Cytokine & Growth Factor Reviews 20 (2009) 203–212

Survey

Emerging role of bone morphogenetic proteins in angiogenesis

Laurent David a,b,c,1, Jean-Jacques Feige a,b,c,2, Sabine Bailly a,b,c,*a Institut National de la Sante et de la Recherche Medicale, U878, 17 rue des Martyrs, 38054 Grenoble, Franceb Commissariat a l’Energie Atomique, Institut de Recherches en Technologies et Sciences pour le Vivant/LAPV, Grenoble, Francec Universite Joseph Fourier, Grenoble, France

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

2. BMPs and BMP receptors: structural features and sites of expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204

3. The role of BMPs in angiogenesis: lessons from human vascular diseases and from knockout animal models . . . . . . . . . . . . . . . . . . . . . . . 206

3.1. Knowledge from human vascular diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

3.2. Knowledge from knockout animals: mice and zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

3.2.1. BMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

3.2.2. BMP receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

3.2.3. The Smads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

3.2.4. Other proteins related to BMPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

4. Effects of BMPs signalling through ALK2, ALK3 and ALK6: implication in the activation phase of angiogenesis . . . . . . . . . . . . . . . . . . . . . . 207

5. Effects of BMPs signalling through ALK1 (BMP9 and 10): implication in the maturation phase of angiogenesis . . . . . . . . . . . . . . . . . . . . . . 208

6. Smad-dependent and Smad-independent BMP signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

7. BMP antagonists and co-receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

7.1. Noggin/chordin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

7.2. BMPER (CV2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

7.3. Chordin-like 1 (CHL1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

7.4. Gremlin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

8. Conclusion and possible therapeutic applications of BMPs in pathological angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

A R T I C L E I N F O

Article history:

Available online 6 June 2009

Keywords:

BMP

Receptors

Angiogenesis

HHT

PAH

A B S T R A C T

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming

growth factor b (TGFb) superfamily. Recent observations clearly emphasize the emerging role of BMPs in

angiogenesis: (i) two genetic vascular diseases (hereditary hemorrhagic telangiectasia (HHT) and

pulmonary arterial hypertension (PAH)) are caused by mutations in genes encoding components of the

BMP signalling pathway (endoglin, ALK1 and BMPRII). (ii) BMP9 has been identified as the physiological

ligand of the endothelial receptor ALK1 in association with BMPRII. This review will focus on the diverse

functions of BMPs in angiogenesis. We will propose a model that distinguishes the BMP2, BMP7 and

GDF5 subgroups from the BMP9 subgroup on the basis of their functional implication in the two phases

of angiogenesis (activation and maturation).

� 2009 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Cytokine & Growth Factor Reviews

journa l homepage: www.e lsev ier .com/ locate /cytogfr

* Corresponding author at: U878, iRTSV/LAPV, 17 rue des Martyrs, 38054

Grenoble, France. Tel.: +33 4 38 78 92 14; fax: +33 4 38 78 50 58.

E-mail addresses: lizardcore@gmail.com (L. David), jjfeige@cea.fr (J.-J. Feige),

sbailly@cea.fr (S. Bailly).1 Present address: Center for Systems Biology, Samuel Lunenfeld Research

Institute, 1078 Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5,

Canada. Tel.: +1 416 586 4800x23063; fax: +1 416 586 4800x8869.2 Tel.: +33 4 38 78 45 60; fax: +33 4 38 78 50 58.

1359-6101/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.cytogfr.2009.05.001

1. Introduction

Angiogenesis is the formation of new blood capillaries from apre-existing capillary network. It is active during embryonicdevelopment and is normally quiescent at the adult stage exceptfor oestrous cycle-dependent vascularization of the ovaries anduterus and placental development during pregnancy. However,

Fig. 1. Angiogenesis results from an activation phase and a maturation phase. NO: nitric oxide, VEGF: vascular endothelial growth factor, Ang2: angiopoietin 2, Ang1:

angiopoietin 1, MMPs: matrix metalloproteinases, EGF: epidermal growth factor, FGF: fibroblast growth factor, TGFb: transforming growth factor beta, PDGF: platelet-

derived growth factor and TNFa: tumour necrosis factor alpha.

