402 Arterioscler Thromb Vasc Biol. March 2004
Downloaded from http://atvb.ahajournals.org/ by guest on April 30, 2018
Figure 2. Factors affecting the activation of TGF-␤1. Members
of the TGF-␤ superfamily are produced as pre-pro-proteins that
dimerize and lose the signal sequence during secretion. The
resulting pro-protein dimer must then undergo a 2-stage activa-
tion process to yield the active TGF-␤ ligand. In the first step, a
member of the pro-protein convertase family, such as furin,
cleaves the polypeptide backbone at a tetrabasic site between
the pro-peptide (termed the latency associated peptide or LAP)
and the mature TGF-␤ dimer. The LAP dimer and mature TGF-␤
dimer remain noncovalently associated, and this structure
(termed latent TGF-␤) has no known biological activity. In a sec-
ond step, the LAP becomes dissociated from the mature TGF-␤
dimer, which is thereby released to interact with the signaling
receptors. The precise mechanism of this second step is still
poorly understood, but it is known to be promoted by a wide
range of factors (including proteases, physiochemical mediators,
and the binding of other proteins). Several inhibitors of this acti-
vation step have also been described. It is likely that the LAP is
acting as a “sensor” component of the complex, integrating the
levels of a range of environmental factors to determine how
much of the “effector” mature TGF-␤ should be released. A third
component of the complex (namely various TGF-␤ binding pro-
teins) that acts as a “localizer” to regulate the tissue distribution
of the complex is not shown for clarity. MMP indicates matrix
metalloproteinases; M-6-P receptor, cation-independent
mannose-6-phosphate receptor.
integrating the activity of multiple environmental factors to
tightly regulate the release of the “effector” TGF-␤ complex.
Thus, factors such as PAI-1 that are known to be elevated
during atherogenesis,34 or urokinase and plasmin activity
(which are known to be suppressed34), are likely to contribute
to local suppression of TGF-␤ activity at the site of vascular
lipid lesion formation.
Various mechanisms also link defects in lipid metabolism
to suppression of TGF-␤ activity. After a standardized
high-fat meal, TGF-␤ activation is transiently suppressed.
Several mechanisms may contribute to this. PAI-1 production
is stimulated by dietary fat load, to an extent that depends on
the 4G/5G genotype in the PAI-1 promoter.35 Suppression of
TGF-␤ activation was similarly associated with the PAI-1
promoter genotype, suggesting that PAI-1 may contribute to
the reduction in TGF-␤ activity. Additionally, the hydropho-
bic TGF-␤ protein can be sequestered into triglyceride-rich
lipoprotein particles, reducing its ability to interact with its
signaling receptors,36 which may exacerbate the reduction in
TGF-␤ activity seen after dietary fat loading.
TGF-␤ activity may also be suppressed at regions of low
shear stress (for example, at vessel branch points37). These
preliminary observations in human studies have been repli-
cated in studies of the rodent vasculature.26 Again, several
molecular mechanisms may contribute, including a direct
shear response element in the tgfb1 gene promoter38 and a
flow-dependency in the expression pattern of the inhibitory
Smad proteins, Smad6 and Smad7.39
As in mice, the definitive experiment to implicate TGF-␤
in the atherogenesis process in humans would be to identify
an association between tgfb1 genotype and disease. Although
the experiments are more technically demanding, there have
been a number of genetic studies that have attempted to
determine whether polymorphisms in the tgfb1 gene locus are
associated with either myocardial infarction (a measure of
plaque stability as well as plaque load) or angiographically
defined luminal stenosis (a measure of plaque load). The first
study, by Cambien et al40 in the ECTIM cohort, found an
association between polymorphisms in the TGF-␤1 signal
peptide region of the gene and myocardial infarction but no
association with angiographically defined arterial stenosis. A
follow-up study confirmed the lack of association between
several tgfb1 polymorphisms and angiographically defined
disease.41 However, a recent study in Japanese men42 found a
larger association between tgfb1 polymorphisms and myocar-
dial infarction than that originally reported by Cambien.
Taken together, these studies provide tentative support for the
protective cytokine hypothesis in humans: polymorphisms
associated with lower TGF-␤1 secretion are over-represented
among men with myocardial infarction in several popula-
tions.43 The lack of association with angiographically defined
vessel stenosis41 may suggest that in humans, the role of
TGF-␤ is more associated with the switch between stable
matrix-rich lesions and unstable inflammatory lesions (the
“second hit” of reduced TGF-␤) than in determining the total
atherosclerotic plaque load (the “first hit”).17,19,20
Unfortunately, a more clear-cut resolution of this question
is unlikely to come from genetic studies of the tgfb1 gene.
