extracellular control of tgf signalling in vascular development and disease

*Molecular Cell Biology, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands.Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle University, NE1 3BZ, UK.emails: [email protected]; [email protected]:10.1038/nrm2262Published online 26 September 2007

Extracellular control of TGF signalling in vascular development and diseasePeter ten Dijke* and Helen M. Arthur

Abstract | The intracellular mechanism of transforming growth factor (TGF) signalling via kinase receptors and SMAD effectors is firmly established, but recent studies of human cardiovascular syndromes such as Marfan syndrome and preeclampsia have refocused attention on the importance of regulating the availability of active extracellular TGF. It seems that elastic extracellular matrix (ECM) components have a crucial role in controlling TGF signalling, while soluble and membrane bound forms of TGF coreceptors add further layers of regulation. Together, these extracellular interactions determine the final bioavailability of TGF to vascular cells, and dysregulation is associated with an increasing number of vascular pathologies.

Transforming growth factor1 (TGF1) is the prototypic member of a large family of evolutionarily conserved pleio-tropic secreted cytokines, which also includes the activins and bone morphogenetic proteins (BMPs). Individual family members have crucial roles in multiple processes throughout development and in the maintenance of tissue homeostasis in adult life1,2. Not surprisingly, therefore, subversion of signalling by TGF family members has been implicated in many human diseases, including cancer, fibrosis, autoimmune and vascular diseases3.

The TGF family of ligands mediate their effects by binding specific transmembrane type I and type II Ser/Thr kinase receptors. The type I receptors act downstream of type II receptors and determine the signalling specificity within the receptor complex. Upon ligandinduced heteromeric complex formation, the type II receptor transphosphorylates and activates the type I receptor, which subsequently propagates the signal by phosphorylating specific receptorregulated (R) SMAD transcription factors at the two Cterminal Ser residues (FIG. 1). On activation, RSMADs form heteromeric complexes with a related partner molecule, the CoSMAD (SMAD4 in mammals), and accumulate in the nucleus where they participate in the transcriptional control of target genes1,2.

Despite the large number and distinct functions of TGF family members (33 in mammals), there is an enormous convergence in signalling to only five type II receptors, seven type I receptors (also described as activin receptorlike kinases (ALKs)) and two main SMAD intracellular pathways1,2 (FIG. 1). So, how are signalling specificity and diversity generated, especially as the signalling receptors and SMADs are broadly coexpressed? Extracellular

regulation of TGF signalling by coreceptors (which control the access of ligands to signalling receptors) and interplay between TGF ligands and extracellular molecules that regulate the activity of these ligands could be important mechanisms that control signalling specificity. Consistent with these potential mechanisms, all three TGF isoforms (TGF1, TGF2 and TGF3 in mammals) are secreted in latent forms that need to be activated before they can bind to signalling receptors4. Furthermore, the biological activities of TGF family members are regulated by specific secreted inhibitors (see below) that sequester ligands and block binding to receptors.

One of the compelling themes to emerge in recent years is the crucial role of elastic extracellular matrix (ECM) proteins in regulating TGF bioavailability in the vascular system5. Defects in the ECM, which were initially thought to affect the physical properties of vessel walls and thereby compromise the vasculature, are now linked to enhanced TGFSMAD signalling. In this Review, we focus on recent insights into the mechanisms that regulate extracellular TGF activity in an appropriate spatio temporal manner and how this contributes to its pivotal role in vascular development and disease. For reviews addressing other related aspects of TGF family signalling at the intracellular level and dysregulated signalling in disease, see REFS 13, 68.

Secretion and ECM interactionSecretion and storage in the ECM.The three TGF isoforms in mammals TGF1, TGF2 and TGF3 are multifunctional and act in an autocrine, paracrine and sometimes endocrine manner to regulate diverse

Pleiotropic Influencing multiple different traits.

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EndoglinTGF

TGFBR2

ALK5 ALK1,2,3,6

Sol-Endo Sol-EndoBetaglycan

BMPR2SMAD2/3 SMAD1/5/8

SMAD4

P P

PP

PP

PP

SMAD7 SMAD6

Geneexpression

PP Gene

expression

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BMP

Convertase family of endoproteases A group of enzymes that make an internal cut in a polypeptide chain to convert it from an inactive to an active form.

FibrillinAn extracellular matrix glycoprotein that is a structural component of microfibrils.

MicrofibrilA fibre component (10 nm in diameter) of the extracellular matrix that is essential for the integrity of elastic fibres, which are particularly abundant in the aorta.

developmental processes and maintain tissue homeostasis in the adult. The bioavailability of active TGF is regulated at multiple levels, including secretion and interaction with ECM components, and each step in the activation pathway is under tight control (FIG. 2). TGFs are synthesized as precursor proteins that are proteolytically processed. The signal peptide is removed from the preproTGF during transit through the rough endoplasmic reticulum and, following dimerization, another cleavage occurs by the convertase family of endoproteases9. These proteases cleave the precursor into the Cterminal mature peptide and the Nterminal precursor remnant (also known as latency associated peptide (LAP)) within the secretory vesicles or in the extracellular space10. Control and/or localization of convertase activity may represent an important level of regulation of TGF ligands. After cleavage, mature TGF

and LAP remain attached via noncovalent bonds to form the small latent complex (SLC). LAP shields the receptorinteracting epitopes in the mature protein and this keeps TGF in its latent form4.

The SLC can covalently attach to the large latent TGFbinding protein (LTBP) to form the large latent complex (LLC)11,12 (FIG. 2). Most cell types secrete TGF as part of LLC, although some cells (such as the bone cell line UMR106) secrete SLC13. Four different LTPBs have been identified, of which LTBP1, LTBP3 and, to a lesser degree, LTBP4 covalently bind to LAPs of all three TGF isoforms14. LTBPs contain multiple epidermalgrowth factor (EGF)like repeats and Cysrich domains that are also found in fibrillins, which are extracellular proteins that are required for elastic fibre formation. The Cterminal region of LTBP1 binds to the Nterminal region of fibrillin1, linking LLC to elastic microfibrils15. After secretion, LLC binds to the ECM via the Nterminal domain of LTBP and this interaction is further supported by covalent transglutaminaseinduced crosslinks16. Antibodies to LTBP1 and inhibitors of transglutaminase activity inhibit the activation of latent TGF, which demonstrates that localization of LTBP to the ECM is required for effective TGF activation16,17.

LTBPs might have multiple functions: they target the latent TGF complex to specific sites, including structural components within the elastic fibres, where they may be stored for later use. This targeting is determined by binding to ECM components, such as fibronectin, via the variable Nterminal regions of LTBPs. In addition, LTPBs stabilize latent TGF complexes and regulate their activation at the cell surface (discussed below)18. Important evidence for these roles comes from mouse models that are either deficient in Ltbp3 or have a hypomorphic mutation in Ltbp4. These mice are viable but have multiple phenotypes that are related to decreased TGF signalling and defects in elastic fibres19,20 (TABLES 1,2). These results indicate an important role for LTBPs in connective tissue deposition and as a local regulator of TGF availability.

Release from microfibrils and ECM.In order for latent TGF to become activated and function at adjacent and neighbouring cells, the LLC must be liberated from microfibrils and ECM (FIG. 2). A recent study revealed that LLC release can be initiated with the displacement of LTBP bound to fibrillin1 (REF. 21). Degradation of microfibrils by inflammatory proteolytic enzymes (such as elastase) releases fragments of fibrillin1, including an internal fibrillin fragment that efficiently binds to the Nterminal region of fibrillin1 and displaces LTBP. This releases LLC from microfibrils and contributes to local TGF activation21.

Several proteases, including plasmin, mastcell chymase and thrombin, release LLC from the ECM4. Cleavage of LTBP1 occurs in a sensitive hinge region such that the Nterminal fragment remains bound to ECM, but the remainder of LLC is released (FIG. 2). In a recent study, BMP1like matrix metalloproteases (MMPs) were shown to cleave LTBP1 at two specific sites in the hinge region to release LLC22, and facilitate the subsequent MMPdependent LAP cleavage. In the absence of BMP1,

Figure 1 | Signal transduction by TgF family members. Canonical signalling by transforminggrowth factor (TGF) superfamily members can be divided into two main intracellular pathways according to the SMAD mediators: either SMAD2/3 or SMAD1/5/8. Members of the TGF family bind to specific Ser/Thr kinase type II and type I receptors; in most cells, TGF signals via TGFBR2 and ALK5 (also known as TGF receptor1; TGFBR1), and bone morphogenetic proteins (BMPs) signal via the BMP type II receptor (BMPR2) and ALK1, 2, 3 and 6. The accessory receptors betaglycan and endoglin can modulate signalling via the type II and type I receptors. Betaglycan enhances TGF2 binding to TGF receptors, whereas endoglin may perform a similar function for selected TGF family members and their receptors. Soluble endoglin (SolEndo) is thought to sequester ligand and thereby inhibits receptor binding95; however, the exact mechanism through which this occurs is not known as endoglin requires TGFBR2 for TGF binding113. Activated type I receptors induce the phosphorylation of specific receptor regulated (R) SMADs, which are the intracellular effectors of TGF family members. In most cell types, TGF induces SMAD2/3 phosphorylation and BMPs induce SMAD1/5/8 phosphorylation. Activated RSMADs form heteromeric complexes with SMAD4 that accumulate in the nucleus, where they regulate the expression of target genes such as SERPINE1 (plasminogen activator inhibitor) and ID1 (inhibitor of DNA binding1) in cooperation with transcription factors, coactivators and corepressors1,2. Inhibitory SMADs, such as SMAD6 and SMAD7, can antagonize TGF signalling by inhibiting the activation of RSMADs.