L. David et al. / Cytokine & Growth Factor Reviews 20 (2009) 203–212204

angiogenesis is re-activated during wound repair and underseveral pathological conditions, such as tumour growth andmetastasis and cardiovascular disorders [1]. Angiogenesis is amultistep process that can be roughly divided into two phases(Fig. 1). During the initial activation phase, the perivascularbasement membrane is degraded, endothelial cells migrate intothe extracellular space, proliferate, form capillary sprouts andself-organize into tubular structures. This is followed by amaturation/stabilization phase, when endothelial cells ceasemigration and proliferation, the basement membrane is recon-stituted and smooth muscle cells/pericytes are recruited andapposed to the neo-vessels, thereby reconstituting vessel wallintegrity. A balance of pro- and anti-angiogenic factors verytightly regulates angiogenesis. Important pro-angiogenic factorsinclude vascular endothelial growth factor (VEGF) and basicfibroblast growth factor (FGF2), which stimulate proliferationand migration of endothelial cells. The angiopoietins (Ang) alsoplay an important role but their effects are more complex andthey are involved in the two phases: Ang1, through its receptorTie2, is involved in vascular quiescence while Ang2, byantagonizing Ang1 binding to Tie2, is involved in the activationphase of angiogenesis. Platelet-derived growth factor (PDGF) is astabilizing factor that mediates the vascular recruitment ofpericytes and smooth muscle cells. Transforming growth factorbeta (TGFb) has also been shown to be involved in extracellularmatrix accumulation as well as in the regulation of endothelialcell functions and smooth muscle differentiation. Several recentreviews have discussed the role of TGFb and TGFb familymembers in angiogenesis [2,3]. In the present review, we willfocus our interest on the subgroup of the TGFb familyconstituted by the bone morphogenetic proteins (BMPs),

as recent data clearly demonstrate their direct roles inangiogenesis.

2. BMPs and BMP receptors: structural features and sites ofexpression

Bone morphogenetic proteins (BMPs) are members of the TGFbsuperfamily, which includes TGFbs, activins/inhibins, nodal, myos-tatin, anti-Mullerian hormone (AMH) and growth and differentia-tion factors (GDFs). More than 15 BMP-related proteins have beenidentified and subdivided into several groups based on their aminoacid sequence similarity and their functions [4]. BMP2, BMP4, andthe Drosophila dpp form one subgroup (BMP2/4 subgroup).Members of this subgroup have a wide range of biological activitiesand play pivotal roles during embryonic development. BMP5, BMP6,BMP7, BMP8, and the Drosophila gbb, form another subgroup (BMP7subgroup). GDF5, GDF6, and GDF7 form a third group (GDF5subgroup). Genetic disruption of the members of these two lastsubgroups are not embryonic lethal, but many result in varioustissue defects including skeletal dysplasias. BMP9 and BMP10 form afourth subgroup and little was known about these two factors untilrecently. Monomers of all these dimeric BMPs possess sevencysteine residues, which form three intrachain disulfide bonds andone disulfide bond with a second monomer. In contrast to TGFbsthat are secreted in an inactive form, BMPs are secreted in an activeform and are regulated through reversible interactions withextracellular antagonists, including noggin, chordin, and DAN [5].These interactions determine the bioavailability of the differentBMPs for their receptors.

Like other members of the TGFb family, BMPs elicit their effectsthrough activation of different receptor complexes, each composed

L. David et al. / Cytokine & Growth Factor Reviews 20 (2009) 203–212 205

of two type I and two type II serine/threonine kinase receptors(Fig. 2A). There are seven type I receptors termed activin receptor-like kinase (ALK1 to ALK7 and five type II receptors (ActRIIA,ActRIIB, BMPRII, TGFbRII and AMHRII). Upon ligand binding, thetype II receptors phosphorylate and activate the type I receptors ina highly conserved TTSGSGSG motif termed the GS domain. Theactivated type I receptors then propagate the signal by phosphor-ylating a family of transcription factors, called Smads [6]. BMPsbind to specific heterotetrameric complexes comprising, eitherBMPRII, ActRIIA or ActRIIB as type II receptor, and either ALK3(BMPR1A), ALK6 (BMPR1B), ALK2 or ALK1 as type I receptors. Themode of interaction of BMPs with their receptors is different fromthat of TGFb with its receptors (Fig. 2B) [7,8]. Unlike TGFb andactivin, which display a higher affinity for type II receptors and donot stably interact with type I receptors alone, BMPs bindindependently to both type I and type II receptors. Some BMPshave a higher affinity for their type I receptor (i.e. ALK3-BMP2) andothers for their type II receptor (i.e. ActRII-BMP7). The first receptorto recruit the ligand is probably that with the highest affinity.These receptor complexes can also contain a type III receptors alsotermed a co-receptors (betaglycan, endoglin or RMG-a, b, c) that

Fig. 2. BMP signalling. (A): BMP binding to type I and type II receptors induces Smad1

transcription of target genes. (B): Phylogenetic tree of the different BMP sub-families o

modulates ligand affinity for its type I and type II receptors [6].Endoglin and betaglycan can both be shed from the cell surfacesuggesting that they can modulate TGFb and BMP functions [9].

The BMP2, BMP7 and GDF5 ligands have been found to bind toALK3, ALK6 and, in some cases, to ALK2 (Fig. 2B). The ALK1 receptorhas long been considered an orphan receptor. A model has beenproposed in which TGFb binds to a TGFbRII/ALK5/ALK1 hetero-complex on endothelial cells, with the presence of ALK1antagonizing the biological response to ALK5 [10]. However, thismodel is still debated in the literature: (i) a non-overlappingexpression pattern of ALK1 and ALK5 was found in Alk5(lacZ) andAlk1(lacZ) knockin mice [11]. (ii) Recently, vascular-specificgenetic invalidations of Alk1, TGFbR2, and Alk5 using theendothelium-specific promoter (ALK1) were reported [12]; Alk1

deletion induced vessel dilation, decreased wall thickness, andreduced and irregularly located vascular smooth muscle cells. Incontrast, TGFbR2 or Alk5 deletion in endothelial cells did not affectvascular morphogenesis. In parallel, another group published thespecific invalidation of TGFbR2 and Alk5 in endothelial cells usinganother endothelium-specific promoter (Tie1 promoter) thatdrives earlier vascular expression [13]. They found that both

/5/8 phosphorylation and Smad-independent signalling pathways, which regulate

btained by alignment of the human mature protein sequences.

