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Transforming Growth Factor-␤s and Vascular Disorders
Alex Bobik
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Abstract—Transforming growth factor-␤ (TGF-␤) superfamily members, TGF-␤ and bone morphogenetic proteins
(BMPs), are potent regulatory cytokines with diverse functions on vascular cells. They signal through heteromeric
type I and II receptor complexes activating Smad-dependent and Smad-independent signals, which regulate
proliferation, differentiation, and survival. They are potent regulators of vascular development and vessel
remodeling and play key roles in atherosclerosis and restenosis, regulating endothelial, smooth muscle cell,
macrophage, T cell, and probably vascular calcifying cell responses. In atherosclerosis, TGF-␤ regulates lesion
phenotype by controlling T-cell responses and stimulating smooth muscle cells to produce collagen. It contributes
to restenosis by augmenting neointimal cell proliferation and collagen accumulation. Defective TGF-␤ signaling
in endothelial cells attributable to mutations in endoglin or the type I receptor ALK-1 leads to hereditary
hemorrhagic telangiectasia, whereas defective BMP signaling attributable to mutations in the BMP receptor II has
been associated with development of primary pulmonary hypertension. The development of mouse models with
either cell type–specific or general inactivation of TGF-␤/BMP signaling has started to reveal the importance of
the regulatory network of TGF-␤/BMP pathways in vivo and their significance for atherosclerosis, hereditary
hemorrhagic telangiectasia, and primary pulmonary hypertension. This review highlights recent findings that have
advanced our understanding of the roles of TGF-␤ superfamily members in regulating vascular cell responses and
provides likely avenues for future research that may lead to novel pharmacological therapies for the treatment or
prevention of vascular disorders. (Arterioscler Thromb Vasc Biol. 2006;26:1712-1720.)
Key Words: TGF-␤ 䡲 BMP 䡲 signaling 䡲 vascular cells 䡲 atherosclerosis 䡲 restenosis
䡲 hereditary hemorrhagic telangiectasia 䡲 pulmonary hypertension
he transforming growth factor-␤ (TGF-␤) superfamily is
composed of many multifunctional cytokines, including
TGF-␤s, bone morphogenetic proteins (BMPs), activins,
inhibins, and mycostatin. They participate in a wide range of
processes, from tissue differentiation during development
through to regulation of mesenchymal and immune cell
functions. Two members of the TGF-␤ superfamily, TGF-␤
and BMPs, have been the most extensively studied members
in the vasculature, affecting key cell functions and have been
implicated in vascular disorders such as atherosclerosis,
pulmonary hypertension, hereditary hemorrhagic telangiectasia (HHT), and restenosis. TGF-␤ isoform TGF-␤, TGF-␤2,
and TGF-␤3, as well as several BMPs, in particular BMP-2,
BMP-4, BMP-6, and BMP-7, are expressed by cells in the
vessel wall and are capable of modulating vascular development and remodeling by altering cell differentiation, proliferation, migration, extracellular matrix production, and the
activities of immune cells.
This review discusses the vascular effects of these TGF-␤
superfamily members, their signaling systems, and how their
actions in multiple cell types including those of the immune
system regulate processes involved in the development of
T
atherosclerosis, restenosis, vascular development, and remodeling associated with diseases such as HHT and primary
pulmonary hypertension (PPH).
TGF-␤ Superfamily Members and Effects on
Vascular Cells
TGF-␤ isoforms and several BMPs, BMP-2, BMP-4, BMP-6,
and BMP-7 are expressed by ⱖ1 vascular cells, endothelial
cells, vascular smooth muscle cells (VSMCs), macrophages,
and various types of lymphocytes. TGF-␤ isoforms are
produced as inactive dimeric latent precursors and are subsequently processed extracellularly to yield active receptor
binding ligands. In the vessel wall, inactive TGF-␤ can be
activated via the cooperation between the mannose-6phosphate/insulin-like growth factor II receptor and the
urokinase-type plasminogen activator receptor, plasmin,
thrombospondin, and furin-like proprotein convertases; the
latter are responsible for TGF-␤ activation after arterial
injury.1 In contrast, BMPs are secreted in an active form and
regulated through reversible interactions with extracellular
antagonists, including noggin, chordin, and DAN.2 These
interactions determine the bioavailability of different TGF-
Original received January 3, 2006; final version accepted April 24, 2006.
From the Cell Biology Laboratory, Baker Heart Research Institute, Melbourne, Australia.
