Keywords

1 TGFb Molecule Family: Sources, Activation, and Regulation of Transcription

1.1 The TGFb Family

The transforming growth factor beta (TGFβ) superfamily consists of 33 members, most of which are dimeric, secreted polypeptides that regulate proliferation, survival/apoptosis, migration, adhesion, invasiveness, and self-renewal properties in responsive cells [1, 2]. Depending on the cell and tissue type, modulation of these cellular properties by TGFβ superfamily members will regulate different processes ranging from gastrulation to formation of a functional vascular system during embryonic development, as well as organ morphogenesis and homeostasis at various postnatal stages [1].

The TGFβ superfamily is conserved through metazoan evolution and includes TGFβs (1–3), bone morphogenic proteins (BMPs 1–20), growth and differentiation factors (GDFs including myostatin), activins (A and B), inhibins (A and B), nodal, leftys (1 and 2), and Mullerian inhibiting substances (MIS) [2]. The TGFβ family, the focus of this review, comprises three different isoforms encoded by separate genes: TGFβ1, 2, and 3. Human TGFβ2 and TGFβ3 have a 70 % homology with TGFβ1 [3]. TGFβ1 is the predominant and most ubiquitous isoform, while the other two are expressed in a more limited spectrum of cells and tissues. The three isoforms have overlapping functions in vitro; however, mice deficient in individual isoforms show nonoverlapping phenotypes suggesting that each TGFβ isoform has distinct functions in vivo [4].

1.2 TGFb Sources

TGFβ ligands are not specific to a particular cell type, as they have been shown to be synthesized by a variety of normal and malignant cells. In addition, almost every cell in the body expresses TGFβ receptors (TGFβRs) and so is capable of responding to TGFβ [5]. TGFβ was initially isolated from platelets, due to their high content of the ligand. However, bone cells, particularly osteoblasts, are the highest producers of TGFβ currently known. Strong intracellular TGFβ staining has also been reported in adrenal cortex, megakaryocytes, cardiac myocytes, chondrocytes, renal distal tubules, ovarian glandular cells, and chorionic cells of mouse placenta, among others (reviewed in [6]). In carcinomas, as well as in sites of wound healing, TGFβ is expressed by epithelial cells, associated fibroblasts and myofibroblasts, and infiltrating immune cells, such as macrophages and T lymphocytes [7].

1.3 Synthesis and Activation of TGFb

All TGFβ ligands are synthesized as precursor polypeptides, containing a longer 25 kDa N-terminal pro-peptide, followed by a C-terminal, 12.5 kDa mature polypeptide. Two of these precursors form a dimer via disulfide bonds. Pro-peptide and mature peptide are cleaved by furin-like proteases while trafficking via the exocytic pathway, but remain associated by the disulfide bonds. Once cleaved, the pro-peptide becomes the “latency associated peptide” (LAP), which acts as a chaperone during exocytosis of the complex. LAP also aids in TGFβ deposition into the extracellular matrix (ECM) and keeps TGFβ inactive within its core once the complex is secreted [1]. LAP-TGFβ is known as the small latent complex (SLC) that often exists in association with the latent TGFβ binding protein (LTBP), which, together with the SLC, forms the large latent complex (LLC) [5]. LAP’s direct interaction with LTBP as well as with ECM components such as fibronectin and fibrillin, among others, mediates TGFβ’s deposition into the ECM [1]. Cleavage-dependent activation of the mature C-terminal dimeric TGFβ ligand from its ECM-deposited form is mediated by a number of proteases including thrombospondin 1 (TSP-1), plasmin, cathepsin D, matrix metalloproteinase (MMP) 2 and 9, calpain, chymase, elastase, endoglycosidase F, and kallikrein. In addition, acidic environment, reactive oxygen species (ROS), heat, and sheer stress have also been shown to activate TGFβ (reviewed in [2, 5, 8]). However, in many physiological situations, integrins have been shown to be the critical players in TGFβ activation. An RGD sequence present in TGFβ’s LAP mediates its binding to all αv integrins, and αvβ3, αvβ5, αvβ6, and αvβ8 have all been shown to release active TGFβ via both proteolysis-independent and proteolysis-dependent mechanisms [5].

