Extracellular Control of TGF Signalling in Vascular Development and Disease

REVIEWS
Extracellular control of TGF signalling

in vascular development and disease
Peter ten Dijke* and Helen M. Arthur
Abstract | The intracellular mechanism of transforming growth factor (TGF) signalling

via kinase receptors and SMAD effectors is firmly established, but recent studies of human
cardiovascular syndromes such as Marfan syndrome and pre-eclampsia have refocused
attention on the importance of regulating the availability of active extracellular TGF.
It seems that elastic extracellular matrix (ECM) components have a crucial role in controlling
TGF signalling, while soluble and membrane bound forms of TGF co-receptors add
further layers of regulation. Together, these extracellular interactions determine the final
bioavailability of TGF to vascular cells, and dysregulation is associated with an increasing
number of vascular pathologies.
Pleiotropic
Influencing multiple different
traits.
*Molecular Cell Biology,

Leiden University Medical
Center, Postbus 9600, 2300
RC Leiden, The Netherlands.
Institute of Human Genetics,

International Centre for Life,
Central Parkway, Newcastle
University, NE1 3BZ, UK.
emails: p.ten_dijke@lumc.nl;
helen.arthur@newcastle.ac.uk
doi:10.1038/nrm2262
Published online
26 September 2007
Transforming growth factor1 (TGF1) is the prototypic

member of a large family of evolutionarily conserved pleiotropic secreted cytokines, which also includes the activins
and bone morphogenetic proteins (BMPs). Individual
family members have crucial roles in multiple processes
throughout development and in the maintenance of
tissue homeostasis in adult life1,2. Not surprisingly, there
fore, subversion of signalling by TGF family members
has been implicated in many human diseases, including
cancer, fibrosis, autoimmune and vascular diseases3.
The TGF family of ligands mediate their effects by
binding specific transmembrane typeI and typeII Ser/Thr
kinase receptors. The typeI receptors act downstream of
typeII receptors and determine the signalling specificity
within the receptor complex. Upon ligand-induced hetero
meric complex formation, the typeII receptor transphos
phorylates and activates the typeI receptor, which
subsequently propagates the signal by phosphorylating
specific receptor-regulated (R) SMAD transcription fac
tors at the two C-terminal Ser residues (FIG.1). On activation,
RSMADs form heteromeric complexes with a related
partner molecule, the Co-SMAD (SMAD4 in mammals),
and accumulate in the nucleus where they participate
in the transcriptional control of target genes1,2.
Despite the large number and distinct functions of
TGF family members (33 in mammals), there is an
enormous convergence in signalling to only five typeII
receptors, seven typeI receptors (also described as activin
receptor-like kinases (ALKs)) and two main SMAD intra
cellular pathways1,2 (FIG.1). So, how are signalling specificity
and diversity generated, especially as the signalling recep
tors and SMADs are broadly co-expressed? Extracellular
nature reviews | molecular cell biology
regulation of TGF signalling by co-receptors (which

control the access of ligands to signalling receptors) and
interplay between TGF ligands and extracellular mole
cules that regulate the activity of these ligands could be
important mechanisms that control signalling specificity.
Consistent with these potential mechanisms, all three
TGF isoforms (TGF1, TGF2 and TGF3 in mammals)
are secreted in latent forms that need to be activated before
they can bind to signalling receptors4. Furthermore, the
biological activities of TGF family members are regulated
by specific secreted inhibitors (see below) that sequester
ligands and block binding to receptors.
One of the compelling themes to emerge in recent
years is the crucial role of elastic extracellular matrix
(ECM) proteins in regulating TGF bioavailability in the
vascular system5. Defects in the ECM, which were initially
thought to affect the physical properties of vessel walls
and thereby compromise the vasculature, are now linked
to enhanced TGFSMAD signalling. In this Review, we
focus on recent insights into the mechanisms that regu
late extracellular TGF activity in an appropriate spatiotemporal manner and how this contributes to its pivotal
role in vascular development and disease. For reviews
addressing other related aspects of TGF family signal
ling at the intracellular level and dysregulated signalling
in disease, see REFS13, 68.
Secretion and ECM interaction

Secretion and storage in the ECM.The three TGF
isoforms in mammals TGF1, TGF2 and TGF3
are multifunctional and act in an autocrine, paracrine
and sometimes endocrine manner to regulate diverse
volume 8 | november 2007 | 857
2007 Nature Publishing Group
REVIEWS
Betaglycan
Sol-Endo
TGF
Sol-Endo
Endoglin
BMP
Plasma membrane
ALK5
TGFBR2
ALK1,2,3,6
SMAD2/3
BMPR2
SMAD1/5/8
SMAD7
SMAD6
P
P
SMAD4
P
P
P
P
P
P
Gene
expression
P
P
Gene
expression
Nucleus
Figure 1 | Signal transduction by TGF family members. Canonical signalling by

Nature Reviews
Molecular
Biology
transforming-growth factor (TGF) superfamily members
can be |divided
intoCell
two
main intracellular pathways according to the SMAD mediators: either SMAD2/3 or
SMAD1/5/8. Members of the TGF family bind to specific Ser/Thr kinase typeII and
typeI receptors; in most cells, TGF signals via TGFBR2 and ALK5 (also known as TGF
receptor-1; TGFBR1), and bone morphogenetic proteins (BMPs) signal via the BMP
type II receptor (BMPR2) and ALK1, -2, -3 and -6. The accessory receptors betaglycan and
endoglin can modulate signalling via the typeII and typeI receptors. Betaglycan
enhances TGF2 binding to TGF receptors, whereas endoglin may perform a similar
function for selected TGF family members and their receptors. Soluble endoglin (SolEndo) is thought to sequester ligand and thereby inhibits receptor binding95; however,
the exact mechanism through which this occurs is not known as endoglin requires
TGFBR2 for TGF binding113. Activated typeI receptors induce the phosphorylation of
specific receptor regulated (R) SMADs, which are the intracellular effectors of TGF
family members. In most cell types, TGF induces SMAD2/3 phosphorylation and BMPs
induce SMAD1/5/8 phosphorylation. Activated RSMADs form heteromeric complexes
with SMAD4 that accumulate in the nucleus, where they regulate the expression of
target genes such as SERPINE1 (plasminogen activator inhibitor) and ID1 (inhibitor
of DNA binding-1) in cooperation with transcription factors, co-activators and
co-repressors1,2. Inhibitory SMADs, such as SMAD6 and SMAD7, can antagonize
TGF signalling by inhibiting the activation of RSMADs.
Convertase family of
endoproteases
A group of enzymes that make
an internal cut in a polypeptide
chain to convert it from an
inactive to an active form.
Fibrillin
An extracellular matrix
glycoprotein that is a structural
component of microfibrils.
Microfibril
A fibre component (10nm in
diameter) of the extracellular
matrix that is essential for the
integrity of elastic fibres, which
are particularly abundant in
the aorta.
developmental processes and maintain tissue homeostasis

in the adult. The bioavailability of active TGF is regulated
at multiple levels, including secretion and interaction with
ECM components, and each step in the activation path
way is under tight control (FIG. 2). TGFs are synthesized as
precursor proteins that are proteolytically processed. The
signal peptide is removed from the pre-pro-TGF dur
ing transit through the rough endoplasmic reticulum and,
following dimerization, another cleavage occurs by the
convertase family of endoproteases9. These proteases cleave
the precursor into the Cterminal mature peptide and the
Nterminal precursor remnant (also known as latency
associated peptide (LAP)) within the secretory vesicles
or in the extracellular space10. Control and/or localization
of convertase activity may represent an important level of
regulation of TGF ligands. After cleavage, mature TGF
858 | november 2007 | volume 8
and LAP remain attached via non-covalent bonds to form

