Review

Signaling by members of the TGF-b

family in vascular morphogenesis
and disease
Evangelia Pardali, Marie-Jose Goumans and Peter ten Dijke
Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Center, The Netherlands

Members of the transforming growth factor-b (TGF-b)

family play pivotal roles in development and disease.
These cytokines elicit their pleiotropic effects on cells,
including endothelial and mural cells, through specific
type I and type II serine/threonine kinase receptors and
intracellular Smad transcription factors. This review
highlights recent progress in our understanding of
TGF-b signaling in vascular development and angiogenesis and of how perturbed TGF-b signaling might contribute to vascular pathologies, tumor angiogenesis and
tumor progression. Recent research has provided exciting insights into the role of the TGF-b type I receptor
(ALK1) in tumor angiogenesis and the curative effects of
thalidomide on vascular malformations in hereditary
hemorrhagic telangiectasia (HHT). These advances provide opportunities for the development of new therapies
for diseases with vascular abnormalities.
Introduction
Tissue homeostasis is dependent on an adequate supply of
oxygen and nutrients and removal of waste products
through blood vessels [1]. The development and proper
function of the vascular system (Box 1, Figure I) is essential for survival of all higher organisms. The vascular
system plays an important role in embryonic development
but also later during life. During embryo development the
formation of new blood vessels depends on vasculogenesis
and angiogenesis. Vasculogenesis refers to the de novo
formation of blood vessels. Angiogenesis is the formation
of new blood vessels from pre-existing ones and is controlled by a number of growth factors and signaling pathways and the balance between pro- and anti-angiogenic
factors (Box 2, Figure I) [2]. Angiogenesis takes also place
in adult life to maintain physiological homeostasis and
tissue integrity during wound healing, inflammation and
during the female menstrual cycle. Deregulation of vasculogenesis and angiogenesis has been implicated in a multitude of pathological situations.
TGF-b (TGFB13) is the prototype of the extended TGFb family of cytokines which also includes activins/inhibins,
Nodal, bone morphogenetic proteins (BMPs) and growth
differentiation factors (GDFs) [17,18]. TGF-b family members play crucial roles in embryonic development, adult
tissue homeostasis and the pathogenesis of a variety of
diseases. Research over the past two decades into the
Corresponding author: ten Dijke, P. (p.ten_dijke@lumc.nl).

556

mechanisms of TGF-b signaling has led to a well-accepted

canonical signaling cascade involving heteromeric cell-surface complexes of receptor kinases together with Smad
transcription factors (named from C. elegans Sma and
Drosophila Mad (mothers against decapentaplegic)) that
act as intracellular signaling effectors (Box 3, Figure I)
[17,18]. In addition to this highly conserved signaling core,
TGF-b family members can regulate the activity of a
number of other signaling pathways (non-Smad signaling
pathways; Box 3, Figure I) [19]. Thus, cellular responses to
TGF-b signaling result from a dynamic regulation of Smad
and non-Smad cascades.
Although several in vitro and in vivo studies provide
strong evidence for the important role of the TGF-b and
BMP (bone morphogenetic protein) signaling pathways in
vasculogenesis and angiogenesis, there is still confusion in
the field, generated by reports of opposite effects on angiogenesis by specific family members. Both pro- and antiangiogenic effects of TGF-b, BMP9 and ALK1 (activin
receptor-like kinase 1) have been reported. In addition
the role of TGF-b type I and II receptors on EC (endothelial
cell) function was questioned by some studies. Much of this
confusion stems seemingly from the remarkable diversity
and context-dependent effects of TGF-b family members on
the multistep and intricately regulated process of blood
vessel formation. Here, we review recent insights into the
role of TGF-b signaling in vascular morphogenesis and
dysfunction. The mechanisms by which TGF-b family
members control the function and interplay between endothelial and smooth muscle cells will be discussed, and how
these new advances could be exploited for restoring the
vascular bed in HHT or for anti-angiogenic therapy in
cancer.
Role of TGF-b signaling in vasculogenesis and
angiogenesis
Genetic studies in mouse and human have provided much
evidence for the importance of components of the TGF-b
signaling pathway in vascular morphogenesis and dysfunction (Table 1). Deletion of Tgfb1 in the mouse results
in embryo lethality because of defective yolk sac vasculogenesis. Interestingly, Tgfb1 deletion leads to vascular
abnormalities only in a specific genetic background,
suggesting the involvement of other factors (modifiers)
in the development of vascular abnormalities due to
defects in TGF-b signaling. Similar phenotypes have been
observed in mice deficient for Tgfbr2 and Tgfbr1 (Alk5),

0962-8924/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2010.06.006 Trends in Cell Biology 20 (2010) 556567

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Box 1. Vascular morphogenesis

During embryo development, blood vessels develop de novo
through differentiation of mesodermal progenitor cells, the hemangioblasts, into endothelial cells (ECs) that generate a primitive
vascular network in a process defined as vasculogenesis (Figure Ia)
[1]. Subsequently, maturation and remodeling of this primitive
plexus, by a process termed angiogenesis, results in a hierarchically
branched vascular system [1]. New blood vessels are formed from
this primary capillary network either by sprouting angiogenesis,
through end-to-end fusion of endothelial sprouts, or by intussusceptive vascular growth, a variant of angiogenesis in which an
individual capillary subdivides into two separate vessels (Figure Ib)
[1,3]. Vasculogenesis can also take place in adult life because
endothelial progenitor cells, by incorporating into the neovessels,
can contribute to vessel formation [4]. Maturation of nascent blood
vessels requires recruitment of mural cells [pericytes and smooth
muscle cells (SMCs)] and deposition of extracellular matrix, and this
contributes to vessel stabilization (Figure Ic) [5]. Pericytes, which
cover capillaries, provide structural support and protect ECs from

