Signaling by members of the TGF-β family in vascular morphogenesis and disease

Review

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

Members of the transforming growth factor-b (TGF-b) family play pivotal roles in development and disease. These cytokines elicit their pleiotropic effects on cells, including endothelial and mural cells, through specific type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors. This review highlights recent progress in our understanding of TGF-b signaling in vascular development and angiogenesis and of how perturbed TGF-b signaling might contribute to vascular pathologies, tumor angiogenesis and tumor progression. Recent research has provided exciting insights into the role of the TGF-b type I receptor (ALK1) in tumor angiogenesis and the curative effects of thalidomide on vascular malformations in hereditary hemorrhagic telangiectasia (HHT). These advances provide opportunities for the development of new therapies for diseases with vascular abnormalities. Introduction Tissue homeostasis is dependent on an adequate supply of oxygen and nutrients and removal of waste products through blood vessels [1]. The development and proper function of the vascular system (Box 1, Figure I) is essential for survival of all higher organisms. The vascular system plays an important role in embryonic development but also later during life. During embryo development the formation of new blood vessels depends on vasculogenesis and angiogenesis. Vasculogenesis refers to the de novo formation of blood vessels. Angiogenesis is the formation of new blood vessels from pre-existing ones and is controlled by a number of growth factors and signaling pathways and the balance between pro- and anti-angiogenic factors (Box 2, Figure I) [2]. Angiogenesis takes also place in adult life to maintain physiological homeostasis and tissue integrity during wound healing, inflammation and during the female menstrual cycle. Deregulation of vasculogenesis and angiogenesis has been implicated in a multitude of pathological situations. TGF-b (TGFB1–3) is the prototype of the extended TGFb family of cytokines which also includes activins/inhibins, Nodal, bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs) [17,18]. TGF-b family members play crucial roles in embryonic development, adult tissue homeostasis and the pathogenesis of a variety of diseases. Research over the past two decades into the Corresponding author: ten Dijke, P. ([email protected]).

556

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

0962-8924/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2010.06.006 Trends in Cell Biology 20 (2010) 556–567

Review

Trends in Cell Biology

Vol.20 No.9

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

[(Box_1)TD$FIG]

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

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

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

highly reminiscent of those described in patients with HHT [20], an autosomal dominant vascular disorder characterized by fragile blood vessels which lead to telangiectases and arteriovenous malformations (AVMs). HHT-1 and HHT-2 arise from heterozygous mutations in ENG and ACVRL1 (ALK1) genes, respectively. A subset of patients with juvenile polyposis, carrying mutations in the SMAD4 gene, can also develop HHT [31]. It has been postulated that genetic background and/or environmental factors (second hits), in addition to the genetic mutations in the ENG or ACVRL1 genes, play an important role in the development of vascular malformations in HHT patients. Park and colleagues demonstrated recently using Acvrl1-deficient mice that a second hit, excisional skin wounding, is essential for the development of AVMs in HHT. Their results provide new insights for understanding the pathogenesis of HHT [32]. Despite the identification of the genes responsible for HHT, the underlying molecular mechanisms for the pathogenesis of HHT remain obscure. Genetic studies in mouse demonstrated that deletion of Acvrl1 (Alk1) or Eng results in loss of arteriovenous specification and the development of AVMs between major arteries and veins [33]. Zebrafish 557

Review

Trends in Cell Biology Vol.20 No.9

Box 2. Angiogenesis Sprouting angiogenesis comprises two phases: activation and resolution (Figure I) [10]. The activation phase is characterized by changes in EC shape, EC junction rearrangement, degradation of the basement membrane (BM) and extracellular matrix, detachment of pericytes, destabilization of the vessel, and increased permeability (Figure Ib). Activated ECs start to proliferate and migrate into the perivascular space towards the angiogenic stimulus. During the resolution phase, ECs stop proliferating and migrating, SMCs and pericytes are recruited to the new sprout, and the BM reconstitutes to ensure stabilization and maturation of the newly formed vessels (Figure Ic). Finally, blood vessels become quiescent (Figure Id). Recent advances in vascular biology have suggested that specialized ECs with distinct cellular specifications and functions contribute to the formation of new blood vessels. During the sprouting process a selected EC, the ‘tip cell’, leads each sprout [11]. Tip cells are migratory cells that do not proliferate, instead they sense the angiogenic stimulus and invade the surrounding tissue by extending numerous filopodia. Tip cells have a changed polarity and do not form a lumen (Figure Ib). Tip cells rely on the ‘stalk cells’, the ECs that follow the tip cell; these can proliferate and form lumens because they are fully polarized, but do not form many filopodia [11,12]. Stalk-cell proliferation ensures sprout elongation and the recruitment of support cells. Finally, the newly formed branch connects with another branch by means of tip-cell to tip-cell fusion, and a new vascular

[(Box_2)TD$FIG]