L. David et al. / Cytokine & Growth Factor Reviews 20 (2009) 203–212206

knockouts induced defects in yolk sac vasculogenesis. However,when ALK5 was replaced by a mutant Alk5 defective in Smad2/3activation, that retained the ability to transactivate ALK1, thedefect in yolk sac vasculature was still observed suggesting thatthis defect was not due to deficient ALK1 signalling. (iii) Finally, itwas described this year by two groups, that TGFb couldphosphorylate Smad1/5/8 in the absence of ALK1 [14,15]. Thesefindings challenge the ALK1/ALK5 balance model. Two years ago,we described that BMP9, and the closely related BMP10, bind ALK1in association with BMPRII or ActRIIA [16]. This was rapidlyconfirmed by another group who reported that BMP9 could alsobind to ALK2 and ActRIIB under certain conditions [17]. BMP9binding to ALK1 and BMPRII was also reported using biosensor-immobilized ALK1and BMPRII extracellular domains [18]. BMP10was also found to bind to ALK3 and ALK6 and BMP9 to ALK6although with a much lower affinity than to ALK1 [16,19]; weshowed that BMP9 triggers Smad1/5/8 phosphorylation inendothelial cells with an EC50 around 50 pg/ml (2 pM). This is amuch higher affinity than that of the other BMPs for their type Ireceptors, ALK3 and ALK6 (BMP2 has an apparent Kd of 0.9 nM forALK3, 3.6 nM for ALK6) [20]. This unusually high affinity of BMP9for ALK1 suggests that BMP9 binding to its receptor might occurthrough a different kind of molecular interaction than that used byclassical BMPs. Interestingly, ALK1, in contrast to all other type Ireceptors, is missing the residue Phe85, which was shown to beinvolved in the hydrophobic interaction between classical BMPsand their type I receptors [7,8].

Within these heterocomplexes, it is the type I receptor thatdetermines the specificity of the signal. The type I receptorinitiates signal propagation by phosphorylation of the receptor-activated Smad (R-Smads). BMP receptors (ALK1, ALK2, ALK3and ALK6) activate Smad1, Smad5 and Smad8, whereas Smad2and Smad3 are phosphorylated by the activin and the TGFbreceptors (ALK4, ALK5 and ALK7). Remarkably, ALK3 and ALK6can activate all three Smads [1,5,8], but ALK2 only phosphor-ylates Smad1 and 5 [4]. In addition to R-Smads, two other typesof Smads have been identified, the common mediator Smad (co-Smad: Smad4) and the inhibitory Smads (I-Smads: Smad6 andSmad7). Activated R-Smads assemble into a heteromericcomplex with Smad4 in the cytoplasm, which translocates tothe nucleus where it participate directly in the modulation oftarget gene expression. These complexes do not have a highaffinity for DNA but bind to target gene promoters in associationwith other transcription factors [4]. BMPs mainly activate theSmad pathway but other non-Smad signalling pathways havebeen described for BMPs (Fig. 2A). These pathways will bediscussed later in this review. The choice of the signallingcascade (Smad or non-Smad) has been proposed to be regulatedby different endocytic pathways, or different types of receptorcomplexes (preformed receptor complexes initiate the Smadpathway whereas BMP2-induced signalling complexes result inthe activation of p38-MAPK) [21].

Phosphorylated Smad1/5/8 is detectable in endothelial cellsfrom freshly-isolated mouse aorta and pulmonary endothelium,suggesting that endothelial cells are physiologically activated byBMPs [22,23]. An important point in understanding how BMPsregulate angiogenesis is to know their receptor localization. ALK2,ALK3, ALK6 and BMPRII are expressed in almost all cells, while theexpression of ALK1 and endoglin is mostly restricted to endothelialcells. ALK1 is primarily expressed in arterial endothelial cellsduring development and, in adult life, remains highly expressed inthe lung vasculature [11,24–26]. In the developing embryo,endoglin is more widely expressed throughout the vascular bed,but is downregulated in adult life, with the notable exception ofpulmonary vessels and tissues undergoing neovascularization[9,27]. A recent study reported that, in the adult, ALK1 and

endoglin have distinct expression profiles in the mouse pulmonaryvasculature [28].

3. The role of BMPs in angiogenesis: lessons from humanvascular diseases and from knockout animal models

3.1. Knowledge from human vascular diseases

A clear demonstration of the implication of BMPs in angiogen-esis comes from the causal implication of mutations of BMPreceptors in several human vascular genetic diseases.