[(Box_1)TD$FIG]

apoptosis, whereas SMCs, in arteries and veins, endow the vessels

with vasomotor properties. As blood starts to flow and tissues
differentiate, the primary vascular plexus is remodeled into a
network of arteries, capillaries and veins. Both genetic mechanisms
and local environmental factors, for example hemodynamic forces
such as blood pressure and blood flow, dictate the differentiation of
a vessel towards an arterial or venous character [6,7]. VEGF, Notch
and ephrinB signaling play important roles in arteriovenous
specification (Figure Id) [8]. Angiogenesis can also occur in the
adult, in physiological settings (after wound healing, inflammation,
ischemia and during the female reproductive cycle), to maintain
physiological homeostasis and the integrity of growing or healing
tissues. Angiogenesis is tightly regulated by a balance between proand anti-angiogenic signals, including VEGF, bFGF, PDGF and TGF-b
[1]. Alteration of this equilibrium results in dysregulated vessel
growth and can result in different pathologies. In addition, angiogenesis plays a crucial role in tumor growth, and inhibition of tumor
angiogenesis can suppress tumor growth [9,10].

Figure I. Development of the vascular system. (a) During vasculogenesis, mesodermal precursors, the hemangioblasts, differentiate into ECs and form a primary
vascular plexus. (b) Angiogenesis involves the formation of new vessels from pre-existing ones, either by sprouting angiogenesis or by intussusceptive angiogenesis,
and is regulated by several angiogenic factors including VEGF, the angiopoietin system, PDGF and TGF-b. (c) Maturation and stabilization of the nascent plexus relies on
the recruitment of pericytes and SMCs and deposition of extracellular matrix under the control of the coordinated action of PDGF, Ang2 (angiopoietin 2) and TGF-b
signaling. (d) Finally, the primary vascular plexus is remodeled into a network of arteries, capillaries and veins. VEGF, Notch and ephrinB signaling play an important
role in arteriovenous specification.

suggesting an important role for these receptors in EC

function. Recent studies suggested that the effects of
TGFBR2 or TGFBR1 loss on angiogenesis are not due to
their role on ECs but are due to defects in smooth muscle
cell (SMC) function [27]. Using an Acvrl1 (Alk1)-derived
promoter to delete Tgfbr2 or Tgfbr1 in ECs, no effects on
vascular morphogenesis were observed. In addition, it was
suggested that ALK5 is expressed only on SMCs but not in
ECs [28]. By using the tyrosine-protein kinase 1 Tie1
vascular EC-specific promoter, another group has clearly
shown that both receptors play important roles in EC
function [29,30]. Tie-1 is a vascular endothelial-specific
receptor tyrosine kinase essential for the regulation of
vascular network formation and remodeling. EC deletion
of Tgfbr2 or Tgfbr1 results in embryo lethality at embryonic day (E) 10.5 due to vascular defects. The discrepancies
between the two studies are most probably attributable to
temporal regulation of the promoters used. These results
further support the notion that the role of TGF-b signaling
and its receptors is context-dependent.
Targeted deletions of Acvrl1 (Alk1), Tgfbr1 (Alk5),
Tgfbr2 and Eng (endoglin, a TGF-b coreceptor highly
expressed on EC) in mice result in vascular abnormalities

highly reminiscent of those described in patients with HHT

[20], an autosomal dominant vascular disorder characterized by fragile blood vessels which lead to telangiectases
and arteriovenous malformations (AVMs). HHT-1 and
HHT-2 arise from heterozygous mutations in ENG and
ACVRL1 (ALK1) genes, respectively. A subset of patients
with juvenile polyposis, carrying mutations in the SMAD4
gene, can also develop HHT [31]. It has been postulated
that genetic background and/or environmental factors (second hits), in addition to the genetic mutations in the ENG
or ACVRL1 genes, play an important role in the development of vascular malformations in HHT patients. Park and
colleagues demonstrated recently using Acvrl1-deficient
mice that a second hit, excisional skin wounding, is essential for the development of AVMs in HHT. Their results
provide new insights for understanding the pathogenesis
of HHT [32].
Despite the identification of the genes responsible for
HHT, the underlying molecular mechanisms for the pathogenesis of HHT remain obscure. Genetic studies in mouse
demonstrated that deletion of Acvrl1 (Alk1) or Eng results
in loss of arteriovenous specification and the development
of AVMs between major arteries and veins [33]. Zebrafish
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Box 2. Angiogenesis
Sprouting angiogenesis comprises two phases: activation and
resolution (Figure I) [10]. The activation phase is characterized by
changes in EC shape, EC junction rearrangement, degradation of the
basement membrane (BM) and extracellular matrix, detachment of
pericytes, destabilization of the vessel, and increased permeability
(Figure Ib). Activated ECs start to proliferate and migrate into the
perivascular space towards the angiogenic stimulus. During the
resolution phase, ECs stop proliferating and migrating, SMCs and
pericytes are recruited to the new sprout, and the BM reconstitutes to
ensure stabilization and maturation of the newly formed vessels
(Figure Ic). Finally, blood vessels become quiescent (Figure Id).
Recent advances in vascular biology have suggested that specialized ECs with distinct cellular specifications and functions contribute
to the formation of new blood vessels. During the sprouting process a
selected EC, the tip cell, leads each sprout [11]. Tip cells are
migratory cells that do not proliferate, instead they sense the
angiogenic stimulus and invade the surrounding tissue by extending
numerous filopodia. Tip cells have a changed polarity and do not
form a lumen (Figure Ib). Tip cells rely on the stalk cells, the ECs that
follow the tip cell; these can proliferate and form lumens because they
are fully polarized, but do not form many filopodia [11,12]. Stalk-cell
proliferation ensures sprout elongation and the recruitment of
support cells. Finally, the newly formed branch connects with another
branch by means of tip-cell to tip-cell fusion, and a new vascular