Correspondence to Professor Alexander Bobik, Cell Biology Laboratory, Baker Heart Research Institute, PO Box 6492, St Kilda Rd Central,
Melbourne, Victoria 8008, Australia. E-mail [email protected]
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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DOI: 10.1161/01.ATV.0000225287.20034.2c
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Figure 1. TGF-␤ and BMP transduce signals through Smad-dependent and Smad-independent pathways in vascular cells. Dimeric
TGF-␤ becomes active after dissociation from its complex of the latency-associated protein (LAP) and latent TGF-␤ binding protein-1
(LTBP), whereas BMP free of antagonists activate their respective tetrameric receptor complexes with or without endoglin. Subsequently, the type I receptors (ALK-5 or ALK-1 for TGF-␤ and ALK-1, ALK-2, ALK-3, and ALK-6 for BMP) phosphorylate respective
receptor-activated Smads (R-Smads; -1,-2,-3,-5, and -8), which translocate to the nucleus as a trimeric complex with Smad4. The
complex interacts with other transcription factors (TF), coactivators, or corepressors and the Smad binding element (SBE). When complexed with transcriptional coactivators (eg, CBP/p300) target genes are activated), and when complexed with corepressors (eg, Sno),
transcription is repressed. Smad-independent signals generated by TGF-␤/BMPs are boxed and include ERK1/2, p38MAPK, JNK, and
phosphatidylinositol 3-kinase (PI3K).
␤s/BMPs for binding to their receptors. Endothelial cells,
smooth muscle cells, mesenchymal cells, macrophages, and
lymphocytes respond to these TGF-␤ family members by
their interaction with a heteromeric complex of type I and
type II transmembrane serine/threonine kinase receptors.
Multiple type I and II receptors have been identified that
exhibit specificities in signaling mediated by TGF-␤ isoforms
or BMPs (Figure 1). TGF-␤ isoforms, BMP-6, and BMP-7
have a high affinity for the type II receptors, and on binding,
a type I receptor is recruited, forming a heteromeric complex
resulting in phosphorylation of the type I receptor in a
conserved region, the GS domain, changing its conformation;
BMP-2 and BMP-4 exhibit a higher affinity for the type I
receptor but also signal through the heteromeric type I/type II
complex.3 Signal propagation to the nucleus then occurs by
the type I receptor phosphorylating Smad proteins, the major
nuclear effectors for TGF-␤ receptors.4 There are 3 distinct
types of Smads: receptor regulated, common mediator
Smads, and inhibitory Smads. Smad1, Smad2, Smad3,
Smad5, and Smad8 are receptor regulated and phosphorylated
at their extreme C-terminal serines followed by interaction
with the common mediator Smad, Smad4, and translocation
to the nucleus. Inhibitory Smads, Smad6, and Smad7 compete
with receptor-activated Smads for receptor interaction and
recruit ubiquitin ligases or phosphatases to activated receptors, promoting their degradation or dephosphorylation.5
TGF-␤ and BMPs exert multiple effects on endothelial
cells. TGF-␤ exerts bifunctional effects on cell proliferation,
stimulating proliferation and migration at low concentrations,
whereas at higher concentrations, it inhibits these processes.
It regulates the activation state of endothelial cells via 2 type
I receptors, ALK-5, and ALK-1.6 The TGF-␤/ALK-1 pathway stimulates endothelial cell proliferation and migration,
whereas the TGF-␤/ALK-5 pathway inhibits these processes.
Constitutively active forms of ALK-1 and ALK-5 initiate
differential patterns of gene expression.7 ALK-1 stimulates
the expression of Id-1, an inhibitor of basic helix-loop-helix
proteins that promotes endothelial cell proliferation, migration, and tube formation,8 whereas ALK-5 induces expression
of plasminogen activator inhibitor-1, a negative regulator of
endothelial cell migration and angiogenesis. When ALK-5
kinase is inhibited by SB-431542, TGF-␤1 only stimulates
endothelial cell proliferation.9 Studies on signaling mechanisms initiated by ALK-1 and ALK-5 indicate a complex
interaction between the receptors. ALK-5 deficiency not only
impairs TGF-␤/ALK-5 signaling but also reduces TGF-␤/
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ALK-1 responses, suggesting that ALK-5 is essential for
efficient ALK-1 activation and recruitment into a TGF-␤
receptor complex.10 ALK-1 not only induces responses opposite to ALK-5 but also antagonizes ALK-5/Smad3 signaling. This dual receptor system in endothelial cells provides an
intricate mechanism for switching between different TGF-␤–
induced responses. When ALK-5 is inhibited by SB-431542,
TGF-␤1 also affects vascular permeability by upregulating
the expression of Claudin-5, an endothelial cell–specific
component of tight junctions,9 and inhibits the expression of
adhesion molecules. The TGF-␤ type III receptor endoglin is
also important for TGF-␤ responses by endothelial cells. Both
the extracellular and intracellular domains of endoglin interact with T␤R-II and ALK-5 and its cytoplasmic domain,
which is phosphorylated by ALK-5 and T␤R-II.11 Deletion of
endoglin from endothelial cells potentiates the inhibitory
effects of TGF-␤ on endothelial cell migration and growth,
suggesting that it is a negative regulator of TGF-␤/ALK-5
signaling. Endoglin stimulates TGF-␤/ALK-1 signaling and
indirectly inhibits TGF-␤/ALK-5 signaling. It also stimulates
the expression of endothelial NO. In addition to activating
genes in endothelial cells, TGF-␤ also represses gene expression, particularly genes that promote inflammation,
interleukin-6 (IL-6), monocyte chemoattractant protein-1,
and granulocyte– colony-stimulating factor.12 Such effects
may be tissue specific (see below).