The first vascular disease related to BMPs is the Rendu–Osler–Weber syndrome also known as hemorrhagic hereditary telan-giectasia (HHT). HHT is an autosomal dominant vascular disorder,in which the patients develop mucosal and skin telangiectasia,pulmonary, cerebral and hepatic malformations, and hemorrhagesassociated with these vascular lesions. Three genes are causallyrelated to HHT: the ENG gene encoding the co-receptor endoglinwas first reported to be mutated in some HHT patients, thusdefining HHT1 [29]; then, the ACVRL1 gene encoding the type Ireceptor, ALK1 was found to be mutated in another subset of HHTpatients, thus defining HHT2 [30], and more recently, mutations inthe SMAD4 gene were found to cause a mixed syndrome consistingof both juvenile polyposis and HHT [31].

BMPR2, and later on, ACVRL1 mutations, were linked topulmonary arterial hypertension (PAH) and very recently a SMAD8

mutation was described in a patient with idiopathic PAH [32–34].PAH is a progressive disorder characterized by raised pulmonaryartery pressures with pathological changes in small pulmonaryarteries. The explanation that is proposed for this disorder is anabnormal proliferation of vascular smooth muscle cells and alteredproliferation and survival of endothelial cells. This leads toocclusions on the artery side of the pulmonary vasculature,progressing to severe pulmonary hypertension and right-sidedheart failure.

Preeclampsia, which involves a rise in systemic blood pressureduring pregnancy, is a major source of maternal, foetal andneonatal mortality. Soluble endoglin, formed by proteolyticcleavage, has recently been shown to contribute to the pathogen-esis of preeclampsia and has been proposed as a diagnostic markerin preeclampsia together with soluble Flt1 (VEGFR1 receptor) [35].

3.2. Knowledge from knockout animals: mice and zebrafish

A large number of mice with genetic disruption (conditional ornot) of genes encoding BMP ligands, receptors or antagonists havebeen generated and many of these exhibit cardiovasculardevelopmental abnormalities [36]. Here, we will focus mainlyon the animal models that exhibit defective angiogenesis linked toBMP signalling.

3.2.1. BMPs

Not much has been described concerning BMP invalidation andangiogenesis. This might be due to BMPs redundancy.

BMP4 knockout embryos die between E7.5 and E9.5 withdefects in mesoderm formation and patterning and those thatsurvive show severe defects in blood island formation [36]. Thissuggests that BMP4 is involved in vasculogenesis.

Radar (GDF6a) morpholinos in the zebrafish induce an aberrantvascular development [37].

3.2.2. BMP receptors

3.2.2.1. The type I receptors. The genetic invalidation of ALK1 inzebrafish and mice leads to embryonic lethality due to angiogen-esis defects [38–40]. In mice, the vascular plexus gets formed

L. David et al. / Cytokine & Growth Factor Reviews 20 (2009) 203–212 207

normally but, both in the yolk sac and the embryo, the vascularnetwork fails to develop and to mature into a vascular tree. There isalso a defect in smooth muscle cell recruitment, and some arterio-veinous shunts are observed in the embryo. Disruption of ALK1(violet beauregarde, vbg) in the zebrafish leads to an abnormalcirculation pattern in which most blood cells flow through alimited number of dilated cranial vessels (due to an increase inendothelial cell number) and fail to perfuse the trunk [40]. It isinteresting to note that the Acvrl1+/� mice develop symptomssimilar to the HHT pathology after 12 months of age [41].Endothelial-specific ALK1 gene deletion results in dilated yolk sacvessels with convoluted and tortuous morphology and abnormaldirect connections between arteries and veins without connectingcapillary beds, thus mimicking the main pathological features ofHHT [12].

The conditional invalidation of ALK3 in endothelial cells (flk1-Cre) leads to defects in vessel remodelling and VSMC formation orrecruitment [42].

3.2.2.2. The type II receptors. Mice lacking BMPRII die during earlyembryogenesis due to abnormal mesoderm formation [36]. Theheterozygous mice exhibit increased pulmonary vascular resis-tance and thickened-muscularized arteries [43]. One grouprecently developed a transgenic mouse expressing an shRNAtargeting BMPR2; this allows maintaining a low basal level ofBMPRII to circumvent embryonic lethality of the null mutation[44]. These mice had no increase in pulmonary arterial resistancebut had severe mucosal hemorrhage and incomplete mural cellcoverage of vessel walls. The expression of endothelial guidancemolecules was also severely impaired demonstrating that BMPRIIregulates vascular remodelling during angiogenesis.

3.2.2.3. The type III receptors. Genetic disruption of either of thetwo type III receptors leads to embryonic lethality with vasculardefects. Betaglycan knockout mice die at E14.5 because of a lack ofcoronary vessels [45]. Endoglin disruption gives a more dramaticphenotype, quite similar to that found in the Alk1 null mice [46,47].Embryos die between E10 and E11.5. As for ALK1, they showdefects in both the yolk sac and in the embryo vasculatures and thevascular smooth muscle cells (VSMC) are affected. Endoglin

heterozygous mice develop also some symptoms of HHT disease,with an average onset of 6 months [48,49].

