Advances in the Cellular and Molecular Biology of Angiogenesis
Angiogenesis regulation by TGFβ signalling: clues
from an inherited vascular disease
Marwa Mahmoud*, Paul D. Upton† and Helen M. Arthur*1
*Institute of Genetic Medicine, Centre for Life, Newcastle University, Newcastle upon Tyne NE1 3BZ, U.K., and †Division of Respiratory Medicine, Department
of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QQ, U.K.
Abstract
Studies of rare genetic diseases frequently reveal genes that are fundamental to life, and the familial
vascular disorder HHT (hereditary haemorrhagic telangiectasia) is no exception. The majority of HHT patients
are heterozygous for mutations in either the ENG (endoglin) or the ACVRL1 (activin receptor-like kinase 1)
gene. Both genes are essential for angiogenesis during development and mice that are homozygous for
mutations in Eng or Acvrl1 die in mid-gestation from vascular defects. Recent development of conditional
mouse models in which the Eng or Acvrl1 gene can be depleted in later life have confirmed the importance of
both genes in angiogenesis and in the maintenance of a normal vasculature. Endoglin protein is a co-receptor
and ACVRL1 is a signalling receptor, both of which are expressed primarily in endothelial cells to regulate
TGFβ (transforming growth factor β) signalling in the cardiovasculature. The role of ACVRL1 and endoglin
in TGFβ signalling during angiogenesis is now becoming clearer as interactions between these receptors
and additional ligands of the TGFβ superfamily, as well as synergistic relationships with other signalling
pathways, are being uncovered. The present review aims to place these recent findings into the context of
a better understanding of HHT and to summarize recent evidence that confirms the importance of endoglin
and ACVRL1 in maintaining normal cardiovascular health.
Hereditary haemorrhagic telangiectasia
HHT (hereditary haemorrhagic telangiectasia) is an inherited
vascular disorder associated with altered TGFβ (transforming
growth factor β) signalling in endothelial cells. It is inherited
in an autosomal dominant fashion and affects approximately
1 in 5000 individuals. Clinically, HHT is characterized by
multiple red spots known as telangiectases that develop
on the skin, in the mucocutaneous lining of the oronasal
and GI (gastrointestinal) tract, and are prone to severe
haemorrhage [1]. The telangiectases form due to local vascular
abnormalities: the emergence of direct connections between
a small artery and vein and associated loss of a capillary bed
[2,3]. Larger AVMs (arteriovenous malformations), forming
as a result of abnormal connections between more substantial
arteries and veins, may occur in the lung, brain and liver of
HHT patients, causing major clinical problems [1].
The majority of HHT patients carry mutations in one
of two genes: ENG (endoglin), a co-receptor for ligands of
the TGFβ superfamily [4], or ACVRL1 (activin receptor-like
Key words: activin receptor-like kinase 1 (ACVRL1), angiogenesis, endoglin, hereditary
haemorrhagic telangiectasia (HHT), transforming growth factor β (TGFβ), vascular disease.
Abbreviations used: ACTRII, activin type II receptor; ACVRL1, activin receptor-like kinase 1;
ALK5, activin receptor-like kinase 5; AVM, arteriovenous malformation; BMP, bone morphogenetic
protein; BMPR2, type II BMP receptor; Co-SMAD, common-mediator SMAD; EC, extracellular; ENG,
endoglin; ERK, extracellular-signal-regulated kinase; GI, gastrointestinal; GS, glycine/serine-rich;
HHT, hereditary haemorrhagic telangiectasia; IC, intracellular; L-endoglin, large endoglin; ID1,
inhibitor of DNA binding 1; MAPK, mitogen-activated protein kinase; R-SMAD, receptor-regulated
SMAD; S-endoglin, short endoglin; Sol-Eng, soluble endoglin; TGFβ, transforming growth factor
β; TGFBR, TGFβ receptor; TM, transmembrane; VEGF, vascular endothelial growth factor; ZP, zona
pellucida.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2011) 39, 1659–1666; doi:10.1042/BST20110664
kinase 1, also known as ALK1), a type I TGFβ superfamily
receptor [5]. Mutations in other (as yet unidentified) genes
have also been mapped in other HHT families [1], and a rare
combined syndrome of juvenile polyposis and HHT is due
to mutations in SMAD4 [6].