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

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

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

Review

Trends in Cell Biology

Vol.20 No.9

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

[(Box_3)TD$FIG]

are intracellular mediators for the TGF-b family and are classified into three groups: receptor-regulated (R-Smad), common-mediator (CoSmad), and inhibitory (I-Smad) [17,18]. Upon activation, the type I receptor recruits and phosphorylates R-Smads at two serine residues in their extreme C-termini. ALK4 and 5 (and 7) mediate phosphorylation of R-Smads 2 and 3, whereas ALK1,2,3,6 mediate phosphorylation of R-Smad1,5,8. Activated R-Smads interact with Smad4 and translocate into the nucleus, where, together with other transcription factors, they regulate target gene expression (Figure I). I-Smads (Smads 6,7) can inhibit the activation of R-Smads by competing with R-Smads for type I receptor interaction and by recruiting specific ubiquitin ligases or phosphatases to the activated receptor complex, thereby targeting it for proteasomal degradation or dephosphorylation, respectively (Figure I) [17,18,20]. Non-Smad signaling pathway: TGF-b and BMP receptor activation results in activation of several other non-Smad signaling pathways in a context-dependent manner. Non-Smad signaling pathways can involve the TGF-b-activated kinase-1 (TAK-1), ERK, JNK, p38, Rho GTPases and the PI3K–AKT pathway, and these can crosstalk with the Smad pathways (Figure I) [19].

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

other proteins are involved [33]. The Notch family of receptors and their ligands are expressed in arterial ECs, promote artery-specific ephrin B2 expression and have been implicated as potential regulators of arteriovenous fate. However, no expression differences were observed in Acvrl1- or Eng-deficient embryos based on in situ hybridization of Notch signaling-pathway-related

genes [33]. Paradoxically, although Eng deletion in mouse embryos results in arterial expression of the venousspecific marker COUPTFII (chicken ovalbumin upstream promoter-transcription factor II) [35], endothelial-cellspecific deletion of endoglin did not affect expression of arterial jagged-1 and ephrin B2, or venous markers Ephb4 and Aplnr (G protein-coupled apelin receptor) in neonatal 559

Review

Trends in Cell Biology Vol.20 No.9

Table 1. Defects in components of TGF-b signaling pathways lead to vascular abnormalities in human and mouse Gene (mouse/human) Ligands Tgfb1/TGFB1

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

Accessory receptors Eng/ENG (endoglin)

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

Animal model

Human disease

Refs

KO: embryonic lethal with vascular defects or postnatal lethality from autoimmune disease

Camurati– Engelmann disease a unknown

[20]

unknown

[20]

MFS2a, LDS a

HHT b

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

PAH b

[20,23,24]

HHT

[20]

Pre-eclampsia Unknown

[25] [20]

Unknown

[20]

JPb and HHT Unknown

[20] [20]

Unknown

[20]

Unknown

[26]

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

LDS

[20]

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

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

Vascular endothelial growth factor (VEGF) signaling plays a crucial role in angiogenesis and its dysregulation leads to defects in angiogenesis. It has been shown that VEGF levels are elevated in skin telangiectatic lesions of HHT patients and that anti-angiogenic drugs, such as thalidomide and bevacizumab (anti-VEGF antibody), are effective in treating gastrointestinal bleedings and liver AVMs, respectively [41–43]. By contrast, inhibition of ALK1/endoglin signaling using the soluble chimeric proteins ALK1-Fc and endoglin-Fc (containing either the ALK1 or the endoglin extracellular domains fused to the Fc part of IgG that sequester ligands from binding to endogenous endoglin and ALK1) hindered VEGF-induced EC sprouting in vitro [44,45]. ALK1-Fc also inhibited VEGF/ basic fibroblast growth factor (bFGF)-induced angiogenesis in an in vivo matrigel plug assay [44]. Although these results clearly suggest that there is crosstalk between VEGF and ALK1/endoglin signaling in angiogenesis, the exact molecular mechanisms underlying this interplay remain to be elucidated. TGF-b signaling and endothelial cell function Several studies have revealed that the effect of TGF-b on angiogenesis is context dependent [46,47]. TGF-b was