Because TGF-␤1 is involved in such a wide range of
physiological and pathological functions, polymorphisms or
mutations that caused significant changes to the temporal,
spatial, or quantitative production of TGF-␤ would likely
have effects that manifested themselves much earlier in life
than atherosclerosis. Unlike in mice, in which definitive
experiments were possible, it is likely that a fuller under-
standing of the role of TGF-␤ in human cardiovascular
disease will have to come from the weight of evidence
yielded by an array of experimental approaches.
How Important Is TGF-␤ Signaling
in Atherogenesis?
Recent studies from a range of groups have confirmed that in
rodents, suppression of TGF-␤ activity results in increased
lipid lesion deposition during early lesion development and a
switch from a stable, matrix-rich plaque phenotype to an
unstable, proinflammatory plaque at risk for rupture.19 –21 The
available evidence suggest that similar pathways are likely to

Figure 3. A 3-pathway model for atherogenesis. Epidemiology
and reverse genetic approaches have unequivocally demon-
strated that there are at least 3 major pathways (represented as
colored circles) contributing to the risk of myocardial infarction.
For some individuals, a major defect in a single pathway is
Downloaded from http://atvb.ahajournals.org/ by guest on April 30, 2018
dominantly responsible. For individual 1, a major defect in
lipoprotein metabolism is the major contributor to their risk. For
individual 2, a major defect in fibrinolysis might be responsible.
We propose, however, that for most individuals with coronary
artery disease (represented by individual 3), it is a combination
of mild defects in all 3 pathways that results in high risk for
myocardial infarction. Effective preventative therapy will depend
on targeting each of the pathways contributing to the risk for a
given individual.
be operating in humans. Thus, defects in TGF-␤ metabolism
join an ever-growing list of pathways that have been plausi-
bly linked to the development of atherosclerosis resulting in
myocardial infarction, including defects in lipoprotein metab-
olism and imbalance between thrombotic and fibrinolytic
cascades.
It is no longer sufficient merely to ask whether defects in
particular pathways might (under certain circumstances) con-
tribute to the an increased risk for myocardial infarction.
Instead, we need to ask how much of the variation in
susceptibility to myocardial infarction across the human
population can be attributed to each of these pathways. It is
likely that for different individuals, different pathways may
dominate (Figure 3). For some individuals, such as those with
familial hypercholesterolemia caused by genetic defects in
the LDL receptor protein and some individuals with less
severe lipoprotein metabolism defects, the lipoprotein traf-
ficking and metabolism defect may be the dominant cause of
the increased risk of myocardial infarction by increasing the
atherosclerotic burden. For others, increased susceptibility to
intravascular coagulation may be the dominant cause of their
increased risk. Yet for the majority, it seems plausible that the
development of atherosclerosis and, ultimately, its progres-
sion to yield myocardial infarction result from the combined
effects of milder defects in a number of contributing path-
ways. Increased inflammatory responses, whether systemic or
local to the vessel wall, seem likely to contribute to the total
atherosclerotic burden and to the tendency to the develop-
ment of unstable, leukocyte-rich plaques that predispose to
myocardial infarction.
Our studies to date suggest that reduced TGF-␤ activity is
a common consequence of a range of environmental and
Grainger Protective Cytokine Hypothesis 403
genetic factors associated with development of atherosclero-
sis. It is possible, therefore, that it occupies an important
nexus in the web of interacting pathways controlling blood
vessel wall structure. The challenge now lies in quantifying
the contribution of aberrant TGF-␤ activity to the develop-
ment of clinically significant atherosclerosis in the human
population. Unfortunately, there are no simple experiments
that will conclusively determine the relative importance of
suppressed TGF-␤ activity as a risk factor for myocardial
infarction. Instead, a gradual understanding is likely to evolve
as genetic and epidemiological evidence accumulates over a
period of years.
This task has more than academic importance. Identifica-
tion of the most prevalent pathways contributing to athero-
genesis is a key step to improving current clinical manage-
ment of coronary heart disease. The widespread clinical
benefit of treatment with lipid-lowering therapies and anti-
thrombotic agents is now well-established, yet tens of thou-
sands of myocardial infarction events still occur each year in
subjects receiving the current best therapy. By identifying
further pathways, independent of lipid metabolism and
thrombosis, which contribute to the disease process, we can
develop additional therapeutic options that may be effective
in at least a proportion of the population. To this end, we and
others have proposed the use of triphenylethylene-derived
drugs, such as tamoxifen44 or raloxifene,45 which stimulate
the production of TGF-␤46,47 as therapies for coronary artery
disease. The recent articles19 –21 describing direct evidence for
the protective cytokine hypothesis in rodents provide yet
further impetus toward clinical trials of these TGF-␤–stimu-
lating drugs in humans.