Endoglin and ACVRL1 protein structures
Endoglin and ACVRL1 are transmembrane proteins, found
primarily on the surface of endothelial cells, that play
an important role in TGFβ superfamily signalling [7].
The endoglin protein has a large highly glycosylated EC
(extracellular) domain, containing a ZP (zona pellucida)
domain that, together with the orphan domain, forms a domelike structure on the cell surface [8] (Figure 1). The ZP domain
is also a feature of the related accessory protein betaglycan,
suggesting an important role for this region in TGFβ
signalling [9]. The IC (intracellular) domain of endoglin
terminates with a PDZ domain-binding motif that interacts
with the scaffold protein GIPC [GAIP (Gα -interacting
protein)-interacting protein C-terminus, also known as
synectin] to modulate downstream signalling and regulate
endothelial cell migration [10]. The IC domain of endoglin
is constitutively phosphorylated on particular serine residues
and, to a minor extent, on threonine residues [11]. The type
II TGFβ receptor, TGFBR2, preferentially phosphorylates
endoglin on Ser634 and Ser635 , which subsequently permits
ACVRL1 to phosphorylate endoglin on three threonine
residues, Thr640 , Thr647 and Thr654 [12]. In addition, ALK5
(activin receptor-like kinase 5), the type I TGFβ receptor (also
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Figure 1 ACVRL1 and endoglin proteins
ACVRL1 (A) and endoglin (B and C) are transmembrane receptors composed of 503 and 658 amino acids respectively. Their
protein structures (A and B) can be divided into four distinct regions: the signal peptide (SP), which is removed during
processing to generate the mature protein; the EC domain, important for ligand binding; and the TM and IC domains. The IC
domain of ACVRL1 contains the GS domain at the juxtamembrane position and the kinase responsible for phosphorylation of
SMAD1/5/8 (as well as selected threonine residues in endoglin). Endoglin has a large glycosylated EC domain and a short
non-signalling IC domain with a PDZ-binding motif (SSMA) at its C-terminus. Alternative splicing generates two different IC
domains for endoglin, L-Eng and S-Eng, and various serine and threonine residues are phosphorylated in L-Eng by receptor
kinases (see the text). (C) The three-dimensional structure of the EC domain of endoglin comprises the N- and C-terminal
regions of the ZP domain (ZP-N and ZP-C), and the third is an orphan domain of no known homology [8]. Endoglin normally
forms a disulfide-linked homodimer at the cell surface and the dome consists of two antiparallel oriented monomers, but
only one monomer is depicted for simplicity.
known as TGFBR1) phosphorylates endoglin on Ser646 and
Ser649 [13] (Figure 1). The precise role of phosphorylation
of the IC domain of endoglin is not understood, but it is
known to affect TGFβ family signalling and endothelial cell
migration, and may also affect interactions between endoglin
and cytoskeletal or signalling proteins. The IC domain of
endoglin interacts with the focal adhesion proteins zyxin
and zyxin-related protein to remodel the actin cytoskeleton
[14,15]. There may also be effects on the microtubule
machinery as a result of endoglin interacting with the
dynein-related protein Tctex2β [16]. Furthermore, the IC
domain of endoglin interacts with β-arrestin2 in a manner
that depends on Thr650 of endoglin and leads to increased
endoglin internalization and altered ERK (extracellularsignal-regulated kinase) signalling [17].