Although VSMCs express multiple type I receptors as well as
the type II TGF-␤ receptor,13 most of the effects of TGF-␤ on
VSMC function appear to be mediated via the ALK-5/T␤R-II
complex. Subsequent downstream signaling is complex, not
only involving Smad2/3 but also kinases such as p38 mitogenactivated protein kinase (MAPK), p42/p44, and JNK as well as
transcription factors, which are all dependent on ALK-5 (Figure
1).14 TGF-␤ is a potent vascular smooth muscle differentiating
factor, increasing the expression of ␣-smooth muscle actin,
smooth muscle myosin, and calponin. At low concentrations, it
stimulates proliferation by inducing platelet-derived growth
factor (PDGF)–AA. TGF-␤1 can also attenuate VSMC activation by opposing the effects of mitogenic growth factors,
proinflammatory cytokines, and genes that affect vascular remodeling. Both Smad-dependent and Smad-independent mechanisms contribute to its ability to inhibit proliferation,15 whereas
its ability to activate genes such as ␣2 type I collagen and
inhibitor of metalloproteinase-1 is Smad3 dependent. TGF-␤/
Smad3-dependent signaling also prevents cytokine-mediated
activation of smooth muscle cells by Smad3 binding directly to
the CCAT/enhancer-binding protein-␤ and also reducing the
expression of CCAT/enhancer-binding protein-␦.16 Other signaling cascades are also important, for example, increases in
plasminogen activator inhibitor type I gene expression by
TGF-␤1 is dependent on transactivation of the epidermal growth
factor receptor and c-Src activation.17 Increases in fibronectin
expression and the synthesis of vascular endothelial growth
factor are also Smad independent and regulated via disabled-2
association with TAK1, leading to JNK activation and
p38MAPK activation, respectively.18 Mechanistically, other effects of TGF-␤1 on VSMCs, such as stimulating proteoglycan
synthesis, are yet to be defined.
TGF-␤1 also exerts important effects on immune cells.
Macrophages produce and are highly responsive to TGF-␤1.
TGF-␤ can activate and also deactivate these cells depending on
the local cytokine environment.19 Signaling cascades appear
similar to those in VSMCs. TGF-␤1 promotes monocyte adhesion to type IV collagen, laminin, and fibronectin20 and stimulates monocyte chemotaxis via Smad3,21 increasing expression
of receptors for multiple chemokines.22 TGF-␤ may also potentiate inflammation by inducing the secretion of IL-1 and IL6.19,23 It protects macrophages from apoptosis by stimulating
extracellular-signal regulated kinase (ERK) and attenuates macrophage foam cell formation by downregulating the expression
of CD36; it also increases cholesterol efflux, the expression of
the ATP-binding cassette transporter-1,24 and inhibits lipoprotein
lipase expression via mechanisms involving Sp1-binding sites.25
TGF-␤ exerts anti-inflammatory effects by attenuating macrophage activation. It reduces cytokine-stimulated inducible NO
synthase expression in a Smad3-dependent manner by recruiting
the limited amounts of p300/CREB-binding protein from nuclear factor ␬B and AP-1 to Smad3.26 It also promotes inducible
NO synthase protein degradation27 and inhibits cytokinemediated increases in metalloelastase and the expression of
chemokines KC and MIP-2 by destabilizing their mRNAs via
p38MAPK-dependent mechanism.28
TGF-␤ affects T-cell proliferation, differentiation, and
survival. This important function of TGF-␤ is exemplified in
vivo, in which animals expressing the dominant-negative
TGF-␤ type II receptor specifically in T cells develop severe
autoimmunity characterized by uncontrolled T-cell activation.29 Similarly, blockade of TGF-␤/Smad signaling in T
cells by overexpression of Smad7 also enhances inflammation.30 Like the other vascular cell types, in lymphocytes,
TGF-␤ signals by recruiting type II and type I (ALK-5)
receptors followed by phosphorylation of receptor-regulated
Smads. Endoglin is also expressed on T-cells and acts to
counteract TGF-␤–mediated suppression. TGF-␤ inhibits
proliferation by inhibiting IL-2 expression, c-myc, and cyclins D2 and E. Inhibition is influenced by differentiation state
and is most apparent in naive cells; it has minimal effects on
activated T cells, which express low amounts of the type II
receptor. Upregulation of the type II receptor by IL-10
treatment restores TGF-␤ responsiveness on activated T
cells.31 Other effects include inhibition of Th1 and Th2
differentiation,32 the latter via GATA3, inhibition of
interferon-␥ (IFN-␥) production, upregulation of tristetraprolin, a protein involved in tumor necrosis factor-␣ degradation,
inhibition of IL-2 production, and T-cell proliferation.33
TGF-␤ inhibits Th1 development in part via suppression of
T-bet, an IFN-␥–induced transcription factor that promotes
Th1 differentiation (Figure 2).34 TGF-␤ also inhibits Fas
ligand expression and subsequent activation-induced cell
death by downregulating c-myc. Other effects of TGF-␤
include inhibition of CD1d expression on dendritic cells;35
CD1d is essential for lipid antigen recognition by NKT cells.
