signaling by members of the tgf-β family in vascular morphogenesis and disease

Signaling by members of the TGF-bfamily in vascular morphogenesisand diseaseEvangelia Pardali, Marie-Jose Goumans and Peter ten Dijke

Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Center, The Netherlands

Review

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 specifictype I and type II serine/threonine kinase receptors andintracellular Smad transcription factors. This reviewhighlights recent progress in our understanding ofTGF-b signaling in vascular development and angiogen-esis and of how perturbed TGF-b signaling might con-tribute to vascular pathologies, tumor angiogenesis andtumor progression. Recent research has provided excit-ing insights into the role of the TGF-b type I receptor(ALK1) in tumor angiogenesis and the curative effects ofthalidomide on vascular malformations in hereditaryhemorrhagic telangiectasia (HHT). These advances pro-vide opportunities for the development of new therapiesfor diseases with vascular abnormalities.

IntroductionTissue homeostasis is dependent on an adequate supply ofoxygen and nutrients and removal of waste productsthrough blood vessels [1]. The development and properfunction of the vascular system (Box 1, Figure I) is essen-tial for survival of all higher organisms. The vascularsystem plays an important role in embryonic developmentbut also later during life. During embryo development theformation of new blood vessels depends on vasculogenesisand angiogenesis. Vasculogenesis refers to the de novoformation of blood vessels. Angiogenesis is the formationof new blood vessels from pre-existing ones and is con-trolled by a number of growth factors and signaling path-ways and the balance between pro- and anti-angiogenicfactors (Box 2, Figure I) [2]. Angiogenesis takes also placein adult life to maintain physiological homeostasis andtissue integrity during wound healing, inflammation andduring the female menstrual cycle. Deregulation of vascu-logenesis and angiogenesis has been implicated in a multi-tude of pathological situations.

TGF-b (TGFB1–3) is the prototype of the extended TGF-b family of cytokines which also includes activins/inhibins,Nodal, bone morphogenetic proteins (BMPs) and growthdifferentiation factors (GDFs) [17,18]. TGF-b family mem-bers play crucial roles in embryonic development, adulttissue homeostasis and the pathogenesis of a variety ofdiseases. Research over the past two decades into the

Corresponding author: ten Dijke, P. ([email protected]).

556 0962-8924/$ – see front matter � 2010 Elsevier Ltd. A

mechanisms of TGF-b signaling has led to a well-acceptedcanonical signaling cascade involving heteromeric cell-sur-face complexes of receptor kinases together with Smadtranscription factors (named from C. elegans Sma andDrosophila Mad (mothers against decapentaplegic)) thatact 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 anumber of other signaling pathways (non-Smad signalingpathways; Box 3, Figure I) [19]. Thus, cellular responses toTGF-b signaling result from a dynamic regulation of Smadand non-Smad cascades.

Although several in vitro and in vivo studies providestrong evidence for the important role of the TGF-b andBMP (bone morphogenetic protein) signaling pathways invasculogenesis and angiogenesis, there is still confusion inthe field, generated by reports of opposite effects on angio-genesis by specific family members. Both pro- and anti-angiogenic effects of TGF-b, BMP9 and ALK1 (activinreceptor-like kinase 1) have been reported. In additionthe role of TGF-b type I and II receptors on EC (endothelialcell) function was questioned by some studies. Much of thisconfusion stems seemingly from the remarkable diversityand context-dependent effects of TGF-b familymembers onthe multistep and intricately regulated process of bloodvessel formation. Here, we review recent insights into therole of TGF-b signaling in vascular morphogenesis anddysfunction. The mechanisms by which TGF-b familymembers control the function and interplay between endo-thelial and smooth muscle cells will be discussed, and howthese new advances could be exploited for restoring thevascular bed in HHT or for anti-angiogenic therapy incancer.