Two protein isoforms of human endoglin have been
characterized. L (large)-endoglin is the predominant isoform
with a cytoplasmic domain of 47 residues. In contrast,
S (short)-endoglin, the minor isoform, has a cytoplasmic
domain of only 14 residues and arises by alternative splicing
[18]. These isoforms have opposing roles in angiogenesis,
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Authors Journal compilation with S-endoglin having anti-angiogenic effects [19], whereas
L-endoglin is pro-angiogenic [20]. The increased S/L ratio
of endoglin isoforms in senescent human endothelial cells
is regulated by the ASF (alternative splicing factor)/SF2
(splicing factor-2) and is consistent with an anti-angiogenic
role for S-endoglin [21,22].
Endoglin protein is also expressed in the placental
syncytiotrophoblasts and can be shed from the cell surface
to generate Sol-Eng (soluble endoglin). Increased circulating
levels of Sol-Eng are associated with pre-eclampsia [23],
and Sol-Eng also inhibits angiogenesis [24]. The regulatory
mechanisms involved in endoglin shedding are not yet
fully understood, but exposure of endothelial cells to the
inflammatory cytokine TNFα (tumour necrosis factor α)
leads to increased shedding of endoglin protein in vitro and
this may be through increased expression of MMP14 (matrix
metalloproteinase 14) which promotes cleavage of endoglin
protein at residues 586–587 [24].
The ACVRL1 receptor, similar to other type I receptors
of the TGFβ family, is composed of a cysteine-rich EC
domain, a single TM (transmembrane) region and an IC
Advances in the Cellular and Molecular Biology of Angiogenesis
Figure 2 TGFβ family signalling: basic canonical pathway
(A) TGFβ superfamily ligands mediate their effects by binding to specific, constitutively phosphorylated, type II receptors.
This induces the recruitment and phosphorylation of specific type I receptors which leads to phosphorylation of specific
R-SMAD signal transduction proteins. Activated R-SMADs form a complex with the Co-SMAD SMAD4, and this SMAD complex
translocates to the nucleus where it binds DNA and regulates the transcription of specific target genes. Endoglin and
β-glycan co-receptors (also called type III receptors) can be recruited to the TGFβ receptor complex and modulate signalling.
(B) Translocation of SMAD2 protein to the nucleus of endothelial cells over the 60 min following treatment with 5 ng/ml
TGFβ1 ligand. Images courtesy of Leon Jonker (Newcastle University).
domain containing a serine/threonine kinase as well as a
highly conserved GS (glycine/serine-rich) domain that plays
an important regulatory role in kinase activity (Figure 1).
Essentially, phosphorylation of the GS domain of a type
I receptor such as ACVRL1 by a TGFβ family type II
receptor is required to activate its kinase signalling activity
[25]. There are various splice variants for human ACVRL1 in
the Ensembl database, but little is known about the function
of the different isoforms.
TGFβ family signalling: the role of
endoglin and ACVRL1
TGFβ superfamily ligands mediate their effects through
heteromeric complexes of type I and type II receptors
(Figure 2A). Following ligand binding and receptor activation, the TGFβ type I receptor phosphorylates specific
R (receptor-regulated)-SMADs that can then interact with
the Co (common-mediator)-SMAD4 and translocate to
the nucleus to regulate transcription of specific target
genes, usually in complex with other transcription factors
[26,27] (Figure 2). Another class of SMAD proteins,
the I (inhibitory)-SMADs (SMAD6 and SMAD7), inhibit
activated R-SMADs by competing with them for receptor
interaction, promoting the proteosomal degradation of the
activated type I receptor or by recruiting phosphatases to
dephosphorylate the activated type I receptor [28].
In endothelial cells, TGFβ1 can propagate signalling
via two distinct type I receptors with opposing effects
on angiogenesis [29] (Figure 3). TGFβ1 predominantly
signals via the type I receptor ALK5, leading to activation
of SMAD2/3 and transcription of target genes such as
PAI-1 (plasminogen-activator inhibitor 1) associated with
endothelial cell quiescence. TGFβ1 can also signal via
ACVRL1, which results in the activation of R-SMAD1/5/8
and the transcription of target genes such as ID1 (inhibitor of
DNA binding 1) associated with endothelial cell proliferation
and angiogenesis. ALK5 mediates the TGFβ-dependent
recruitment of ACVRL1 to the receptor complex and is
required for optimal ACVRL1 activation [30]. Endoglin
recruitment to the TGFBR2–ACVRL1–ALK5 receptor
complex promotes signalling via the SMAD1/5/8 pathway
and this indirectly inhibits SMAD2/3 signalling via the
ALK5/SMAD2/3 pathway [30,31].