the small latent complex (SLC). LAP shields the receptorinteracting epitopes in the mature protein and this keeps
TGF in its latent form4.
The SLC can covalently attach to the large latent
TGF-binding protein (LTBP) to form the large latent
complex (LLC)11,12 (FIG.2). Most cell types secrete TGF
as part of LLC, although some cells (such as the bone
cell line UMR106) secrete SLC13. Four different LTPBs
have been identified, of which LTBP1, LTBP3 and,
to a lesser degree, LTBP4 covalently bind to LAPs of
all three TGF isoforms14. LTBPs contain multiple
epidermal-growth factor (EGF)-like repeats and Cysrich domains that are also found in fibrillins, which are
extracellular proteins that are required for elastic fibre
formation. The Cterminal region of LTBP1 binds to the
Nterminal region of fibrillin1, linking LLC to elastic
microfibrils15. After secretion, LLC binds to the ECM via
the Nterminal domain of LTBP and this interaction is
further supported by covalent transglutaminase-induced
crosslinks16. Antibodies to LTBP1 and inhibitors of
transglutaminase activity inhibit the activation of latent
TGF, which demonstrates that localization of LTBP to
the ECM is required for effective TGF activation16,17.
LTBPs might have multiple functions: they target the
latent TGF complex to specific sites, including structural
components within the elastic fibres, where they may
be stored for later use. This targeting is determined by
binding to ECM components, such as fibronectin, via the
variable N-terminal regions of LTBPs. In addition, LTPBs
stabilize latent TGF complexes and regulate their acti
vation at the cell surface (discussed below)18. Important
evidence for these roles comes from mouse models that
are either deficient in Ltbp3 or have a hypomorphic muta
tion in Ltbp4. These mice are viable but have multiple
phenotypes that are related to decreased TGF signalling
and defects in elastic fibres19,20 (TABLEs1,2). These results
indicate an important role for LTBPs in connective tissue
deposition and as a local regulator of TGF availability.
Release from microfibrils and ECM.In order for latent
TGF to become activated and function at adjacent and
neighbouring cells, the LLC must be liberated from
microfibrils and ECM (FIG.2). A recent study revealed
that LLC release can be initiated with the displacement
of LTBP bound to fibrillin1 (Ref.21). Degradation of
microfibrils by inflammatory proteolytic enzymes (such
as elastase) releases fragments of fibrillin1, including
an internal fibrillin fragment that efficiently binds to
the Nterminal region of fibrillin1 and displaces LTBP.
This releases LLC from microfibrils and contributes to
local TGF activation21.
Several proteases, including plasmin, mast-cell
chymase and thrombin, release LLC from the ECM4.
Cleavage of LTBP1 occurs in a sensitive hinge region such
that the Nterminal fragment remains bound to ECM,
but the remainder of LLC is released (FIG.2). In a recent
study, BMP1-like matrix metalloproteases (MMPs) were
shown to cleave LTBP1 at two specific sites in the hinge
region to release LLC22, and facilitate the subsequent
MMP-dependent LAP cleavage. In the absence of BMP1,
www.nature.com/reviews/molcellbio
REVIEWS
a Synthesis and secretion
b Activation and receptor binding
1 Pre-pro-TGF
N
5
C
2 Pro-TGF
Furin
Emilin
3 SLC
Elastase
6
LAP
Cytoskeleton
Mature
protein
Integrin
Plasma
membrane
4 LLC
7
Hinge
region
LTBP
(and other
fibrillin fragments)
BMP1
MMP2
8
LLC bound
to ECM
and elastic
microfibrils
LAP
remnants
Plasma
membrane
N
N
ECM
(fibronectin)
Fibrillin-1
ALK5
TGFBR2
Intracellular signaling
Figure 2 | Regulation of TGF bioavailability. Schematic model of synthesis, secretion

Nature
Reviews
Molecular
Cell Biology
and matrix deposition of transforming growth factor
(TGF)
(a) |and
activation
and
TGF receptor binding (b). TGF is synthesized as a pre-pro-protein, which undergoes
proteolytic processing in the rough endoplasmic reticulum (1). Two monomers of TGF
dimerize through disulphide bridges (2). The pro-TGF dimer is then cleaved by furin
convertase to yield the small latent TGF complex (SLC), in which the latency-associated
peptide (LAP; orange) and the mature peptide (red) are connected by non-covalent
bonds (3). This processing step is inhibited by emilin1. The large latent TGF complex
(LLC) is formed by covalent attachment of the large latent TGF binding protein (LTBP,
shown in blue; 4). The Nterminal and hinge region of LTBP interact with extracellular
matrix (ECM) components such as fibronectin; this interaction can be covalent owing to
crosslinking by transglutaminase. The Cterminal region of LTBP (blue) interacts noncovalently with the Nterminal region of fibrillin1 (green; 4). As part of TGF activation
and receptor binding (b), an internal fragment of fibrillin1 (indicated in purple in 5) can
be released by proteolysis (mediated by elastases at sites indicated by black arrowheads
in 5) and interacts with Nterminal region of fibrillin1 to displace LTBP and release LLC
(6). The LLC can be targeted to the cell surface by binding to integrins via RGD
sequences (blue regions) in LAP (6). Bone morphogenetic protein-1 (BMP1) can cleave
two sites in the hinge region of LTBP (arrowheads in 6), which results in the release of
LLC (7). Matrix metalloprotease2 (MMP2) (and other proteases) can cleave LAP (black
arrowheads in 7) to release the mature TGF (red). Mature TGF can then bind to its
cognate receptors, TGFBR2 and ALK5 (8).
cells have increased fibrillar structures that contain

LTBP1 and show reduced TGF1 activity22. Consistent
with this interaction, impairment in TGF signalling or
deficiency in BMP1 result in similar defects in the frontal
skull bones of mice23.
Activation and receptor binding. In order for TGF to

bind to its cognate cell-surface receptors, the mature
peptide needs to be released from LAP (FIG.2). The
mechanism by which latent TGF is converted into
active TGF varies according to cell type and context,
but all activating mechanisms directly target LAP4.
Invitro, physical conditions such as exposure to
extremes of pH or high temperature denature LAP
but not mature TGF4. Invivo, it is thought that bind
ing of the multifunctional secreted thrombospondin1
(THBS1) to LAP disrupts the non-covalent interactions
between LAP and mature TGF24. Activation can also
occur by proteases that cleave LAP to release bioactive
TGF. These processes couple matrix turnover to the
production of a strong inducer of ECM accumulation
and may be crucial in maintaining a balance of matrix
components.
Another important activation mechanism for latent
TGF is through binding of v6 and v8 integrins
to the RGD sequence in LAP 25. The mechanism is
unclear, but interaction with the RGD domain of LAP
may induce a conformational change that leads to liber
ation or exposure of TGF. Knock-in mice, in which
the RGD sequence in TGF1-LAP was replaced by a
non-functional Arg-Gly-Glu sequence, recapitulate all
the main features of Tgfb1-null mice, pointing to the
importance of RGD in TGF1 activation26. It should be
noted that the LAP of TGF2 does not contain an RGD
sequence and may be activated by another mechanism
that remains to be determined25. Activation of latent
TGF by v8 integrins involves the membrane type I
(MT1) matrix metalloprotease (MMP), and is therefore
sensitive to MMP inhibitors; however, it is currently
unclear whether it requires LTBP27. By contrast, v6mediated activation is resistant to MMP inhibitors and
requires a direct interaction of fibronectin with LTBP1
that targets LLC to the ECM28. As a result, latent TGF
is inefficiently activated in cells that lack fibronectin or
its receptor, 51 (Ref.28).
In line with these roles, inactivation of some of the
murine genes that encode putative activators leads to
defects that are reminiscent of mice deficient in TGF
signalling components (TABLES1,2). For example, Thbs1deficient mice have a similar, albeit milder, phenotype
to the Tgfb1 knockout24. Also, mice that lack v or 8
integrins have defects in vascular and palatal develop
ment that phenocopy the defects of Tgfb1- and Tgfb3knockout mice, respectively29,30. These phenotypes
support the idea that extracellular activation of TGF
is a major controlling step before receptor binding
invivo.
Cell-surface receptors. Once the active TGF family
member is released from the ECM, it signals via specific
complexes of typeI and typeII Ser/Thr kinase receptors
(FIG.1). The typeI and typeII receptors are structurally
similar with small Cys-rich extracellular domains, single
transmembrane-spanning regions and intracellular parts
that mainly comprise kinase domains. TGF signals via
TGF typeII receptor (TGFBR2) and TGFBR1 (also
known as ALK5; a typeI receptor). Other combinations
REVIEWS
Table 1 | Human syndromes and animal models associated with inactivation or misexpression of TGF signalling components*
Gene
Human syndrome
Animal models
Refs
Extracellular regulation of TGF signalling