[(Box_2)TD$FIG]

lumen is formed. Both genetic and environmental factors regulate

tip-/stalk-cell specialization. Tip-cell migration and filopodia formation
depend on VEGF gradients and increased VEGFR2 and PDGFB
expression [11,13]. Stalk-cell proliferation depends on VEGF concentration and low-affinity VEGFR2 signaling [13]. Deltalike ligand 4
(Dll4)/Notch signaling plays an important role in the specification of
ECs. High expression of Dll4 by the tip cells results in increased Notch
signaling in the stalk cells, and consequently in reduced VEGFR2
expression in the stalk cells [14,15]. Finally, ECs acquire a quiescent
phenotype and become phalanx EC. Phalanx cells do not migrate and
proliferate but contribute to vessel stabilization by depositing a
basement membrane (Figure Id). The molecular mechanisms underlying the endothelial phalanx phenotype remain to be explored.
Although ECs are the major players in angiogenesis, they require
mural cell support to complete mature vessel formation. Interactions
between endothelial and mural cells play an important role in proper
vessel assembly, and failure of these interactions will result in severe
vascular defects. Crosstalk between endothelial and mural cells is
under the control of several signaling pathways. Signaling by PDGFB/
PDGFRb acts in an endothelial-to-pericyte paracrine fashion and is
necessary for pericyte recruitment [5], whereas the angiopoietinTie2
signaling pathway acts in the opposite orientation from mural cells
to the endothelium and plays an important role in vessel
stabilization [5,16].

Figure I. Regulation of angiogenesis. Angiogenesis comprises two phases, the activation and the resolution phase. Following EC activation by an angiogenic stimulus
(VEGF, bFGF, TGF-b) (Figure Ia), BM is degraded and the tip cell at the forefront of the sprout invades the surrounding tissue by extending numerous filopodia (Figure
Ib). The new sprouts elongate through proliferation of the stalk cells and the new branches connect through tip-celltip-cell fusion (Figure Ib). Finally, ECs cease
proliferation and sprout maturation occurs by reconstitution of BM and pericyte/SMC recruitment (Figure Ic) and acquire a quiescent phenotype (phalanx EC) (Figure Id).

acvrl1 mutants (violet beauregarde, or vbg) display a

similar phenotype to that seen in Acvrl1 null mice with
dilated vessels and enlarged cranial arteries that abnormally connect directly to veins [34]. Acvrl1 deficiency
558

results in decreased expression of the arterial marker

ephrin B2, an Eph receptor ligand, known to be associated
with arterial identity. Interestingly, ephrin B2 is
expressed normally in Eng-deficient mice, suggesting that

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Box 3. TGF-b signaling

Members of the TGF-b family of proteins are generated as inactive
precursor dimers that are subsequently cleaved by proteases, and this
determines the bioavailability of TGF-b ligands for their receptors
[17,18,20]. BMPs are secreted in an active form, and their bioavailability is regulated through reversible interactions with extracellular
antagonists [21].
Canonical Smad signaling pathway: TGF-b family members elicit
their cellular effects by inducing heterotetrameric complexes of type I
and type II serine/threonine-kinase transmembrane receptors. Five
type II receptors and seven type I receptors, also termed activin
receptor-like kinases (ALKs) have been identified. TGF-b signals in
most cells through the TGF-b type I (TGFBR1/ALK5) and type II
(TGFBR2) receptors, and activins through activin receptors types IIA
(ACVR2A) and IIB and ALK4, and BMPs through BMP type II receptor
(BMPR2), ACVR2 s and ALK1, 2, 3 and 6 [17,18,20]. There are two
accessory receptors endoglin and betaglycan that regulate ligand
receptor interactions [20,22].
Upon ligand binding the constitutively active type II receptor
phosphorylates the type I receptor on specific serine and threonine
residues in the intracellular juxtamembrane region. Smad proteins

[(Box_3)TD$FIG]

are intracellular mediators for the TGF-b family and are classified into
three groups: receptor-regulated (R-Smad), common-mediator (CoSmad), and inhibitory (I-Smad) [17,18]. Upon activation, the type I
receptor recruits and phosphorylates R-Smads at two serine residues
in their extreme C-termini. ALK4 and 5 (and 7) mediate phosphorylation of R-Smads 2 and 3, whereas ALK1,2,3,6 mediate phosphorylation of R-Smad1,5,8. Activated R-Smads interact with Smad4 and
translocate into the nucleus, where, together with other transcription
factors, they regulate target gene expression (Figure I). I-Smads
(Smads 6,7) can inhibit the activation of R-Smads by competing with
R-Smads for type I receptor interaction and by recruiting specific
ubiquitin ligases or phosphatases to the activated receptor complex,
thereby targeting it for proteasomal degradation or dephosphorylation, respectively (Figure I) [17,18,20].
Non-Smad signaling pathway: TGF-b and BMP receptor activation
results in activation of several other non-Smad signaling pathways in
a context-dependent manner. Non-Smad signaling pathways can
involve the TGF-b-activated kinase-1 (TAK-1), ERK, JNK, p38, Rho
GTPases and the PI3KAKT pathway, and these can crosstalk with the
Smad pathways (Figure I) [19].