ACVRL1 can also bind to BMP9 (bone morphogenetic
protein 9) with high affinity in association with BMPR2
(type II BMP receptor) or ACTRII (activin type II receptor)
to activate SMAD1/5/8 [32,33] (Figure 3). Unlike TGFβ1
signalling via ACVRL1, which promotes endothelial cell
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Figure 3 Role of endoglin and ACVRL1 in modulating TGFβ family signalling in endothelial cells
TGFβ superfamily signalling depends on ligand context and the composition of the receptor complex. (A) TGFβ1 can activate
two distinct signalling pathways in endothelial cells. Low levels of circulating TGFβ1 ligand promote signalling through the
ACVRL1–ALK5–TGFBR2 receptor complex and activation of SMAD1/5/8 which leads to the transcription of pro-angiogenic
target genes. On the other hand, high levels of TGFβ1 ligand induce signalling through the ALK5–TGFBR2 receptor complex
and activation of SMAD2/3, which leads to the transcription of anti-angiogenic target genes. Endoglin promotes activation
of SMAD1/5/8 and indirectly inhibits activation of SMAD2/3. Note that TGFβ1 may also exert anti-angiogenic effects on the
underlying mural cells via ALK5–TGFBR2 and R-SMAD2/3 signalling. (B) BMP9 ligand can also activate two distinct signalling
pathways in endothelial cells via two distinct type II receptors. BMP9 signalling via the ACVRL1–BMPR2 and ACVRL1–ACTRII
receptor complexes both activate R-SMAD1/5/8, with loss of one type II receptor compensated for by the other. This
response promotes the transcription of target genes required for maintaining endothelial cell quiescence. BMP9 signalling
via the ACVRL1–ACTRII receptor complex also activates SMAD2, with BMPR2 contributing to this response. The opposing roles
of ID1 as pro- or anti-angiogenic (compare A and B) is likely to depend on the longevity of the ID1 signal and how it is
integrated with other transcriptional mediators. ECM, extracellular matrix; IL8, interleukin 8; PAI-1, plasminogen-activator
inhibitor 1.
proliferation and angiogenesis [29,31], BMP9 signalling
via the BMPR2–ACVRL1 receptor complex inhibits FGF
(fibroblast growth factor)-induced cell proliferation, VEGF
(vascular endothelial growth factor)-induced angiogenesis
and maintains endothelial cell quiescence [32,34]. Conversely,
BMP9 can also act in a pro-angiogenic capacity in
combination with TGFβ1, as both ligands act synergistically
to improve the endothelial cell response to VEGF [35].
BMP9 can also activate signalling via ACVRL1 in vascular
endothelial cells independently of ALK5 and endoglin [36]
(Figure 3), leading to the phosphorylation of SMAD1/5/8,
induction of E-selectin and IL-8 (interleukin 8) expression
and the promotion of endothelial cell quiescence. These
findings point to a dual role for ACVRL1 in promoting or
inhibiting endothelial cell activation dependent on cytokine
context and the composition of the signalling receptor
complex.
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Authors Journal compilation Although not a core receptor for TGFβ ligands, endoglin
can indirectly modulate signalling by refining the selectivity
of ligand binding [37]. Endoglin binds to TGFβ isoforms 1
and 3 in the presence of TGFBR2, and to BMP9 and BMP10 in
a receptor-independent manner, and, in both cases, promotes
signalling through ACVRL1 [31,32,38]. Circulating Sol-Eng,
on the other hand, can sequester ligands of the TGFβ
superfamily and thereby has the potential to reduce ligand
availability at the endothelial cell surface [23,39]. Thus
endoglin can modulate TGFβ family signalling at a number
of different levels.