Role of TGF-b signaling in vasculogenesis andangiogenesisGenetic studies in mouse and human have provided muchevidence for the importance of components of the TGF-bsignaling pathway in vascular morphogenesis and dys-function (Table 1). Deletion of Tgfb1 in the mouse resultsin embryo lethality because of defective yolk sac vasculo-genesis. Interestingly, Tgfb1 deletion leads to vascularabnormalities only in a specific genetic background,suggesting the involvement of other factors (modifiers)in the development of vascular abnormalities due todefects in TGF-b signaling. Similar phenotypes have beenobserved in mice deficient for Tgfbr2 and Tgfbr1 (Alk5),

ll rights reserved. doi:10.1016/j.tcb.2010.06.006 Trends in Cell Biology 20 (2010) 556–567

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http://dx.doi.org/10.1016/j.tcb.2010.06.006

Box 1. Vascular morphogenesis

During embryo development, blood vessels develop de novo

through differentiation of mesodermal progenitor cells, the heman-

gioblasts, 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 intussus-

ceptive 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

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 pro-

and 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, angio-

genesis plays a crucial role in tumor growth, and inhibition of tumor

angiogenesis can suppress tumor growth [9,10].[(Box_1)TD$FIG]

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.

Review Trends in Cell Biology Vol.20 No.9

suggesting an important role for these receptors in ECfunction. Recent studies suggested that the effects ofTGFBR2 or TGFBR1 loss on angiogenesis are not due totheir role on ECs but are due to defects in smooth musclecell (SMC) function [27]. Using an Acvrl1 (Alk1)-derivedpromoter to delete Tgfbr2 or Tgfbr1 in ECs, no effects onvascular morphogenesis were observed. In addition, it wassuggested that ALK5 is expressed only on SMCs but not inECs [28]. By using the tyrosine-protein kinase 1 Tie1vascular EC-specific promoter, another group has clearlyshown that both receptors play important roles in ECfunction [29,30]. Tie-1 is a vascular endothelial-specificreceptor tyrosine kinase essential for the regulation ofvascular network formation and remodeling. EC deletionof Tgfbr2 or Tgfbr1 results in embryo lethality at embryo-nic day (E) 10.5 due to vascular defects. The discrepanciesbetween the two studies are most probably attributable totemporal regulation of the promoters used. These resultsfurther support the notion that the role of TGF-b signalingand its receptors is context-dependent.

Targeted deletions of Acvrl1 (Alk1), Tgfbr1 (Alk5),Tgfbr2 and Eng (endoglin, a TGF-b coreceptor highlyexpressed on EC) in mice result in vascular abnormalities

highly reminiscent of those described in patients withHHT[20], an autosomal dominant vascular disorder character-ized by fragile blood vessels which lead to telangiectasesand arteriovenous malformations (AVMs). HHT-1 andHHT-2 arise from heterozygous mutations in ENG andACVRL1 (ALK1) genes, respectively. A subset of patientswith juvenile polyposis, carrying mutations in the SMAD4gene, can also develop HHT [31]. It has been postulatedthat genetic background and/or environmental factors (sec-ond hits), in addition to the genetic mutations in the ENGor ACVRL1 genes, play an important role in the develop-ment of vascularmalformations inHHT patients. Park andcolleagues demonstrated recently using Acvrl1-deficientmice that a second hit, excisional skin wounding, is essen-tial for the development of AVMs in HHT. Their resultsprovide new insights for understanding the pathogenesisof HHT [32].

Despite the identification of the genes responsible forHHT, the underlying molecular mechanisms for the patho-genesis of HHT remain obscure. Genetic studies in mousedemonstrated that deletion of Acvrl1 (Alk1) or Eng resultsin loss of arteriovenous specification and the developmentof 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 specia-

lized 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

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 concen-

tration 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 under-

lying 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 angiopoietin–Tie2

signaling pathway acts in the opposite orientation – from mural cells

to the endothelium – and plays an important role in vessel

stabilization [5,16].[(Box_2)TD$FIG]

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-cell–tip-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 asimilar phenotype to that seen in Acvrl1 null mice withdilated vessels and enlarged cranial arteries that abnor-mally connect directly to veins [34]. Acvrl1 deficiency

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results in decreased expression of the arterial markerephrin B2, an Eph receptor ligand, known to be associatedwith arterial identity. Interestingly, ephrin B2 isexpressed normally in Eng-deficient mice, suggesting that

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 bioavail-

ability 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

are intracellular mediators for the TGF-b family and are classified into

three groups: receptor-regulated (R-Smad), common-mediator (Co-

Smad), 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 phosphoryla-

tion of R-Smads 2 and 3, whereas ALK1,2,3,6 mediate phosphoryla-

tion 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 dephosphoryla-

tion, 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 PI3K–AKT pathway, and these can crosstalk with the

Smad pathways (Figure I) [19].[(Box_3)TD$FIG]