3.2.2.4. Extracellular regulators of BMPs. CYR61 (CCN1) is a memberof the CCN family of secreted matricellular proteins that functionsas a ligand of integrin receptors and BMPs. Cyr61 invalidationshows that this factor is essential for placental development andvascular integrity [50].

BMP-binding endothelial cell precursor-derived regulator(BMPER), which is the mouse ortholog of Drosophila crossveinless2, is an extracelllular regulator of BMPs, which was originally,identified in a screen for differentially expressed proteins inembryonic endothelial precursor cells [51]. It was recently shownin the zebrafish that morpholinos directed against BMPER disturbthe intersomitic vasculature [52].

3.2.3. The Smads

Disruption of the gene encoding the BMP receptor-activatedtranscription factor Smad5 also leads to angiogenesis impairmentwith defects in the yolk sac vasculature and enlarged embryonicvessels, similarly to the Alk1 and Eng phenotypes [36]. However,deletion of Smad5 in endothelial or smooth muscle cells did notperturb the organization of the embryonic and extra-embryonicvasculatures [53].

Smad1-deficient mice display defects in chorion-allantoicfusion, do not develop a placenta and die between E9.5 and

E10.5. Although overall haematopoietic and vascular develop-ments appears to be normal, some Smad1-deficient embryosdisplay defects in yolk sac angiogenesis [36].

Endothelial specific Smad4 knockout mice die at E10.5 due tocardiovascular defects, including attenuated vessels sprouting andremodelling [54].

3.2.4. Other proteins related to BMPs

Invalidation of other genes encoding proteins linked to the BMPpathway also lead to defect in angiogenesis. Inhibitory differentia-tion factor-1 and -3 (Id1 and Id3) are members of a family of helix–loop–helix proteins that inhibit lineage commitment withinmultiple cell types through sequestration of bHLH transcriptionfactors. Id1 and Id3 are direct transcriptional targets for BMPreceptors. Id1/Id3 double knockout mice show abnormal angio-genesis forming enlarged dilated blood vessels [55].

The TGFb activated kinase-1 (Tak1) knockout mice display aphenotype highly similar to the Alk1�/�, Smad5�/� and Eng�/�

phenotypes, suggesting that TAK1 is involved in this pathway [56].Moreover, the same group showed that a constitutively activatedform of TAK1 can partially rescue ALK1 invalidation in zebrafish[56].

As this review is focused on BMPs and angiogenesis, the resultsof TGFb signalling gene inactivations have not been presentedhere. However, it is important to keep in mind that, althoughTGFb1, Alk5 and TGFbR2 gene disruption leads to angiogenesisdefects, these defects are restricted to the yolk sac. In contrast,ALK1, ENG and Smad5 gene knockout leads to angiogenic defects inthe embryos as well as in the yolk sac, defining thereby twofunctionally distinct groups [36].

All these in vivo data clearly demonstrate an important role ofthe BMP pathway in angiogenesis, and particularly of the endoglin/ALK1/BMPRII signalling pathway.

4. Effects of BMPs signalling through ALK2, ALK3 and ALK6:implication in the activation phase of angiogenesis

GDF5 was the first BMP reported to have a role in angiogenesis.GDF5 enhances angiogenesis in the chorio-allantoic membrane(CAM) assay and in the rabbit cornea assay [57]. In the same study,it was found that the addition of GDF5 accelerated the migration ofbovine aortic endothelial cells (BAEC) while it did not affect theirproliferation.

In contrast, BMP2 was initially described to have no effect in theCAM and in the rabbit cornea assays [57]. Later on, another groupfound that BMP2 stimulated blood vessel formation in tumoursformed from A549 cells subcutaneously implanted into nude miceand enhanced angiogenesis in Matrigel plugs containing A549 cells[58]. In accordance with this study, it was reported that BMP2induces vascularization in the mouse sponge assay and that BMP2overexpression in the MCF-7 breast tumour cell line leads tovascularized tumours [59]. In vitro, BMP2 stimulates proliferationof human aortic endothelial cells (HAEC) but not of humanumbilical vein endothelial cells (HUVEC) while it has no effect onhuman umbilical arterial endothelial cells (HUAEC) migration butincreases tube formation in both cell types [58]. It also increasesmigration and tube formation of human microvascular endothelialcells (HMEC) and chemotaxis of circulating endothelial cellprecursors (EPC) [60,61] and it was found to promote pulmonaryaortic endothelial cell (PAEC) survival and proliferation [62,63]. Inone study it was found to have no effect on HUVEC chemotaxis[64].

BMP4 induces angiogenesis in the CAM assay [65,66]. GraftingBMP4-releasing beads into the paraxial mesoderm in the quailembryo, also increased the number of blood vessels [67]. In vitro,BMP4 induces the proliferation and migration of mouse embryonic

L. David et al. / Cytokine & Growth Factor Reviews 20 (2009) 203–212208

stem cell-derived endothelial cells (MESEC) and HMECs [68]. It alsoincreases migration and tube formation of HMEC and mouse aorticendothelial cells (MAEC) [60,66]. In contrast, it was shown byanother group that transcorneal injection of BMP4 in rats mediatedapoptosis of endothelial cells of the pupillary membrane [69]. Thesame group found that capillary and venous endothelial cells wereresponsive to BMP4-induced apoptosis while arterial endothelialcells were resistant to BMP4 and this was due to differentialexpression of inhibitory Smads [70].