TGF-␤ also influences CD4⫹CD25⫹ T-cell development by
regulating the expression of Foxp3, a transcription factor
essential for their generation and function.36
BMPs BMP-2, BMP-4, BMP-6, and BMP-7 also exert
important effects on endothelial and smooth muscle cells.
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Figure 2. TGF-␤ and T-cell responses. TGF-␤ inhibits T-cell proliferation by inhibiting IL-2 production and suppressing c-myc, cyclins, and cyclin-dependent kinase 4 (CDK4). It inhibits differentiation
of Th1 and Th2 cells by suppressing the expression or function of
T-bet and GATA3 but stimulates expression of Foxp3, increasing
the suppressive activities of regulatory CD4⫹CD25⫹ T cells.
TGF-␤ can also prevent T-cell activation–induced apoptosis by
inhibiting c-myc. Dysregulation of TGF-␤ signaling in T cells
increases atherosclerosis severity. Adapted from Li et al.34
BMPs interact with 3 type 2 receptors, BMP receptor II
(BMPR-II), ActR-II, and ActR-IIB, to activate ⱖ1 of 4 different
type I receptors, ALK-1, ALK-2, ALK-3, and ALK-6, which
then signal in a manner similar to TGF-␤ using specific
receptor-regulated Smads (Figure 1). BMP-2 stimulates endothelial cell migration and angiogenesis via Id1 and the
p38MAPK pathway,37 whereas BMP-4 stimulates production of
reactive oxygen species and monocyte adhesion.38 In some
endothelial cell types, BMP-4 induces apoptosis.39 BMPs are
expressed by endothelial cells and influence smooth muscle cell
phenotype. BMP-2 expression by endothelial cells is regulated
via a hydrogen peroxide/nuclear factor ␬B pathway, activated by
proinflammatory cytokines and high pressure; oscillatory shear
stress regulates BMP-4 expression. The effects of BMPs on
VSMCs include inhibition of proliferation and modulation of
smooth muscle cell differentiation markers; BMP-2 decreases,
whereas BMP-7 increases expression of smooth muscle cell
markers.40 In pulmonary VSMCs, BMP-4 activates Smad1,
p38MAPK, and ERK1/2 pathways.41 Inhibition of proliferation
by BMP-4 is dependent on the Smad1 pathway and elevations in
p21WAF1; activation of Smad1 also downregulates the antiapoptotic protein Bcl-2, making these cells more sensitive to
apoptosis. In contrast, p38MAPK/ERK promotes proliferation
and is antiapoptotic. BMP-2 also stimulates smooth muscle cell
migration by undefined mechanisms.