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 R-

Smads. 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 ofreceptors and their ligands are expressed in arterialECs, promote artery-specific ephrin B2 expression andhave been implicated as potential regulators of arteriove-nous fate. However, no expression differences wereobserved in Acvrl1- or Eng-deficient embryos based on insitu hybridization of Notch signaling-pathway-related

genes [33]. Paradoxically, although Eng deletion in mouseembryos results in arterial expression of the venous-specific marker COUPTFII (chicken ovalbumin upstreampromoter-transcription factor II) [35], endothelial-cell-specific deletion of endoglin did not affect expression ofarterial jagged-1 and ephrin B2, or venous markers Ephb4and 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) Animal model Human disease Refs

Ligands

Tgfb1/TGFB1 KO: embryonic lethal with vascular defects or

postnatal lethality from autoimmune disease

Camurati–

Engelmann

diseasea

[20]

Tgfb2/TGFB2 KO: aortic arch defects, cardiac septal defects,

perinatal lethality

unknown [20]

Tgfb3/TGFB3 KO: cleft palate, delayed lung maturation,

die shortly after birth

unknown [20]

Receptors

Tgfbr2/TGFBR2 KO: embryonic lethal, vascular defects MFS2a, LDSa [20]

SM22-Cre-Tgfb2fl/fl KO: embryonic lethal, vascular defects [20]

Tie1-Cre-Tgfbr2fl/fl KO: embryonic lethal, vascular defects [20]

Tgfbr1 (Alk5)/TGFBR1 (ALK5) KO: embryonic lethal, angiogenesis defects LDS [20]

Tie1-Cre-Tgfbr1fl/fl KO: embryonic lethal, angiogenesis defects [20]

Acvrl1 (Alk1) / ACVRL1 (ALK1) KO: embryonic lethal, reduced VSMC

differentiation, dilated vessels, AVMs.

HHTb [20]

Bmpr2/BMPR2 KO: embryonic lethal (pre-angiogenesis)

lethality. Transgenic BMPR2-mutant allele:

pulmonary hypertension

PAHb [20,23,24]

Accessory receptors

Eng/ENG (endoglin) KO: embryonic lethal due to vascular defects,

reduced VSMC differentiation, heart defects.

Het: vascular lesions similar to HHT

HHT [20]

Soluble endoglin Pre-eclampsia [25]

Tgfbr3/TGFBR3 (betaglycan) KO: poorly formed cardiac septa, incomplete

compaction of ventricular walls

Unknown [20]

Smads

Smad1/SMAD1 KO: embryonic lethal due to defects in

chorioallantoic circulation

Unknown [20]

Smad4/SMAD4 KO: embryonic lethal JPb and HHT [20]

Smad5/SMAD5 KO: embryonic lethal due to angiogenesis

defects

Unknown [20]

Smad6/SMAD6 KO: heart abnormalities, aortic ossification

and elevated blood pressure

Unknown [20]

Smad7/SMAD7 KO: embryonic lethal due to cardiovascular

defects

Unknown [26]

Abbreviations: AVMs arteriovenous malformations; Het heterozygote; JP Juvenile polyposis; KO knockout; LDS Loeys–Dietz syndrome; MFS2 Marfan syndrome type 2; HHT

hereditary hemorrhagic telangiectasia; PAH pulmonary arterial hypertension; VSMC vascular smooth muscle cell.aDue to hyperactivation of TGF-b signaling.bDue to attenuated TGF-b signaling.


retinas [36]. Interestingly, AVMs in these mice expressedEphb4 and Aplnr, suggesting that they have venouscharacteristics [36]. Previous studies have suggested theexistence of synergism between Notch and TGF-b [37,38]or BMP6 signaling [39], and that Notch signaling modu-lates the balance between TGF-b/ALK1 and TGF-b/ALK5signaling pathways [40]. Future studies are awaited toelucidate the crosstalk between ALK1/endoglin and othersignaling pathways involved in arteriovenous fate. Failureto establish or maintain proper arterial–venous bound-aries might be related to abnormalities in sproutingmechanisms during angiogenesis and in tip/stalk ECdetermination. Endoglin has been shown to be expressedin tip cells and in tip cell filopodia. Endothelial-cell-specificEng depletion did not affect filopodia formation in the tipcells or the numbers of filopodia in tip and stalk ECs inneonatal mouse retinas [36]. Future research is needed tofurther characterize the exact role of endoglin and ALK1 inarteriovenous specification and tip, stalk and phalanx ECfunction. The molecular pathways by which Eng andAcvrl1 deficiency lead to AVM are still not known, andtheir crosstalk with Notch, ephrinB or additional factorsinvolved in the molecular determination of vein identityremains to be elucidated.