BMP6 was initially shown to induce migration and tubeformation of BAECs [22]. Then, another group showed thatBMP6 induced mouse embryonic endothelial cell (MEC) prolifera-tion, migration, and network assembly and microvessel outgrowthin aortic rings [71].

BMP7 was shown to induce angiogenesis in the CAM assay[65,66]. In vitro, BMP7 stimulates proliferation of HPAECs [62].

It was shown that the constitutively active forms of ALK2, ALK3and ALK6 promote endothelial cell migration and tube formation[22].

VSMC differentiation and functions are also influenced byBMPs. It was first described that BMP2 inhibited rat VSMCproliferation in vitro and in vivo, while BMP7 had no effect in vitroon VSMC proliferation [72]. BMP2 was also shown to inducehuman aortic smooth muscle cell (HASMC) chemotaxis while ithad no significant effect on thymidine incorporation [64]. BMP2,BMP4 and BMP7 suppressed pulmonary artery smooth musclecells (PASMC) and HASMC proliferation, induced apoptosis,increased VSMC migration and induced the expression of smoothmuscle differentiation markers [73–75]. BMP4 was shown toinhibit proliferation of proximal PASMCs but to stimulateproliferation of peripheral PASMCs [76], suggesting that theeffects of BMPs might depend on the source of VSMCs studiedas well as their local environment.

BMPs can also induce angiogenesis indirectly through induc-tion of angiogenic factor: BMP7 was found to induce VEGF-Aexpression in primary cultures of foetal rat calvaria cells andBMP2, BMP4 and BMP6 in osteoblasts and preosteoblast-like celllines [77,78].

5. Effects of BMPs signalling through ALK1 (BMP9 and 10):implication in the maturation phase of angiogenesis

For a number of years, in the absence of a specific ligand forALK1, several groups have bypassed this problem by expressing aconstitutively active form of ALK1 (ALK1ca) in primary endothe-lial cells and have reached different conclusions. Using HUVECs,Ota et al. found that ALK1ca expression inhibited endothelial cellproliferation while it had no effect on tube formation in collagengels [79]. Goumans et al., using MECs, showed that ALK1caexpression increased migration [80]. In contrast, we found thatALK1ca expression inhibited proliferation and migration ofdermal HMECs, as well as endothelial sprouting in culturedembryoid bodies derived from mouse embryonic stem cells[81,82].

More recently, as BMP9 has been shown to be the specificligand of ALK1 [16,17], the role of ALK1 has been readdressed. Itwas shown by us and confirmed by another group that BMP9inhibited proliferation and migration of dermal HMECs andBAECs [16,17]. Very recently, it was also shown that BMP9inhibited HPAEC mitogenicity via ALK1 and BMPRII [83]. Theinhibitory effect of BMP9 was confirmed in the ex vivo metatarsalculture model [17]. Furthermore, it was shown that BMP9inhibited neo-angiogenesis, in vivo, in the mouse sponge assayand inhibited blood circulation in the CAM assay [84]. BMP10 wasalso shown to inhibit proliferation and migration of dermalHMVECs [16].

6. Smad-dependent and Smad-independent BMP signalling

Taken together these data demonstrate that BMPs, signallingthrough ALK3 and ALK6, inhibit VSMC proliferation and increaseVSMC migration while they inhibit endothelial cell proliferationand migration. In contrast, BMP9/10, signalling more specificallythrough ALK1, inhibit endothelial cell proliferation and migration.As these receptors all induce the Smad1/5/8 pathway, it wouldsuggest that Smad-independent signalling pathways are alsoinvolved. BMPs have been found to regulate a variety of Smad-independent pathways including the p38 and ERK MAPK signallingpathways [4]. It was also shown that induction of HUVEC sproutingby BMP4 is dependent on ERK signalling and not on Smadsignalling [85]. The pro-proliferative effect of BMP4 on PASMC wasfound to be p38-dependent [76]. It was recently reported thatBMP2 induces pulmonary angiogenesis via the WNT pathway [63].Interestingly, we showed that the ALK1ca-mediated inhibition ofendothelial cell migration is Smad-independent and may involvethe Jun N-terminal kinase (JNK) and to a lesser extent the ERKpathways [86]. It was also described that ALK1ca could directlyphosphorylate endoglin and this phosphorylation was involved inHUVEC proliferation and adhesion [87]. The BMP type II receptorhas a long carboxy terminal tail (508 amino acids), a unique featureamongst TGFb receptors, that allows signalling through non-type Ireceptor substrates: it was shown that its C-terminal domainmodulates the actin cytoskeleton regulatory kinase LIMK1 [4].Interestingly, mutations in BMPRII cytoplasmic tail were shown todisrupt Smad-mediated signalling but also to lead to constitutivep38 MAPK activation and to affect the phosphorylation of ERK andTctex-1, a light chain of the motor dynein or the c-Src tyrosinekinase, which are two proteins that have been shown to directlyassociate with BMPRII [88–92].