BMP1
Unknown
KO: reduced skull ossification, abnormal collagen fibrils in amnion, die at birth.
23
EMILIN1
Hypertension
KO: reduced arterial diameter, increased vascular resistance, emphysema,

increased TGF signalling in vascular wall.
93
FBN1 (fibrillin-1)
MFS1
KO: ruptured aortic aneurysm, impaired pulmonary function, die at birth.
FBN2 (fibrillin-2)
Contractural
arachnodactyly
KO: bilateral syndactyly.
109
FN1 (fibronectin-1)
EhlersDanlos
syndrome, type X
KO: deformed heart and embryonic vessels, defective extra-embryonic

vasculature.
114
EFEMP2 (fibulin-4)
Cutis laxa
KO: perinatal lethality, arterial narrowing, aortic rupture.
FBLN5 (fibulin-5, DANCE)
Cutis laxa
KO: tortuosity and elongation of the aorta, loose skin.
LTBP1
Unknown
KO: persistent truncus arteriosus.
LTBP3
Unknown
KO: craniofacial malformations, osteosclerosis and osteoarthritis.
LTBP4
Unknown
Hypomorphic: emphysema, cardiomyopathy and colorectal cancer.
THBS1 (thrombospondin-1)
Unknown
KO: pneumonia, alveolar haemorrhage, vSMC hyperplasia.
ITGB8 (integrin, 8)
Unknown
KO: embryonic lethal with vascular defects and/or perinatal lethality and
cerebral haemorrhage.
29
ITGAV (integrin, aV)
Unknown
KO: embryonic lethal placental defects or perinatal lethal with intracerebral

and intestinal haemorrhages and cleft palate.
30
ITGB6 (integrin, 6)
Unknown
KO: inflammation of lungs and skin.
TGFB1
CamuratiEngelmann
disease||
KO: embryonic lethal with vascular defects or postnatal lethality from

autoimmune disease (phenotype is modifier-dependent).
KI of RGD/E mutation: recapitulates KO phenotypes.
26
TGFB2
Unknown
KO: aortic arch defects, cardiac septal defects, perinatal lethality.
40
TGFB3
Unknown
KO: cleft palate, delayed lung maturation, die shortly after birth.
40
83, 84
Het for missense mutation (C1039G): aortic aneurysm, emphysema.
90, 91
Hypomorphic: tortuous aorta, aortic aneurysm, increased TGF activation.
88, 89
124
19
20
24, 115
116
40, 44
*Continued in Table 2. Animal models are murine, unless otherwise indicated, and may be heterozygous (Het) or homozygous for the targeted allele (knockout
(KO) for null allele; knock-in (KI) for hypomorphic/mutant allele) or transgenic. Cutis laxa maps to several genes and is characterized by pendulous inelastic skin,
which is sometimes combined with aortic aneurysm and tortuous arteries; however, the specific genes associated with the cardiovascular features are not yet clear.
||
CamuratiEngelmann disease is caused by an activating mutation in TGF1. BMP1, bone morphogenetic protein-1; LTBP, large latent TGF-binding protein;
MFS1, Marfan syndrome type 1; TGF, transforming growth factor; vSMC, vascular smooth muscle cell.
Matrix metalloprotease
One of a family of structurally
related extracellular
Ca2+-dependent zinccontaining proteases involved
in tissue remodelling and ECM
degradation.
RGD sequence
An amino acid sequence
(Arg-Gly-Asp) found in
extracellular matrix proteins
that directly binds to integrins.
of typeI and typeII receptors respond to other TGF

family ligands, for example, the BMP typeII receptor
(BMPR2) responds to BMP ligands in combination with
ALK1, -2, -3 or -6 typeI receptors.
TGF receptors are expressed on endothelial cells
(ECs) and vascular smooth muscle cells (vSMCs), but
also on many other cell types31,32. This, together with
the highly context-dependent responses, generates a
multifaceted role for TGF family members in vascu
lar biology33. In ECs, TGF can transduce signals via
ALK1 in addition to ALK5, and is thought to contribute
to the bifunctional effects of TGF on angiogenesis34.
ALK5 is required for TGFALK1 activation, whereas
ALK1 inhibits intracellular ALK5SMAD signalling.
The differential activation of these two distinct typeI
receptor pathways by TGF provides the ECs with an
intricate mechanism to precisely regulate, and even
switch between, TGF-induced biological responses. For
example, TGFALK1 activation leads to stimulation of
EC proliferation and migration, whereas TGFALK5
activation inhibits these responses34.
TypeIII (or auxiliary) receptors such as betaglycan

and endoglin (ENG) present another mechanism
through which signalling specificity is regulated. TGF2,
which has a low intrinsic affinity for TGFBR2, requires
betaglycan for efficient signalling35. In addition, beta
glycan can be shed by cells upon proteolysis in the juxta
membrane region to sequester mature TGF and inhibit
TGF signalling36. Proliferating ECs express endoglin,
which is required for efficient TGFALK1 signalling37,38;
endoglin may also be proteolytically shed (see below).
Thus, different combinations of ligands, receptors and
co-receptors, either on the membrane or shed from it,
may result in complex patterns of TGF activity.
TGF regulates vascular development

New blood vessels develop by a combination of vasculo
genesis and angiogenesis39 (BOX1). These processes are
regulated by cytokines and growth factors in a highly
orchestrated manner during embryogenesis. The vital
importance of TGF signalling in vascular development
was recognized following the identification of mutations
REVIEWS
Table 2 | Human syndromes and animal models associated with inactivation or misexpression of TGF signalling components*
Gene
Human syndrome
Animal models
Refs
HHT1
Het: vascular lesions similar to HHT.
TGF auxiliary receptors

ENG (endoglin)
40, 59
KO: embryonic lethal, reduced vSMC differentiation, heart defects.

Elevated soluble endoglin
Pre-eclampsia
None
TGFBR3 (betaglycan)
Unknown
KO: poorly formed cardiac septa, incomplete compaction of ventricular walls.
95
HHT2 (rarely PAH)
Het: vascular lesions similar to HHT.
117
TGF signalling receptors

ACVRL1 (ALK1, vbg)
60, 118
KO: embryonic lethal, reduced vSMC differentiation, dilated vessels, AVMs.
40, 64
Zebrafish KO: dilated vessels, AVMs.

TGFBR2
MFS2, LDS
KO: embryonic lethal, vascular defects.
TGFBR1 (ALK5)
LDS
KO: embryonic lethal, angiogenesis defects.
BMPR2
PAH
KO: pre-angiogenesis lethality.
51
40, 123
119
120, 121
Het: mild pulmonary hypertension.