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Vascular endothelial growth factor (VEGF) signalingplays a crucial role in angiogenesis and its dysregulationleads to defects in angiogenesis. It has been shown thatVEGF levels are elevated in skin telangiectatic lesions ofHHT patients and that anti-angiogenic drugs, such asthalidomide and bevacizumab (anti-VEGF antibody), areeffective in treating gastrointestinal bleedings and liverAVMs, respectively [41–43]. By contrast, inhibition ofALK1/endoglin signaling using the soluble chimericproteins ALK1-Fc and endoglin-Fc (containing either theALK1 or the endoglin extracellular domains fused to the Fcpart of IgG that sequester ligands from binding to endogen-ous endoglin and ALK1) hindered VEGF-induced ECsprouting in vitro [44,45]. ALK1-Fc also inhibited VEGF/basic fibroblast growth factor (bFGF)-induced angiogen-esis in an in vivo matrigel plug assay [44]. Although theseresults clearly suggest that there is crosstalk betweenVEGF and ALK1/endoglin signaling in angiogenesis, theexact molecular mechanisms underlying this interplayremain to be elucidated.

TGF-b signaling and endothelial cell functionSeveral studies have revealed that the effect of TGF-b onangiogenesis is context dependent [46,47]. TGF-b was

[(Figure_1)TD$FIG]

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 lowconcentrations, whereas high concentrations had the oppo-site effect [46–49]. Low concentrations of TGF-b enhancedthe angiogenic effects of bFGF or VEGF in a 3D fibrin orcollagenassay,whereashighconcentrationswere inhibitory[48]. Treatment of bovine capillary endothelial (BCE) cellswith TGF-b initially induces apoptosis by inducing VEGFexpression because TGF-b signaling converts the VEGF/VEGFR2-activated p38 (MAPK) into a proapoptotic signal[50]. However, protracted treatment of BCE cells with TGF-b results in EC remodeling and formation of cord-like struc-tures [51]. Inhibition of the TGF-b–ALK5 pathway by anALK5 kinase inhibitor resulted in sustained proliferationand maintenance of human embryonic stem cell (hESC)-derived ECs by sustaining Id1 expression [52]. Similarly,addition of an ALK5 kinase inhibitor in mouse ESCsincreased EC growth and integrity through upregulationof the tight junction component claudin-5 [53]. It was alsoshown that suboptimal doses of VEGF and ALK5 kinaseinhibitor synergistically induceECmigrationand sproutingin vitro by inducing integrin a5 expression. In addition,ALK5kinase inhibition inducedangiogenesis and enhancedVEGF/bFGF-induced angiogenesis in amatrigel-plug assayin vivo, an effect that could be inhibited by an antibody thatneutralizes the a5 integrin [54].

The initial view of TGF-b family signaling as a simplelinear cascade, where TGF-b/Nodal/activin induce phos-phorylation of Smads 2 and 3 and BMP/GDFs inducephosphorylation of Smads 1, 5 and 8, has been re-evalu-ated. 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 inactivation of Smad2,3 and Smad1,5,8, respectively(Figure 1) [47,55]. Although TGF-b/ALK5/Smad2,3 sig-naling was found to antagonize TGF-b/ALK1/Smad1 sig-naling, ALK5 is essential for ALK1 recruitment into theTGF-b receptor complex and for its activation. It wasshown that TGF-b/ALK1 signaling potentiates and TGF-b/ALK5 inhibits EC proliferation and migration of ECs[47,55]. It was thus suggested that the balance betweenTGF-b/ALK1 versus TGF-b/ALK5 will determine the pro-or anti-angiogenic effects of TGF-b. In addition to beingpresent in ECs, the TGF-b/ALK1 pathway has also beenobserved in neurons, chondrocytes and hepatic stellatecells [56–58]. Moreover, in tumor cells, TGF-b was shownto signal by means of the Smad1 pathway. In one of thestudies, TGF-b induced Smad1 phosphorylation throughALK2 and ALK3 [59], whereas two studies suggested thatALK5 can directly interact with and phosphorylate Smad1[60,61]. The mechanistic differences between these studiesare probably attributable to different receptors expressedon different cell types as well as the relative expressionlevels of each receptor.