7. BMP antagonists and co-receptors

Ligand-receptor interaction can be inhibited by secretedantagonists including noggin, chordin, follistatin and the DAN/Cerberus family of proteins or can be activated through co-receptors. Some of these extracellular regulators appear to havedual functions and are therefore difficult to classify into onespecific group. Some extracellular regulators have been recentlydescribed to play a role in angiogenesis [93].

7.1. Noggin/chordin

Noggin protects endothelial cells of the papillary membrane innewborn rat eyes from death [69]. In contrast, noggin and chordinwere shown to be negative regulators of developmental angiogen-esis through inhibition of the BMP4 response in the notochord[67,94]. Interestingly, BMP9 signalling, in contrast to that of otherBMPs, was found not to be inhibited by noggin, suggesting thatnoggin would principally inhibit the activation phase of angiogen-esis [84].

7.2. BMPER (CV2)

The extracellular BMP antagonist, BMPER, is ascribed both pro-and anti-BMP activities [93]. A dose-dependent effect of BMPERhas recently been described in angiogenesis; low BMPERconcentrations enhance sprouting and vasculature formation,whereas high concentrations inhibit it [52].

7.3. Chordin-like 1 (CHL1)

CHL1 is upregulated by hypoxia in human retina and wasshown to antagonize the anti-angiogenic effects of BMP4 [95].

L. David et al. / Cytokine & Growth Factor Reviews 20 (2009) 203–212 209

7.4. Gremlin

Gremlin belongs to the DAN family of cysteine-knot secretedproteins that exert potent BMP antagonist activities by bindingBMP2, BMP4, and BMP7 and preventing their interaction with theircell surface receptors. Gremlin was shown to be upregulated inresponse to hypoxia and to inhibit lung HMECs responses to BMPstimulation [96]. However, gremlin has also been shown to becapable of pro-angiogenic activity through BMP-independentmechanisms: in dermal HMECs, gremlin interacts with anunknown membrane protein to induce the phosphorylation offocal adhesion kinase and paxillin [66].

The CCN family proteins (Cyr16, CTGF and NOV) are a class ofextracellular mediators that have been shown to interact with IGF,BMPs, TGFb, VEGF and integrins. They are involved in angiogenesismainly through their interactions with integrins [97].

8. Conclusion and possible therapeutic applications of BMPs inpathological angiogenesis

The data that we have discussed in this review clearly placeBMPs as important players in angiogenesis. Overall, BMPs from theGDF5, BMP2 and BMP7 subgroups, signalling through ALK2, ALK3or ALK6, increase endothelial cell proliferation and migration whilethey inhibit VSMC proliferation, induce VSMC migration andmaintain VSMC differentiation. These data suggest that theseBMPs, through their actions on endothelial cells, are importantactors of the activation phase of angiogenesis and, through theiractions on VSMCs, also play a role in the maturation phase ofangiogenesis. On the other hand, the BMP9 subgroup, signallingmainly through ALK1, inhibits endothelial cell proliferation andmigration, suggesting that it is involved in the maturation phase(Fig. 3). Interestingly, in vitro, in endothelial cells, 10 pg/ml ofBMP9/BMP10 is enough to induce Smad1/5/8 phosphorylation

Fig. 3. Relative roles of the different BMP subgroups in the angiogenic balance. The BM

actions on endothelial cells) and in the maturation phase (through their actions on VSM

(probably due to the presence of ALK1) while at least 10 ng/ml ofthe other BMPs are needed for the same effect [16,22,83]. In vivo,BMPs from the GDF5, BMP2 and BMP7 subgroups seem to induceangiogenesis but we cannot distinguish if this is due to a directeffect of these BMPs on endothelial cells or an indirect effect,through the induction of pro-angiogenic factors such as VEGF[77,78]. We found that BMP9 is present in serum and plasma at aconcentration of around 5 ng/ml [84] and this was recentlyconfirmed by another group [98]. BMP4 has also been described inhuman serum but at a lower level (<1 ng/ml) [98]. Interestingly,BMP9 is the only BMP circulating at a higher concentration than itsEC50 (50 pg/ml for ALK1). Furthermore, in contrast to most of theother BMPs, we showed that BMP9 is not inhibited by thecirculating antagonist noggin [84]. We speculate from these data,that BMP9 is the major endothelial-stimulating BMP and thatBMP9 is responsible for Smad1/5/8 phosphorylation seen in situ inendothelial cells [22,23]. Considering its inhibitory effects onangiogenesis, we propose that BMP9 is a major circulating vascularquiescence factor. The other BMPs would act principally on VSMCand as such might play an important role in the maturation phaseof angiogenesis.