(Figure_1)TD$IG][ Review

Trends in Cell Biology

Vol.20 No.9

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

shown to promote EC proliferation and migration at low concentrations, whereas high concentrations had the opposite effect [46–49]. Low concentrations of TGF-b enhanced the angiogenic effects of bFGF or VEGF in a 3D fibrin or collagen assay, whereas high concentrations were inhibitory [48]. Treatment of bovine capillary endothelial (BCE) cells with TGF-b initially induces apoptosis by inducing VEGF expression because TGF-b signaling converts the VEGF/ VEGFR2-activated p38 (MAPK) into a proapoptotic signal [50]. However, protracted treatment of BCE cells with TGFb results in EC remodeling and formation of cord-like structures [51]. Inhibition of the TGF-b–ALK5 pathway by an ALK5 kinase inhibitor resulted in sustained proliferation and maintenance of human embryonic stem cell (hESC)derived ECs by sustaining Id1 expression [52]. Similarly, addition of an ALK5 kinase inhibitor in mouse ESCs increased EC growth and integrity through upregulation of the tight junction component claudin-5 [53]. It was also shown that suboptimal doses of VEGF and ALK5 kinase inhibitor synergistically induce EC migration and sprouting in vitro by inducing integrin a5 expression. In addition, ALK5 kinase inhibition induced angiogenesis and enhanced VEGF/bFGF-induced angiogenesis in a matrigel-plug assay in vivo, an effect that could be inhibited by an antibody that neutralizes the a5 integrin [54]. The initial view of TGF-b family signaling as a simple linear cascade, where TGF-b/Nodal/activin induce phosphorylation of Smads 2 and 3 and BMP/GDFs induce phosphorylation of Smads 1, 5 and 8, has been re-evaluated. Studies on ECs revealed that TGF-b can bind to and

signal through two distinct types of receptors in these cells – TGFBR1 (ALK5) and ACVRL1 (ALK1) – resulting in activation of Smad2,3 and Smad1,5,8, respectively (Figure 1) [47,55]. Although TGF-b/ALK5/Smad2,3 signaling was found to antagonize TGF-b/ALK1/Smad1 signaling, ALK5 is essential for ALK1 recruitment into the TGF-b receptor complex and for its activation. It was shown that TGF-b/ALK1 signaling potentiates and TGFb/ALK5 inhibits EC proliferation and migration of ECs [47,55]. It was thus suggested that the balance between TGF-b/ALK1 versus TGF-b/ALK5 will determine the proor anti-angiogenic effects of TGF-b. In addition to being present in ECs, the TGF-b/ALK1 pathway has also been observed in neurons, chondrocytes and hepatic stellate cells [56–58]. Moreover, in tumor cells, TGF-b was shown to signal by means of the Smad1 pathway. In one of the studies, TGF-b induced Smad1 phosphorylation through ALK2 and ALK3 [59], whereas two studies suggested that ALK5 can directly interact with and phosphorylate Smad1 [60,61]. The mechanistic differences between these studies are probably attributable to different receptors expressed on different cell types as well as the relative expression levels of each receptor. Although the studies discussed above suggest an important role for ALK1 in the activation phase of angiogenesis, overexpression studies demonstrated that ALK1 signaling inhibits the proliferation and migration of a human microvascular EC line, implying that ALK1 promotes the resolution phase of angiogenesis [62], consistent with the phenotype described in Alk1-deficient mice 561

Review – fragile blood vessels and increased mRNA levels for genes involved in the activation phase of angiogenesis [23]. The discrepancies could be due to different cell types being used in the studies or to the adaptive processes that take place in the Alk1-deficient embryos. Alternatively, ALK1 could be involved in both the activation and resolution phases of angiogenesis. In ECs, BMP9 and BMP10 have been shown to signal through ALK1 and ALK2, induce Smad1 phosphorylation, and inhibit EC proliferation and migration [63,64]. In human pulmonary artery endothelial cells (HPAECs), BMP9 was shown to induce phosphorylation of Smad1-5 and Smad2 through BMPR2/ALK1 and the activin type II receptors (ACVR2) [65] (Figure 1). Although both BMPR2 and ACVR2 were required for Smad1 phosphorylation, Smad2 activation was mainly mediated through ACVR2. The significance of this differential BMP9 signaling in angiogenesis and in vascular pathologies such as HHT and primary arterial hypertension (PAH, a vascular disorder associated with missregulated BMP signaling due to mutations in BMPR2) remains to be elucidated. BMP9 signaling through ALK1 was shown to inhibit VEGF expression [66], whereas increased VEGF levels were reported in Acvrl1-deficient embryos and HHT-2 patients [67,68]. Although BMP9 and BMP10 were considered to have an anti-angiogenic action, BMP10 was shown to induce angiogenesis in the chorioallantoic membrane (CAM) assay [69] and BMP9 to induce proliferation of ECs in vitro and in vivo [70]. In addition, BMP9 in combination with TGF-b was shown to potentiate VEGFinduced proliferation of ECs in vitro and VEGF/bFGFinduced angiogenesis in vivo [44]. Several studies have provided evidence that the TGF-b coreceptor endoglin plays an important role in balancing the TGF-b–ALK1 and TGF-b–ALK5 pathways [22]. Endoglin was shown to potentiate the TGF-b–ALK1 pathway and to inhibit the TGF-b–ALK5 pathway in ECs as well as in other cell types [71]. It was also shown that endoglin plays an important role in BMP9 signaling in ECs [63]. The molecular mechanisms by which endoglin regulates TGF-b and BMP signaling are not fully understood. A recent study suggested that ALK5 phosphorylates the cytoplasmic domain of endoglin on serines 646 and 649. S646A is required for activation of TGF-b–ALK1–Smad1,5,8 signaling, whereas both S646 and S649 are essential for BMP9-induced Smad1,5,8 phosphorylation [72]. Analysis of mouse retinal vessels showed that endothelial-cellspecific deletion of Eng does not affect levels of phosphorylated Smad2 or Smad1,5,8 [36]; however, deletion of Eng results in loss of Smad1,5,8 phosphorylation in the quiescent pulmonary vasculature [73]. As with ALK1, the role of endoglin in EC function is not completely understood; in vitro studies have suggested that knockdown of endoglin results in decreased EC proliferation and migration, whereas Eng deletion in the mouse results in a dramatic increase in EC proliferation [36]. BMPs 2, 4, 6 and 7 have been shown to induce EC proliferation and angiogenesis by inducing VEGF expression [46,74]. In mouse embryonic stem cells (ESCs), BMP4 exerts its angiogenic effects by activating the VEGF/ VEGFR2 and angiopoietin-1/Tie2 signaling cascades in 562