TGF-␤ and Atherogenesis
TGF-␤ and receptors are abundantly expressed by smooth
muscle cells, macrophages, and T cells in human atherosclerotic lesions during development of fatty streaks and subsequent atheroma.42 Because TGF-␤ stimulates leukocyte chemotaxis21 and proteoglycan production by smooth muscle
cells, it is possible that TGF-␤ contributes to early macrophage migration and lipid accumulation. TGF-␤ appears to
determine the extent to which developing atherosclerotic
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lesions are stabilized by a collagen-rich fibrous cap. Smooth
muscle cells in stable lesions express greater amounts of
TGF-␤ than unstable lesions.43 Smooth muscle cells from
lesions dominantly express the type I receptor, whereas
smooth muscle cells from normal vessels dominantly express
the type II receptor.44 Cells dominantly expressing type I
receptors respond to TGF-␤ by producing copious amounts of
extracellular matrix. Smooth muscle cells within the fibrous
cap of atherosclerotic lesions and fibrofatty lesions also
appear to differ in their responsiveness to TGF-␤. Smooth
muscle cells within fibrous plaques express Smad proteins,
which are essential for TGF-␤–mediated increases in collagen gene expression, but in macrophage-rich fibrofatty lesions, smooth muscle cells do not express Smad proteins,
suggesting that their ability to produce collagen in response to
TGF-␤ is impaired.45 In fibrofatty lesions, IFN-␥ produced by
T cells and macrophages can also contribute to reduced
collagen levels by inhibiting collagen production.46 Treatment of apolipoprotein E– deficient (apoE⫺/⫺) mice with an
anti–TGF-␤ neutralizing antibody reduces collagen by 50%,
also supporting an important role for TGF-␤ in collagen
production in lesions.47 This was associated with reduced
Smad2 phosphorylation and an increase in lesion size but no
change in the smooth muscle cell numbers. Inhibiting TGF-␤
with a recombinant soluble TGF-␤ receptor fusion protein
was associated not only with increased inflammation but also
intraplaque hemorrhage.48 At least a component of the inflammatory response to TGF-␤ depletion appears because of
T-lymphocyte deregulation; adventitial lymphocytes increase
in number after treatment with the neutralizing antibody.47
Furthermore, disruption of TGF-␤ signaling specifically in T
cells also results in increased lesion size and development of
an unstable phenotype.49 Lesions in the transgenic mice
overexpressing dominant-negative type II receptors in T cells
contain more macrophages and lymphocytes and less collagen, similar to mice treated with neutralizing antibodies;
IFN-␥, which was also increased, could contribute to collagen
reductions in the lesions. The recent identification of markers
of CD4⫹CD25⫹ regulatory T cells in lesions raises the
question as to whether the effect of impairing TGF-␤ signaling in T lymphocytes is at least in part a consequence of
reduced CD4⫹CD25⫹ regulatory T-cell activity.50 Depletion
of CD4⫹CD25⫹ regulatory T cells increases lesion size in
apoE⫺/⫺ mice.51 These cells, which constitute 5% to 10% of
peripheral CD4⫹ lymphocytes, are capable of inhibiting
CD4⫹ and CD8⫹ T-cell responses via TGF-␤– dependent
mechanisms; T cells that do not respond to TGF-␤ escape
control by CD4⫹CD25⫹ regulatory T cells.52 TGF-␤ supports Foxp3 expression in these cells, their regulatory function, and homeostasis. Foxp3 is a master control gene for the
development and function of CD4⫹CD25⫹ regulatory T
cells and is upregulated in atherosclerotic lesions of
apoE⫺/⫺ mice, suggesting an important role for these cells
in regulating atherosclerosis.50 Depletion of TGF-␤ greatly
reduces their numbers in the periphery.53 Furthermore, inhibition of TGF-␤ signaling markedly reduces Foxp3 expression, impairing their suppressor activity. Together, these
studies indicate that TGF-␤ promotes a stable lesion phenotype by ⱖ2 mechanisms, stimulating collagen biosynthesis by
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VSMCs and inhibiting T-cell activation either directly or
indirectly via CD4⫹CD25⫹ regulatory T cells.
Evidence for a role for BMPs in atherogenesis is more
indirect. BMP-2, BMP-4, and BMP-6 are expressed in advanced
atherosclerotic lesions and associated with lesion calcification.54,55 BMP-2 not only induces VSMC cycle arrest but also
stimulates the loss of smooth muscle cell markers56 and the gain
of an osteoblastic gene expression profile related to Msx-2 and
Runx, transcription factors that promote osteogenic gene expression.57 BMP-2 may also induce apoptosis; apoptosis appears to
be critical to the initiation and propagation of calcification by
calcifying vascular cells. Reductions in atherosclerotic lesions of
matrix Gla protein would promote calcification by a BMP-2–
dependent mechanism.58 It will be of interest to determine
whether overexpression of BMP antagonists such as noggin
reduces calcification of advanced atherosclerotic lesions in
apoE⫺/⫺ mice.59 Recently, BMP-7 has been shown to reduce
calcification of lesions.60 The mechanism by which BMP-7
reduces calcification may be related its ability to promote a
contractile phenotype in smooth muscle cells and reduce serum
phosphate levels.
TGF-␤ and Restenosis
Restenosis attributable to excessive intimal fibrocellular proliferation and inward remodeling is the main limitation to
therapeutic revascularization, angioplasty, stenting, or
atherectomy used to treat obstructive atherosclerotic lesions.