Our hypothesis for explaining the aetiology of HHT is that adeficient BMP9/ALK1/endoglin pathway would lead to re-activa-tion of angiogenesis leading to endothelial hyperproliferation andtherefore vasodilation, endothelial cell hypermigration and AVMformation. These processes have already been observed in thezebrafish, where disruption of Acvrl1 induces endothelial hyper-proliferation and in mice, where Acvrl1 knockout induces AVMsthat can be attributed to a defect in endothelial cell migration[39,40,77]. Taken together, these data lead us to propose that HHTcould be due to a defect in the angiogenic balance. In accordancewith this hypothesis, it has been recently reported two cases ofHHT patients who were successfully treated with an anti-angiogenic therapy [99,100]. Following this idea further, several

P2, BMP7 and GDF5 subgroups are involved in the activation phase (through their

Cs), while the BMP9 subgroup is involved in the maturation phase of angiogenesis.

L. David et al. / Cytokine & Growth Factor Reviews 20 (2009) 203–212210

clinical trials in HHT patients are currently underway with anti-angiogenesis agents in order to inhibit bleeding and other vascularmalformations associated with HHT (http://www.hht.org/).

PAH is characterized by a progressive muscularization of small,peripheral pulmonary arteries due to abnormal proliferation ofVSMCs and altered apoptosis/growth of endothelial cells that reducethe diameter of the vessels. BMP9 has been identified as a ligand forthe receptor complex ALK1/BMPRII or/ActRIIA providing newinsight into the link between PAH and HHT. Interestingly, identicalALK1 mutations can give HHT and/or PAH, while BMPRII mutationsonly lead to PAH. As ALK1 expression is limited to endothelial cells,this would suggest that the starting point of this disease occurs inendothelial cells. In accordance, it was shown that BMPRII isexpressed at a higher level in endothelial cells and to a lesser extentin VSMCs within the intact pulmonary vasculature [23].

As BMP9 has been shown to bind to endoglin in the absence of itssignalling receptors [17], we could also speculate that some of theclinical manifestations observed in preeclampsia could be due to theneutralization of circulating BMP9 by soluble endoglin, as solubleendoglin has been reported to be increased in preeclampsia [35].

The presence of BMP9 in blood at a biologically activeconcentration suggests that BMP9 could be a target for pro-angiogenic treatment in diseases such as ischemia that needangiogenesis to be re-activated. One could envision then to inhibitthe circulating quiescence factor BMP9 in order to re-activateangiogenesis. For this, neutralizing anti-BMP9, anti-ALK1 anti-bodies or soluble ALK1 receptor could be interesting therapeutictools. In conclusion, we are just beginning to understand howBMPs are involved in angiogenesis. Forthcoming works willcertainly reinforce the interest for BMPs as new therapeutictargets for the treatment of diseases involving the remodelling ofthe vasculature.

Acknowledgments

We apologize to those whose work was not cited due to spacelimitation. Our studies are supported by INSERM, CEA and by ARC(Association pour la Recherche sur le Cancer).

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Laurent David obtained his MSc and PhD degrees inCellular and Molecular Biology at Universite JosephFourier (Grenoble, France), respectively in 2003 and2007. During his PhD, he characterized the specificligands of the orphan receptor ALK1: BMP9 andBMP10. After that, he demonstrated that BMP9 was acirculating factor and a negative regulator of angiogen-esis. Those results lead to the conclusion that BMP9might be a regulator of endothelium quiescence. He isnow a post-doctoral fellow in Jeff Wrana’s Lab (Toronto,Canada), where he is working on TGFb signalling incancer and embryonic stem cells.

Jean-Jacques Feige is a senior researcher in angiogenesisand growth factor research at the Institut National de laSante et de la Recherche Medicale (INSERM) in Grenoble,France and Head of the INSERM Unit 878. He graduatedfrom the Institut National des Sciences Appliquees in Lyonin 1977 and obtained his PhD at the University of Gre-noble-1 in 1981. He was appointed by INSERM in 1984.Between 1986 and 1988, Dr Feige visited the Departmentof Cell Biology at the University of California San Diego(USA) and the Salk Institute for Biological Studies (La Jolla,CA, USA, Laboratory of Pr R. Guillemin). In 2001, heestablished his own lab in Grenoble. He has a long-stand-ing scientific experience in the field of growth factor

research and, for many years, he has been working on the role of growth factors(FGF-2, TGFb, VEGF, EG-VEGF and BMPs) in the control of endocrine functions, fibrosisand essentially angiogenesis. He has published more than 120 scientific publicationsand reviews in peer-reviewed journals.

Sabine Bailly is a senior researcher in growth factorresearch and angiogenesis at the Institut National de laSante et de la Recherche Medicale (INSERM U878) inGrenoble, France. She graduated from the University ofSciences of Paris-Sud and obtained her PhD in 1991 in X.Bichat Hospital in Paris on inflammatory cytokines. Sheperformed a post-doctoral training in the Department ofMolecular Biology at the Royal Hallamshire Hospital inSheffield (England) between 1992 and 1993. She wasappointed by INSERM in 1994. In 1995, she joined thelaboratory of Dr Jean-Jacques Feige in Grenoble and hasworked since on the function and signal transductionpathways of growth factors of the TGFb family, first in

endocrinology and more recently in angiogenesis. She has published 38 scientificpublications and reviews on peer-reviewed journals.