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N

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cells have increased fibrillar structures that contain LTBP1 and show reduced TGF1 activity22. Consistent with this interaction, impairment in TGF signalling or deficiency in BMP1 result in similar defects in the frontal skull bones of mice23.

Activation and receptor binding. In order for TGF to bind to its cognate cellsurface receptors, the mature peptide needs to be released from LAP (FIG. 2). The mechanism by which latent TGF is converted into active TGF varies according to cell type and context, but all activating mechanisms directly target LAP4. Invitro, physical conditions such as exposure to extremes of pH or high temperature denature LAP but not mature TGF4. Invivo, it is thought that binding of the multifunctional secreted thrombospondin1 (THBS1) to LAP disrupts the noncovalent interactions between LAP and mature TGF24. Activation can also occur by proteases that cleave LAP to release bioactive TGF. These processes couple matrix turnover to the production of a strong inducer of ECM accumulation and may be crucial in maintaining a balance of matrix components.

Another important activation mechanism for latent TGF is through binding of v6 and v8 integrins to the RGD sequence in LAP25. The mechanism is unclear, but interaction with the RGD domain of LAP may induce a conformational change that leads to liberation or exposure of TGF. Knockin mice, in which the RGD sequence in TGF1LAP was replaced by a nonfunctional ArgGlyGlu sequence, recapitulate all the main features of Tgfb1null mice, pointing to the importance of RGD in TGF1 activation26. It should be noted that the LAP of TGF2 does not contain an RGD sequence and may be activated by another mechanism that remains to be determined25. Activation of latent TGF by v8 integrins involves the membrane type I (MT1) matrix metalloprotease (MMP), and is therefore sensitive to MMP inhibitors; however, it is currently unclear whether it requires LTBP27. By contrast, v6mediated activation is resistant to MMP inhibitors and requires a direct interaction of fibronectin with LTBP1 that targets LLC to the ECM28. As a result, latent TGF is inefficiently activated in cells that lack fibronectin or its receptor, 51 (REF. 28).

In line with these roles, inactivation of some of the murine genes that encode putative activators leads to defects that are reminiscent of mice deficient in TGF signalling components (TABLES 1,2). For example, Thbs1deficient mice have a similar, albeit milder, phenotype to the Tgfb1 knockout24. Also, mice that lack v or 8 integrins have defects in vascular and palatal development that phenocopy the defects of Tgfb1 and Tgfb3knockout mice, respectively29,30. These phenotypes support the idea that extracellular activation of TGF is a major controlling step before receptor binding invivo.

Cell-surface receptors. Once the active TGF family member is released from the ECM, it signals via specific complexes of type I and type II Ser/Thr kinase receptors (FIG. 1). The type I and type II receptors are structurally similar with small Cysrich extracellular domains, single transmembranespanning regions and intracellular parts that mainly comprise kinase domains. TGF signals via TGF type II receptor (TGFBR2) and TGFBR1 (also known as ALK5; a type I receptor). Other combinations

Figure 2 | regulation of TgF bioavailability. Schematic model of synthesis, secretion and matrix deposition of transforming growth factor (TGF) (a) and activation and TGF receptor binding (b). TGF is synthesized as a preproprotein, which undergoes proteolytic processing in the rough endoplasmic reticulum (1). Two monomers of TGF dimerize through disulphide bridges (2). The proTGF dimer is then cleaved by furin convertase to yield the small latent TGF complex (SLC), in which the latencyassociated peptide (LAP; orange) and the mature peptide (red) are connected by noncovalent bonds (3). This processing step is inhibited by emilin1. The large latent TGF complex (LLC) is formed by covalent attachment of the large latent TGF binding protein (LTBP, shown in blue; 4). The Nterminal and hinge region of LTBP interact with extracellular matrix (ECM) components such as fibronectin; this interaction can be covalent owing to crosslinking by transglutaminase. The Cterminal region of LTBP (blue) interacts noncovalently with the Nterminal region of fibrillin1 (green; 4). As part of TGF activation and receptor binding (b), an internal fragment of fibrillin1 (indicated in purple in 5) can be released by proteolysis (mediated by elastases at sites indicated by black arrowheads in 5) and interacts with Nterminal region of fibrillin1 to displace LTBP and release LLC (6). The LLC can be targeted to the cell surface by binding to integrins via RGD sequences (blue regions) in LAP (6). Bone morphogenetic protein1 (BMP1) can cleave two sites in the hinge region of LTBP (arrowheads in 6), which results in the release of LLC (7). Matrix metalloprotease2 (MMP2) (and other proteases) can cleave LAP (black arrowheads in 7) to release the mature TGF (red). Mature TGF can then bind to its cognate receptors, TGFBR2 and ALK5 (8).

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Matrix metalloprotease One of a family of structurally related extracellular Ca2+-dependent zinc-containing proteases involved in tissue remodelling and ECM degradation.

RGD sequence An amino acid sequence (Arg-Gly-Asp) found in extracellular matrix proteins that directly binds to integrins.

of type I and type II receptors respond to other TGF family ligands, for example, the BMP type II receptor (BMPR2) responds to BMP ligands in combination with ALK1, 2, 3 or 6 type I receptors.

TGF receptors are expressed on endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), but also on many other cell types31,32. This, together with the highly contextdependent responses, generates a multifaceted role for TGF family members in vascular biology33. In ECs, TGF can transduce signals via ALK1 in addition to ALK5, and is thought to contribute to the bifunctional effects of TGF on angiogenesis34. ALK5 is required for TGFALK1 activation, whereas ALK1 inhibits intracellular ALK5SMAD signalling. The differential activation of these two distinct type I receptor pathways by TGF provides the ECs with an intricate mechanism to precisely regulate, and even switch between, TGFinduced biological responses. For example, TGFALK1 activation leads to stimulation of EC proliferation and migration, whereas TGFALK5 activation inhibits these responses34.

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

TGF regulates vascular developmentNew blood vessels develop by a combination of vasculogenesis and angiogenesis39 (BOX 1). These processes are regulated by cytokines and growth factors in a highly orchestrated manner during embryogenesis. The vital importance of TGF signalling in vascular development was recognized following the identification of mutations

Table 1 | Human syndromes and animal models associated with inactivation or misexpression of TGF signalling components*gene Human syndrome animal models refs

Extracellular regulation of TGF signallingBMP1 Unknown KO: reduced skull ossification, abnormal collagen fibrils in amnion, die at birth. 23

EMILIN1 Hypertension KO: reduced arterial diameter, increased vascular resistance, emphysema, increased TGF signalling in vascular wall.

93

FBN1 (fibrillin1) MFS1 KO: ruptured aortic aneurysm, impaired pulmonary function, die at birth. 83, 84

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

FBN2 (fibrillin2) Contractural arachnodactyly

KO: bilateral syndactyly. 109

FN1 (fibronectin1) EhlersDanlos syndrome, type X

KO: deformed heart and embryonic vessels, defective extraembryonic vasculature.

114

EFEMP2 (fibulin4) Cutis laxa KO: perinatal lethality, arterial narrowing, aortic rupture. 90, 91

Hypomorphic: tortuous aorta, aortic aneurysm, increased TGF activation.FBLN5 (fibulin5, DANCE) Cutis laxa KO: tortuosity and elongation of the aorta, loose skin. 88, 89

LTBP1 Unknown KO: persistent truncus arteriosus. 124

LTBP3 Unknown KO: craniofacial malformations, osteosclerosis and osteoarthritis. 19

LTBP4 Unknown Hypomorphic: emphysema, cardiomyopathy and colorectal cancer. 20

THBS1 (thrombospondin1) Unknown KO: pneumonia, alveolar haemorrhage, vSMC hyperplasia. 24, 115

ITGB8 (integrin, 8) Unknown KO: embryonic lethal with vascular defects and/or perinatal lethality and cerebral haemorrhage.

29

ITGAV (integrin, V) Unknown KO: embryonic lethal placental defects or perinatal lethal with intracerebral and intestinal haemorrhages and cleft palate.

30

ITGB6 (integrin, 6) Unknown KO: inflammation of lungs and skin. 116TGFB1 CamuratiEngelmann

disease||KO: embryonic lethal with vascular defects or postnatal lethality from autoimmune disease (phenotype is modifierdependent).

40, 44

KI of RGD/E mutation: recapitulates KO phenotypes. 26

TGFB2 Unknown KO: aortic arch defects, cardiac septal defects, perinatal lethality. 40

TGFB3 Unknown KO: cleft palate, delayed lung maturation, die shortly after birth. 40

*Continued in Table 2. Animal models are murine, unless otherwise indicated, and may be heterozygous (Het) or homozygous for the targeted allele (knockout (KO) for null allele; knockin (KI) for hypomorphic/mutant allele) or transgenic. Cutis laxa maps to several genes and is characterized by pendulous inelastic skin, which is sometimes combined with aortic aneurysm and tortuous arteries; however, the specific genes associated with the cardiovascular features are not yet clear. ||CamuratiEngelmann disease is caused by an activating mutation in TGF1. BMP1, bone morphogenetic protein1; LTBP, large latent TGFbinding protein; MFS1, Marfan syndrome type 1; TGF, transforming growth factor; vSMC, vascular smooth muscle cell.