The TGF-␤ system is rapidly upregulated after balloon
catheter injury to vessels with increases in TGF-␤ isoforms,
together with the type II TGF-␤ receptor and ALK-5.61 After
endothelial denudation, platelets attached to the site of injury
degranulate, releasing large amounts of TGF-␤;62 this can
further enhance platelet aggregation63 and stimulate apoptosis
of surviving endothelial cells64 and promote smooth muscle
cell survival.65 The TGF-␤ inhibitory binding protein vasorin
is also downregulated, enabling TGF-␤ to participate in the
healing process.66 Two lines of evidence suggest that TGF-␤
contributes to restenosis, (1) its overexpression stimulates
neointimal hyperplasia and extracellular matrix accumulation,67 and (2) targeting TGF-␤1 mRNA degradation with
chimeric DNA–RNA hammerhead ribozymes attenuates neointima growth.68 Pharmacological inhibition of TGF-␤1 signaling also attenuates neointima growth and remodeling after
stenting.69 The mechanisms by which TGF-␤ promotes proliferation under these circumstances most likely involve
TGF-␤–stimulating release of mitogenic growth factors. Low
concentrations of TGF-␤ stimulate PDGF-AA expression,
PDGF-B expression, and release of extracellular fibroblast
growth factor-2.70,71 TGF-␤1 may also enhance proliferation
by upregulating Nox4, a gp91phox homologue of the
NADPH oxidase system, and increasing reactive oxygen
species production with transient oxidative inactivation of
phosphatases and augmentation of growth signaling cascades.72 Inhibition of TGF-␤1 in addition to reducing intimal
proliferation also reduces collagen accumulation in the neointima. These combined results suggest that TGF-␤1 is a
potential target to reduce restenosis. It will be of interest to
determine whether the recently developed ALK-5 antago-
nists,73 adsorbed onto stents, delivered by microparticles or
administered systemically can prevent clinical restenosis.
TGF-␤ Signaling and HHT
HHT is an autosomal dominant disorder affecting ⬇1 in 8000
individuals. Serious complications of HHT include pulmonary, cerebral, and hepatic arteriovenous malformations,
which can lead to severe hemorrhage, stroke, and brain
abscess. Mutations in 2 receptors of the TGF-␤ family have
been causally linked to HHT: endoglin for type I HHT and
ALK-1 for type 2 HHT.74,75 Mutations of all types are
distributed through both genes, and severity of diseases does
not correlate with any aspecific mutations. Mutations in
endoglin include deletions, insertions, and missense mutations and splice site changes, the majority representing null
alleles, which lead to reduced levels of endoglin protein on
the surface of endothelial cells. More than 150 mutations
have been reported in endoglin, mostly within exons 1 to 12,
which encode the extracellular domain, and none in exons 13
and 14, which encode the transmembrane and cytoplasmic
domains, respectively. More than 120 mutations have been
reported in ALK-1 with more than half being missense
mutations and the remaining being small deletions, insertions,
splice site mutations, and nonsense mutations frequently
leading to truncated proteins.76 The most common mutations
occur in both the extracellular region and kinase domain with
frequencies highest in the kinase domain. Mutations in the
kinase domain lead to alterations in polarity, charge, and
hydrophobicity, as well as misfolding of the protein. HHT1
has a higher prevalence of pulmonary arteriovenous malformations, and HHT2 families generally exhibit a milder
phenotype and later onset of disease.77
It still remains to be determined how reductions in endoglin or ALK-1 predispose to HHT and what causes vascular
lesions to develop in selective vascular beds. Some insights
into mechanisms responsible for the disease have been
obtained from endoglin⫹/⫺ and ALK-1⫹/⫺ mice.78,79
Endoglin-deficient mice die at midgestation of angiogenic
and cardiovascular defects. Vasculogenesis in these mice is
normal, but angiogenesis is impaired along with remodeling
of the primary vascular plexus. The mice exhibit poor VSMC
development that results in dilatation and rupture of the
vascular channels. In the heterozygous mice, disease severity
increases with age and can include rupture of major vessels.