Figure I. Signal transduction by TGF-b family members. TGF-b and BMP dimers induce heteromeric complex formation between specific type II and type I receptors.
The type II receptors then transphosphorylate the type I receptors, leading to their activation. Subsequently, the type I receptor propagates the signal into the cell by
phosphorylating R-Smads, which then form heteromeric complexes with Smad4 (Co-Smad). These Smad complexes translocate in the nucleus where by interacting
with other transcription factors they can regulate gene transcriptional responses (canonical Smad signaling pathway). I-Smads 6 and 7 inhibit receptor activation of RSmads. In addition, the activated type I receptors can activate non-Smad pathways (non-Smad signaling pathway). ALK, activin receptor-like kinase; BMP, bone
morphogenetic protein; BMPR, BMP receptor; ERK, early response kinase; PI3K, phosphoinositide 3-kinase; TAK, TGF-b-activated kinase; TGF-b, transforming growth
factor b; TGFBR, TGF-b receptor.

other proteins are involved [33]. The Notch family of

receptors and their ligands are expressed in arterial
ECs, promote artery-specific ephrin B2 expression and
have been implicated as potential regulators of arteriovenous fate. However, no expression differences were
observed in Acvrl1- or Eng-deficient embryos based on in
situ hybridization of Notch signaling-pathway-related

genes [33]. Paradoxically, although Eng deletion in mouse

embryos results in arterial expression of the venousspecific marker COUPTFII (chicken ovalbumin upstream
promoter-transcription factor II) [35], endothelial-cellspecific deletion of endoglin did not affect expression of
arterial jagged-1 and ephrin B2, or venous markers Ephb4
and Aplnr (G protein-coupled apelin receptor) in neonatal
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Table 1. Defects in components of TGF-b signaling pathways lead to vascular abnormalities in human and mouse
Gene (mouse/human)
Ligands
Tgfb1/TGFB1

Tgfb2/TGFB2
Tgfb3/TGFB3
Receptors
Tgfbr2/TGFBR2
SM22-Cre-Tgfb2fl/fl
Tie1-Cre-Tgfbr2fl/fl
Tgfbr1 (Alk5)/TGFBR1 (ALK5)
Tie1-Cre-Tgfbr1fl/fl
Acvrl1 (Alk1) / ACVRL1 (ALK1)
Bmpr2/BMPR2

Accessory receptors
Eng/ENG (endoglin)

Soluble endoglin
Tgfbr3/TGFBR3 (betaglycan)
Smads
Smad1/SMAD1
Smad4/SMAD4
Smad5/SMAD5
Smad6/SMAD6
Smad7/SMAD7

Animal model

Human disease

Refs

KO: embryonic lethal with vascular defects or

postnatal lethality from autoimmune disease

Camurati
Engelmann
disease a
unknown

[20]

unknown

[20]

MFS2a, LDS a

HHT b

[20]
[20]
[20]
[20]
[20]
[20]

PAH b

[20,23,24]

HHT

[20]

Pre-eclampsia
Unknown

[25]
[20]

Unknown

[20]

JPb and HHT

Unknown

[20]
[20]

Unknown

[20]

Unknown

[26]

KO: aortic arch defects, cardiac septal defects,

perinatal lethality
KO: cleft palate, delayed lung maturation,
die shortly after birth
KO: embryonic lethal, vascular defects
KO: embryonic lethal, vascular defects
KO: embryonic lethal, vascular defects
KO: embryonic lethal, angiogenesis defects
KO: embryonic lethal, angiogenesis defects
KO: embryonic lethal, reduced VSMC
differentiation, dilated vessels, AVMs.
KO: embryonic lethal (pre-angiogenesis)
lethality. Transgenic BMPR2-mutant allele:
pulmonary hypertension
KO: embryonic lethal due to vascular defects,
reduced VSMC differentiation, heart defects.
Het: vascular lesions similar to HHT
KO: poorly formed cardiac septa, incomplete
compaction of ventricular walls
KO: embryonic lethal due to defects in
chorioallantoic circulation
KO: embryonic lethal
KO: embryonic lethal due to angiogenesis
defects
KO: heart abnormalities, aortic ossification
and elevated blood pressure
KO: embryonic lethal due to cardiovascular
defects

LDS

[20]

Abbreviations: AVMs arteriovenous malformations; Het heterozygote; JP Juvenile polyposis; KO knockout; LDS LoeysDietz syndrome; MFS2 Marfan syndrome type 2; HHT
hereditary hemorrhagic telangiectasia; PAH pulmonary arterial hypertension; VSMC vascular smooth muscle cell.
a
Due to hyperactivation of TGF-b signaling.
b
Due to attenuated TGF-b signaling.

retinas [36]. Interestingly, AVMs in these mice expressed

Ephb4 and Aplnr, suggesting that they have venous
characteristics [36]. Previous studies have suggested the
existence of synergism between Notch and TGF-b [37,38]
or BMP6 signaling [39], and that Notch signaling modulates the balance between TGF-b/ALK1 and TGF-b/ALK5
signaling pathways [40]. Future studies are awaited to
elucidate the crosstalk between ALK1/endoglin and other
signaling pathways involved in arteriovenous fate. Failure
to establish or maintain proper arterialvenous boundaries might be related to abnormalities in sprouting
mechanisms during angiogenesis and in tip/stalk EC
determination. Endoglin has been shown to be expressed
in tip cells and in tip cell filopodia. Endothelial-cell-specific
Eng depletion did not affect filopodia formation in the tip
cells or the numbers of filopodia in tip and stalk ECs in
neonatal mouse retinas [36]. Future research is needed to
further characterize the exact role of endoglin and ALK1 in
arteriovenous specification and tip, stalk and phalanx EC
function. The molecular pathways by which Eng and
Acvrl1 deficiency lead to AVM are still not known, and
their crosstalk with Notch, ephrinB or additional factors
involved in the molecular determination of vein identity
remains to be elucidated.
560