Trends in Cell Biology Vol.20 No.9

addition to the Smad signaling pathway because BMP4 induces phosphorylation not only of Smad1 but also Tie2 and VEGFR2 in these cells [75]. The pro-angiogenic effects of BMP6 have been shown to be mediated by the upregulation of Id1 and cyclooxygenase-2 expression in ECs [76,77]. Recently it was also shown that Myo10 plays a crucial role in BMP6-induced angiogenesis. Myo10 is upregulated in EC in response to BMP6 and that it is required for BMP6induced filopodia formation and migration [78]. Although it is apparent that TGF-b and BMP signaling components play crucial roles in EC function and angiogenesis, we have only started to unravel the molecular mechanisms by which these molecules regulate vascular system. Cellular context, local concentration of the different ligands, receptors, coreceptors, antagonists and their interplay play crucial roles in the apparently contradictory actions of TGF-b signaling during the different stages of angiogenesis. The identification and characterization of additional molecules and mechanisms involved in vascular signaling by TGF-b family members will help us to understand what determines the pro- or anti-angiogenic functions of TGF-b, ALK1 and BMP9, and their interplay with other factors involved in EC function and angiogenesis. TGF-b signaling and SMC function Vessel formation and stabilization cannot be completed unless pericytes and vascular SMCs are recruited to complete vessel assembly. Proper communications between ECs and mural cells play a crucial role in vessel formation, and these are tightly controlled by a number of signaling pathways [16] (Boxes 1, 2). TGF-b regulates SMC muscle differentiation by increasing the expression of alpha smooth-muscle actin and smooth-muscle myosin through the Smad3 and p38/MAPK pathways [79]. TGF-b treatment of ESC-derived cultures or embryoid bodies potentiates their differentiation into SMCs [53,80]. TGF-b can also induce proliferation and migration of SMCs. Genetic studies in the mouse (Table 1) revealed the important role of TGF-b signaling in SMC cell development and recruitment for the formation of stable vessels. EC- and SMC-specific deletions of Tgfbr2 show similar phenotypes. Deletion of Tgfbr2 in SMCs results in vascular defects in the yolk sac and embryo lethality between E12.5 and E16.5. Those results suggest that TGF-b signaling on ECs and SMCs plays an important role in SMC differentiation and function and, as a consequence, in proper EC–SMC interaction [29,30]. In addition, mice with neural-crest-specific ablation of Tgfbr2 develop a phenotype akin to DiGeorge syndrome because neural crest derivatives fail to differentiate into SMCs in the cardiac outflow tract [81]. Both Eng and Acvrl1 (Alk1) depletion result in fragile blood vessels due to impaired mural cell development, as shown by the absence or inappropriate association of SMCs with ECs [33,82–84]. Conditional endoglin expression in Eng-null embryos, using either SMC- or EC-specific promoters, can partially rescue SMC recruitment to the dorsal aorta, suggesting that endoglin plays distinct and cellautonomous roles in SMC recruitment [35]. Telangiectatic lesions in HHT patients are characterized by abnormal endothelial cell proliferation and SMC recruitment.

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

[(Figure_2)TD$IG]

Trends in Cell Biology

Vol.20 No.9

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

Figure 2. Targeting TGF-b signaling in tumor angiogenesis. Tumors cannot grow to more than 1–2 mm3 if supply of oxygen and nutrients is limited. Many tumors can be dormant for years. Tumor angiogenesis is essential to escape this period of dormancy. This process, also known as the angiogenic switch, is regulated by a variety of proand anti-angiogenic factors such as VEGF, bFGF, P1GF, TGF-b and BMP. Endoglin and ALK1 play important roles in tumor angiogenesis and tumor growth. Genetic deletion or inhibition of endoglin and ALK1 function by endoglin-neutralizing antibodies or by ALK1-Fc (an ALK1 ligand trap) results in reduced tumor angiogenesis and tumor growth. ECD, extracellular domain; Fc, antibody Fc region.