Non-canonical TGFβ signalling
In addition to canonical TGFβ signalling via the SMAD
proteins, TGFβ can activate several non-canonical signalling
pathways such as the ERK, MAPK (mitogen-activated
Advances in the Cellular and Molecular Biology of Angiogenesis
protein kinase), Rho-like GTPase and PI3K (phosphoinositide 3-kinase)/Akt pathways [25]. The extent to which
endoglin and ACVRL1 play a role in these non-canonical
pathways is not clear. However, the highly similar defects
reported for the ACVRL1, endoglin and TAK1 (TGFβactivated kinase 1) constitutive knockout mice indicates
possible cross-talk between these signalling proteins and
involvement of the MAPK pathway [40–42]. Constitutively
active Acvrl1 inhibited activation of JNK (c-Jun Nterminal kinase) and ERK in microvascular endothelial cells
[43], whereas the interaction of endoglin with β-arrestin2
discussed above [17] is enhanced by ACVRL1 and results
in the internalization and accumulation of endoglin and βarrestin2 in endocytic vesicles. This appears to have no effect
on SMAD activation, but down-regulates ERK activation
and alters the subcellular distribution of activated ERK in
endothelial cells.
Expression of endoglin and ACVRL1
ACVRL1 and endoglin are both expressed in the developing
vasculature of the mouse embryo [44,45]. Endoglin appears
to have a broader expression pattern in the developing
endocardium and cardiac cushions of the heart, as well as
all blood vessel types, whereas ACVRL1 expression appears
to be more restricted to the arteries. Both are expressed
at lower levels in the lymphatic endothelial cells, and
recent evidence suggests ACVRL1 is important in lymphatic
development and remodelling [46]. In the adult, ACVRL1
exhibits preferential expression in the blood vessels of the
lung, where it is predominantly expressed in the pulmonary
capillaries and pre-capillary arterioles [47]. Endoglin is also
highly expressed in pulmonary blood vessels in the adult
mouse and has been shown to co-localize with ACVRL1
in the distal regions of the pulmonary vasculature, where
associated activation of SMAD1/5/8 is observed [47]. The
expression of both the Eng and Acvrl1 genes is up-regulated
during periods of active angiogenesis during dermal wound
healing, heart repair and tumour angiogenesis [48–51], where
hypoxia, which leads to up-regulation of endoglin, is likely
to be an important trigger [52].
In addition to their expression in vascular endothelium,
endoglin and ACVRL1 are also found in other cell types.
Endoglin is expressed in bone marrow with a reported role
in erythropoiesis; in myofibroblasts with effects on fibrosis;
in macrophages with a likely role in inflammation; and in
mesenchymal cells as a marker of mesenchymal stem cells
[53]. ACVRL1 has been reported in hepatic stellate cells [54]
and in chondrocytes [55] where it is thought to regulate the
balance of ALK5 and ACVRL1 signalling.
Role of endoglin and ACVRL1 in vascular
development
Mice that are homozygous for loss-of-function mutations
in Acvrl1 or Eng develop severe vascular abnormalities
and die at mid-gestation, consistent with a critical role for
both genes during angiogenesis [40,41,56,57]. In addition,
Eng-null embryos exhibit heart defects that may be a
consequence of reduced endothelial–mesenchymal transition
of the endocardial cells overlying the cardiac cushions
[41,58]. Examination of the vascular defects in the yolk
sac of Eng-null embryos reveals reduced TGFβ signalling
‘cross-talk’ between endothelial cells and adjacent periendothelial support cells, leading to a failure in muscle
maturation [59]. This reduced muscularization is also seen
in the vessels of Eng-heterozygous mice [60], and can
be rescued by thalidomide treatment, which increases the
expression of PDGFβ (platelet-derived growth factor β) by
endothelial cells, potentially compensating for the reduced
TGFβ signalling [61]. Importantly, thalidomide treatment has
been used successfully in a small group of HHT patients to
reduce bleeding, confirming the validity of these findings [61].