Although the studies discussed above suggest animportant role for ALK1 in the activation phase of angio-genesis, overexpression studies demonstrated that ALK1signaling inhibits the proliferation and migration of ahuman microvascular EC line, implying that ALK1promotes the resolution phase of angiogenesis [62], con-sistent with the phenotype described inAlk1-deficientmice

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– fragile blood vessels and increasedmRNA levels for genesinvolved in the activation phase of angiogenesis [23]. Thediscrepancies could be due to different cell types being usedin the studies or to the adaptive processes that take placein the Alk1-deficient embryos. Alternatively, ALK1 couldbe involved in both the activation and resolution phases ofangiogenesis.

In ECs, BMP9 and BMP10 have been shown to signalthrough ALK1 and ALK2, induce Smad1 phosphorylation,and inhibit EC proliferation and migration [63,64]. Inhuman pulmonary artery endothelial cells (HPAECs),BMP9 was shown to induce phosphorylation of Smad1-5and Smad2 through BMPR2/ALK1 and the activin type IIreceptors (ACVR2) [65] (Figure 1). Although both BMPR2and ACVR2 were required for Smad1 phosphorylation,Smad2 activation was mainly mediated through ACVR2.The significance of this differential BMP9 signaling inangiogenesis and in vascular pathologies such as HHTand primary arterial hypertension (PAH, a vascular dis-order associated with missregulated BMP signaling due tomutations in BMPR2) remains to be elucidated. BMP9signaling through ALK1 was shown to inhibit VEGFexpression [66], whereas increased VEGF levels werereported in Acvrl1-deficient embryos and HHT-2 patients[67,68]. Although BMP9 and BMP10 were considered tohave an anti-angiogenic action, BMP10 was shown toinduce angiogenesis in the chorioallantoic membrane(CAM) assay [69] and BMP9 to induce proliferation ofECs in vitro and in vivo [70]. In addition, BMP9 in com-bination with TGF-b was shown to potentiate VEGF-induced proliferation of ECs in vitro and VEGF/bFGF-induced angiogenesis in vivo [44].

Several studies have provided evidence that the TGF-bcoreceptor endoglin plays an important role in balancingthe TGF-b–ALK1 and TGF-b–ALK5 pathways [22]. Endo-glin was shown to potentiate the TGF-b–ALK1 pathwayand to inhibit the TGF-b–ALK5 pathway in ECs as well asin other cell types [71]. It was also shown that endoglinplays an important role in BMP9 signaling in ECs [63]. Themolecular mechanisms by which endoglin regulates TGF-bandBMP signaling are not fully understood. A recent studysuggested that ALK5 phosphorylates the cytoplasmicdomain of endoglin on serines 646 and 649. S646A isrequired for activation of TGF-b–ALK1–Smad1,5,8 sig-naling, whereas both S646 and S649 are essential forBMP9-induced Smad1,5,8 phosphorylation [72]. Analysisof mouse retinal vessels showed that endothelial-cell-specific deletion of Eng does not affect levels of phosphory-lated Smad2 or Smad1,5,8 [36]; however, deletion of Engresults in loss of Smad1,5,8 phosphorylation in the quies-cent pulmonary vasculature [73]. As with ALK1, the role ofendoglin in EC function is not completely understood; invitro studies have suggested that knockdown of endoglinresults in decreased EC proliferation and migration,whereas Eng deletion in the mouse results in a dramaticincrease in EC proliferation [36].

BMPs 2, 4, 6 and 7 have been shown to induce ECproliferation and angiogenesis by inducing VEGF expres-sion [46,74]. In mouse embryonic stem cells (ESCs), BMP4exerts 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 BMP4induces phosphorylation not only of Smad1 but also Tie2and VEGFR2 in these cells [75]. The pro-angiogenic effectsof BMP6 have been shown to be mediated by the upregula-tion of Id1 and cyclooxygenase-2 expression in ECs [76,77].Recently it was also shown that Myo10 plays a crucial rolein BMP6-induced angiogenesis. Myo10 is upregulated inEC in response to BMP6 and that it is required for BMP6-induced filopodia formation and migration [78].

Although it is apparent that TGF-b and BMP signalingcomponents play crucial roles in EC function and angio-genesis, we have only started to unravel the molecularmechanisms by which these molecules regulate vascularsystem. Cellular context, local concentration of the differ-ent ligands, receptors, coreceptors, antagonists and theirinterplay play crucial roles in the apparently contradictoryactions of TGF-b signaling during the different stages ofangiogenesis. The identification and characterization ofadditional molecules andmechanisms involved in vascularsignaling by TGF-b family members will help us to under-stand what determines the pro- or anti-angiogenic func-tions of TGF-b, ALK1 and BMP9, and their interplay withother factors involved in EC function and angiogenesis.