563

Review therapeutic responses against tumor growth and tumor angiogenesis. Endoglin is upregulated in the tumor-associated endothelium and its expression correlates with poor prognosis [22]. Several studies have considered endoglin as a therapeutic target in anti-angiogenic therapies because tumor vascularization and growth are diminished in Eng-heterozygous mice [22]. Endoglin-neutralizing antibodies can target tumor vasculature and inhibit tumor growth in mouse tumor models (Figure 2) [22]. Recent studies suggested that soluble endoglin (sEng) can interfere with the function of endogenous endoglin on ECs and inhibit spontaneous cord formation in human umbilical vein endothelial cells (HUVECs) and VEGF-induced EC sprout formation [25,45]. These results suggest that sEng has great potential in anti-angiogenic cancer therapy by interfering with tumor angiogenesis and tumor growth. The exact role of ALK1 in angiogenesis is not fully understood. Although some studies suggest that ALK1 inhibits EC proliferation, and perturbation of ALK1 signaling results in increased VEGF signaling and enhanced angiogenesis [23,62,66], ALK1 was also shown to promote EC proliferation and migration [55]. Recent studies have revealed an important role for ALK1 in tumor angiogenesis and growth [44]. Expression analysis in mice suggested that expression of ALK1 and its ligands TGF-b and BMP9 is increased during tumor growth. Deletion of one Acvrl1 (Alk1) allele resulted in reduced tumor growth and progression by inhibition of angiogenesis in the RIP1-Tag2 transgenic mouse model of multistep tumorigenesis [44]. Pharmacological inhibition of ALK1 signaling using an ALK1-ligand trap (ALK1-Fc), resulted in reduced tumor angiogenesis and tumor growth in the RIP1-Tag2 transgenic mouse model of pancreatic islet carcinomas as well as in a breast cancer orthotopic tumor model [44,69]. In addition, ALK1-Fc treatment of RIP-Tag2 mice resulted in increased pericyte coverage of tumor vessels [44]. ALK1Fc inhibits tumor angiogenesis by interfering with the angiogenic activity of proangiogenic factors such as VEGF and bFGF (Figure 2). Interestingly, TGF-b and BMP9 can synergistically induce the pro-angiogenic effects of VEGF and bFGF. ALK1-Fc could efficiently interfere with this synergistic effect both in vitro and in vivo [44]. Those results suggest that ALK1 provides a valuable target for anti-angiogenic therapy and that ALK1-Fc is a powerful anti-angiogenic agent capable of reducing tumor angiogenesis and tumor growth (Figure 2). The therapeutic potential of human ALK1-Fc and humanized ALK1 and endoglin-neutralizing antibodies is currently being evaluated in clinical cancer trials [96–98]. Concluding remarks The role of TGF-b signaling in angiogenesis has been highly controversial, with numerous studies showing that it is either pro-angiogenic or, conversely, anti-angiogenic in a context-dependent manner. Recent studies emphasize the growing appreciation that the pleiotropic effects of TGF-b signaling are the outcome of multiple and fine-tuned signaling cascades rather than the result of a simple linear signal-transduction pathway. Some of these discrepancies might be explained by variations in ligand concentrations, 564

Trends in Cell Biology Vol.20 No.9

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

Box 4. Questions for future research  What determines the pro- and anti-angiogenic effects of TGF-b family members? How can different ligand concentrations have different effects on EC function and activation?  Are TGF-b family members and their receptors involved in both the activation and resolution phases of angiogenesis? How are their effects influenced by the angiogenic microenvironment?  How do mutations in ENG and ACVRL1 lead to AVMs in HHT? What is the role of modifier genes and what are the second hits in the development of HHT?  Can the anti-tumorigenic effects of endoglin- and ALK1-inhibitors be enhanced by combination therapy with anti-angiogenic agents or conventional chemotherapeutic drugs?