Acknowledgments
I am grateful to Profs Jim Metcalfe and Peter Weissberg (University
of Cambridge, UK) for their support during the formulation of the
protective cytokine hypothesis, and to Dr David Mosedale for
helpful discussions over many years. D.J.G. is a British Heart
Foundation Senior Research Fellow.
References
1. Engelse MA, Neele JM, van Achterberg TA, van Aken BE, van Schaik
RH, Pannekoek H, de Vries CJ. Human activin-A is expressed in the
atherosclerotic lesion and promotes the contractile phenotype of smooth
muscle cells. Circ Res. 1999;85:931–939.
2. Dhore CR, Cleutjens JP, Lutgens E, Cleutjens KB, Geusens PP, Kitslaar
PJ, Tordoir JH, Spronk HM, Vermeer C, Daemen MJ. Differential
expression of bone matrix regulatory proteins in human atherosclerotic
plaques. Arterioscler Thromb Vasc Biol. 2001;21:1998 –2003.
3. Willette RN, Gu JL, Lysko PG, Anderson KM, Minehart H, Yue T.
BMP-2 gene expression and effects on human vascular smooth muscle
cells. J Vasc Res. 1999;36:120 –125.
4. Metcalfe JC, Grainger DJ. Transforming growth factor-beta and the
protection from cardiovascular injury hypothesis. Biochem Soc Trans.
1995;23:403– 406.
5. Grainger DJ, Metcalfe JC. A pivotal role for TGF-beta in atherogenesis?
Biol Rev Camb Philos Soc. 1995;70:571–596.
6. Stouffer GA, Owens GK. Angiotensin II-induced mitogenesis of sponta-
neously hypertensive rat-derived cultured smooth muscle cells is
dependent on autocrine production of transforming growth factor-beta.
Circ Res. 1992;70:820 – 828.
7. Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross R. TGF-beta
induces bimodal proliferation of connective tissue cells via complex
control of an autocrine PDGF loop. Cell. 1990;63:515–524.
 404 Arterioscler Thromb Vasc Biol. March 2004
8. Bjorkerud S. Effects of transforming growth factor-beta 1 on human
arterial smooth muscle cells in vitro. Arterioscler Thromb. 1991;11:
892–902.
9. Grainger DJ, Kemp PR, Witchell CM, Weissberg PL, Metcalfe JC.
Transforming growth factor beta decreases the rate of proliferation of rat
vascular smooth muscle cells by extending the G2 phase of the cell cycle
and delays the rise in cyclic AMP before entry into M phase. Biochem J.
1994;299(Pt 1):227–35.
10. Owens GK, Geisterfer AA, Yang YW, Komoriya A. Transforming
growth factor-beta-induced growth inhibition and cellular hypertrophy in
cultured vascular smooth muscle cells. J Cell Biol. 1988;107:771–780.
11. Kojima S, Harpel PC, Rifkin DB. Lipoprotein (a) inhibits the generation
of transforming growth factor beta: an endogenous inhibitor of smooth
muscle cell migration. J Cell Biol. 1991;113:1439 –1445.
12. Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC,
Roberts AB, Sporn MB, Ward JM, Karlsson S. Transforming growth
factor beta 1 null mutation in mice causes excessive inflammatory
response and early death. Proc Natl Acad Sci U S A. 1993;90:770 –774.
13. Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen
R, Sidman C, Proetzel G, Calvin D, et al. Targeted disruption of the
mouse transforming growth factor-␤ 1 gene results in multifocal inflam-
matory disease. Nature. 1992;359:693– 699.
14. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999;
Downloaded from http://atvb.ahajournals.org/ by guest on April 30, 2018
340:115–126.
15. Gamble JR, Khew-Goodall Y, Vadas MA. Transforming growth
factor-beta inhibits E-selectin expression on human endothelial cells.
J Immunol. 1993;150:4494 – 4503.
16. Argmann CA, Van Den Diepstraten CH, Sawyez CG, Edwards JY,
Hegele RA, Wolfe BM, Huff MW. Transforming growth factor-beta1
inhibits macrophage cholesteryl ester accumulation induced by native and
oxidized VLDL remnants. Arterioscler Thromb Vasc Biol. 2001;21:
2011–2018.
17. Mallat Z, Tedgui A. The role of transforming growth factor beta in
atherosclerosis: novel insights and future perspectives. Curr Opin Lipidol.