TGF-b signaling and SMC functionVessel formation and stabilization cannot be completedunless pericytes and vascular SMCs are recruited to com-plete vessel assembly. Proper communications betweenECs and mural cells play a crucial role in vessel formation,and these are tightly controlled by a number of signalingpathways [16] (Boxes 1, 2).

TGF-b regulates SMCmuscle differentiation by increas-ing the expression of alpha smooth-muscle actin andsmooth-muscle myosin through the Smad3 and p38/MAPKpathways [79]. TGF-b treatment of ESC-derived culturesor embryoid bodies potentiates their differentiation intoSMCs [53,80]. TGF-b can also induce proliferation andmigration of SMCs. Genetic studies in the mouse(Table 1) revealed the important role of TGF-b signalingin SMC cell development and recruitment for the for-mation of stable vessels. EC- and SMC-specific deletionsof Tgfbr2 show similar phenotypes. Deletion of Tgfbr2 inSMCs results in vascular defects in the yolk sac andembryo lethality between E12.5 and E16.5. Those resultssuggest that TGF-b signaling on ECs and SMCs plays animportant role in SMC differentiation and function and, asa consequence, in proper EC–SMC interaction [29,30]. Inaddition, mice with neural-crest-specific ablation of Tgfbr2develop a phenotype akin to DiGeorge syndrome becauseneural crest derivatives fail to differentiate into SMCs inthe cardiac outflow tract [81].

Both Eng and Acvrl1 (Alk1) depletion result in fragileblood vessels due to impaired mural cell development, asshown by the absence or inappropriate association of SMCswith ECs [33,82–84]. Conditional endoglin expression inEng-null embryos, using either SMC- or EC-specific pro-moters, can partially rescue SMC recruitment to the dorsalaorta, suggesting that endoglin plays distinct and cell-autonomous roles in SMC recruitment [35]. Telangiectaticlesions in HHT patients are characterized by abnormalendothelial cell proliferation and SMC recruitment.


Thalidomide treatment of HHT patients was shown toenhance blood vessel stabilization and reduce nosebleedfrequency [85]. Vessel maturation induced by thalidomidetook place by mural cell recruitment in Eng-heterozygousmice and HHT patients, partly by inducing the expressionof the platelet-derived growth factor B subunit (PDGFB) inECs, further supporting the important role of endoglin inEC–SMC interactions [85].

BMPs also play a role in SMC differentiation and func-tion. BMP7 inhibits SMC growth induced by PDGF-BB(the dimer of PDFGB) and by TGF-b1, whereas it main-tains the expression of markers that maintain the SMCphenotype [86]. BMP2 was shown to induce SMCmigration and to inhibit PDGF-induced proliferation ofSMCs [87]. BMP pathway activation through BMPR2 isnecessary for growth and differentiation control in SMCs[88,89]. Mutations in BMPR2 result in PAH, a vasculardisorder characterized by uncontrolled remodeling of thepulmonary arteries due to increased proliferation of SMCsand increased pulmonary EC apoptosis [24]. Interestingly,certain HHT2 patients develop a PAH-like syndrome,suggesting that ACVRL1 (ALK1) mutations are also likelyto be involved in PAH [90,91].

In summary, both TGF-b and BMP signaling play cru-cial roles in the regulation of SMC function and in properEC–SMCs interactions, and disruption of these pathwaysin SMCs leads to vascular abnormalities. Vascular defectsdue to misregulated TGF-b and BMP pathways might notonly be due to their direct effects on SMC function, but alsodue to effects on other signaling pathways such as thePDGF pathway, or to disruption of the balance betweenTGF-b and BMP signals. It has been suggested that loss ofBMPR2 could lead to unregulated TGF-b/ALK5 activity inSMCs from patients with idiopathic PAH and this might beimportant in mediating disease progression [92]. Interest-ingly, systemic inhibition of TGF-b/ALK5 signaling signifi-cantly reversed pulmonary arterial pressure in a model of
[(Figure_2)TD$FIG]
Figure 2. Targeting TGF-b signaling in tumor angiogenesis. Tumors cannot grow to mo

dormant for years. Tumor angiogenesis is essential to escape this period of dormancy.

and anti-angiogenic factors such as VEGF, bFGF, P1GF, TGF-b and BMP. Endoglin and AL

or inhibition of endoglin and ALK1 function by endoglin-neutralizing antibodies or by

growth. ECD, extracellular domain; Fc, antibody Fc region.

experimental PAH, thus providing new strategies for dis-ease management. Further characterization of the mol-ecular mechanisms by which TGF-b family membersregulate SMC function and EC–SMC interaction couldprovide us with targets for the development of new thera-peutic strategies against vascular abnormalities.