HHT exhibits an age-related penetrance, suggesting that
haploinsufficiency for the ACVRL1 or ENG alleles is not
sufficient to drive disease, and that a further somatic insult
affecting the intact allele is required. In support of this, AVMs
that are typical of HHT have been successfully modelled
in mice, but a conditional knockout approach is required
to remove both copies of the target gene. For example,
loss of ACVRL1 in endothelial cells of the brain and lung
leads to multiple arteriovenous fistulas and haemorrhage in
pups, causing lethality at 5 days of age. Also, removing
ACVRL1 from adult mice leads to a rapid development of
multiple AVMs and associated micro-haemorrhage in lung
and GI tract, whereas AVM formation in the skin requires a
wounding stimulus, suggesting that an angiogenic trigger is
required [62]. Furthermore, local depletion of Acvrl1 in the
adult mouse brain also leads to severe AVMs in the presence
of an angiogenic stimulus [63]. In zebrafish, loss of ACVRL1
lead to AVMs that are dependent on blood flow [64].
Similarly, loss of endothelial endoglin in the adult mouse
results in the formation of AVMs. Using the neonatal retinal
vascular plexus as a model of angiogenesis, AVMs appear to
develop by gradual vessel enlargement over several days; they
retain a venous identity and are associated with increased
endothelial cell proliferation [65]. These studies all suggest
that pathological or developmental angiogenesis is required
to trigger the formation of AVMs in the absence of endoglin
or ACVRL1, and that blood flow may be required to maintain
an AVM. Thus the heterogeneity seen in HHT patients may
be due to somatic loss of the second allele in combination with
an angiogenic insult such as may occur during inflammation
[65].
Endoglin, ACVRL1 and disease therapies
Some small-scale drug trials have been completed in HHT
patients with positive preliminary outcomes. Thalidomide
treatment promotes vascular stability and reduced bleeding
in a small group of HHT patients [61]. Preliminary studies
also suggest that anti-VEGF targeting is successful in treating
HHT patients, probably due to its anti-angiogenic effects.
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On a broader scale, ACVRL1 and endoglin are important
for angiogenesis in a wide range of disorders that depend
on new blood vessel growth. For example, drugs that inhibit
tumour angiogenesis are currently being used to treat cancer
patients, and endoglin and ACVRL1 both represent new
and potentially valuable anti-angiogenic cancer targets. Even
haploinsufficient levels of endoglin or ACVRL1 lead to
reduced angiogenesis in preclinical mouse models [35,66].
Anti-endoglin therapy is being tested in clinical trials [67],
whereas anti-ACVRL1 therapy also appears to be a promising
approach in anti-angiogenic and anti-lymphatic tumour
therapy [35,46].
Conclusions and future perspectives
Studies of HHT have revealed that endoglin and ACVRL1
are essential for cardiovascular development and play key
roles in angiogenesis. Work using mouse models has shown
that loss of either gene in adult life leads to major vascular
pathologies in the context of an angiogenic insult, and
these mice are proving to be valuable models of HHT. In
addition, spontaneous AVMs, the most frequent cause of
cerebral haemorrhage in the young, may relate to abnormal
function of ENG or ACVRL1 genes. In the context of general
cardiovascular health, circulating Sol-Eng is not only an
important biomarker of pre-eclampsia, but also a predictor
of cardiovascular events in chronic coronary artery disease
patients [68]. It is almost certain that further fundamental
roles of endoglin and ACVRL1 in the cardiovasculature will
be revealed in the near future. For example, a recent genomewide association study has revealed an association between
an endoglin haplotype and bicuspid aortic valve, one of the
most common cardiac malformations [69]. For HHT patients,
the challenge for the next decade is to better understand the
molecular basis of the disease and to develop more effective
therapies. Fortunately, we now have excellent mouse models
with which to make rapid progress in these important fields
of research.
Funding
M.M. is supported by the Wellcome Trust; P.D.U. and H.M.A. are
supported by the British Heart Foundation.
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Received 19 July 2011
doi:10.1042/BST20110664