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

References 1 Adams, R.H. and Alitalo, K. (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat. Rev. Mol. Cell Biol. 8, 464–478 2 Carmeliet, P. (2005) Angiogenesis in life, disease and medicine. Nature 438, 932–936 3 Folkman, J. and D’Amore, P.A. (1996) Blood vessel formation: what is its molecular basis? Cell 87, 1153–1155 4 Ribatti, D. (2007) The discovery of endothelial progenitor cells. An historical review. Leuk. Res. 31, 439–444 5 Betsholtz, C. et al. (2005) Role of pericytes in vascular morphogenesis. EXS 94, 115–125 6 Risau, W. (1997) Mechanisms of angiogenesis. Nature 386, 671– 674 7 Stehbens, W.E. (1996) Structural and architectural changes during arterial development and the role of hemodynamics. Acta Anat. (Basel) 157, 261–274 8 Swift, M.R. and Weinstein, B.M. (2009) Arterial–venous specification during development. Circ. Res. 104, 576–588 9 Carmeliet, P. and Jain, R.K. (2000) Angiogenesis in cancer and other diseases. Nature 407, 249–257 10 Hanahan, D. and Folkman, J. (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353–364 11 Gerhardt, H. et al. (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell Biol. 161, 1163–1177 12 Kamei, M. et al. (2006) Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 442, 453–456 13 Stalmans, I. et al. (2002) Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J. Clin. Invest. 109, 327– 336 14 Hellstrom, M. et al. (2007) Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445, 776–780 15 Suchting, S. et al. (2007) The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc. Natl. Acad. Sci. U. S. A. 104, 3225–3230 16 von Tell, D. et al. (2006) Pericytes and vascular stability. Exp. Cell Res. 312, 623–629 17 Heldin, C.H. et al. (1997) TGF-b signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465–471 18 Schmierer, B. and Hill, C.S. (2007) TGFb–SMAD signal transduction: molecular specificity and functional flexibility. Nat. Rev. Mol. Cell Biol. 8, 970–982 19 Guo, X. and Wang, X.F. (2009) Signaling cross-talk between TGF-beta/ BMP and other pathways. Cell Res. 19, 71–88 20 ten Dijke, P. and Arthur, H.M. (2007) Extracellular control of TGFb signalling in vascular development and disease. Nat. Rev. Mol. Cell Biol. 8, 857–869 21 Constam, D.B. and Robertson, E.J. (1999) Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases. J. Cell Biol. 144, 139–149 22 ten Dijke, P. et al. (2008) Endoglin in angiogenesis and vascular diseases. Angiogenesis 11, 79–89 23 Oh, S.P. et al. (2000) Activin receptor-like kinase 1 modulates transforming growth factor-b1 signaling in the regulation of angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 97, 2626–2631

Trends in Cell Biology

Vol.20 No.9

24 Beppu, H. et al. (2004) BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia. Am. J. Physiol. Lung Cell Mol. Physiol. 287, L1241–L1247 25 Venkatesha, S. et al. (2006) Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat. Med. 12, 642–649 26 Chen, Q. et al. (2009) Smad7 is required for the development and function of the heart. J. Biol. Chem. 284, 292–300 27 Park, S.O. et al. (2008) ALK5- and TGFBR2-independent role of ALK1 in the pathogenesis of hereditary hemorrhagic telangiectasia type 2. Blood 111, 633–642 28 Seki, T. et al. (2006) Nonoverlapping expression patterns of ALK1 and ALK5 reveal distinct roles of each receptor in vascular development. Lab. Invest. 86, 116–129 29 Carvalho, R.L. et al. (2004) Defective paracrine signalling by TGFb in yolk sac vasculature of endoglin mutant mice: a paradigm for hereditary hemorrhagic telangiectasia. Development 131, 6237– 6247 30 Carvalho, R.L. et al. (2007) Compensatory signalling induced in the yolk sac vasculature by deletion of TGFb receptors in mice. J. Cell Sci. 120, 4269–4277 31 Gallione, C.J. et al. (2004) A combined syndrome of juvenile polyposis and hereditary hemorrhagic telangiectasia associated with mutations in MADH4 (SMAD4). Lancet 363, 852–859 32 Park, S.O. et al. (2009) Real-time imaging of de novo arteriovenous malformation in a mouse model of hereditary hemorrhagic telangiectasia. J. Clin. Invest. 119, 3487–3496 33 Sorensen, L.K. et al. (2003) Loss of distinct arterial and venous boundaries in mice lacking endoglin, a vascular-specific TGFb coreceptor. Dev. Biol. 261, 235–250 34 Roman, B.L. et al. (2002) Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels. Development 129, 3009– 3019 35 Mancini, M.L. et al. (2009) Endoglin plays distinct roles in vascular smooth muscle cell recruitment and regulation of arteriovenous identity during angiogenesis. Dev. Dyn. 238, 2479–2493 36 Mahmoud, M. et al. (2010) Pathogenesis of arteriovenous malformations in the absence of endoglin. Circ. Res. 106, 1425– 1433 37 Blokzijl, A. et al. (2003) Cross-talk between the Notch and TGF-b signaling pathways mediated by interaction of the Notch intracellular domain with Smad3. J. Cell Biol. 163, 723–728 38 Niimi, H. et al. (2007) Notch signaling is necessary for epithelial growth arrest by TGF-b. J. Cell Biol. 176, 695–707 39 Itoh, F. et al. (2004) Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells. EMBO J. 23, 541– 551 40 Fu, Y. et al. (2009) Differential regulation of transforming growth factor b signaling pathways by Notch in human endothelial cells. J. Biol. Chem. 284, 19452–19462 41 Bauditz, J. et al. (2004) Thalidomide for treatment of severe intestinal bleeding. Gut 53, 609–612 42 Bauditz, J. and Lochs, H. (2007) Angiogenesis and vascular malformations: antiangiogenic drugs for treatment of gastrointestinal bleeding. World J. Gastroenterol. 13, 5979–5984 43 Bose, P. et al. (2009) Bevacizumab in hereditary hemorrhagic telangiectasia. N. Engl. J. Med. 360, 2143–2144 44 Cunha, S.I. et al. (2010) Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis. J. Exp. Med. 207, 85 45 Hawinkels, L.J.A.C. et al. (2010) MMP-14 (MT1-MMP) mediated endoglin shedding regulates tumor angiogenesis. Canc. Res. 70, 4141–4150 46 Pardali, E. and ten Dijke, P. (2009) Transforming growth factor-b signaling and tumor angiogenesis. Front. Biosci. 14, 4848–4861 47 Goumans, M.J. et al. (2002) Balancing the activation state of the endothelium via two distinct TGF-b type I receptors. EMBO J. 21, 1743–1753 48 Pepper, M.S. (1997) Transforming growth factor-b: vasculogenesis, angiogenesis, and vessel wall integrity. Cytokine Growth Factor Rev. 8, 21–43 49 Serrati, S. et al. (2009) TGFb1 antagonistic peptides inhibit TGFb1dependent angiogenesis. Biochem. Pharmacol. 77, 813–825