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in TGF receptor genes in familial vascular pathologies (discussed below). Furthermore, studies in mouse models showed that, in the absence of key TGF receptors, angiogenesis stalls in the yolk sac at an early stage with fatal consequences40 (FIG. 3).

Role of TGF ligands in angiogenesis.The requirement for TGF receptors for angiogenesis raises the question of which TGF ligand(s) regulates new vessel formation. Of the three mammalian TGF isoforms, TGF1 is localized to ECs during embryogenesis41, suggesting that it is the most likely of the three to be involved in angiogenesis. TGF2 and TGF3 seem to be less important than TGF1 in angiogenesis because mice that lack either of these ligands show relatively normal angiogenesis42,43. However, there may be redundancy between all three TGF ligands, and multiple or conditional knockout mice are required to investigate this possibility. Interestingly, loss of Tgfb1 in a null mouse can result in different phenotypes depending on the genetic background. For example, in C57BL/6 mice, angiogenesis is abnormal and embryos die at embryonic day (E)10.5, whereas in the NIH background the cardiovascular

system tends to develop normally but neonates die from an uncontrolled autoimmune assault40. The dramatic influence of genetic background results from three unlinked genetic modifiers44. These genes and their functions are unknown, but one possibility is that one or more may regulate TGF activation.

Invitro studies have demonstrated the importance of regulating the availability of active TGF. Low extracellular TGF1 concentrations promote the cell proliferation and migration that is associated with the active proliferation of new vessels in angiogenesis37. By contrast, high levels of extracellular TGF1 lead to cytostasis and synthesis of ECM proteins that are associated with mature or differentiating vessels. TGF family members can also act in a paracrine manner by stimulating the production of proangiogenic cytokines, such as vascular endothelial growth factor (vEGF), TGF and monocyte chemoattractant protein1 (MCP1)45,46,47. Moreover, TGFs can affect the function of other factors; for example, TGF converts the prosurvival function of vEGF into an apoptotic factor for ECs48. The resultant interplay results in a refined and cellcontextspecific response to TGF stimulation.

Table 2 | Human syndromes and animal models associated with inactivation or misexpression of TGF signalling components*gene Human syndrome animal models refs

TGF auxiliary receptorsENG (endoglin) HHT1 Het: vascular lesions similar to HHT. 40, 59

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

Elevated soluble endoglin Preeclampsia None 95

TGFBR3 (betaglycan) Unknown KO: poorly formed cardiac septa, incomplete compaction of ventricular walls. 117

TGF signalling receptorsACVRL1 (ALK1, vbg) HHT2 (rarely PAH) Het: vascular lesions similar to HHT. 60, 118

KO: embryonic lethal, reduced vSMC differentiation, dilated vessels, AVMs. 40, 64

Zebrafish KO: dilated vessels, AVMs. 51

TGFBR2 MFS2, LDS KO: embryonic lethal, vascular defects. 40, 123

TGFBR1 (ALK5) LDS KO: embryonic lethal, angiogenesis defects. 119

BMPR2 PAH KO: preangiogenesis lethality. 120, 121

Het: mild pulmonary hypertension.

Transgenic inducible BMPR2mutant allele: pulmonary hypertension. 74

Intracellular TGF signalling moleculesSMAD1 Unknown KO: preangiogenesis lethality, defects in chorionallantoic circulation. 40

SMAD2 Unknown KO: preangiogenesis lethality. 40

SMAD3 Unknown KO: viable. Impaired immunity, colon cancer. 40

SMAD4 Juvenile polyposis +/ HHT

KO: preangiogenesis lethality. 40, 122

Het: polyposis of the glandular stomach and duodenum.

SMAD5 Unknown KO: embryonic lethal, angiogenesis defects. 40

SMAD6 Unknown KO: cardiac defects, aortic ossification, elevated blood pressure. 40

MAP3K7 (TAK1) Unknown KO: embryonic lethal, reduced numbers of vSMCs, dilated vessels, AVMs. 50

*Continued from Table 1. Animal models are murine, unless otherwise indicated, and may be heterozygous (Het) or homozygous for the targeted allele (knockout (KO) for null allele; knockin (KI) for hypomorphic/mutant allele) or transgenic. ALK1, activin receptorlike kinase1; AVMs, arteriovenous malformations; BMPR2, bone morphogenetic protein receptor2; HHT, hereditary haemorrhagic telangiectasia; LDS, LoeysDietz syndrome; MFS2, Marfan syndrome type 2; PAH, pulmonary arterial hypertension; TGF, transforming growth factor; TAK1, TGF activated kinase1; TGFBR, TGF receptor; vSMC, vascular smooth muscle cell.

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Intracellular TGF signalling in angiogenesis.Because canonical TGF signalling occurs through SMAD activation, one might expect similarities in the phenotypes of the SMADknockout and TGF receptorknockout mice. In fact, SMAD1, SMAD2 and SMAD4 are required during early embryonic development before angiogenesis presumably because they are central to multiple TGF family ligands40. SMAD3 regulates mucosal immunity and does not appear to be required for cardiovascular development49. Similarities in the phenotypes of the Smad5null mouse and TGF receptor knockouts point to a proangiogenesis role for SMAD5. The most striking similarities have been observed in TGF activated kinase1 (TAK1) and TGF receptor mutant mouse phenotypes50. TAK1 (also known as MAP3K7) is activated by TGF and inflammatory cytokines and is central to several signalling pathways that control immune and stress responses. Mutations in acvrl1(also known as vbg, the zebrafish homologue of ALK1) result in the formation of dilated cranial vessels that contain increased numbers of ECs51, whereas morpholino knockdown experiments indicate a synergistic interaction between the zebrafish homologues of TAK1 and ALK1 (REF. 50). This invivo data from both mouse and zebrafish models suggest that TGF signalling through TAK1 is important in the regulation of vascular development.

TGF and blood-vessel myogenesis.One of the best understood roles of TGF signalling in angiogenesis is that of promoting vessel muscularization. vSMCs (arteries and veins) or pericytes (capillaries) cover new vessels and are required for both vessel stability and function. Muscularization is achieved when ECs promote paracrine TGF1 signalling to neighbouring mesenchymal cells to promote SMC or pericyte differentiation. Establishment of cellcell contacts, at the level of gapjunction communication between endothelial and mesenchymal cells, is required for TGF activation and SMC differentiation52. TGF signalling between ECs and vSMCs is also dependent on endoglin53. Ultimately, TGF activation stimulates the differentiation of mesenchymal cells

to SMCs through the combinatorial activation of several SMC genes such as SMactin (ACTA2) and transgelin (TAGLN; also known as SM22)54. In line with their vascular myogenesis role, specific loss of the TGF receptors, TGFBR2 or ALK5, in vSMCs results in vascular defects and embryonic lethality55.

Importantly, TGF signalling in vascular development is not confined to SMCs. Recent analysis of mice with ECspecific loss of either Tgfbr2 or Tgfbr1(which encodes ALK5) has clearly shown that both TGF receptors also have essential angiogenesisrelated roles in ECs56,53. The role of TGF receptors in ECs may be crucial in development earlier than in vSMCs, as mutants with ECspecific loss of Tgfbr2 die from vascular defects 2 days sooner (at E10.5) than those with vSMCspecific loss (E12.5)55. However, a recent report suggests that Tgfbr1 is not expressed in ECs57. This discrepancy may be explained by the limitations of knockin lacZ reporters for monitoring endogenous protein expression, and further work is required to determine the relative contributions of TGF signalling receptors to EC function invivo. Nonetheless, the central importance of properly regulated TGF signalling in the vasculature is clearly demonstrated by the growing number of familial cardiovascular disorders that are associated with mutations affecting TGF family signalling.

Vascular disease and TGF signallingOver recent years, numerous familial vascular diseases have been mapped to receptors of the TGF family and, in many cases, further understanding has been reached through studies of mouse models58. A common theme is that TGF signalling is important in bloodvessel morphogenesis and stability. Recent data also provide insights into the role of TGF signalling in hypertension and preeclampsia. Furthermore, recognition of the importance of TGF signalling in angiogenesis has opened up new possibilities for targeting cancer by modulating this pathway in the tumour vasculature.

Hereditary haemorrhagic telangiectasia (HHT). The vascular disorder hereditary haemorrhagic telangiectasia type 1 (HHT1) results from mutations in ENG that lead tohaploinsufficiency of endoglin59. The closely related disorder HHT type 2 (HHT2) is caused by loss of function or dominant negative mutations in ACVRL1 (which encodes ALK1)60,61. Endoglin is expressed in ECs of all developing blood vessels, and ALK1 is primarily expressed in arterial ECs57,62, which points to defects in ECs as the primary cause of HHT.

The main clinical features of HHT are bleeding from small vascular lesions (telangiectases) in the mucocutaneous tissues, and the presence of arteriovenous malformations (AvMs) in the lung, liver and/or cerebral vasculature. The typical onset of symptoms (usually major bleeding from nasal telangiectases) is during puberty, and AvMs may be present that become larger and symptomatic as patients age. Several possible mechanisms have been proposed to explain the development of AvMs, including loss of vSMCs, dysregulated vascular tone or apoptosis of capillary ECs (FIG. 4).