Vascular endothelial growth factor (VEGF) signaling

plays a crucial role in angiogenesis and its dysregulation
leads to defects in angiogenesis. It has been shown that
VEGF levels are elevated in skin telangiectatic lesions of
HHT patients and that anti-angiogenic drugs, such as
thalidomide and bevacizumab (anti-VEGF antibody), are
effective in treating gastrointestinal bleedings and liver
AVMs, respectively [4143]. By contrast, inhibition of
ALK1/endoglin signaling using the soluble chimeric
proteins ALK1-Fc and endoglin-Fc (containing either the
ALK1 or the endoglin extracellular domains fused to the Fc
part of IgG that sequester ligands from binding to endogenous endoglin and ALK1) hindered VEGF-induced EC
sprouting in vitro [44,45]. ALK1-Fc also inhibited VEGF/
basic fibroblast growth factor (bFGF)-induced angiogenesis in an in vivo matrigel plug assay [44]. Although these
results clearly suggest that there is crosstalk between
VEGF and ALK1/endoglin signaling in angiogenesis, the
exact molecular mechanisms underlying this interplay
remain to be elucidated.
TGF-b signaling and endothelial cell function
Several studies have revealed that the effect of TGF-b on
angiogenesis is context dependent [46,47]. TGF-b was

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Vol.20 No.9

Figure 1. A working model for TGF-b and BMP9 signaling in ECs. TGF-b signals through two distinct pathways in ECs. TGF-b binds to TGFBR2, and this subsequently
recruits and phosphorylates TGFBR1 (ALK5) and ACVRL1 (ALK1) in a common complex. Activated ALK5 recruits and phosphorylates Smad2,3, whereas ALK1 induces
Smad1,5 phosphorylation, resulting in activation of ALK5- and ALK1-specific target genes, respectively. ALK1 and ALK5 have opposite effects on EC migration and
proliferation. Endoglin is needed for efficient TGF-b/ALK1 signaling, whereas ALK1 can indirectly inhibit ALK5-induced Smad-dependent transcriptional responses. BMP9
can induce both Smad1 and Smad2 phosphorylation in ECs through the BMPR2/ ACVR2/ALK1,2 pathways. ALK, activin receptor-like kinase; TGF-b, transforming growth
factor b; TGFBR, TGFb receptor.

shown to promote EC proliferation and migration at low

concentrations, whereas high concentrations had the opposite effect [4649]. Low concentrations of TGF-b enhanced
the angiogenic effects of bFGF or VEGF in a 3D fibrin or
collagen assay, whereas high concentrations were inhibitory
[48]. Treatment of bovine capillary endothelial (BCE) cells
with TGF-b initially induces apoptosis by inducing VEGF
expression because TGF-b signaling converts the VEGF/
VEGFR2-activated p38 (MAPK) into a proapoptotic signal
[50]. However, protracted treatment of BCE cells with TGFb results in EC remodeling and formation of cord-like structures [51]. Inhibition of the TGF-bALK5 pathway by an
ALK5 kinase inhibitor resulted in sustained proliferation
and maintenance of human embryonic stem cell (hESC)derived ECs by sustaining Id1 expression [52]. Similarly,
addition of an ALK5 kinase inhibitor in mouse ESCs
increased EC growth and integrity through upregulation
of the tight junction component claudin-5 [53]. It was also
shown that suboptimal doses of VEGF and ALK5 kinase
inhibitor synergistically induce EC migration and sprouting
in vitro by inducing integrin a5 expression. In addition,
ALK5 kinase inhibition induced angiogenesis and enhanced
VEGF/bFGF-induced angiogenesis in a matrigel-plug assay
in vivo, an effect that could be inhibited by an antibody that
neutralizes the a5 integrin [54].
The initial view of TGF-b family signaling as a simple
linear cascade, where TGF-b/Nodal/activin induce phosphorylation of Smads 2 and 3 and BMP/GDFs induce
phosphorylation of Smads 1, 5 and 8, has been re-evaluated. Studies on ECs revealed that TGF-b can bind to and