During development, both endoglin- and ALK-1– deficient
mice lose structural, molecular, and functional distinctions
between arteries and veins, but only ALK-1– deficient mice
exhibit profound vessel dilatation.79,80 Endoglin⫹/⫺ mice
generally exhibit a greater number of dilated venules, a
greater number of capillaries across the dermis, and large
numbers of irregular or thinned vessels with reduced numbers
of smooth muscle cells. The reduction in smooth muscle cell
numbers appears at least in part attributable to defective
vessel formation in early development, probably the consequence of reductions in the production of autoinducible
TGF-␤ by endothelial cells.81 Recruitment of mesenchymal
cells into new vessels is achieved in part by TGF-␤. On
contact of mesenchymal cells with the endothelium, latent
TGF-␤ is activated, inducing differentiation of mesenchymal
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cells into pericytes and smooth muscle cells.82 In endoglindeficient mice, differentiation of smooth muscle cells can be
rescued with exogenous TGF-␤1.81 Endothelial cells from
subjects with type I HHT also exhibit reduced production of
TGF-␤1.83 These cells also exhibit disorganized cytoskeleton
and fail to form endothelial cell tubes. A disorganized
cytoskeleton is prone to cell breaking with changes in shear
stress and blood pressure. The latter defect may be the
consequence of reduced endothelial NO synthase/Hsp90 association and uncoupling endothelial NO synthase activity,
resulting in increased endothelial NO synthase– derived superoxide anion production rather than NO production.84
Although endoglin and ALK-1 mutations are strongly
associated with development of HHT, the clinical manifestations of HHT are highly heterogenous between families as
well as within a given family with the same mutation; also in
mice, the high prevalence of HHT in endoglin⫹/⫺ 129/Ola
mice compared with endoglin⫹/⫺ C57BL6 mice suggests an
involvement of modifier genes.85 Thus, although defective
TGF-␤ signaling in endothelial cells attributable to mutations
in endoglin and ALK-1 are clearly important in HHT, other
yet to be identified genetic and epigenetic factors also
contribute. Identifying these genes, which could involve
genes regulating downstream TGF-␤ signaling and also
understanding how mutations in ALK-1 and endoglin contribute to abnormalities in vessel development, should provide further insights into this vascular disorder. Recently,
mutations in smad4 have been associated with the combined
syndrome of juvenile polyposis and HHT, suggesting a role
for downstream TGF-␤ signaling components in HHT.86
Vessel Remodeling in PPH
PPH is characterized by a marked and sustained elevation in
pulmonary vascular resistance. Typical pathological features
of this disorder include enhanced cell proliferation, muscularization, the obliteration of small pulmonary arteries, and
formation of plexiform lesions. PPH has been strongly linked
to mutations in the BMPR-II gene, suggesting that impaired
BMP signaling greatly increases susceptibility to this disorder.87 Most of the mutations in the BMPR-II gene are
predicted to lead to premature termination codons, but some
missense mutations have also been identified, suggesting that
the mutations can contribute to PPH via haploinsufficiency or
dominant-negative mechanisms. Mutations include nonsense
or frame shift mutations in the extracellular domain, which
can lead to premature truncation of the transcripts and the
absence of production of transmembrane BMPR-II proteins.
Missense mutations involving conserved cysteine residues in
the extracellular domain and missense or frame shift mutations in the kinase domain or frame shift or nonsense
mutations within the cytoplasmic tail result in cytoplasmic
truncation of the receptor.88 However, although mutations in
BMPR-II have been strongly linked to PPH, additional
environmental and genetic factors are also required for
development of clinically significant PPH. Family members
with identical mutations in BMPR-II have a 15% to 20%
chance of developing PPH.88 In support of the importance of
environmental factors, mice carrying 1 mutant allele (BMPRII⫹/⫺) exhibit increased pulmonary vascular resistance and
1717
Figure 3. BMPR-II mutations in pulmonary hypertension. Cysteine mutations in either the extracellular ligand binding or
kinase domains prevent receptor trafficking to the cell membrane. Noncysteine mutations in the kinase domain result in
impaired Smad phosphorylation and Smad-mediated signaling.
Mutations in the C-terminal domain leads to impaired Smadindependent signaling by reducing interactions with cell cycle
regulators (eg, c-Src). All mutations result in an impaired ability
of BMPs to inhibit cell proliferation.