Box 1 | Vasculogenesis and angiogenesis

Vasculogenesis is the earliest step in the development of new blood vessels and involves the differentiation of angioblasts into endothelial cells (ECs) and their assembly into a primary vascular plexus. Angiogenesis is the process during which blood vessels develop from existing capillaries by sprouting, pruning and/or splitting (intussusception). These processes are driven by a complex interaction of growth factors (for example, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and transforming growth factor (TGF)) and their receptors. Live imaging of angiogenesis in zebrafish mutants suggests that the layout of the primitive vascular network is genetically determined. The directionality of new vessel growth is driven by leading endothelial tip cells in response to guidance molecules, whereas the lagging ECs form lumens by intercellular fusion of endothelial vacuoles112. During maturation, pericytes or vascular smooth muscle cells (vSMCs) are recruited to capillaries and larger vessels, respectively. Plateletderived growth factor (PDGF) signalling is important for the initial recruitment of mesenchymal cells that differentiate to vSMCs in response to TGF signalling. As the animal grows, the vascular network continues to remodel by pruning and branching in response to a combination of growth factors, hypoxic triggers and blood flow to form a complex treelike structure of arteries, veins and capillaries.

MorpholinoChemically synthesized oligonucleotide analogues used to knock down gene expression by specifically binding to target transcripts to inhibit RNA splicing or translation.

Vessel muscularization The development of smooth muscle cells around a vessel to support and stabilize it.

PericyteA smooth muscle-like cell that is intimately associated with endothelial cells of small blood vessels.

Mesenchymal cellA member of a heterogeneous multipotent cell population that arises mainly from embryonic mesoderm.

Hypertension Elevated blood pressure.

Arteriovenous malformation(AVM). Abnormal communication between an artery and a vein producing dilated vessels.

Intussusceptive angiogenesis The process of blood vessel growth by splitting the wall of an existing blood vessel extends into the lumen to split a single vessel in two.

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Angioblast cells

Primitive vascular plexus

Intussusceptiveangiogenesis

Vasculogenesis

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Tip cellVacuole fusion and lumen

formation

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Progression blocked in yolk sacin the absence ofTGF1, TGFBR2, endoglin,ALK1, ALK5 or SMAD5

Mesenchymalcell

Gap-junction formation,TGF1 activation,SMC differentiation andmutual inhibition ofproliferation

Endothelial cell

PDGF

PDGF

Sprouting angiogenesis The process by which endothelial cells migrate and proliferate into the surrounding matrix to form new vessel branches in response to an angiogenic stimulus.

To investigate abnormalities in ECs derived from patients with HHT, cultured circulating endothelial progenitor cells were used to generate blood outgrowth ECs. These had a disorganized actin cytoskeleton that may contribute to vascular fragility in HHT63. Further investigations in mouse models of HHT suggest that there are reduced levels of TGF signalling from ECs to vSMCs or pericytes, which results in decreased SMC differentiation.

This defect generates weaker vessels that are prone to bleeding, a main characteristic of HHT53. In addition, development of AvMs early in embryogenesis may be due to loss of arteriovenous identity. Arterialspecific and venousspecific signalling molecules such as ephrin B2 (EFNB2) and EPHB4, respectively, are thought to ensure that these vessel types remain distinct and separate during development. AvMs frequently develop in Acvrl1null mouse embryos, although to a lesser extent in Engnull mice, and may be due to downregulation of EFNB2 in the Acvrl1/ mice64,65.

There may also be systemic defects in TGF signalling in HHT1, as reduced endoglin levels have been shown to cause decreased levels of plasma TGF in both mouse models and patients with HHT1 (REF. 66). However, this remains to be clarified because raised TGF1 levels have also been reported67. The exact molecular changes leading to HHT are not yet clear. New insights are anticipated following the recent identification of BMP9 as a ligand for both endoglin and ALK1 (REF. 68), and newly derived conditional knockout Eng and Acvrl1 mouse models69 (S.P. Oh, unpublished observations).

Pulmonary arterial hypertension (PAH).The devastating lung disease pulmonary arterial hypertension (PAH) is characterized by pathological changes that include hypertrophy (enlargement) of the medial smooth muscle layers and intimal thickening in the precapillary arterioles (FIG. 4). This leads to increased pulmonary artery resistance that can ultimately result in rightsided heart failure. In addition, plexiform lesions that comprise multiple capillary channels and proliferating ECs may develop near occluded regions. The average age of onset is in the third decade of life in women and the fourth decade in men, but can occur at any age.

Familial PAH (fPAH) is associated with lossoffunction mutations in the BMP receptor2 gene (BMPR2). Reduced levels of this receptor in pulmonary artery SMCs and ECs in patients with PAH lead to reduced SMAD1 activation and/or increased activation of the p38 mitogenactivated protein kinase (MAPK) that may contribute to vSMC hyperproliferation70,71. It appears that BMPR2deficient cells may respond abnormally to BMP ligands by redirecting signalling through the type II activin responsive receptor ActRIIA (in association with either ALK2 or ALK3)72. In addition, the long cytoplasmic tail of the BMPR2 protein, which is truncated in many patients with fPAH, is required for proper regulation of the cytoskeleton, and misregulation may contribute to the aetiology of PAH73. Mouse models of fPAH with inactivated Bmpr2 have yielded a lack of PAH symptoms in normoxic conditions. However, postnatal expression of a dominantnegative BMPR2mutation in vSMCs leads to pulmonary hypertension and modest muscularization of distal arteries. These data suggest that aberrant BMPR2 signalling in vSMCs is sufficient to produce the pulmonary hypertensive phenotype and provides a useful animal model for further investigation74.

The link between dysregulated BMP signalling and progression of this devastating disease remains to be determined, but an important clue may come from the

Figure 3 | TgF signalling in vasculogenesis and angiogenesis. Vasculogenesis and two types of angiogenesis are shown: intussusceptive and sprouting angiogenesis (BOX 1). Vasculogenesis involves the differentiation of endothelial cells (ECs) from precursor angioblast cells to form a primitive plexus of capillaries, which remodel and grow by angiogenesis. Intussusceptive angiogenesis involves the splitting and growing of vessels in situ in a metabolically efficient manner, and is found, for example, in the developing yolk sac and lung. Vessel splitting occurs by the formation of translumen pillars (arrowheads) but the molecular mechanisms are not well understood. In sprouting angiogenesis, endothelial cells proliferate behind the tip cell of a growing branch in response to cytokines such as vascular endothelial growth factor (VEGF) and lumens can form by vacuole fusion. Both forms of angiogenesis require the recruitment of smooth muscle cells (SMCs) to stabilize the nascent vessels. Neighbouring mesenchymal cells migrate towards the neovessel in response to plateletderived growth factor (PDGF) and then differentiate into vascular SMCs (vSMCs) in response to transforming growth factor (TGF) signalling. The continued tight intercellular association between ECs and vSMCs promotes sustained TGF activation and mutual inhibition of cell proliferation. When TGF signalling is defective, for example in TGFreceptor knockout mice, smooth muscle differentiation fails to proceed and angiogenesis stalls. ALK, activin receptorlike kinase; TGFBR2, TGF type II receptor.

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Terminal bronchiole and alveoli

Pre-capillary pulmonary arteriole

Pulmonary venule

Capillaries

ArterioleVenule

Site of partialocclusion

Endothelial cellSmooth muscle cellStromal cell

a b

c

d e

fact that patients with HHT who have ACVRL1mutations are predisposed to the development of PAH75. As ALK1 is expressed in pulmonary ECs, this finding suggests that aberrations in TGF family signalling in the endothelium can also be the primary defect in PAH. Recent identification of BMP9 as an ALK1 ligand suggests that reduced ALK1 levels may lead to altered BMP9 signalling68, although any link to PAH remains to be determined.

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

thoracic aneurysm disorder (AAT3) and LoeysDietz syndrome (LDS)7678. MFS is primarily associated with mutations in FBN1(fibrillin1), whereas LDS is associated with mutations in TGFBR1or TGFBR2. Aortic aneurysms are insidious malformations with a variable age of onset which, if left untreated, carry the risk of aortic dissection, rupture and sudden death. The vulnerability of the aortic root in these disorders may relate to the two different developmental origins (neural crest and secondary heart field) of vSMCs at the region of the aortic valve, which have different responses to TGF signalling79. This results in two closely abutting rings of different vSMC populations and it is possible that the junction between them represents a susceptible site for aortic dissection80 (FIG. 5).

Anastomosis A naturally occurring arteriovenous connection that may be dynamically regulated and is particularly frequent in thermoregulatory vascular beds.