1.4 Regulation of Transcription by TGFb

Once activated TGFβ initiates signaling by inducing the activity of specific serine/threonine kinase type I and type II receptor heterotetrameric complexes. These in turn phosphorylate specific effector proteins called Smads, which translocate to the nucleus and modulate transcription of targets genes (discussed in more detail below). Nuclear Smad complexes bind to chromatin and, together with other transcription factors, regulate gene expression. A list of Smad target genes has been published elsewhere [1]. Among these, the inhibitors of differentiation (Id) family of transcription factors, vascular endothelial growth factor (VEGF), and thrombospondin-1 (TSP-1) are important modulators of angiogenesis. TGFβ also signals in a noncanonical manner to modulate the level and function of effector proteins in the absence of changes in gene transcription [9]. Misregulation of TGFβ signaling plays roles in a number of pathologies, including autoimmune and cardiovascular disorders, and cancer [10]. Cardiovascular disorders resulting from abnormal TGFβ signaling include hereditary hemorrhagic telangiectasia (HHT), cardiac remodeling/fibrosis, and pulmonary arterial hypertension, among others [11].

1.5 TGFb’s Role in Angiogenesis

Genetic studies in mouse and human have provided evidence for the importance of components of the TGFβ signaling pathway in vascular morphogenesis, including formation of the primitive vascular plexus, and the recruitment of pericytes/smooth muscle cells necessary for vessel wall integrity [11]. Deletion of TGFβ1 in the mouse results in embryonic lethality because of defective yolk sac vasculogenesis. Targeted deletion of ALK1, ALK5, TGβRII, and endoglin results in similar phenotypes. All of these knockout embryos die during mid-gestation due to hyper-dilated, impaired, leaky vessels [12]. These vascular abnormalities are similar to those described in patients with HHT [11]. Endothelial and smooth muscle cell-specific targeting of TGFβRII and ALK5 suggests that TGFβ signaling in both compartments is required for proper vessel development, but likely at different stages [11, 13].

In the next sections, we present a detailed overview of current knowledge on TGFβ signaling in endothelial and associated vascular cells, such as pericytes and smooth muscle cells, and the role of this signaling at the various stages of the angiogenesis process. We also provide some evidence of differential TGFβ signaling in physiologic vs. pathologic angiogenesis and discuss potential applicability in therapeutic intervention.

2 TGFb Receptors and Signaling

TGFβ members signal through type I and type II serine/threonine kinase receptors. There are seven members of the type I receptor family, also known as activin receptor-like kinases (ALK) 1–7, and five members of the type II receptor family (TGFβRII, BMPRII, ActRIIA, ActRIIB, and MISRII) [14]. TGFβ also signals via accessory, type III, TGFβ receptors: endoglin and betaglycan (discussed in a later section). Reflective of its role in signaling a multitude of cell types, there are relatively few studies devoted specifically to TGFβ signaling in vascular endothelium. In the sections below, except where explicitly stated, the signaling events and outcomes described have not yet been validated in endothelial cells.

In most cell types TGFβ 1–3 isoforms signal through an ALK5-TGFβRII complex; however, endothelial cells also express and signal through an ALK1-TGFβRII complex [15]. The balance between activation of these two signaling pathways regulates endothelial cell functions such as proliferation and migration, and this balance is believed to regulate the switch of endothelial cells from quiescent mature vessels into activated angiogenic sprouts. In fact, genetic mutants of TGFβ receptors, ALK5 and endoglin, inhibit angiogenesis in vitro and result in embryonic lethality in mice due to vascular defects [12].

In the absence of ligand, type I and II receptors form homodimers with themselves, which upon ligand binding complex with each other to form a heterotetramer [16]. Formation of this tetrameric complex brings together the constitutively active type II receptor with the type I receptor, resulting in auto- and transphosphorylation at various serine residues in the receptors’ GS domain. Once activated by these phosphorylation events, the TGFβ receptors become functional serine/threonine kinases and subsequently phosphorylate and activate several intracellular signaling molecules [17].

Although TGFβ receptors are classically referred to as serine/threonine kinases, upon ligand binding TGFβRII also becomes autophosphorylated at multiple tyrosine residues [18]. These phosphorylated tyrosines then act as docking sites for various Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domain containing scaffolding/adaptor molecules, such as Shc, and growth factor-binding protein 2 (Grb2) [19]. These adaptor molecules function as scaffolding proteins, bringing together the TGFβ receptor’s tyrosine kinase function with various protein substrates [20].