thickened muscularized arteries.89 However, vascular obliteration or plexiform lesions typically found in patients with
severe PPH is absent. Similarly, mice engineered to conditionally express a human dominant-negative BMPR-II mutation in smooth muscle cells also exhibit elevated pulmonary
artery pressure but only mild pulmonary arterial muscularization.90 The small increase in muscularization suggests that
deficiencies in BMPR-II signaling in additional cell types,
such as endothelial cells and possibly fibroblasts, may also be
important for induction of the vascular changes associated
with PPH; BMPR-II in normal lung is predominately expressed in endothelial cells and to a lesser extent on smooth
muscle cells. The apparent disparity between the rise in
pulmonary pressure and extent of vessel muscularization
suggests enhanced pulmonary vasoconstriction in mice carrying the dominant-negative BMPR-II mutation and is consistent with a 2-phase hypothesis for PPH in which early
disease is characterized by enhanced vasoconstriction and
minimal vascular remodeling and later disease by progressive
remodeling and little vasoconstriction.91
Although it is clear that factors in additional to mutations
in BMPR-II, possibly serotonin,92 are required to initiate the
proliferation that leads to the vascular obliteration in pulmonary hypertension, defective BMP signaling allows proliferation and intimal smooth muscle cell accumulation to proceed
largely unabated, obliterating small arteries. In pulmonary
arterial smooth muscle cells from subjects with PPH, the
actions of BMP such as induction of apoptosis, suppression of
DNA synthesis and inhibition of proliferation are markedly
attenuated.93 The mechanism by which BMPR-II mutants
disrupt BMP/Smad signaling is heterogenous and mutation
specific. Mutations in the extracellular ligand-binding domain
of BMPR-II, which results in substitution of cysteine residues, prevent receptors from reaching the cell membrane,
effectively reducing the number of functional receptors (Figure 3). Missense mutations in the kinase domain lead to
reductions in Smad1 signaling and unopposed p38MAPK/
ERK signaling, leading to abnormal vascular cell proliferation.41 Mutations in the cytoplasmic tail of BMPR-II, includ-
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ing those responsible for truncating its unusually long
cytoplasmic tail, affect other BMP signaling cascades that can
also affect proliferation. Mutations in the cytoplasmic tail resulting in exon 12 deletion does not affect Smad phosphorylation or
p38MAPK activation, rather it affects the phosphorylation of
Tctex-1, a light chain of the motor complex Dynein; in normal
lung, BMPR-II and Tctex-1 colocalize in endothelial and smooth
muscle cells.94 c-Src is also an interacting partner of the
C-terminal domain of BMPR-II; truncation of the C terminus
disrupts their interaction, whereas missense mutation in the
kinase domain reduces their interaction, impairing the ability of
BMPR-II to inhibit c-Src activating phosphorylation.95 In pulmonary artery, smooth muscle serotonin activates c-Src, resulting in phosphorylation of PDGF receptor and ERK1/2 MAPK
and activating cell cycle regulators, such as cyclins D and E,
leading to cell proliferation. The C-terminal domain also interacts with LIM kinase 1, which regulates actin dynamics by
phosphorylating cofilin. Mutations in BMPR-II, which result in
even small deletions in the C-terminal region, fail to bind or
inhibit LIM kinase 1, suggesting that alterations in the dynamics
of the actin cytoskeleton may contribute to PPH susceptibility.96
Recently, other proteins have been shown to associate with the
C-terminal tail of BMPR-II, including regulators of the cell
cycle, apoptosis, cytoskeleton, and kinases, but their functional
significance for PPH is yet to be elucidated. Although the
functional consequences of mutations in BMPR-II are providing
new insights as to how mutations in BMPR-II may predispose to
PPH, many questions remain unanswered, particularly in relation to functional consequences of loss of BMPR-II signaling in
other cell types such as endothelium. Mutations in ALK-1,
which has been associated with HHT, have also recently been
linked to PPH.97
Conclusions
In conclusion, evidence from animal models and correlative
data from human studies implicate TGF-␤ superfamily members TGF-␤ and BMPs in the regulation or development of a
wide range of vascular disorders. Additional studies are
required to more clearly define how alterations in TGF-␤/
BMP signaling contribute to these disorders and design
appropriate therapeutic strategies. Studies focusing on mechanisms underlying disease heterogeneity in HHT should
provide important insights as to how mutations in endoglin
and ALK-1 are involved in its development. In atherosclerosis, TGF-␤ appears to protect against the development of
unstable lesions by stimulating VSMCs to produce collagen
and suppressing the activity of T cells either directly or
indirectly by CD4⫹CD25⫹ regulatory T cells producing
TGF-␤. In the future, it will be of interest to determine
whether therapeutic activation of the TGF-␤ system is sufficient to alter the phenotype of an atherosclerotic lesion by
promoting the transition of an unstable lesion into a stabilized
lesion. Recently, a novel immunomodulating agent has been
introduced: FTY720. Because FTY720 mimics TGF-␤ responses, activating Smads and the expression of extracellular
matrix proteins, this compound may be useful for testing such
a hypothesis.98 In pulmonary hypertension attributable to
mutations in BMPR-II, it will be important to definitively
identify common downstream signaling mechanisms affected
by the reported mutations. Their identification could become
the staging platform for therapeutically targeting PPH in
susceptible individuals through stimulators of the pathway. In
restenosis, attenuating TGF-␤ actions in the injured vessel
wall, using novel agents such as the recently developed
inhibitors of TGF-␤ receptor kinases73 or a Smad3 inhibitor,99
may be therapeutically useful.
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Transforming Growth Factor-βs and Vascular Disorders
Alex Bobik
Arterioscler Thromb Vasc Biol. 2006;26:1712-1720; originally published online May 4, 2006;
doi: 10.1161/01.ATV.0000225287.20034.2c
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