Transgenic inducible BMPR2-mutant allele: pulmonary hypertension.
74
Intracellular TGF signalling molecules

SMAD1
Unknown
KO: pre-angiogenesis lethality, defects in chorion-allantoic circulation.
40
SMAD2
Unknown
40
SMAD3
Unknown
KO: viable. Impaired immunity, colon cancer.
SMAD4
Juvenile polyposis
+/ HHT
SMAD5
Unknown
KO: embryonic lethal, angiogenesis defects.
40
SMAD6
Unknown
KO: cardiac defects, aortic ossification, elevated blood pressure.
40
MAP3K7 (TAK1)
Unknown
KO: embryonic lethal, reduced numbers of vSMCs, dilated vessels, AVMs.
50
40
40, 122
Het: polyposis of the glandular stomach and duodenum.
*Continued from Table 1. Animal models are murine, unless otherwise indicated, and may be heterozygous (Het) or homozygous for the targeted allele (knockout
(KO) for null allele; knock-in (KI) for hypomorphic/mutant allele) or transgenic. ALK1, activin receptor-like kinase-1; AVMs, arteriovenous malformations; BMPR2,
bone morphogenetic protein receptor-2; HHT, hereditary haemorrhagic telangiectasia; LDS, LoeysDietz syndrome; MFS2, Marfan syndrome type 2; PAH,
pulmonary arterial hypertension; TGF, transforming growth factor; TAK1, TGF activated kinase1; TGFBR, TGF receptor; vSMC, vascular smooth muscle cell.
in TGF receptor genes in familial vascular pathologies

(discussed below). Furthermore, studies in mouse models
showed that, in the absence of key TGF receptors, angio
genesis stalls in the yolk sac at an early stage with fatal
consequences40 (FIG.3).
Role of TGF ligands in angiogenesis. The require
ment for TGF receptors for angiogenesis raises the
question of which TGF ligand(s) regulates new vessel
formation. Of the three mammalian TGF isoforms,
TGF1 is localized to ECs during embryogenesis41, sug
gesting that it is the most likely of the three to be involved
in angiogenesis. TGF2 and TGF3 seem to be less
important than TGF1 in angiogenesis because mice
that lack either of these ligands show relatively normal
angiogenesis42,43. However, there may be redundancy
between all three TGF ligands, and multiple or con
ditional knockout mice are required to investigate this
possibility. Interestingly, loss of Tgfb1 in a null mouse can
result in different phenotypes depending on the genetic
background. For example, in C57BL/6 mice, angiogenesis
is abnormal and embryos die at embryonic day (E)10.5,
whereas in the NIH background the cardiovascular
system tends to develop normally but neonates die from

an uncontrolled autoimmune assault40. The dramatic
influence of genetic background results from three
unlinked genetic modifiers44. These genes and their func
tions are unknown, but one possibility is that one or more
may regulate TGF activation.
Invitro studies have demonstrated the importance
of regulating the availability of active TGF. Low extra
cellular TGF1 concentrations promote the cell prolifer
ation and migration that is associated with the active
proliferation of new vessels in angiogenesis37. By contrast,
high levels of extracellular TGF1 lead to cytostasis and
synthesis of ECM proteins that are associated with mature
or differentiating vessels. TGF family members can also
act in a paracrine manner by stimulating the production
of pro-angiogenic cytokines, such as vascular endothelial
growth factor (VEGF), TGF and monocyte chemo
attractant protein1 (MCP1)45,46,47. Moreover, TGFs
can affect the function of other factors; for example,
TGF converts the pro-survival function of VEGF into
an apoptotic factor for ECs48. The resultant interplay
results in a refined and cell-context-specific response to
TGF stimulation.
REVIEWS
Box 1 | Vasculogenesis and angiogenesis
Vasculogenesis is the earliest step in the development of new blood vessels and involves
the differentiation of angioblasts into endothelial cells (ECs) and their assembly into a
primary vascular plexus. Angiogenesis is the process during which blood vessels develop
from existing capillaries by sprouting, pruning and/or splitting (intussusception).
These processes are driven by a complex interaction of growth factors (for example,
vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and
transforming growth factor (TGF)) and their receptors. Live imaging of angiogenesis
in zebrafish mutants suggests that the layout of the primitive vascular network is
genetically determined. The directionality of new vessel growth is driven by leading
endothelial tip cells in response to guidance molecules, whereas the lagging ECs form
lumens by intercellular fusion of endothelial vacuoles112. During maturation, pericytes or
vascular smooth muscle cells (vSMCs) are recruited to capillaries and larger vessels,
respectively. Platelet-derived growth factor (PDGF) signalling is important for the initial
recruitment of mesenchymal cells that differentiate to vSMCs in response to TGF
signalling. As the animal grows, the vascular network continues to remodel by pruning
and branching in response to a combination of growth factors, hypoxic triggers and
blood flow to form a complex tree-like structure of arteries, veins and capillaries.
Morpholino
Chemically synthesized
oligonucleotide analogues
used to knock down gene
expression by specifically
binding to target transcripts to
inhibit RNA splicing or
translation.
Vessel muscularization
The development of smooth
muscle cells around a vessel to
support and stabilize it.
Pericyte
A smooth muscle-like cell that
is intimately associated with
endothelial cells of small blood
vessels.
Mesenchymal cell
A member of a heterogeneous
multipotent cell population
that arises mainly from
embryonic mesoderm.
Hypertension
Elevated blood pressure.
Arteriovenous malformation
(AVM). Abnormal
communication between an
artery and a vein producing
dilated vessels.
Intussusceptive
angiogenesis
The process of blood vessel
growth by splitting the wall
of an existing blood vessel
extends into the lumen to split
a single vessel in two.
Intracellular TGF signalling in angiogenesis. Because

canonical TGF signalling occurs through SMAD activ
ation, one might expect similarities in the phenotypes
of the SMAD-knockout and TGF receptor-knockout
mice. In fact, SMAD1, SMAD2 and SMAD4 are required
during early embryonic development before angiogenesis
presumably because they are central to multiple TGF
family ligands40. SMAD3 regulates mucosal immunity
and does not appear to be required for cardiovascular
development49. Similarities in the phenotypes of the
Smad5-null mouse and TGF receptor knockouts point
to a pro-angiogenesis role for SMAD5. The most strik
ing similarities have been observed in TGF activated
kinase1 (TAK1) and TGF receptor mutant mouse
phenotypes50. TAK1 (also known as MAP3K7) is acti
vated by TGF and inflammatory cytokines and is cen
tral to several signalling pathways that control immune
and stress responses. Mutations in acvrl1 (also known
as vbg, the zebrafish homologue of ALK1) result in the
formation of dilated cranial vessels that contain increased
numbers of ECs51, whereas morpholino knockdown
experiments indicate a synergistic interaction between
the zebrafish homologues of TAK1 and ALK1 (Ref.50).
This invivo data from both mouse and zebrafish models
suggest that TGF signalling through TAK1 is important
in the regulation of vascular development.
TGF and blood-vessel myogenesis. One of the best
understood roles of TGF signalling in angiogenesis is
that of promoting vessel muscularization. vSMCs (arter
ies and veins) or pericytes (capillaries) cover new vessels
and are required for both vessel stability and function.
Muscularization is achieved when ECs promote paracrine
TGF1 signalling to neighbouring mesenchymal cells to
promote SMC or pericyte differentiation. Establishment
of cellcell contacts, at the level of gap-junction com
munication between endothelial and mesenchymal
cells, is required for TGF activation and SMC differ
entiation52. TGF signalling between ECs and vSMCs is
also dependent on endoglin53. Ultimately, TGF activa
tion stimulates the differentiation of mesenchymal cells
to SMCs through the combinatorial activation of several