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

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Tunica intimaTunica mediaTunica adventitia

a b c

d

LTBP

LAPTGF1

Microfibrils Few microfibrils orsecondary proteolysis

e

MFS

Normal elastin fibres Fragmented elastin fibres

PT

It was initially thought that aortic aneurysms in MFS were due to structural defects in the aorta resulting from a failure to stabilize elasticfibre structure when fibrillin1 was limiting. The resultant elastin fragmentation in the aortic wall would make the aorta susceptible to injury from haemodynamic forces. Recently, it has become clear that an important function of fibrillin1 is to control TGF bioavailability (FIG. 2). Reduced fibrillin1 may result in incorrect LLC sequestration and excessive activation of TGF signalling, a major contributory factor in the vascular pathology of MFS81,82. Mice deficient in Fbn1,but not mice overexpressing mutant Fbn1, develop MFS phenotypic manifestations83,84. This suggests that a deficiency in microfibrils, as opposed to a transdominant effect of mutant fibrillin1, is a major determinant of MFS. It has also been proposed that the progressive proteolytic damage that is characteristic of MFS may lead to the formation of fibrillin fragments that further increase the bioavailability of TGF21. In the case of LDS, when TGFBR2 levels are reduced owing to pathologic mutation, compensatory mechanisms may lead to an overshoot of TGF signalling in the aortic media. For example, increased TGF expression and increased fibrosis have been observed in transgenic mice that overexpress a dominant negative form of Tgfbr2(REF. 85). Similar, but as yet unknown, mechanisms may explain the upregulation of TGFSMAD signalling and fibrosis that is seen in the aortae of patients with LDS, who carry heterozygous inactivating mutations in either TGFBR2 or TGFBR1(REF. 77).

whatever the cause of enhanced TGF signalling, work using animal models of MFS clearly showed that aortic aneurysms were prevented by administration of neutralizing antibodies to TGF, confirming the contribution of excess TGF signalling to the vascular pathology82. In the absence of approved antiTGF clinical therapies, attention turned to angiotensin, a potent vasoconstrictor that interacts with angiotensin receptors on blood vessels and induces SMAD2/3 phosphorylation86. This change of focus was rewarded when the angiotensin type I receptor antagonist, losartan (Cozaar; Merck), showed a strikingly similar protective effect in the development of aortic aneurysms in animal models of MFS82. However, the mechanism by which losartan protects against aortic aneurysm and reduces SMAD2 activation is not fully understood. One possibility is the involvement of THBS1, an important target downstream of angiotensin signalling87 that can activate latent TGF. whatever the mechanism, losartan has rapidly moved to Phase III clinical trials in patients with MFS (see the US National Marfan Foundation web site).

Taken together, the evidence indicates that misregulation of TGF signalling owing to defects in extracellular proteins is centrally important to the development of aortic aneurysms, and there is now a realistic hope for patient therapies. This view has now replaced the previous idea that aortic aneurysms were simply due to a structural deficiency of the elastin matrix in the aorta. It is notable in this context that mice lacking the extracellular protein fibulin5 have fragmented elastin and tortuous elongated aortae, but do not develop aortic

Figure 5 | TgF-associated defects in marfan and loeysDietz syndromes. a | An aorta with two seams of vascular smooth muscle cells (vSMCs) at the aortic root and pulmonary trunk (PT) that are derived from different developmental origins cardiac neuralcrestderived SMCs (dark pink), secondary heartfieldderived SMCs (purple) and secondary heartfieldderived myocardial muscle (blue). These areas may be sites that are vulnerable to dissection. Heart and pulmonary arteries are shown as dotted lines. b | An aortic root aneurysm that is typical of Marfan syndrome (MFS) and LoeysDietz syndrome. c | Ascending aortic aneurysm. d | Descending aortic aneurysm. e | A crosssection of aorta shows the three layers (tunics) of the vessel: tunica adventitia (which contains collagen, fibroblasts, nerves and capillaries), tunica media (which contains SMCs, elastic fibres/microfibrils, collagen and proteoglycan) and tunica intima (which contains endothelial cells and basal lamina). Fragmented elastin fibres in the aorta are seen in MFS and are associated with release of the large latent transforming growth factor (TGF) complex and active TGF1. LAP, latencyassociated peptide; LTBP, large latent TGF binding protein.

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Placental syncytiotrophoblast cellA multinucleated cell found at the boundary of the fetal and maternal layers of the placenta.

aneurysms or dissections88,89. It may be that the aortae of these mice have normal TGF activity and, hence, do not progress to aneurismal changes. By contrast, mutant mice that have lost the related Efemp2 gene (which encodes fibulin4) show a striking aortic rupture phenotype and perinatal lethality90. This defect precluded analysis of this gene in mature vasculature, but mice that are homozygous for a hypomorphic allele with reduced fibulin4 expression survive to adulthood with a milder phenotype. These mice have aortic dilatation, aneurysms and aortic valve dysfunction associated with abnormal TGF signalling in the aorta91. These different mouse models offer the opportunity to unravel the complex interaction between aortic integrity and ECM regulation of TGF activity.

TGF and hypertension.A link between increased levels of circulating TGF and hypertension has been known for some time92, but new findings are throwing light on the nature of this interaction. Emilin1, an ECM protein that is associated with the microfibrils of the elastic matrix in the aortic media, regulates TGF availability and arterial diameter93. By interacting with unprocessed TGF, emilin1 protects it from proteolytic processing by the endoprotease furin convertase (FIG. 2). Loss of emilin1 therefore results in the increased conversion of proTGF to the mature form and a subsequent increase in TGF signalling. This leads to a reduction in the arterial lumen diameter with a resultant increase in vascular resistance and hypertension93. A possible mechanism is that excessive levels of active TGF causes premature cytostasis of the vSMCs, and subsequently restricts vessel size. Alternatively, reduced arterial diameter may be a secondary consequence of vascular remodelling in these mice94. This phenotype can be rescued genetically by crossing in a single Tgfb1null allele to reduce TGF levels.

A related disease that involves raised blood pressure is preeclampsia, a serious disease of late pregnancy that threatens the health of both mother and baby. Patients with preeclampsia have increased circulating levels of a soluble form of vEGF receptor1 (vEGFR1; also known as sFLT1), which is thought to function as a sink for vEGF ligands and reduce the access of vEGF ligands to vEGFRs on ECs. Recently, elevated levels of soluble endoglin were also found to cooperate with soluble vEGFR1 in the pathogenesis of preeclampsia95. Soluble endoglin is probably formed by proteolytic cleavage of fulllength endoglin, and is possibly derived from placental syncytiotrophoblast cells. This soluble form may function in an analogous way to soluble vEGFR1 by binding circulating TGF, which therefore inhibits TGFBR2 and ALK5dependent signalling95. This is thought to reduce the level of nitric oxide synthase3 (NOS3, eNOS) activation and attenuate vasorelaxation responses, leading to preeclampsia.

The mechanisms that regulate the production of soluble endoglin and a detailed understanding of its molecular role in patients with preeclampsia remain to be determined. A recent report showed that haem oxygenase1, an antiinflammatory enzyme required

for successful pregnancy, inhibits the release of soluble vEGFR1 and soluble endoglin, a role that might be used in the future treatment of preeclampsia96. However, circulating levels of soluble endoglin may already be a useful diagnostic marker to prioritize patients for treatment before the onset of symptoms97.

Once again, the role of TGF in the regulation of hypertension appears to be contradictory. Increased levels of activated TGF in emilin1-null mice result in hypertension, whereas reduced TGF signalling in ECs may contribute to increased blood pressure in preeclampsia93,95. However, the underlying mechanisms are different. There is no detectable change in vasoregulatory responses in arteries in emilin1-null mice instead, there is a reduction in vessel size. By contrast, in preeclampsia there is a transient defect in vasoregulatory mechanisms, but the blood pressure returns to normal levels once the fetus is born. In addition, there may be other ligands involved in the preeclampsia model, as endoglin binds other ligands of the TGF family, including BMP9 (REF. 68).

TGF and tumour angiogenesis.The importance of understanding the regulation of angiogenesis by TGF family members is crucial because many of these processes are recapitulated in a disorganized fashion in neoplastic disease. Several smallmolecule TGF receptor kinase inhibitors have been developed for cancer treatment98. However, there may also be unwanted effects of TGF receptor inhibition, such as the possibility that the rate of progression of some cancers will increase when the tumoursuppressive effects of TGF are inhibited99. In light of this possibility, it is promising that there appears to be no predisposition to cancer in patients with LDS who have reduced TGF receptor levels100. Rather, the paradoxical enhancement of TGF signalling in aortic aneurysms of patients with LDS raises the possibility that inhibiting TGF receptors may lead to a signalling imbalance.

A recent study took advantage of the effects of a TGF receptor inhibitor on the neovasculature that mirrored the vascular defects seen in TGFreceptor knockout mice. Shortterm treatment with an ALK5 inhibitor in mice that had intractable solid tumours led to reduced vSMC coverage in neovessels, making them leaky and promoting the accumulation of anticancer drugs in the tumour tissue101. Parallel experiments targeting endoglin, which is strongly upregulated in angiogenic tumour ECs, suggests that endoglin may also represent a useful antiangiogenesis target for cancer treatment102,103,104. Equally important is the development of proangiogenic therapies used to treat ischaemic disease; manipulating TGF signalling may represent a valuable therapeutic approach and preclinical data have shown that endoglin promotes tissue repair by proangiogenic circulating blood cells105.

Conclusions and future perspectivesIn the pursuit of molecular mechanisms that underlie the vascular pathology of MFS and related diseases, an important additional role for elastic ECM components in controlling growth factor signalling was discovered5.

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Truncus arteriosusA single vessel that forms early in development and then septates to form the aorta and pulmonary trunk. A persistent truncus arteriosus is one that has failed to septate, compromising the separation of pulmonary and systemic circulations.