2002;13:523–529.
18. Gojova A, Brun V, Esposito B, Cottrez F, Gourdy P, Ardouin P, Tedgui
A, Mallat Z, Groux H. Specific abrogation of transforming growth factor-
{beta} signaling in T cells alters atherosclerotic lesion size and compo-
sition in mice. Blood. 2003.
19. Mallat Z, Gojova A, Marchiol-Fournigault C, Esposito B, Kamate C,
Merval R, Fradelizi D, Tedgui A. Inhibition of transforming growth
factor-beta signaling accelerates atherosclerosis and induces an unstable
plaque phenotype in mice. Circ Res. 2001;89:930 –934.
20. Lutgens E, Gijbels M, Smook M, Heeringa P, Gotwals P, Koteliansky
VE, Daemen MJ. Transforming growth factor-beta mediates balance
between inflammation and fibrosis during plaque progression. Arte-
rioscler Thromb Vasc Biol. 2002;22:975–982.
21. Grainger DJ, Mosedale DE, Metcalfe JC, Bottinger EP. Dietary fat and
reduced levels of TGF␤1 act synergistically to promote activation of the
vascular endothelium and formation of lipid lesions. J Cell Sci. 2000;
113:2355–2361.
22. Robbie L, Libby P. Inflammation and atherothrombosis. Ann N Y Acad
Sci. 2001;947:167–180.
23. McCaffrey TA, Consigli S, Du B, Falcone DJ, Sanborn TA, Spokojny
AM, Bush HL, Jr. Decreased type II/type I TGF-beta receptor ratio in
cells derived from human atherosclerotic lesions. Conversion from an
antiproliferative to profibrotic response to TGF-␤1. J Clin Invest. 1995;
96:2667–2675.
24. Diebold RJ, Eis MJ, Yin M, Ormsby I, Boivin GP, Darrow BJ, Saffitz JE,
Doetschman T. Early-onset multifocal inflammation in the transforming
growth factor beta 1-null mouse is lymphocyte mediated. Proc Natl Acad
Sci U S A. 1995;92:12215–12219.
25. Tang B, Bottinger EP, Jakowlew SB, Bagnall KM, Mariano J, Anver MR,
Letterio JJ, Wakefield LM. Transforming growth factor-␤1 is a new form
of tumor suppressor with true haploid insufficiency. Nat Med. 1998;4:
802– 807.
26. Grainger DJ, Metcalfe JC, Grace AA, Mosedale DE. Transforming
growth factor-beta dynamically regulates vascular smooth muscle differ-
entiation in vivo. J Cell Sci. 1998;111:2977–2988.
27. Scanu AM, Lawn RM, Berg K. Lipoprotein(a) and atherosclerosis. Ann
Intern Med. 1991;115:209 –218.
28. Grainger DJ, Kirschenlohr HL, Metcalfe JC, Weissberg PL, Wade DP,
Lawn RM. Proliferation of human smooth muscle cells promoted by
lipoprotein(a). Science. 1993;260:1655–1658.
29. Takami S, Yamashita S, Kihara S, Ishigami M, Takemura K, Kume N,
Kita T, Matsuzawa Y. Lipoprotein(a) enhances the expression of inter-
cellular adhesion molecule-1 in cultured human umbilical vein endothe-
lial cells. Circulation. 1998;97:721–728.
30. Grainger DJ, Kemp PR, Liu AC, Lawn RM, Metcalfe JC. Activation of
transforming growth factor-beta is inhibited in transgenic apoli-
poprotein(a) mice. Nature. 1994;370:460 – 462.
31. Lawn RM, Pearle AD, Kunz LL, Rubin EM, Reckless J, Metcalfe JC,
Grainger DJ. Feedback mechanism of focal vascular lesion formation in
transgenic apolipoprotein(a) mice. J Biol Chem. 1996;271:31367–31371.
32. Grainger DJ, Metcalfe JC. Transforming growth factor-beta: the key to
understanding lipoprotein(a)? Curr Opin Lipidol. 1995;6:81– 85.
33. Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFbeta
activation. J Cell Sci. 2003;116:217–224.
34. Martin-Paredero V, Vadillo J, Diaz J, Espinosa A, Berga C, Segura J,
Sanchez J, Barbod A, Villaverde C, Richard CM. Fibrinogen and fibri-
nolysis in blood and in the arterial wall: its role in advanced athero-
sclerotic disease. Cardiovasc Surg. 1998;6:457– 462.