SMC genes such as SM-actin (ACTA2) and transgelin
(TAGLN; also known as SM22)54. In line with their vascu
lar myogenesis role, specific loss of the TGF receptors,
TGFBR2 or ALK5, in vSMCs results in vascular defects
and embryonic lethality55.
Importantly, TGF signalling in vascular develop
ment is not confined to SMCs. Recent analysis of mice
with ECspecific loss of either Tgfbr2 or Tgfbr1 (which
encodes ALK5) has clearly shown that both TGF recep
tors also have essential angiogenesis-related roles in
ECs56,53. The role of TGF receptors in ECs may be crucial
in development earlier than in vSMCs, as mutants with
ECspecific loss of Tgfbr2 die from vascular defects
2 days sooner (at E10.5) than those with vSMC-specific
loss (E12.5)55. However, a recent report suggests that
Tgfbr1 is not expressed in ECs57. This discrepancy may
be explained by the limitations of knock-in lacZ reporters
for monitoring endogenous protein expression, and
further work is required to determine the relative contri
butions of TGF signalling receptors to EC function
invivo. Nonetheless, the central importance of properly
regulated TGF signalling in the vasculature is clearly
demonstrated by the growing number of familial cardio
vascular disorders that are associated with mutations
affecting TGF family signalling.
Vascular disease and TGF signalling

Over recent years, numerous familial vascular diseases
have been mapped to receptors of the TGF family and,
in many cases, further understanding has been reached
through studies of mouse models58. A common theme
is that TGF signalling is important in blood-vessel
morphogenesis and stability. Recent data also provide
insights into the role of TGF signalling in hypertension
and pre-eclampsia. Furthermore, recognition of the
importance of TGF signalling in angiogenesis has
opened up new possibilities for targeting cancer by
modulating this pathway in the tumour vasculature.
Hereditary haemorrhagic telangiectasia (HHT). The
vascular disorder hereditary haemorrhagic telangiectasia
type 1 (HHT1) results from mutations in ENG that
lead to haploinsufficiency of endoglin59. The closely
related disorder HHT type 2 (HHT2) is caused by loss
of function or dominant negative mutations in ACVRL1
(which encodes ALK1)60,61. Endoglin is expressed in ECs
of all developing blood vessels, and ALK1 is primarily
expressed in arterial ECs57,62, which points to defects in
ECs as the primary cause of HHT.
The main clinical features of HHT are bleeding from
small vascular lesions (telangiectases) in the muco
cutaneous tissues, and the presence of arteriovenous
malformations (AVMs) in the lung, liver and/or cerebral
vasculature. The typical onset of symptoms (usually
major bleeding from nasal telangiectases) is during
puberty, and AVMs may be present that become larger
and symptomatic as patients age. Several possible
mechanisms have been proposed to explain the develop
ment of AVMs, including loss of vSMCs, dysregulated
vascular tone or apoptosis of capillary ECs (FIG.4).
REVIEWS
Angioblast cells
Vasculogenesis
Primitive vascular plexus
Progression blocked in yolk sac

in the absence of
TGF1, TGFBR2, endoglin,
ALK1, ALK5 or SMAD5
Angiogenesis
Vacuole fusion and lumen

formation
Tip cell
PDGF
Intussusceptive
angiogenesis
Sprouting
angiogenesis
Endothelial cell
PDGF
Mesenchymal
cell
Gap-junction formation,
TGF1 activation,
SMC differentiation and
mutual inhibition of
proliferation
Figure 3 | TGF signalling in vasculogenesis and angiogenesis. Vasculogenesis and

Nature Reviews | Molecular Cell Biology
two types of angiogenesis are shown: intussusceptive and sprouting angiogenesis (BOX1).
Vasculogenesis involves the differentiation of endothelial cells (ECs) from precursor
angioblast cells to form a primitive plexus of capillaries, which remodel and grow by
angiogenesis. Intussusceptive angiogenesis involves the splitting and growing of vessels
insitu in a metabolically efficient manner, and is found, for example, in the developing
yolk sac and lung. Vessel splitting occurs by the formation of translumen pillars
(arrowheads) but the molecular mechanisms are not well understood. In sprouting
angiogenesis, endothelial cells proliferate behind the tip cell of a growing branch in
response to cytokines such as vascular endothelial growth factor (VEGF) and lumens can
form by vacuole fusion. Both forms of angiogenesis require the recruitment of smooth
muscle cells (SMCs) to stabilize the nascent vessels. Neighbouring mesenchymal cells
migrate towards the neovessel in response to platelet-derived growth factor (PDGF)
and then differentiate into vascular SMCs (vSMCs) in response to transforming growth
factor (TGF) signalling. The continued tight intercellular association between ECs
and vSMCs promotes sustained TGF activation and mutual inhibition of cell
proliferation. When TGF signalling is defective, for example in TGF-receptor knockout
mice, smooth muscle differentiation fails to proceed and angiogenesis stalls. ALK, activin
receptor-like kinase; TGFBR2, TGFb type II receptor.
Sprouting angiogenesis
The process by which
endothelial cells migrate and
proliferate into the surrounding
matrix to form new vessel
branches in response to an
angiogenic stimulus.
To investigate abnormalities in ECs derived from patients

with HHT, cultured circulating endothelial progen
itor cells were used to generate blood outgrowth ECs.
These had a disorganized actin cytoskeleton that may
contribute to vascular fragility in HHT63. Further investi
gations in mouse models of HHT suggest that there are
reduced levels of TGF signalling from ECs to vSMCs or
pericytes, which results in decreased SMC differentiation.
This defect generates weaker vessels that are prone to

bleeding, a main characteristic of HHT53. In addition,
development of AVMs early in embryogenesis may be
due to loss of arteriovenous identity. Arterial-specific and
venous-specific signalling molecules such as ephrin B2
(EFNB2) and EPHB4, respectively, are thought to ensure
that these vessel types remain distinct and separate
during development. AVMs frequently develop in
Acvrl1-null mouse embryos, although to a lesser extent
in Eng-null mice, and may be due to downregulation of
EFNB2 in the Acvrl1/ mice64,65.
There may also be systemic defects in TGF signalling
in HHT1, as reduced endoglin levels have been shown to
cause decreased levels of plasma TGF in both mouse
models and patients with HHT1 (ref. 66). However, this
remains to be clarified because raised TGF1 levels have
also been reported67. The exact molecular changes lead
ing to HHT are not yet clear. New insights are anticipated
following the recent identification of BMP9 as a ligand
for both endoglin and ALK1 (Ref.68), and newly derived
conditional knockout Eng and Acvrl1 mouse models69
(S.P. Oh, unpublished observations).
Pulmonary arterial hypertension (PAH). The devastat
ing lung disease pulmonary arterial hypertension (PAH)
is characterized by pathological changes that include
hypertrophy (enlargement) of the medial smooth muscle
layers and intimal thickening in the pre-capillary arter
ioles (FIG.4). This leads to increased pulmonary artery
resistance that can ultimately result in right-sided heart
failure. In addition, plexiform lesions that comprise
multiple capillary channels and proliferating ECs may
develop near occluded regions. The average age of onset
is in the third decade of life in women and the fourth
decade in men, but can occur at any age.
Familial PAH (fPAH) is associated with loss-of-func
tion mutations in the BMP receptor2 gene (BMPR2).
Reduced levels of this receptor in pulmonary artery
SMCs and ECs in patients with PAH lead to reduced
SMAD1 activation and/or increased activation of the
p38 mitogen-activated protein kinase (MAPK) that may
contribute to vSMC hyper-proliferation70,71. It appears
that BMPR2-deficient cells may respond abnormally to
BMP ligands by redirecting signalling through the typeII
activin responsive receptor ActRIIA (in association with
either ALK2 or ALK3)72. In addition, the long cytoplasmic
tail of the BMPR2 protein, which is truncated in many
patients with fPAH, is required for proper regulation
of the cytoskeleton, and misregulation may contribute
to the aetiology of PAH73. Mouse models of fPAH with
inactivated Bmpr2 have yielded a lack of PAH symptoms
in normoxic conditions. However, postnatal expression
of a dominant-negative BMPR2 mutation in vSMCs leads
to pulmonary hypertension and modest musculariza
tion of distal arteries. These data suggest that aberrant
BMPR2 signalling in vSMCs is sufficient to produce the
pulmonary hypertensive phenotype and provides a useful
animal model for further investigation74.
The link between dysregulated BMP signalling and
progression of this devastating disease remains to be
determined, but an important clue may come from the
REVIEWS
a
b
Endothelial cell
Smooth muscle cell
Stromal cell
Site of partial
occlusion
Terminal bronchiole and alveoli