Aortopulmonary septation The process whereby the pulmonary trunk and aorta separate during development.

Besides being structural scaffolds that provide tissue integrity, elastic microfibril components have an instructive role in regulating TGF processing and bioavailability. Latent TGF complexes, bound via LTBPs to fibrillin1, are sequestered and provide latent embedded signals that can be rapidly released by proteases in response to tissue perturbations. This allows for rapid and localized changes in TGF activity without the need for denovo protein synthesis106. Dysregulation of TGF family signalling in MFS may provide an explanation for clinical variability, as disease pathogenesis may differ in a genotype and contextdependent manner5. In addition, dysregulated TGF signalling may underlie the pathology of other vascular disorders. For example, mutations in the facilitative glucose transporter GLUT10 were found to cause arterial tortuosity syndrome (ATS), and were associated with upregulation of TGFSMAD signalling in the arterial wall107.

The instructive role for microfibrils has been extended to other TGF family members such as BMP7, which binds via its prodomain directly to fibrillin1, potentially targeting it to the ECM108. Mutations in fibrillin2 (FBN2) are associated with disorganized microfibrils and the Marfanlike disorder, congenital contractural arachnodactyly (TABLE 1), and Fbn2 null mice display bilateral syndactyly of forelimbs and hindlimbs109. Interestingly, mice that are double heterozygous for null Fbn2 and Bmp7 alleles, which alone are phenotypically silent, have impaired digit formation, suggesting a functional interaction between fibrillin2 and BMP7 (REF. 109). The extracellular regulation of BMP signalling by ECM components has been largely overlooked and needs further investigation.

It is possible that fibrillins have dual and opposing roles in regulating TGF activity. They may act positively by concentrating ligand at sites of function, and negatively by sequestering the LLC and inhibiting TGF activation. It is also interesting to note that mice lacking Ltbp1 show persistent truncus arteriosus, a phenotype that is also seen after neuralcrestspecific ablation of TGF signalling110 (TABLE 1). This may reflect a failure to provide a sequestered source of TGF in the absence of LTBP1 during aortopulmonary septation.

Recognizing the importance of extracellular regulation of TGF activity in aortic muscle cells in MFS and LDS has opened up exciting possibilities for treatments of further muscle disorders. Skeletal muscle weakness is an additional feature of MFS that results from increased TGF signalling, which inhibits regeneration of muscle cells and stimulates fibrosis. In parallel to the beneficial effects of losartan on aortic function in a mouse model of MFS, losartan also improves muscle morphology and enhances satellitecell activation and muscle regeneration. This led to the consideration that these benefits might ameliorate another progressive muscle weakness disorder, Duchennes muscular dystrophy, which is caused by familial mutations in dystrophin (DMD). Losartan treatment lowered the abnormally high intramuscular TGF levels, improved muscle histopathology and reduced fibrosis in a Dmd-deficient mouse model of this disease, raising hopes that this treatment may also benefit patients with Duchennes muscular dystrophy111.

The complexities of TGF signalling are challenging to scientists and clinicians alike. However, it is clear that TGF components represent valuable therapeutic targets, even though the changes in blood vessel architecture and blood pressure that are associated with dysregulated TGF signalling indicate the necessity for careful monitoring of these parameters in patients. The effectiveness of losartan for treating dysregulated TGF signalling means that the interaction between angiotensin and TGF signalling pathways is an important area of investigation. There is also an urgent need to improve our understanding of the extracellular regulation of TGF ligand activation and the context dependence of cellular responses. Furthermore, the phenotypes of mouse models with mutations in genes that are associated with extracellular regulation of TGF signalling (TABLE 1) raise the possibility that further human cardiovascular syndromes may eventually be linked to some of these genes. Advances in all of these areas will inform current treatment strategies and open up possibilities for developing new therapies for a range of vascular pathologies.

1. Massague, J. & Gomis, R. R. The logic of TGF signaling. FEBS Lett. 580, 28112820 (2006).

2. Feng, X. H. & Derynck, R. Specificity and versatility in TGF- signaling through Smads. Annu. Rev. Cell Dev. Biol. 21, 659693 (2005).

3. Blobe, G. C., Schiemann, W. P. & Lodish, H. F. Role of transforming growth factor in human disease. N. Engl. J. Med. 342, 13501358 (2000).

4. Annes, J. P., Munger, J. S. & Rifkin, D. B. Making sense of latent TGF activation. J. Cell Sci. 116, 217224 (2003).

5. Robinson, P. N. et al. The molecular genetics of Marfan syndrome and related disorders. J. Med. Genet. 43, 769787 (2006).

6. Grainger, D. J. TGF- and atherosclerosis in man. Cardiovasc. Res. 74, 213222 (2007).

7. Ruiz-Ortega, M., Rodriguez-Vita, J., Sanchez-Lopez, E., Carvajal, G. & Egido, J. TGF- signaling in vascular fibrosis. Cardiovasc. Res. 74, 196206 (2007).

8. Bierie, B. & Moses, H. L. TGF- and cancer. Cytokine Growth Factor Rev. 17, 2940 (2006).

9. Dubois, C. M., Laprise, M. H., Blanchette, F., Gentry, L. E. & Leduc, R. Processing of transforming growth factor 1 precursor by human furin convertase. J. Biol. Chem. 270, 1061810624 (1995).

10. Beck, S. et al. Extraembryonic proteases regulate Nodal signalling during gastrulation. Nature Cell Biol. 4, 981985 (2002).

11. Kanzaki, T. et al. TGF-1 binding protein: a component of the large latent complex of TGF-1 with multiple repeat sequences. Cell 61, 10511061 (1990).

12. Saharinen, J., Taipale, J. & Keski-Oja, J. Association of the small latent transforming growth factor- with an eight cysteine repeat of its binding protein LTBP-1. EMBO J. 15, 245253 (1996).

13. Dallas, S. L. et al. Characterization and autoregulation of latent transforming growth factor (TGF ) complexes in osteoblast-like cell lines. Production of a latent complex lacking the latent TGF -binding protein. J. Biol. Chem. 269, 68156821 (1994).

14. Saharinen, J. & Keski-Oja, J. Specific sequence motif of 8-Cys repeats of TGF- binding proteins, LTBPs, creates a hydrophobic interaction surface for binding of small latent TGF-. Mol. Biol. Cell 11, 26912704 (2000).

15. Isogai, Z. et al. Latent transforming growth factor -binding protein 1 interacts with fibrillin and is a microfibril-associated protein. J. Biol. Chem. 278, 27502757 (2003).

16. Nunes, I., Gleizes, P. E., Metz, C. N. & Rifkin, D. B. Latent transforming growth factor- binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-. J. Cell Biol. 136, 11511163 (1997).

17. Flaumenhaft, R. et al. Role of the latent TGF- binding protein in the activation of latent TGF- by co-cultures of endothelial and smooth muscle cells. J. Cell Biol. 120, 9951002 (1993).

18. Rifkin, D. B. Latent transforming growth factor- (TGF-) binding proteins: orchestrators of TGF- availability. J. Biol. Chem. 280, 74097412 (2005).

19. Dabovic, B. et al. Bone abnormalities in latent TGF-[] binding protein (Ltbp)-3-null mice indicate a role for Ltbp-3 in modulating TGF- bioavailability. J. Cell Biol. 156, 227232 (2002).

20. Sterner-Kock, A. et al. Disruption of the gene encoding the latent transforming growth factor- binding protein 4 (LTBP-4) causes abnormal lung development, cardiomyopathy, and colorectal cancer. Genes Dev. 16, 22642273 (2002).

21. Chaudhry, S. S. et al. Fibrillin-1 regulates the bioavailability of TGF1. J. Cell Biol. 176, 355367 (2007).

R E V I E W S


An internal proteolytic fragment of fibrillin1 is shown to regulate the bioavailability of TGF by inducing the release of the large latent complex bound to microfibrils.

22. Ge, G. & Greenspan, D. S. BMP1 controls TGF1 activation via cleavage of latent TGF-binding protein. J. Cell Biol. 175, 111120 (2006).Bone morphogenetic protein1 (BMP1)like metalloprotease is shown to cleave large latent TGF binding protein 1 (LTBP1) at two specific sites, thereby liberating the large latent TGF complex (LLC) from the extracellular matrix (ECM).

23. Pappano, W. N., Steiglitz, B. M., Scott, I. C., Keene, D. R. & Greenspan, D. S. Use of Bmp1/Tll1 doubly homozygous null mice and proteomics to identify and validate in vivo substrates of bone morphogenetic protein 1/tolloid-like metalloproteinases. Mol. Cell Biol. 23, 44284438 (2003).

24. Crawford, S. E. et al. Thrombospondin-1 is a major activator of TGF-1 in vivo. Cell 93, 11591170 (1998).

25. Sheppard, D. Integrin-mediated activation of latent transforming growth factor . Cancer Metastasis Rev. 24, 395402 (2005).

26. Yang, Z. et al. Absence of integrin-mediated TGF1 activation in vivo recapitulates the phenotype of TGF1-null mice. J. Cell Biol. 176, 787793 (2007).

27. Mu, D. et al. The integrin v8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-1. J. Cell Biol. 157, 493507 (2002).

28. Fontana, L. et al. Fibronectin is required for integrin alphav6-mediated activation of latent TGF- complexes containing LTBP-1. FASEB J. 19, 17981808 (2005).