signal through two distinct types of receptors in these cells

TGFBR1 (ALK5) and ACVRL1 (ALK1) resulting in
activation of Smad2,3 and Smad1,5,8, respectively
(Figure 1) [47,55]. Although TGF-b/ALK5/Smad2,3 signaling was found to antagonize TGF-b/ALK1/Smad1 signaling, ALK5 is essential for ALK1 recruitment into the
TGF-b receptor complex and for its activation. It was
shown that TGF-b/ALK1 signaling potentiates and TGFb/ALK5 inhibits EC proliferation and migration of ECs
[47,55]. It was thus suggested that the balance between
TGF-b/ALK1 versus TGF-b/ALK5 will determine the proor anti-angiogenic effects of TGF-b. In addition to being
present in ECs, the TGF-b/ALK1 pathway has also been
observed in neurons, chondrocytes and hepatic stellate
cells [5658]. Moreover, in tumor cells, TGF-b was shown
to signal by means of the Smad1 pathway. In one of the
studies, TGF-b induced Smad1 phosphorylation through
ALK2 and ALK3 [59], whereas two studies suggested that
ALK5 can directly interact with and phosphorylate Smad1
[60,61]. The mechanistic differences between these studies
are probably attributable to different receptors expressed
on different cell types as well as the relative expression
levels of each receptor.
Although the studies discussed above suggest an
important role for ALK1 in the activation phase of angiogenesis, overexpression studies demonstrated that ALK1
signaling inhibits the proliferation and migration of a
human microvascular EC line, implying that ALK1
promotes the resolution phase of angiogenesis [62], consistent with the phenotype described in Alk1-deficient mice
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fragile blood vessels and increased mRNA levels for genes
involved in the activation phase of angiogenesis [23]. The
discrepancies could be due to different cell types being used
in the studies or to the adaptive processes that take place
in the Alk1-deficient embryos. Alternatively, ALK1 could
be involved in both the activation and resolution phases of
angiogenesis.
In ECs, BMP9 and BMP10 have been shown to signal
through ALK1 and ALK2, induce Smad1 phosphorylation,
and inhibit EC proliferation and migration [63,64]. In
human pulmonary artery endothelial cells (HPAECs),
BMP9 was shown to induce phosphorylation of Smad1-5
and Smad2 through BMPR2/ALK1 and the activin type II
receptors (ACVR2) [65] (Figure 1). Although both BMPR2
and ACVR2 were required for Smad1 phosphorylation,
Smad2 activation was mainly mediated through ACVR2.
The significance of this differential BMP9 signaling in
angiogenesis and in vascular pathologies such as HHT
and primary arterial hypertension (PAH, a vascular disorder associated with missregulated BMP signaling due to
mutations in BMPR2) remains to be elucidated. BMP9
signaling through ALK1 was shown to inhibit VEGF
expression [66], whereas increased VEGF levels were
reported in Acvrl1-deficient embryos and HHT-2 patients
[67,68]. Although BMP9 and BMP10 were considered to
have an anti-angiogenic action, BMP10 was shown to
induce angiogenesis in the chorioallantoic membrane
(CAM) assay [69] and BMP9 to induce proliferation of
ECs in vitro and in vivo [70]. In addition, BMP9 in combination with TGF-b was shown to potentiate VEGFinduced proliferation of ECs in vitro and VEGF/bFGFinduced angiogenesis in vivo [44].
Several studies have provided evidence that the TGF-b
coreceptor endoglin plays an important role in balancing
the TGF-bALK1 and TGF-bALK5 pathways [22]. Endoglin was shown to potentiate the TGF-bALK1 pathway
and to inhibit the TGF-bALK5 pathway in ECs as well as
in other cell types [71]. It was also shown that endoglin
plays an important role in BMP9 signaling in ECs [63]. The
molecular mechanisms by which endoglin regulates TGF-b
and BMP signaling are not fully understood. A recent study
suggested that ALK5 phosphorylates the cytoplasmic
domain of endoglin on serines 646 and 649. S646A is
required for activation of TGF-bALK1Smad1,5,8 signaling, whereas both S646 and S649 are essential for
BMP9-induced Smad1,5,8 phosphorylation [72]. Analysis
of mouse retinal vessels showed that endothelial-cellspecific deletion of Eng does not affect levels of phosphorylated Smad2 or Smad1,5,8 [36]; however, deletion of Eng
results in loss of Smad1,5,8 phosphorylation in the quiescent pulmonary vasculature [73]. As with ALK1, the role of
endoglin in EC function is not completely understood; in
vitro studies have suggested that knockdown of endoglin
results in decreased EC proliferation and migration,
whereas Eng deletion in the mouse results in a dramatic
increase in EC proliferation [36].
BMPs 2, 4, 6 and 7 have been shown to induce EC
proliferation and angiogenesis by inducing VEGF expression [46,74]. In mouse embryonic stem cells (ESCs), BMP4
exerts its angiogenic effects by activating the VEGF/
VEGFR2 and angiopoietin-1/Tie2 signaling cascades in
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addition to the Smad signaling pathway because BMP4

induces phosphorylation not only of Smad1 but also Tie2
and VEGFR2 in these cells [75]. The pro-angiogenic effects
of BMP6 have been shown to be mediated by the upregulation of Id1 and cyclooxygenase-2 expression in ECs [76,77].
Recently it was also shown that Myo10 plays a crucial role
in BMP6-induced angiogenesis. Myo10 is upregulated in
EC in response to BMP6 and that it is required for BMP6induced filopodia formation and migration [78].
Although it is apparent that TGF-b and BMP signaling
components play crucial roles in EC function and angiogenesis, we have only started to unravel the molecular
mechanisms by which these molecules regulate vascular
system. Cellular context, local concentration of the different ligands, receptors, coreceptors, antagonists and their
interplay play crucial roles in the apparently contradictory
actions of TGF-b signaling during the different stages of
angiogenesis. The identification and characterization of
additional molecules and mechanisms involved in vascular
signaling by TGF-b family members will help us to understand what determines the pro- or anti-angiogenic functions of TGF-b, ALK1 and BMP9, and their interplay with
other factors involved in EC function and angiogenesis.
TGF-b signaling and SMC function
Vessel formation and stabilization cannot be completed
unless pericytes and vascular SMCs are recruited to complete vessel assembly. Proper communications between
ECs and mural cells play a crucial role in vessel formation,
and these are tightly controlled by a number of signaling
pathways [16] (Boxes 1, 2).
TGF-b regulates SMC muscle differentiation by increasing the expression of alpha smooth-muscle actin and
smooth-muscle myosin through the Smad3 and p38/MAPK
pathways [79]. TGF-b treatment of ESC-derived cultures
or embryoid bodies potentiates their differentiation into
SMCs [53,80]. TGF-b can also induce proliferation and
migration of SMCs. Genetic studies in the mouse
(Table 1) revealed the important role of TGF-b signaling
in SMC cell development and recruitment for the formation of stable vessels. EC- and SMC-specific deletions
of Tgfbr2 show similar phenotypes. Deletion of Tgfbr2 in
SMCs results in vascular defects in the yolk sac and
embryo lethality between E12.5 and E16.5. Those results
suggest that TGF-b signaling on ECs and SMCs plays an
important role in SMC differentiation and function and, as
a consequence, in proper ECSMC interaction [29,30]. In
addition, mice with neural-crest-specific ablation of Tgfbr2
develop a phenotype akin to DiGeorge syndrome because
neural crest derivatives fail to differentiate into SMCs in
the cardiac outflow tract [81].
Both Eng and Acvrl1 (Alk1) depletion result in fragile
blood vessels due to impaired mural cell development, as
shown by the absence or inappropriate association of SMCs
with ECs [33,8284]. Conditional endoglin expression in
Eng-null embryos, using either SMC- or EC-specific promoters, can partially rescue SMC recruitment to the dorsal
aorta, suggesting that endoglin plays distinct and cellautonomous roles in SMC recruitment [35]. Telangiectatic
lesions in HHT patients are characterized by abnormal
endothelial cell proliferation and SMC recruitment.