Targeting TGF-b signaling in tumor angiogenesisTumor angiogenesis plays a crucial role in tumor initiation,progression and metastasis (Figure 2). Several studieshave focused on the molecular characterization of tumorangiogenesis for the development of anti-angiogenic agentsfor cancer therapy [10,93]. TGF-b signaling plays animportant role in tumor growth and metastasis [46].Increased TGF-b expression has been reported in manycancers, and such expression was shown to correlate withpoor prognosis, increased tumor growth and angiogenesis,whereas administration of TGF-b inhibitors stronglyreduced tumor angiogenesis and tumor growth. TGF-bsignaling antagonists are currently used to prevent growthand metastasis of certain cancers [46]. However, severalstudies have suggested that inhibition of TGF-b signalingcan promote tumor angiogenesis [46]. A combination ofVEGF and a TGFBR1 (ALK5) kinase inhibitor synergisti-cally promoted angiogenesis [54]. Therefore, anti-TGF-b-based therapeutic strategies must be carefully consideredbefore administration because there could be adverseeffects, such as induction of tumor angiogenesis and tumorgrowth. Inhibitors of ALK5 kinase block signaling of bothSmad2 and Smad3. However, recent studies suggestedthat Smad2 has tumor-suppressor and anti-metastaticactivities and inhibits angiogenesis, whereas Smad3 playsan important role in stimulating tumor growth and metas-tasis in part by inducing VEGF expression and promotingtumor angiogenesis [94]. Thus, selective targeting ofSmad3 in tumor cells, for example by halofuginone [95]or small interfering RNAs, could lead to more effective

re than 1–2 mm3 if supply of oxygen and nutrients is limited. Many tumors can be

This process, also known as the angiogenic switch, is regulated by a variety of pro-

K1 play important roles in tumor angiogenesis and tumor growth. Genetic deletion

ALK1-Fc (an ALK1 ligand trap) results in reduced tumor angiogenesis and tumor

563

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?


therapeutic responses against tumor growth and tumorangiogenesis.

Endoglin is upregulated in the tumor-associated endo-thelium and its expression correlates with poor prognosis[22]. Several studies have considered endoglin as a thera-peutic target in anti-angiogenic therapies because tumorvascularization and growth are diminished in Eng-hetero-zygous mice [22]. Endoglin-neutralizing antibodies cantarget tumor vasculature and inhibit tumor growth inmouse tumor models (Figure 2) [22]. Recent studiessuggested that soluble endoglin (sEng) can interfere withthe function of endogenous endoglin on ECs and inhibitspontaneous cord formation in human umbilical vein endo-thelial cells (HUVECs) and VEGF-induced EC sprout for-mation [25,45]. These results suggest that sEng has greatpotential in anti-angiogenic cancer therapy by interferingwith tumor angiogenesis and tumor growth.

The exact role of ALK1 in angiogenesis is not fullyunderstood. Although some studies suggest that ALK1inhibits EC proliferation, and perturbation of ALK1 sig-naling results in increased VEGF signaling and enhancedangiogenesis [23,62,66], ALK1 was also shown to promoteEC proliferation and migration [55]. Recent studies haverevealed an important role for ALK1 in tumor angiogenesisand growth [44]. Expression analysis in mice suggestedthat expression of ALK1 and its ligands TGF-b and BMP9is increased during tumor growth. Deletion of one Acvrl1(Alk1) allele resulted in reduced tumor growth and pro-gression by inhibition of angiogenesis in the RIP1-Tag2transgenic mouse model of multistep tumorigenesis [44].Pharmacological inhibition of ALK1 signaling using anALK1-ligand trap (ALK1-Fc), resulted in reduced tumorangiogenesis and tumor growth in the RIP1-Tag2 trans-genic mousemodel of pancreatic islet carcinomas as well asin a breast cancer orthotopic tumor model [44,69]. Inaddition, ALK1-Fc treatment of RIP-Tag2 mice resultedin increased pericyte coverage of tumor vessels [44]. ALK1-Fc inhibits tumor angiogenesis by interfering with theangiogenic activity of proangiogenic factors such as VEGFand bFGF (Figure 2). Interestingly, TGF-b and BMP9 cansynergistically induce the pro-angiogenic effects of VEGFand bFGF. ALK1-Fc could efficiently interfere with thissynergistic effect both in vitro and in vivo [44]. Thoseresults suggest that ALK1 provides a valuable target foranti-angiogenic therapy and that ALK1-Fc is a powerfulanti-angiogenic agent capable of reducing tumor angiogen-esis and tumor growth (Figure 2). The therapeutic poten-tial of human ALK1-Fc and humanized ALK1 andendoglin-neutralizing antibodies is currently being evalu-ated in clinical cancer trials [96–98].