29. Zhu, J. et al. 8 integrins are required for vascular morphogenesis in mouse embryos. Development 129, 28912903 (2002).

30. Bader, B. L., Rayburn, H., Crowley, D. & Hynes, R. O. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all alpha v integrins. Cell 95, 507519 (1998).

31. Goumans, M. J. et al. Balancing the activation state of the endothelium via two distinct TGF- type I receptors. EMBO J. 21, 17431753 (2002).

32. Massague, J., Cheifetz, S., Boyd, F. T. & Andres, J. L. TGF- receptors and TGF- binding proteoglycans: recent progress in identifying their functional properties. Ann. N. Y. Acad. Sci. 593, 5972 (1990).

33. Lebrin, F., Deckers, M., Bertolino, P. & ten Dijke, P. TGF- receptor function in the endothelium. Cardiovasc. Res. 65, 599608 (2005).

34. Goumans, M. J. et al. Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGF /ALK5 signaling. Mol. Cell 12, 817828 (2003).

35. Sankar, S., Mahooti-Brooks, N., Centrella, M., McCarthy, T. L. & Madri, J. A. Expression of transforming growth factor type III receptor in vascular endothelial cells increases their responsiveness to transforming growth factor 2. J. Biol. Chem. 270, 1356713572 (1995).

36. Lopez-Casillas, F., Payne, H. M., Andres, J. L. & Massague, J. Betaglycan can act as a dual modulator of TGF- access to signaling receptors: mapping of ligand binding and GAG attachment sites. J. Cell Biol. 124, 557568 (1994).

37. Lebrin, F. et al. Endoglin promotes endothelial cell proliferation and TGF-/ALK1 signal transduction. EMBO J. 23, 40184028 (2004).

38. Blanco, F. J. et al. Interaction and functional interplay between endoglin and ALK-1, two components of the endothelial transforming growth factor- receptor complex. J. Cell Physiol. 204, 574584 (2005).

39. Adams, R. H. & Alitalo, K. Molecular regulation of angiogenesis and lymphangiogenesis. Nature Rev. Mol. Cell Biol. 8, 464478 (2007).Excellent review on the molecular mechanisms that underlie the formation and function of blood and lymphatic vessels.

40. Goumans, M. J. & Mummery, C. Functional analysis of the TGF receptor/Smad pathway through gene ablation in mice. Int. J. Dev. Biol. 44, 253265 2000).

41. Akhurst, R. J., Lehnert, S. A., Faissner, A. & Duffie, E. TGF in murine morphogenetic processes: the early embryo and cardiogenesis. Development 108, 645656 (1990).

42. Bartram, U. et al. Double-outlet right ventricle and overriding tricuspid valve reflect disturbances of looping, myocardialization, endocardial cushion differentiation, and apoptosis in TGF-2-knockout mice. Circulation 103, 27452752 (2001).

43. Kaartinen, V. et al. Abnormal lung development and cleft palate in mice lacking TGF-3 indicates defects of epithelial-mesenchymal interaction. Nature Genet. 11, 415421 (1995).

44. Tang, Y. et al. Epistatic interactions between modifier genes confer strain-specific redundancy for Tgfb1 in developmental angiogenesis. Genomics 85, 6070 (2005).

45. Deckers, M. M. et al. Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A. Endocrinology 143, 15451553 (2002).

46. Vinals, F. & Pouyssegur, J. Transforming growth factor 1 (TGF-1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF- signaling. Mol. Cell Biol. 21, 72187230 (2001).

47. Ma, J., Wang, Q., Fei, T., Han, J. D. & Chen, Y. G. MCP-1 mediates TGF- -induced angiogenesis by stimulating vascular smooth muscle cell migration. Blood 109, 987994 (2007).

48. Ferrari, G. et al. VEGF, a prosurvival factor, acts in concert with TGF-1 to induce endothelial cell apoptosis. Proc. Natl Acad. Sci. USA 103, 1726017265 (2006).

49. Yang, X. et al. Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-. EMBO J. 18, 12801291 (1999).

50. Jadrich, J. L., OConnor, M. B. & Coucouvanis, E. The TGF activated kinase TAK1 regulates vascular development in vivo. Development 133, 15291541 (2006).

51. Roman, B. L. et al. Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels. Development 129, 30093019 (2002).

52. Hirschi, K. K., Burt, J. M., Hirschi, K. D. & Dai, C. Gap junction communication mediates transforming growth factor- activation and endothelial-induced mural cell differentiation. Circ. Res. 93, 429437 (2003).

53. Carvalho, R. L. et al. Defective paracrine signalling by TGF in yolk sac vasculature of endoglin mutant mice: a paradigm for hereditary haemorrhagic telangiectasia. Development 131, 62376247 (2004).

54. Kumar, M. S. & Owens, G. K. Combinatorial control of smooth muscle-specific gene expression. Arterioscler. Thromb. Vasc. Biol. 23, 737747 (2003).

55. Carvalho, R. et al. Compensatory mechanisms activated during vasculogenesis in mice by TGF-receptor deletion. J. Cell Sci. (in the press).

56. Jiao, K. et al. Tgf signaling is required for atrioventricular cushion mesenchyme remodeling during in vivo cardiac development. Development 133, 45854593 (2006).

57. Seki, T., Hong, K. H. & Oh, S. P. Nonoverlapping expression patterns of ALK1 and ALK5 reveal distinct roles of each receptor in vascular development. Lab. Invest. 86, 116129 (2006).

58. Harradine, K. A. & Akhurst, R. J. Mutations of TGF signaling molecules in human disease. Ann. Med. 38, 403414 (2006).

59. McAllister, K. A. et al. Endoglin, a TGF- binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nature Genet. 8, 345351 (1994).

60. Johnson, D. W. et al. Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. Nature Genet. 13, 189195 (1996).

61. Gu, Y. et al. Functional analysis of mutations in the kinase domain of the TGF- receptor ALK1 reveals different mechanisms for induction of hereditary hemorrhagic telangiectasia. Blood 107, 19511954 (2006).

62. Jonker, L. & Arthur, H. M. Endoglin expression in early development is associated with vasculogenesis and angiogenesis. Mech. Dev. 110, 193196 (2002).

63. Fernandez, L. A. et al. Blood outgrowth endothelial cells from hereditary haemorrhagic telangiectasia patients reveal abnormalities compatible with vascular lesions. Cardiovasc. Res. 68, 235248 (2005).

64. Urness, L. D., Sorensen, L. K. & Li, D. Y. Arteriovenous malformations in mice lacking activin receptor-like kinase-1. Nature Genet. 26, 328331 (2000).

65. Sorensen, L. K., Brooke, B. S., Li, D. Y. & Urness, L. D. Loss of distinct arterial and venous boundaries in mice lacking endoglin, a vascular-specific TGF coreceptor. Dev. Biol. 261, 235250 (2003).

66. Letarte, M. et al. Reduced endothelial secretion and plasma levels of transforming growth factor-1 in patients with hereditary hemorrhagic telangiectasia type 1. Cardiovasc. Res. 68, 155164 (2005).

67. Sadick, H. et al. Patients with hereditary hemorrhagic telangiectasia have increased plasma levels of vascular endothelial growth factor and transforming growth factor-1 as well as high ALK1 tissue expression. Haematologica 90, 818828 (2005).

68. Scharpfenecker, M. et al. BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis. J. Cell Sci. 120, 964972 (2007).

69. Allinson, K., Carvalho, R. L. C., van den Brink, S., Mummery, C. L. & Arthur, H. M. Generation of a floxed allele of the mouse endoglin gene. Genesis 45, 391395 (2007).

70. Morrell, N. W. et al. Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factor- (1) and bone morphogenetic proteins. Circulation 104, 790795 (2001).

71. Yang, X. et al. Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension. Circ. Res. 96, 10531063 (2005).

72. Yu, P. B., Beppu, H., Kawai, N., Li, E. & Bloch, K. D. 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, 2444324450 (2005).

73. Foletta, V. C. et al. Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. J. Cell Biol. 162, 10891098 (2003).

74. West, J. et al. Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle. Circ. Res. 94, 11091114 (2004).

75. Harrison, R. E. et al. Molecular and functional analysis identifies ALK-1 as the predominant cause of pulmonary hypertension related to hereditary haemorrhagic telangiectasia. J. Med. Genet. 40, 865871 (2003).

76. Mizuguchi, T. et al. Heterozygous TGFBR2 mutations in Marfan syndrome. Nature Genet. 36, 855860 (2004).

77. Loeys, B. L. et al. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nature Genet. 37, 275281 (2005).

78. Pannu, H. et al. Mutations in transforming growth factor- receptor type II cause familial thoracic aortic aneurysms and dissections. Circulation 112, 513520 (2005).

79. Topouzis, S. & Majesky, M. W. Smooth muscle lineage diversity in the chick embryo. Two types of aortic smooth muscle cell differ in growth and receptor-mediated transcriptional responses to transforming growth factor-. Dev. Biol. 178, 430445 (1996).

80. Waldo, K. L. et al. Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart. Dev. Biol. 281, 7890 (2005).

81. Neptune, E. R. et al. Dysregulation of TGF- activation contributes to pathogenesis in Marfan syndrome. Nature Genet. 33, 407411 (2003).