35. Byrne CD, Wareham NJ, Martensz ND, Humphries SE, Metcalfe JC,
Grainger DJ. Increased PAI activity and PAI-1 antigen occurring with an
oral fat load: associations with PAI-1 genotype and plasma active
TGF-beta levels. Atherosclerosis. 1998;140:45–53.
36. Grainger DJ, Byrne CD, Witchell CM, Metcalfe JC. Transforming growth
factor beta is sequestered into an inactive pool by lipoproteins. J Lipid
Res. 1997;38:2344 –2352.
37. Borkowski P, Robinson MJ, Kusiak JW, Borkowski A, Brathwaite C,
Mergner WJ. Studies on TGF-beta 1 gene expression in the intima of the
human aorta in regions with high and low probability of developing
atherosclerotic lesions. Mod Pathol. 1995;8:478 – 482.
38. Ohno M, Cooke JP, Dzau VJ, Gibbons GH. Fluid shear stress induces
endothelial transforming growth factor beta-1 transcription and produc-
tion. Modulation by potassium channel blockade. J Clin Invest. 1995;95:
1363–1369.
39. Topper JN, Cai J, Qiu Y, Anderson KR, Xu YY, Deeds JD, Feeley R,
Gimeno CJ, Woolf EA, Tayber O, Mays GG, Sampson BA, Schoen FJ,
Gimbrone MA, Jr., Falb D. Vascular MADs: two novel MAD-related
genes selectively inducible by flow in human vascular endothelium. Proc
Natl Acad Sci U S A. 1997;94:9314 –9319.
40. Cambien F, Ricard S, Troesch A, Mallet C, Generenaz L, Evans A,
Arveiler D, Luc G, Ruidavets JB, Poirier O. Polymorphisms of the
transforming growth factor-beta 1 gene in relation to myocardial
infarction and blood pressure. The Etude Cas-Temoin de l’Infarctus du
Myocarde (ECTIM) Study. Hypertension. 1996;28:881– 887.
41. Syrris P, Carter ND, Metcalfe JC, Kemp PR, Grainger DJ, Kaski JC,
Crossman DC, Francis SE, Gunn J, Jeffery S, Heathcote K. Transforming
growth factor-␤1 gene polymorphisms and coronary artery disease. Clin
Sci (Lond). 1998;95:659 – 667.
42. Yokota M, Ichihara S, Lin TL, Nakashima N, Yamada Y. Association of
a T29 –⬎C polymorphism of the transforming growth factor-␤1 gene
with genetic susceptibility to myocardial infarction in Japanese. Circu-
lation. 2000;101:2783–2787.
43. Andreotti F, Porto I, Crea F, Maseri A. Inflammatory gene poly-
morphisms and ischaemic heart disease: review of population association
studies. Heart. 2002;87:107–112.
44. Grainger DJ, Metcalfe JC. Tamoxifen: teaching an old drug new tricks?
Nat Med. 1996;2:381–385.
45. Saitta A, Morabito N, Frisina N, Cucinotte D, Corrado F, D’Anna R,
Altavilla D, Squadrito G, Minutoli L, Arcoraci V, Cancellieri F, Squadrito
F. Cardiovascular effects of raloxifene hydrochloride. Cardiovasc Drug
Rev. 2001;19:57–74.
46. Yang NN, Bryant HU, Hardikar S, Sato M, Galvin RJ, Glasebrook AL,
Termine JD. Estrogen and raloxifene stimulate transforming growth
factor-beta 3 gene expression in rat bone: a potential mechanism for
estrogen- or raloxifene-mediated bone maintenance. Endocrinology.
1996;137:2075–2084.
47. Grainger DJ, Weissberg PL, Metcalfe JC. Tamoxifen decreases the rate of
proliferation of rat vascular smooth-muscle cells in culture by inducing
production of transforming growth factor beta. Biochem J.
1993;294:109 –112.
 Transforming Growth Factor β and Atherosclerosis: So Far, So Good for the Protective
Downloaded from http://atvb.ahajournals.org/ by guest on April 30, 2018

Cytokine Hypothesis
David J. Grainger

Arterioscler Thromb Vasc Biol. 2004;24:399-404; originally published online December 29,
2003;
doi: 10.1161/01.ATV.0000114567.76772.33
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2003 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636

The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/24/3/399

Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the
Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for
which permission is being requested is located, click Request Permissions in the middle column of the Web
page under Services. Further information about this process is available in the Permissions and Rights
Question and Answer document.

Reprints: Information about reprints can be found online at:

http://www.lww.com/reprints

Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online
at:
http://atvb.ahajournals.org//subscriptions/