565

Review 50 Ferrari, G. et al. (2006) VEGF, a prosurvival factor, acts in concert with TGF-b1 to induce endothelial cell apoptosis. Proc. Natl. Acad. Sci. U. S. A. 103, 17260–17265 51 Ferrari, G. et al. (2009) Transforming growth factor-b1 (TGF-b1) induces angiogenesis through vascular endothelial growth factor (VEGF)-mediated apoptosis. J. Cell Physiol. 219, 449–458 52 James, D. et al. (2010) Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFb inhibition is Id1 dependent. Nat. Biotechnol. 28, 161–166 53 Watabe, T. et al. (2003) TGF-b receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells. J. Cell Biol. 163, 1303–1311 54 Liu, Z. et al. (2009) VEGF and inhibitors of TGFb type-I receptor kinase synergistically promote blood-vessel formation by inducing a5-integrin expression. J. Cell Sci. 122, 3294–3302 55 Goumans, M.J. et al. (2003) Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFb /ALK5 signaling. Mol. Cell 12, 817–828 56 Finnson, K.W. et al. (2008) ALK1 opposes ALK5/Smad3 signaling and expression of extracellular matrix components in human chondrocytes. J. Bone Miner. Res. 23, 896–906 57 Konig, H.G. et al. (2005) TGF-b1 activates two distinct type I receptors in neurons: implications for neuronal NF-kB signaling. J. Cell Biol. 168, 1077–1086 58 Wiercinska, E. et al. (2006) Id1 is a critical mediator in TGF-betainduced transdifferentiation of rat hepatic stellate cells. Hepatology 43, 1032–1041 59 Daly, A.C. et al. (2008) Transforming growth factor b-induced Smad1/5 phosphorylation in epithelial cells is mediated by novel receptor complexes and is essential for anchorage-independent growth. Mol. Cell Biol. 28, 6889–6902 60 Liu, I.M. et al. (2009) TGFb -stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro-migratory TGFb switch. EMBO J. 28, 88–98 61 Wrighton, K.H. et al. (2009) Transforming growth factor b can stimulate Smad1 phosphorylation independently of bone morphogenic protein receptors. J. Biol. Chem. 284, 9755–9763 62 Lamouille, S. et al. (2002) Activin receptor-like kinase 1 is implicated in the maturation phase of angiogenesis. Blood 100, 4495–4501 63 David, L. et al. (2007) Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells. Blood 109, 1953–1961 64 Scharpfenecker, M. et al. (2007) BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis. J. Cell Sci. 120, 964–972 65 Upton, P.D. et al. (2009) Bone morphogenetic protein (BMP) and activin type II receptors balance BMP9 signals mediated by activin receptor-like kinase-1 in human pulmonary artery endothelial cells. J. Biol. Chem. 284, 15794–15804 66 Shao, E.S. et al. (2009) Expression of vascular endothelial growth factor is coordinately regulated by the activin-like kinase receptors 1 and 5 in endothelial cells. Blood 114, 2197–2206 67 Sadick, H. et al. (2005) Patients with hereditary hemorrhagic telangiectasia have increased plasma levels of vascular endothelial growth factor and transforming growth factor-b1 as well as high ALK1 tissue expression. Haematologica 90, 818–828 68 Sadick, H. et al. (2005) Plasma level and tissue expression of angiogenic factors in patients with hereditary hemorrhagic telangiectasia. Int. J. Mol. Med. 15, 591–596 69 Mitchell, D. et al. (2010) ALK1-Fc inhibits multiple mediators of angiogenesis and suppresses tumor growth. Mol. Cancer Ther. 9, 379–388 70 Suzuki, Y. et al. (2010) BMP-9 induces proliferation of multiple types of endothelial cells in vitro and in vivo. J. Cell Sci. 123, 1684–1692 71 Velasco, S. et al. (2008) L- and S-endoglin differentially modulate TGFb1 signaling mediated by ALK1 and ALK5 in L6E9 myoblasts. J. Cell Sci. 121, 913–919 72 Ray, B.N. et al. (2010) ALK5 phosphorylation of the endoglin cytoplasmic domain regulates Smad1/5/8 signaling and endothelial cell migration. Carcinogenesis 31, 435–441 73 Mahmoud, M. et al. (2009) Endoglin and activin receptor-like-kinase 1 are co-expressed in the distal vessels of the lung: implications for