Review
Thalidomide treatment of HHT patients was shown to
enhance blood vessel stabilization and reduce nosebleed
frequency [85]. Vessel maturation induced by thalidomide
took place by mural cell recruitment in Eng-heterozygous
mice and HHT patients, partly by inducing the expression
of the platelet-derived growth factor B subunit (PDGFB) in
ECs, further supporting the important role of endoglin in
ECSMC interactions [85].
BMPs also play a role in SMC differentiation and function. BMP7 inhibits SMC growth induced by PDGF-BB
(the dimer of PDFGB) and by TGF-b1, whereas it maintains the expression of markers that maintain the SMC
phenotype [86]. BMP2 was shown to induce SMC
migration and to inhibit PDGF-induced proliferation of
SMCs [87]. BMP pathway activation through BMPR2 is
necessary for growth and differentiation control in SMCs
[88,89]. Mutations in BMPR2 result in PAH, a vascular
disorder characterized by uncontrolled remodeling of the
pulmonary arteries due to increased proliferation of SMCs
and increased pulmonary EC apoptosis [24]. Interestingly,
certain HHT2 patients develop a PAH-like syndrome,
suggesting that ACVRL1 (ALK1) mutations are also likely
to be involved in PAH [90,91].
In summary, both TGF-b and BMP signaling play crucial roles in the regulation of SMC function and in proper
ECSMCs interactions, and disruption of these pathways
in SMCs leads to vascular abnormalities. Vascular defects
due to misregulated TGF-b and BMP pathways might not
only be due to their direct effects on SMC function, but also
due to effects on other signaling pathways such as the
PDGF pathway, or to disruption of the balance between
TGF-b and BMP signals. It has been suggested that loss of
BMPR2 could lead to unregulated TGF-b/ALK5 activity in
SMCs from patients with idiopathic PAH and this might be
important in mediating disease progression [92]. Interestingly, systemic inhibition of TGF-b/ALK5 signaling significantly reversed pulmonary arterial pressure in a model of

[(Figure_2)TD$IG]

Trends in Cell Biology

Vol.20 No.9

experimental PAH, thus providing new strategies for disease management. Further characterization of the molecular mechanisms by which TGF-b family members
regulate SMC function and ECSMC interaction could
provide us with targets for the development of new therapeutic strategies against vascular abnormalities.
Targeting TGF-b signaling in tumor angiogenesis
Tumor angiogenesis plays a crucial role in tumor initiation,
progression and metastasis (Figure 2). Several studies
have focused on the molecular characterization of tumor
angiogenesis for the development of anti-angiogenic agents
for cancer therapy [10,93]. TGF-b signaling plays an
important role in tumor growth and metastasis [46].
Increased TGF-b expression has been reported in many
cancers, and such expression was shown to correlate with
poor prognosis, increased tumor growth and angiogenesis,
whereas administration of TGF-b inhibitors strongly
reduced tumor angiogenesis and tumor growth. TGF-b
signaling antagonists are currently used to prevent growth
and metastasis of certain cancers [46]. However, several
studies have suggested that inhibition of TGF-b signaling
can promote tumor angiogenesis [46]. A combination of
VEGF and a TGFBR1 (ALK5) kinase inhibitor synergistically promoted angiogenesis [54]. Therefore, anti-TGF-bbased therapeutic strategies must be carefully considered
before administration because there could be adverse
effects, such as induction of tumor angiogenesis and tumor
growth. Inhibitors of ALK5 kinase block signaling of both
Smad2 and Smad3. However, recent studies suggested
that Smad2 has tumor-suppressor and anti-metastatic
activities and inhibits angiogenesis, whereas Smad3 plays
an important role in stimulating tumor growth and metastasis in part by inducing VEGF expression and promoting
tumor angiogenesis [94]. Thus, selective targeting of
Smad3 in tumor cells, for example by halofuginone [95]
or small interfering RNAs, could lead to more effective

Figure 2. Targeting TGF-b signaling in tumor angiogenesis. Tumors cannot grow to more than 12 mm3 if supply of oxygen and nutrients is limited. Many tumors can be
dormant for years. Tumor angiogenesis is essential to escape this period of dormancy. This process, also known as the angiogenic switch, is regulated by a variety of proand anti-angiogenic factors such as VEGF, bFGF, P1GF, TGF-b and BMP. Endoglin and ALK1 play important roles in tumor angiogenesis and tumor growth. Genetic deletion
or inhibition of endoglin and ALK1 function by endoglin-neutralizing antibodies or by ALK1-Fc (an ALK1 ligand trap) results in reduced tumor angiogenesis and tumor
growth. ECD, extracellular domain; Fc, antibody Fc region.