82. Habashi, J. P. et al. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome. Science 312, 117121 (2006).Aortic aneurysm in a mouse model of MFS is shown to be associated with increased TGF signalling and can be prevented by TGFneutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker, losartan.

83. Carta, L. et al. Fibrillins 1 and 2 perform partially overlapping functions during aortic development. J. Biol. Chem. 281, 80168023 (2006).

84. Judge, D. P. et al. Evidence for a critical contribution of haploinsufficiency in the complex pathogenesis of Marfan syndrome. J. Clin. Invest. 114, 172181 (2004).

85. Denton, C. P. et al. Fibroblast-specific expression of a kinase-deficient type II transforming growth factor (TGF) receptor leads to paradoxical activation of TGF signaling pathways with fibrosis in transgenic mice. J. Biol. Chem. 278, 2510925119 (2003).

86. Wang, W. et al. Essential role of Smad3 in angiotensin II-induced vascular fibrosis. Circ. Res. 98, 10321039 (2006).

87. Zhou, Y., Poczatek, M. H., Berecek, K. H. & Murphy-Ullrich, J. E. Thrombospondin 1 mediates angiotensin II induction of TGF- activation by cardiac and renal cells under both high and low glucose conditions. Biochem. Biophys. Res. Commun. 339, 633641 (2006).

R E V I E W S


88. Yanagisawa, H. et al. Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo. Nature 415, 168171 (2002).

89. Nakamura, T. et al. Fibulin-5/DANCE is essential for elastogenesis in vivo. Nature 415, 171175 (2002).

90. McLaughlin, P. J. et al. Targeted disruption of fibulin-4 abolishes elastogenesis and causes perinatal lethality in mice. Mol. Cell Biol. 26, 17001709 (2006).

91. Hanada, K. et al. Perturbations of vascular homeostasis and aortic valve abnormalities in fibulin-4 deficient mice. Circ. Res. 100, 738746 (2007).

92. Cambien, F. et al. Polymorphisms of the transforming growth factor- 1 gene in relation to myocardial infarction and blood pressure. The Etude Cas-Temoin de lInfarctus du Myocarde (ECTIM) Study. Hypertension 28, 881887 (1996).

93. Zacchigna, L. et al. Emilin1 links TGF- maturation to blood pressure homeostasis. Cell 124, 929942 (2006).Mice lacking emilin1, a Cysrich secreted glycoprotein that prevents maturation of proTGF precursor by furin convertases, display elevated blood pressure due to increased TGF signalling in the vasculature.

94. August, P. & Suthanthiran, M. Transforming growth factor signaling, vascular remodeling, and hypertension. N. Engl. J. Med. 354, 27212723 (2006).

95. Venkatesha, S. et al. Soluble endoglin contributes to the pathogenesis of preeclampsia. Nature Med. 12, 642649 (2006).Soluble endoglin is shown to lead to dysregulated TGF signalling in the vasculature and may act in concert with VEGFR1 to induce severe preeclampsia.

96. Cudmore, M. et al. Negative regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1. Circulation 115, 17891797 (2007).

97. Lopez-Novoa, J. M. Soluble endoglin is an accurate predictor and a pathogenic molecule in pre-eclampsia. Nephrol. Dial. Transplant. 22, 712714 (2007).

98. Akhurst, R. J. Large- and small-molecule inhibitors of transforming growth factor- signaling. Curr. Opin. Investig. Drugs 7, 513521 (2006).

99. Derynck, R., Akhurst, R. J. & Balmain, A. TGF- signaling in tumor suppression and cancer progression. Nature Genet. 29, 117129 (2001).

100. Akhurst, R. J. TGF signaling in health and disease. Nature Genet. 36, 790792 (2004).

101. Kano, M. R. et al. Improvement of cancer-targeting therapy, using nanocarriers for intractable solid tumors by inhibition of TGF- signaling. Proc. Natl Acad. Sci. USA 104, 34603465 (2007).Application of a smallmolecule TGF type I receptor inhibitor at a low dose decreased pericyte coverage of the endothelium specifically in the tumour neovasculature, and promoted accumulation of anticancer nanocarriers in tumours.

102. Jerkic, M. et al. Reduced angiogenic responses in adult endoglin heterozygous mice. Cardiovasc. Res. 69, 845854 (2006).

103. Duff, S. E., Li, C., Garland, J. M. & Kumar, S. CD105 is important for angiogenesis: evidence and potential applications. FASEB J. 17, 984992 (2003).

104. Tan, G. H. et al. Combination of low-dose cisplatin and recombinant xenogeneic endoglin as a vaccine induces synergistic antitumor activities. Int. J. Cancer 112, 701706 (2004).

105. van Laake, L. W. et al. Endoglin has a crucial role in blood cell-mediated vascular repair. Circulation 114, 22882297 (2006).

106. Ramirez, F. & Rifkin, D. B. Cell signaling events: a view from the matrix. Matrix Biol. 22, 101107 (2003).

107. Coucke, P. J. et al. Mutations in the facilitative glucose transporter GLUT10 alter angiogenesis and cause arterial tortuosity syndrome. Nature Genet. 38, 452457 (2006).

108. Gregory, K. E. et al. The prodomain of BMP-7 targets the BMP-7 complex to the extracellular matrix. J. Biol. Chem. 280, 2797027980 (2005).

109. Arteaga-Solis, E. et al. Regulation of limb patterning by extracellular microfibrils. J. Cell Biol. 154, 275281 (2001).

110. Choudhary, B. et al. Cardiovascular malformations with normal smooth muscle differentiation in neural crest-specific type II TGF receptor (Tgfbr2) mutant mice. Dev. Biol. 289, 420429 (2006).

111. Cohn, R. D. et al. Angiotensin II type 1 receptor blockade attenuates TGF--induced failure of muscle regeneration in multiple myopathic states. Nature Med. 13, 204210 (2007).

112. Kamei, M. et al. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 442, 453456 (2006).

113. Barbara, N. P., Wrana, J. L. & Letarte, M. Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor- superfamily. J. Biol. Chem. 274, 584594 (1999).

114. George, E. L., Georges-Labouesse, E. N., Patel-King, R. S., Rayburn, H. & Hynes, R. O. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 119, 10791091 (1993).

115. Lawler, J. et al. Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J. Clin. Invest. 101, 982992 (1998).

116. Huang, X. Z. et al. Inactivation of the integrin 6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lung and skin. J. Cell Biol. 133, 921928 (1996).

117. Stenvers, K. L. et al. Heart and liver defects and reduced transforming growth factor 2 sensitivity in transforming growth factor type III receptor-deficient embryos. Mol. Cell Biol. 23, 43714385 (2003).

118. Srinivasan, S. et al. A mouse model for hereditary hemorrhagic telangiectasia (HHT) type 2. Hum. Mol. Genet. 12, 473482 (2003).

119. Larsson, J. et al. Abnormal angiogenesis but intact hematopoietic potential in TGF- type I receptor-deficient mice. EMBO J. 20, 16631673 (2001).

120. Beppu, H. et al. 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, L1241L1247 (2004).

121. Lane, K. B. et al. Heterozygous germline mutations in BMPR2, encoding a TGF- receptor, cause familial primary pulmonary hypertension. The International PPH Consortium. Nature Genet. 26, 8184 (2000).

122. Gallione, C. J. et al. A combined syndrome of juvenile polyposis and hereditary haemorrhagic telangiectasia associated with mutations in MADH4 (SMAD4). Lancet 363, 852859 (2004).

123. Loeys, B.L. et al. Aneurysm syndromes caused by mutations in the TGF- receptor. N. Engl. J. Med. 355, 788798 (2006).Mutations in either TGFBR1 or TGFBR2 are shown to predispose patients to aggressive and widespread vascular disease.

124. Todorovic, V. et al. Long form of latent TGF- binding protein 1 (Ltbp1L) is essential for cardiac outflow tract septation and remodeling. Development (in the press).

AcknowledgementsResearch in our laboratories is supported by grants from the Dutch Cancer Society, the EC (Angiotargeting and Tumour Host Genomics), the Ludwig Institute for Cancer Research, the Netherlands Organization for Scientific Research, the British Heart Foundation, Newcastle Hospital Trustees, the Cookson Foundation and the Wellcome Trust. We are grateful to our colleagues for valuable discussion, and apologize to those whose contributions have not been cited because of space constraints.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneACVRL1 | BMPR2OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMarterial tortuosity syndrome | Duchennes muscular dystrophy | familial thoracic aneurysm disorder | hereditary haemorrhagic telangiectasia type 1 | HHT type 2 | LoeysDietz syndrome | Marfan syndrome | MFS type 2 | pulmonary arterial hypertensionUniProtKB: http://ca.expasy.org/sprotALK1 | betaglycan | BMP1 | emilin1 | endoglin | fibrillin1 | fibrillin2 | SMAD1 | SMAD2 | SMAD3 | SMAD4 | SMAD5 | TGF1 | TGF2 | TGF3 | TGFBR1 | TGFBR2

FURTHER INFORMATIONPeter ten Dijkes homepage: http://www.lumc.nl/1050/research/signaaltransductie.htmlHelen Arthurs homepage: http://www.ncl.ac.uk/ihg/staff/profile/helen.arthurHHT Foundation International web site: http://www.hht.org/The US National Marfan Foundation web site: http://www.marfan.org

all linkS are acTiVe in THe online PDF

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