Pre-capillary pulmonary arteriole
Pulmonary venule
Capillaries
Arteriole
Venule
Figure 4 | Vascular remodelling in PAH and HHT. a | Terminal alveoli in pulmonary arterial hypertension (PAH) showing
the approximate site of occlusion in the pre-capillary arteriole. b | Cross-section of a Nature
normalReviews
pre-capillary
pulmonary
| Molecular
Cell Biology
arteriole, which shows a typical vessel structure of a single layer of endothelial cells surrounded by supporting smooth
muscle cells. c | Cross-section of a partially occluded pre-capillary arteriole in PAH with proliferating vascular smooth
muscle cells (vSMCs) and endothelial cells (ECs). A plexiform lesion that comprises multiple vascular channels is shown to
the left of the occluded arteriole. d | The normal capillary network that is present in most vascular beds and interconnects
an arteriole and a venule. e | Formation of an arteriovenous malformation in hereditary haemorrhagic telangiectasia (HHT),
which may develop by one or more different mechanisms: the loss of arterial and venous identity during development
would disrupt the normal separation of arteries and veins leading to arteriovenous connections; abnormal vascular
remodelling and dilation following local inflammation or trauma may fail to resolve; apoptosis of the capillary ECs in
regions of hypoxia would remove the natural capillary bed that separates arteries and veins; or gradual dilation of a
naturally occurring anastomosis may occur as a result of loss of SMCs and/or loss of vessel tone leading to capillary
regression due to lack of blood flow.
fact that patients with HHT who have ACVRL1 muta

tions are predisposed to the development of PAH75.
As ALK1 is expressed in pulmonary ECs, this finding
suggests that aberrations in TGF family signalling
in the endothelium can also be the primary defect in
PAH. Recent identification of BMP9 as an ALK1 ligand
suggests that reduced ALK1 levels may lead to altered
BMP9 signalling68, although any link to PAH remains
to be determined.
Anastomosis
A naturally occurring
arteriovenous connection that
may be dynamically regulated
and is particularly frequent in
thermoregulatory vascular
beds.
Enhanced TGF signalling and aortic aneurysms. Aortic

aneurysms, predominantly originating in the aortic
root (FIG.5), are important features of Marfan syndrome
(MFS) and a clinically overlapping disorder that has
been variously termed MFS type 2 (MFS2), familial
thoracic aneurysm disorder (AAT3) and LoeysDietz

syndrome (LDS)7678. MFS is primarily associated with
mutations in FBN1 (fibrillin1), whereas LDS is asso
ciated with mutations in TGFBR1 or TGFBR2. Aortic
aneurysms are insidious malformations with a variable
age of onset which, if left untreated, carry the risk of
aortic dissection, rupture and sudden death. The vulner
ability of the aortic root in these disorders may relate
to the two different developmental origins (neural crest
and secondary heart field) of vSMCs at the region of the
aortic valve, which have different responses to TGF
signalling79. This results in two closely abutting rings of
different vSMC populations and it is possible that the
junction between them represents a susceptible site for
aortic dissection80 (FIG.5).
REVIEWS
a
PT
Tunica adventitia
Tunica media
Tunica intima
Normal elastin fibres
Fragmented elastin fibres

MFS
LAP
TGF1
LTBP
Microfibrils
Few microfibrils or
secondary proteolysis
Figure 5 | TGF-associated defects in Marfan and LoeysDietz syndromes.

Nature Reviews | Molecular Cell Biology
a | An aorta with two seams of vascular smooth muscle
cells (vSMCs) at the aortic
root and pulmonary trunk (PT) that are derived from different developmental origins
cardiac neural-crest-derived SMCs (dark pink), secondary heart-field-derived
SMCs (purple) and secondary heart-field-derived myocardial muscle (blue).
These areas may be sites that are vulnerable to dissection. Heart and pulmonary
arteries are shown as dotted lines. b | An aortic root aneurysm that is typical of
Marfan syndrome (MFS) and LoeysDietz syndrome. c | Ascending aortic aneurysm.
d | Descending aortic aneurysm. e | A cross-section of aorta shows the three layers
(tunics) of the vessel: tunica adventitia (which contains collagen, fibroblasts, nerves
and capillaries), tunica media (which contains SMCs, elastic fibres/microfibrils,
collagen and proteoglycan) and tunica intima (which contains endothelial cells and
basal lamina). Fragmented elastin fibres in the aorta are seen in MFS and are
associated with release of the large latent transforming growth factor (TGF)
complex and active TGF1. LAP, latency-associated peptide; LTBP, large latent TGF
binding protein.
It was initially thought that aortic aneurysms in MFS

were due to structural defects in the aorta resulting from
a failure to stabilize elastic-fibre structure when fibrillin1
was limiting. The resultant elastin fragmentation in the
aortic wall would make the aorta susceptible to injury
from haemodynamic forces. Recently, it has become
clear that an important function of fibrillin1 is to con
trol TGF bioavailability (FIG.2). Reduced fibrillin1
may result in incorrect LLC sequestration and excessive
activation of TGF signalling, a major contributory
factor in the vascular pathology of MFS81,82. Mice defi
cient in Fbn1, but not mice overexpressing mutant Fbn1,
develop MFS phenotypic manifestations83,84. This sug
gests that a deficiency in microfibrils, as opposed to a
transdominant effect of mutant fibrillin1, is a major
determinant of MFS. It has also been proposed that the
progressive proteolytic damage that is characteristic of
MFS may lead to the formation of fibrillin fragments
that further increase the bioavailability of TGF21. In the
case of LDS, when TGFBR2 levels are reduced owing to
pathologic mutation, compensatory mechanisms may
lead to an overshoot of TGF signalling in the aortic
media. For example, increased TGF expression and
increased fibrosis have been observed in transgenic mice
that overexpress a dominant negative form of Tgfbr2
(Ref.85). Similar, but as yet unknown, mechanisms may
explain the upregulation of TGFSMAD signalling and
fibrosis that is seen in the aortae of patients with LDS,
who carry heterozygous inactivating mutations in either
TGFBR2 or TGFBR1 (Ref.77).
Whatever the cause of enhanced TGF signalling,
work using animal models of MFS clearly showed that
aortic aneurysms were prevented by administration
of neutralizing antibodies to TGF, confirming the
contribution of excess TGF signalling to the vascular
pathology82. In the absence of approved anti-TGF
clinical therapies, attention turned to angiotensin, a
potent vasoconstrictor that interacts with angiotensin
receptors on blood vessels and induces SMAD2/3
phosphorylation86. This change of focus was rewarded
when the angiotensin typeI receptor antagonist, losartan
(Cozaar; Merck), showed a strikingly similar protective
effect in the development of aortic aneurysms in animal
models of MFS82. However, the mechanism by which
losartan protects against aortic aneurysm and reduces
SMAD2 activation is not fully understood. One possi
bility is the involvement of THBS1, an important target
downstream of angiotensin signalling87 that can activate
latent TGF. Whatever the mechanism, losartan has rap
idly moved to PhaseIII clinical trials in patients with MFS
(see the US National Marfan Foundation web site).
Taken together, the evidence indicates that misregu
lation of TGF signalling owing to defects in extracellular
proteins is centrally important to the development
of aortic aneurysms, and there is now a realistic hope
for patient therapies. This view has now replaced the
previous idea that aortic aneurysms were simply due to
a structural deficiency of the elastin matrix in the aorta.
It is notable in this context that mice lacking the extra
cellular protein fibulin5 have fragmented elastin and
tortuous elongated aortae, but do not develop aortic
REVIEWS
aneurysms or dissections88,89. It may be that the aortae
of these mice have normal TGF activity and, hence,
do not progress to aneurismal changes. By contrast,
mutant mice that have lost the related Efemp2 gene
(which encodes fibulin4) show a striking aortic rup
ture phenotype and perinatal lethality90. This defect
precluded analysis of this gene in mature vasculature, but
mice that are homozygous for a hypomorphic allele with
reduced fibulin4 expression survive to adulthood
with a milder phenotype. These mice have aortic dilata
tion, aneurysms and aortic valve dysfunction associated
with abnormal TGF signalling in the aorta91. These dif
ferent mouse models offer the opportunity to unravel the
complex interaction between aortic integrity and ECM
regulation of TGF activity.
Placental
syncytiotrophoblast cell
A multinucleated cell found at
the boundary of the fetal and
maternal layers of the
placenta.
TGF and hypertension. A link between increased levels