Concluding remarksThe role of TGF-b signaling in angiogenesis has been highlycontroversial, with numerous studies showing that it iseither pro-angiogenic or, conversely, anti-angiogenic in acontext-dependent manner. Recent studies emphasize thegrowing appreciation that the pleiotropic effects of TGF-bsignaling are the outcome of multiple and fine-tuned sig-naling cascades rather than the result of a simple linearsignal-transduction pathway. Some of these discrepanciesmight be explained by variations in ligand concentrations,

564

receptor and downstream signaling component expression;in addition, the same ligand, such as TGF-b, can induceopposing effects by activating different classes of Smadsthrough the formation of diverse receptor complexes(Figure 1). Interestingly, this signaling flexibility is notrestricted to ECs but applies to other types of cells. BMP9wasalso showntoactivatedifferent classesofSmadproteins(Figure1).The exact role ofBMP9-inducedSmad1orSmad2phosphorylation in angiogenesis remains to be elucidated.

Misregulated TGF-b signaling results in vasculardefects, and the phenotypes of mice lacking different com-ponents of the TGF-b and BMP signaling pathways arequite similar, suggesting that they might act in concert toregulate vessel formation in vivo. TGF-b ligands regulateangiogenesis through their actions either on ECs and/or onmural cells, demonstrating that they play important rolesin both the activation and resolution phases of angiogen-esis. This can explain the contradictory results of differentstudies on the role in angiogenesis of endoglin, ALK1 andALK5. In addition, experimental evidence suggests that, indifferent phases of the multistep angiogenesis process(normal and pathological), there is differential expressionTGF-b family members [44,46]. This could explain thecontroversial results of different studies where TGF-bsignaling components exhibit distinct effects on angiogen-esis depending on the angiogenic microenvironment. Sep-arate treatment with TGF-b or BMP9 inhibits ECproliferation, whereas the combination of the two factorscan enhance EC proliferation. Mutations in ENG andACVRL1 lead to vascular abnormalities in HHT patientsdue to increased EC proliferation and impaired SMCrecruitment. However, deletion of Eng and Acvrl1 in themouse results in reduced tumor angiogenesis and tumorgrowth. It could also involve synergistic and/or antagon-istic interactions of TGF-b–BMP signaling with other sig-naling pathways such as VEGF, PDGF and Notch. Thecombination of TGF-b and BMP9 enhances VEGF/bFGF-induced angiogenesis whereas TGF-b inhibits it. The cross-talk with these pathways is only partly understood, andfuture studies using in vitro and in vivo systems of angio-genesis will be invaluable in elucidating these interactions.

Although there have been new insights into the role ofTGF-b signaling in vascular development and function,the exact mechanisms by which TGF-b family membersregulate angiogenesis are still not fully understood. Manyquestions remain to be answered and additional studiesare required (Box 4) to explain the contradictory but


intriguing role of TGF-b signaling in angiogenesis. Un-derstanding the molecular mechanisms by which TGF-bsignaling exerts its diverse, context-dependent effects onangiogenesis will enable us to develop new therapeuticinterventions to manage pathological vascular malfor-mations, tumor angiogenesis and tumor growth.

AcknowledgementsResearch in our laboratories is supported by grants from the NetherlandsOrganization for Scientific Research, the Dutch Cancer Society, theLudwig Institute for Cancer, the Netherlands Heart foundation(2009B063), the Leducq foundation and the Centre for BiomedicalGenetics. We apologize to those whose work has not been cited becauseof space limitations.

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