563

Review
therapeutic responses against tumor growth and tumor
angiogenesis.
Endoglin is upregulated in the tumor-associated endothelium and its expression correlates with poor prognosis
[22]. Several studies have considered endoglin as a therapeutic target in anti-angiogenic therapies because tumor
vascularization and growth are diminished in Eng-heterozygous mice [22]. Endoglin-neutralizing antibodies can
target tumor vasculature and inhibit tumor growth in
mouse tumor models (Figure 2) [22]. Recent studies
suggested that soluble endoglin (sEng) can interfere with
the function of endogenous endoglin on ECs and inhibit
spontaneous cord formation in human umbilical vein endothelial cells (HUVECs) and VEGF-induced EC sprout formation [25,45]. These results suggest that sEng has great
potential in anti-angiogenic cancer therapy by interfering
with tumor angiogenesis and tumor growth.
The exact role of ALK1 in angiogenesis is not fully
understood. Although some studies suggest that ALK1
inhibits EC proliferation, and perturbation of ALK1 signaling results in increased VEGF signaling and enhanced
angiogenesis [23,62,66], ALK1 was also shown to promote
EC proliferation and migration [55]. Recent studies have
revealed an important role for ALK1 in tumor angiogenesis
and growth [44]. Expression analysis in mice suggested
that expression of ALK1 and its ligands TGF-b and BMP9
is increased during tumor growth. Deletion of one Acvrl1
(Alk1) allele resulted in reduced tumor growth and progression by inhibition of angiogenesis in the RIP1-Tag2
transgenic mouse model of multistep tumorigenesis [44].
Pharmacological inhibition of ALK1 signaling using an
ALK1-ligand trap (ALK1-Fc), resulted in reduced tumor
angiogenesis and tumor growth in the RIP1-Tag2 transgenic mouse model of pancreatic islet carcinomas as well as
in a breast cancer orthotopic tumor model [44,69]. In
addition, ALK1-Fc treatment of RIP-Tag2 mice resulted
in increased pericyte coverage of tumor vessels [44]. ALK1Fc inhibits tumor angiogenesis by interfering with the
angiogenic activity of proangiogenic factors such as VEGF
and bFGF (Figure 2). Interestingly, TGF-b and BMP9 can
synergistically induce the pro-angiogenic effects of VEGF
and bFGF. ALK1-Fc could efficiently interfere with this
synergistic effect both in vitro and in vivo [44]. Those
results suggest that ALK1 provides a valuable target for
anti-angiogenic therapy and that ALK1-Fc is a powerful
anti-angiogenic agent capable of reducing tumor angiogenesis and tumor growth (Figure 2). The therapeutic potential of human ALK1-Fc and humanized ALK1 and
endoglin-neutralizing antibodies is currently being evaluated in clinical cancer trials [9698].
Concluding remarks
The role of TGF-b signaling in angiogenesis has been highly
controversial, with numerous studies showing that it is
either pro-angiogenic or, conversely, anti-angiogenic in a
context-dependent manner. Recent studies emphasize the
growing appreciation that the pleiotropic effects of TGF-b
signaling are the outcome of multiple and fine-tuned signaling cascades rather than the result of a simple linear
signal-transduction pathway. Some of these discrepancies
might be explained by variations in ligand concentrations,
564

Trends in Cell Biology Vol.20 No.9

receptor and downstream signaling component expression;

in addition, the same ligand, such as TGF-b, can induce
opposing effects by activating different classes of Smads
through the formation of diverse receptor complexes
(Figure 1). Interestingly, this signaling flexibility is not
restricted to ECs but applies to other types of cells. BMP9
was also shown to activate different classes of Smad proteins
(Figure 1). The exact role of BMP9-induced Smad1 or Smad2
phosphorylation in angiogenesis remains to be elucidated.
Misregulated TGF-b signaling results in vascular
defects, and the phenotypes of mice lacking different components of the TGF-b and BMP signaling pathways are
quite similar, suggesting that they might act in concert to
regulate vessel formation in vivo. TGF-b ligands regulate
angiogenesis through their actions either on ECs and/or on
mural cells, demonstrating that they play important roles
in both the activation and resolution phases of angiogenesis. This can explain the contradictory results of different
studies on the role in angiogenesis of endoglin, ALK1 and
ALK5. In addition, experimental evidence suggests that, in
different phases of the multistep angiogenesis process
(normal and pathological), there is differential expression
TGF-b family members [44,46]. This could explain the
controversial results of different studies where TGF-b
signaling components exhibit distinct effects on angiogenesis depending on the angiogenic microenvironment. Separate treatment with TGF-b or BMP9 inhibits EC
proliferation, whereas the combination of the two factors
can enhance EC proliferation. Mutations in ENG and
ACVRL1 lead to vascular abnormalities in HHT patients
due to increased EC proliferation and impaired SMC
recruitment. However, deletion of Eng and Acvrl1 in the
mouse results in reduced tumor angiogenesis and tumor
growth. It could also involve synergistic and/or antagonistic interactions of TGF-bBMP signaling with other signaling pathways such as VEGF, PDGF and Notch. The
combination of TGF-b and BMP9 enhances VEGF/bFGFinduced angiogenesis whereas TGF-b inhibits it. The crosstalk with these pathways is only partly understood, and
future studies using in vitro and in vivo systems of angiogenesis will be invaluable in elucidating these interactions.
Although there have been new insights into the role of
TGF-b signaling in vascular development and function,
the exact mechanisms by which TGF-b family members
regulate angiogenesis are still not fully understood. Many
questions remain to be answered and additional studies
are required (Box 4) to explain the contradictory but

Box 4. Questions for future research

 What determines the pro- and anti-angiogenic effects of TGF-b
family members? How can different ligand concentrations have
different effects on EC function and activation?
 Are TGF-b family members and their receptors involved in both
the activation and resolution phases of angiogenesis? How are
their effects influenced by the angiogenic microenvironment?
 How do mutations in ENG and ACVRL1 lead to AVMs in HHT?
What is the role of modifier genes and what are the second hits in
the development of HHT?
 Can the anti-tumorigenic effects of endoglin- and ALK1-inhibitors
be enhanced by combination therapy with anti-angiogenic agents
or conventional chemotherapeutic drugs?

Review
intriguing role of TGF-b signaling in angiogenesis. Understanding the molecular mechanisms by which TGF-b
signaling exerts its diverse, context-dependent effects on
angiogenesis will enable us to develop new therapeutic
interventions to manage pathological vascular malformations, tumor angiogenesis and tumor growth.
Acknowledgements
Research in our laboratories is supported by grants from the Netherlands
Organization for Scientific Research, the Dutch Cancer Society, the
Ludwig Institute for Cancer, the Netherlands Heart foundation
(2009B063), the Leducq foundation and the Centre for Biomedical
Genetics. We apologize to those whose work has not been cited because
of space limitations.

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