566

Trends in Cell Biology Vol.20 No.9

74 75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93 94

95

two familial vascular dysplasias. HHT and PAH. Lab. Invest. 89, 15–25 David, L. et al. (2009) Emerging role of bone morphogenetic proteins in angiogenesis. Cytokine Growth Factor Rev. 20, 203–212 Suzuki, Y. et al. (2008) BMPs promote proliferation and migration of endothelial cells via stimulation of VEGF-A/VEGFR2 and angiopoietin-1/Tie2 signalling. J. Biochem. 143, 199–206 Ren, R. et al. (2007) Gene expression profiles identify a role for cyclooxygenase 2-dependent prostanoid generation in BMP6-induced angiogenic responses. Blood 109, 2847–2853 Valdimarsdottir, G. et al. (2002) Stimulation of Id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells. Circulation 106, 2263–2270 Pi, X. et al. (2007) Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors. Cell Biol. 179, 1569–1582 Seay, U. et al. (2005) Transforming growth factor-b-dependent growth inhibition in primary vascular smooth muscle cells is p38-dependent. J. Pharmacol. Exp. Ther. 315, 1005–1012 Hirschi, K.K. et al. (1998) PDGF, TGF-b, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J. Cell Biol. 141, 805–814 Wurdak, H. et al. (2005) Inactivation of TGFb signaling in neural crest stem cells leads to multiple defects reminiscent of DiGeorge syndrome. Genes Dev. 19, 530–535 Johnson, D.W. et al. (1996) Mutations in the activin receptor-like kinase 1 gene in hereditary hemorrhagic telangiectasia type 2. Nat. Genet. 13, 189–195 McAllister, K.A. et al. (1994) Endoglin, a TGF-b binding protein of endothelial cells, is the gene for hereditary hemorrhagic telangiectasia type 1. Nat. Genet. 8, 345–351 Bot, P.T. et al. (2009) Increased expression of the transforming growth factor-b signaling pathway, endoglin, and early growth response-1 in stable plaques. Stroke 40, 439–447 Lebrin, F. et al. (2010) Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia. Nat. Med. 16, 420–428 Dorai, H. and Sampath, T.K. (2001) Bone morphogenetic protein-7 modulates genes that maintain the vascular smooth muscle cell phenotype in culture. J. Bone Joint Surg. Am. 83, S70–S78 Nakaoka, T. et al. (1997) Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic protein-2. J. Clin. Invest 100, 2824–2832 Yu, P.B. et al. (2005) Bone morphogenetic protein (BMP) type II receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary artery smooth muscle cells. J. Biol. Chem. 280, 24443–24450 Yu, P.B. et al. (2008) Bone morphogenetic protein (BMP) type II receptor is required for BMP-mediated growth arrest and differentiation in pulmonary artery smooth muscle cells. J. Biol. Chem. 283, 3877–3888 Harrison, R.E. et al. (2003) Molecular and functional analysis identifies ALK-1 as the predominant cause of pulmonary hypertension related to hereditary hemorrhagic telangiectasia. J. Med. Genet. 40, 865–871 Trembath, R.C. (2001) Mutations in the TGF-b type 1 receptor, ALK1, in combined primary pulmonary hypertension and hereditary hemorrhagic telangiectasia, implies pathway specificity. J. Heart Lung Transplant 20, 175 Thomas, M. et al. (2009) Activin-like kinase 5 (ALK5) mediates abnormal proliferation of vascular smooth muscle cells from patients with familial pulmonary arterial hypertension and is involved in the progression of experimental pulmonary arterial hypertension induced by monocrotaline. Am. J. Pathol. 174, 380–389 Bergers, G. and Hanahan, D. (2008) Modes of resistance to antiangiogenic therapy. Nat. Rev. Cancer 8, 592–603 Petersen, M. et al. (2010) Smad2 and Smad3 have opposing roles in breast cancer bone metastasis by differentially affecting tumor angiogenesis. Oncogene 29, 1351–1361 Roffe, S. et al. (2010) Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: Effects on myotube fusion. Exp. Cell Res. 316, 1061–1069

Review 96 North, M.A et al. Amgen Fremont Inc and Pfizer Inc. Human monoclonal antibodies to Activin receptor-like kinase 1, WO/2007/ 040912 97 Grinberg, A. et al. Acceleron Pharma, Inc and Ludwig Institute for Cancer Research Ltd. Methods and compositions based on ALK1

Trends in Cell Biology

Vol.20 No.9

antagonists for modulating angiogenesis and pericyte coverage, WO/ 2009/134428 98 Seehra, J. et al. (2009) Acceleron Pharma, Inc. Antagonists of BMP9, BMP10, ALK1 and other ALK1 ligands, and uses therof, WO/2009/ 139891

567