of circulating TGF and hypertension has been known
for some time92, but new findings are throwing light on
the nature of this interaction. Emilin1, an ECM pro
tein that is associated with the microfibrils of the elastic
matrix in the aortic media, regulates TGF availability
and arterial diameter93. By interacting with unprocessed
TGF, emilin1 protects it from proteolytic processing
by the endoprotease furin convertase (FIG.2). Loss of
emilin1 therefore results in the increased conversion
of pro-TGF to the mature form and a subsequent
increase in TGF signalling. This leads to a reduc
tion in the arterial lumen diameter with a resultant
increase in vascular resistance and hypertension 93.
A possible mechanism is that excessive levels of active
TGF causes premature cytostasis of the vSMCs, and
subsequently restricts vessel size. Alternatively, reduced
arterial diameter may be a secondary consequence of
vascular remodelling in these mice94. This phenotype can
be rescued genetically by crossing in a single Tgfb1-null
allele to reduce TGF levels.
A related disease that involves raised blood pressure
is pre-eclampsia, a serious disease of late pregnancy that
threatens the health of both mother and baby. Patients
with pre-eclampsia have increased circulating levels
of a soluble form of VEGF receptor1 (VEGFR1; also
known as sFLT1), which is thought to function as a
sink for VEGF ligands and reduce the access of VEGF
ligands to VEGFRs on ECs. Recently, elevated levels
of soluble endoglin were also found to cooperate with
soluble VEGFR1 in the pathogenesis of pre-eclampsia95.
Soluble endoglin is probably formed by proteolytic
cleavage of full-length endoglin, and is possibly derived
from placental syncytiotrophoblast cells. This soluble form
may function in an analogous way to soluble VEGFR1
by binding circulating TGF, which therefore inhibits
TGFBR2- and ALK5-dependent signalling95. This is
thought to reduce the level of nitric oxide synthase-3
(NOS3, eNOS) activation and attenuate vasorelaxation
responses, leading to pre-eclampsia.
The mechanisms that regulate the production of
soluble endoglin and a detailed understanding of its
molecular role in patients with pre-eclampsia remain
to be determined. A recent report showed that haem
oxygenase1, an anti-inflammatory enzyme required
for successful pregnancy, inhibits the release of soluble

VEGFR1 and soluble endoglin, a role that might be used
in the future treatment of pre-eclampsia96. However,
circulating levels of soluble endoglin may already be
a useful diagnostic marker to prioritize patients for
treatment before the onset of symptoms97.
Once again, the role of TGF in the regulation of
hypertension appears to be contradictory. Increased
levels of activated TGF in emilin1-null mice result
in hypertension, whereas reduced TGF signalling in
ECs may contribute to increased blood pressure in preeclampsia93,95. However, the underlying mechanisms are
different. There is no detectable change in vasoregula
tory responses in arteries in emilin1-null mice instead,
there is a reduction in vessel size. By contrast, in preeclampsia there is a transient defect in vasoregulatory
mechanisms, but the blood pressure returns to normal
levels once the fetus is born. In addition, there may
be other ligands involved in the pre-eclampsia model,
as endoglin binds other ligands of the TGF family,
including BMP9 (Ref.68).
TGF and tumour angiogenesis. The importance of
understanding the regulation of angiogenesis by TGF
family members is crucial because many of these pro
cesses are recapitulated in a disorganized fashion in neo
plastic disease. Several small-molecule TGF receptor
kinase inhibitors have been developed for cancer treat
ment98. However, there may also be unwanted effects of
TGF receptor inhibition, such as the possibility that the
rate of progression of some cancers will increase when
the tumour-suppressive effects of TGF are inhibited99.
In light of this possibility, it is promising that there
appears to be no predisposition to cancer in patients
with LDS who have reduced TGF receptor levels100.
Rather, the paradoxical enhancement of TGF signal
ling in aortic aneurysms of patients with LDS raises the
possibility that inhibiting TGF receptors may lead to a
signalling imbalance.
A recent study took advantage of the effects of a TGF
receptor inhibitor on the neovasculature that mirrored the
vascular defects seen in TGF-receptor knockout mice.
Short-term treatment with an ALK5 inhibitor in mice
that had intractable solid tumours led to reduced vSMC
coverage in neovessels, making them leaky and promot
ing the accumulation of anticancer drugs in the tumour
tissue101. Parallel experiments targeting endoglin, which is
strongly upregulated in angiogenic tumour ECs, suggests
that endoglin may also represent a useful anti-angiogen
esis target for cancer treatment102,103,104. Equally important
is the development of pro-angiogenic therapies used to
treat ischaemic disease; manipulating TGF signalling
may represent a valuable therapeutic approach and preclinical data have shown that endoglin promotes tissue
repair by pro-angiogenic circulating blood cells105.
Conclusions and future perspectives

In the pursuit of molecular mechanisms that underlie
the vascular pathology of MFS and related diseases, an
important additional role for elastic ECM components
in controlling growth factor signalling was discovered5.
REVIEWS
Truncus arteriosus
A single vessel that forms early
in development and then
septates to form the aorta and
pulmonary trunk. A persistent
truncus arteriosus is one that
has failed to septate,
compromising the separation
of pulmonary and systemic
circulations.
Aortopulmonary septation
The process whereby the
pulmonary trunk and aorta
separate during development.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Besides being structural scaffolds that provide tissue

integrity, elastic microfibril components have an
instructive role in regulating TGF processing and bio
availability. Latent TGF complexes, bound via LTBPs
to fibrillin1, are sequestered and provide latent embed
ded signals that can be rapidly released by proteases in
response to tissue perturbations. This allows for rapid
and localized changes in TGF activity without the need
for denovo protein synthesis106. Dysregulation of TGF
family signalling in MFS may provide an explanation
for clinical variability, as disease pathogenesis may differ
in a genotype- and context-dependent manner5. In
addition, dysregulated TGF signalling may underlie
the pathology of other vascular disorders. For example,
mutations in the facilitative glucose transporter GLUT10
were found to cause arterial tortuosity syndrome (ATS),
and were associated with upregulation of TGFSMAD
signalling in the arterial wall107.
The instructive role for microfibrils has been extended
to other TGF family members such as BMP7, which binds
via its prodomain directly to fibrillin1, potentially target
ing it to the ECM108. Mutations in fibrillin2 (FBN2) are
associated with disorganized microfibrils and the Marfanlike disorder, congenital contractural arachnodactyly
(TABLE1), and Fbn2 null mice display bilateral syndactyly
of forelimbs and hindlimbs109. Interestingly, mice that
are double heterozygous for null Fbn2 and Bmp7 alleles,
which alone are phenotypically silent, have impaired digit
formation, suggesting a functional interaction between
fibrillin-2 and BMP7 (Ref.109). The extracellular regula
tion of BMP signalling by ECM components has been
largely overlooked and needs further investigation.
It is possible that fibrillins have dual and oppos
ing roles in regulating TGF activity. They may act
positively by concentrating ligand at sites of function,
and negatively by sequestering the LLC and inhibit
ing TGF activation. It is also interesting to note that
mice lacking Ltbp1 show persistent truncus arteriosus, a
phenotype that is also seen after neural-crest-specific
ablation of TGF signalling110 (table 1). This may reflect
a failure to provide a sequestered source of TGF in the
absence of LTBP1 during aortopulmonary septation.
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Feng, X.H. & Derynck, R. Specificity and versatility in
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transforming growth factor in human disease.
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Recognizing the importance of extracellular regula

tion of TGF activity in aortic muscle cells in MFS and
LDS has opened up exciting possibilities for treatments
of further muscle disorders. Skeletal muscle weakness is
an additional feature of MFS that results from increased
TGF signalling, which inhibits regeneration of muscle
cells and stimulates fibrosis. In parallel to the beneficial
effects of losartan on aortic function in a mouse model
of MFS, losartan also improves muscle morphology
and enhances satellite-cell activation and muscle
regeneration. This led to the consideration that these
benefits might ameliorate another progressive muscle
weakness disorder, Duchennes muscular dystrophy,
which is caused by familial mutations in dystrophin
(DMD). Losartan treatment lowered the abnormally
high intramuscular TGF levels, improved muscle
histopathology and reduced fibrosis in a Dmd-deficient
mouse model of this disease, raising hopes that this
treatment may also benefit patients with Duchennes
muscular dystrophy111.
The complexities of TGF signalling are challeng
ing to scientists and clinicians alike. However, it is clear
that TGF components represent valuable therapeutic
targets, even though the changes in blood vessel archi
tecture and blood pressure that are associated with
dysregulated TGF signalling indicate the necessity
for careful monitoring of these parameters in patients.
The effectiveness of losartan for treating dysregulated
TGF signalling means that the interaction between
angiotensin and TGF signalling pathways is an
important area of investigation. There is also an urgent
need to improve our understanding of the extracellular
regulation of TGF ligand activation and the context
dependence of cellular responses. Furthermore, the
phenotypes of mouse models with mutations in genes
that are associated with extracellular regulation of TGF
signalling (TABLE1) raise the possibility that further
human cardiovascular syndromes may eventually be
linked to some of these genes. Advances in all of these
areas will inform current treatment strategies and open
up possibilities for developing new therapies for a range
of vascular pathologies.
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12. Saharinen, J., Taipale, J. & Keski-Oja, J. Association of
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Acknowledgements
Research in our laboratories is supported by grants from the

Dutch Cancer Society, the EC (Angiotargeting and Tumour
Host Genomics), the Ludwig Institute for Cancer Research, the
Netherlands Organization for Scientific Research, the British
Heart Foundation, Newcastle Hospital Trustees, the Cookson
Foundation and the Wellcome Trust. We are grateful to our
colleagues for valuable discussion, and apologize to those
whose contributions have not been cited because of space
constraints.
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
ACVRL1 | BMPR2
OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=OMIM
arterial tortuosity syndrome | Duchennes muscular
dystrophy | familial thoracic aneurysm disorder | hereditary
haemorrhagic telangiectasia type 1 | HHT type 2 | LoeysDietz syndrome | Marfan syndrome | MFS type 2 | pulmonary
arterial hypertension
UniProtKB: http://ca.expasy.org/sprot
ALK1 | betaglycan | BMP1 | emilin1 | endoglin | fibrillin1 |
fibrillin2 | SMAD1 | SMAD2 | SMAD3 | SMAD4 | SMAD5 |
TGF1 | TGF2 | TGF3 | TGFBR1 | TGFBR2
FURTHER INFORMATION
Peter ten Dijkes homepage:
http://www.lumc.nl/1050/research/signaaltransductie.html
Helen Arthurs homepage:
http://www.ncl.ac.uk/ihg/staff/profile/helen.arthur
HHT Foundation International web site:
http://www.hht.org/
The US National Marfan Foundation web site:
http://www.marfan.org
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REVIEWS

Extracellular control of TGF signalling

Abstract | The intracellular mechanism of transforming growth factor (TGF) signalling

*Molecular Cell Biology,

Institute of Human Genetics,

Transforming growth factor1 (TGF1) is the prototypic

nature reviews | molecular cell biology

regulation of TGF signalling by co-receptors (which

Secretion and ECM interaction

2007 Nature Publishing Group

Figure 1 | Signal transduction by TGF family members. Canonical signalling by

developmental processes and maintain tissue homeostasis

858 | november 2007 | volume 8

and LAP remain attached via non-covalent bonds to form

2007 Nature Publishing Group

b Activation and receptor binding

Figure 2 | Regulation of TGF bioavailability. Schematic model of synthesis, secretion

cells have increased fibrillar structures that contain

Activation and receptor binding. In order for TGF to

2007 Nature Publishing Group

Extracellular regulation of TGF signalling

KO: reduced arterial diameter, increased vascular resistance, emphysema,

KO: ruptured aortic aneurysm, impaired pulmonary function, die at birth.

KO: bilateral syndactyly.

KO: deformed heart and embryonic vessels, defective extra-embryonic

KO: perinatal lethality, arterial narrowing, aortic rupture.

FBLN5 (fibulin-5, DANCE)

KO: tortuosity and elongation of the aorta, loose skin.

KO: persistent truncus arteriosus.

KO: craniofacial malformations, osteosclerosis and osteoarthritis.

Hypomorphic: emphysema, cardiomyopathy and colorectal cancer.

KO: pneumonia, alveolar haemorrhage, vSMC hyperplasia.

ITGAV (integrin, aV)

KO: embryonic lethal placental defects or perinatal lethal with intracerebral

KO: inflammation of lungs and skin.

KO: embryonic lethal with vascular defects or postnatal lethality from

KO: aortic arch defects, cardiac septal defects, perinatal lethality.

Het for missense mutation (C1039G): aortic aneurysm, emphysema.

Hypomorphic: tortuous aorta, aortic aneurysm, increased TGF activation.

of typeI and typeII receptors respond to other TGF

860 | november 2007 | volume 8

TypeIII (or auxiliary) receptors such as betaglycan

TGF regulates vascular development

2007 Nature Publishing Group

Het: vascular lesions similar to HHT.

TGF auxiliary receptors

KO: embryonic lethal, reduced vSMC differentiation, heart defects.

KO: poorly formed cardiac septa, incomplete compaction of ventricular walls.

HHT2 (rarely PAH)

Het: vascular lesions similar to HHT.

TGF signalling receptors

KO: embryonic lethal, reduced vSMC differentiation, dilated vessels, AVMs.

Zebrafish KO: dilated vessels, AVMs.

KO: embryonic lethal, vascular defects.

KO: embryonic lethal, angiogenesis defects.

KO: pre-angiogenesis lethality.

Het: mild pulmonary hypertension.

Intracellular TGF signalling molecules

KO: pre-angiogenesis lethality, defects in chorion-allantoic circulation.

KO: pre-angiogenesis lethality.

KO: viable. Impaired immunity, colon cancer.

KO: pre-angiogenesis lethality.

KO: embryonic lethal, angiogenesis defects.

KO: cardiac defects, aortic ossification, elevated blood pressure.