TGFβ receptors, through their action as both serine/threonine and tyrosine kinases, are able to activate several intracellular signaling cascades, including the canonical Smad signaling pathways, as well as several noncanonical signaling pathways, such as PI3K/Akt, RhoA-dependent, and JNK, p38, and Erk MAPK pathways.

2.1 Canonical Smad Signaling

The Smad family of proteins is composed of three classes: receptor Smads (R-Smads 1, 2, 3, 5, and 8), inhibitory Smads (I-Smads 6 and 7), and the common Smad (Co-Smad4) [14]. R-Smads are recruited to TGFβRI following receptor activation and interact indirectly via auxiliary proteins such as Smad anchor for receptor activation (SARA) [21]. Once recruited R-Smads become phosphorylated by TGFβRI in their C-terminal SSXS domain; R-Smads 1, 5, and 8 are activated by ALKs 1–3 and 6, whereas R-Smads 2–3 are activated by ALKs 4, 5, and 7 [22].

Once activated R-Smads dissociate from TGFβRI and form heterodimers with Co-Smad4 or heterotrimers containing two R-Smads and one Smad4, which translocate to the nucleus [23]. This translocation is guided by nuclear localization sequences (NLSs) in the MH1 domains of Smad3 and 4, which mediate their interaction with importin proteins β1 and α, respectively [24, 25]. In the nucleus these Smad complexes function as transcription factors and regulate transcription through their direct interaction with DNA containing Smad-binding elements (SBEs), as well as corepressors, co-activators (CBP and p300), and other ­transcription factors [26]. The diversity of these transcriptional complexes directs the tissue and dose-dependent regulation of transcription by the Smad proteins.

TGFβ stimulation affects the transcription of several hundred genes [27]. Targets of R-Smad and Co-Smad transcriptional regulation include proteins involved in regulating cellular proliferation, apoptosis, and the epithelial-to-mesenchymal transition (EMT). Additionally, TGFβ signaling results in expression of I-Smads 6 and 7, whose promoters contain SBEs [28]. The I-Smads then establish a negative feedback circuit on TGFβ signaling through their ability to negatively regulate signaling pathway activation on multiple levels, with Smad7 expressed in response to and antagonizing all TGFβ signaling and Smad6 expressed specifically in response to and antagonizing the Smad1, 5, and 8 signaling pathways [29].

I-Smads contain various functional domains that enable their inhibitory function. Through their MH2 domain, Smads 6 and 7 are able to compete with R-Smads for TGFβRI binding, thus inhibiting R-Smad phosphorylation and subsequent Co-Smad4 complex formation [30]. I-Smads are also capable of recruiting E3 ubiquitin ligases, Smurf1 and Smurf2, to activated TGFβRI leading to its polyubiquitination and subsequent proteasomal degradation [31]. Smad7 is also capable of interfering with TGFβ signaling at the level of receptor activation via its ability to recruit the phosphatase GADD34-PP1c to the activated receptor complex [32].

In addition to their functioning in the cytoplasm, at the level of receptor and R-Smads inhibition, I-Smads also function in the nucleus at the level of transcriptional repression. Through its MH2 domain, Smad7 is able to bind directly to DNA to prevent Smad2, 3, and 4 binding [26]. Also, once bound to DNA, I-Smads recruit histone deacetylases (HDACs) to the promoter regions of Smad target genes, leading to chromatin compaction and transcriptional inhibition [33].

2.2 Noncanonical Signaling Pathways

2.2.1 MAPK

TGFβ signaling can also lead to activation of MAPK signaling pathways, including Erk, p38, and JNK MAPK signaling. This activation is likely independent of Smad-­dependent transcription, due to the rapid onset of MAPK phosphorylation (5–15 min) [34], and the ability of cells genetically deficient in Smad activation to maintain their ability to activate MAPK signaling in response to TGFβ [35]. Erk MAPK becomes activated through a receptor tyrosine kinase (RTK)/Ras/Erk pathway. Following TGFβ ligand binding to TGFβRII, type I and II receptors become phosphorylated on three tyrosine residues, Y259, Y336, and Y424, in the receptors’ cytoplasmic domain [18]. These phosphorylated tyrosine residues are then bound by SH2 and PTB domain containing adaptor molecules. Grb2 is an SH2 domain-containing protein that complexes with SOS in the cytoplasm and upon RTK phosphorylation is recruited to the receptor. Once localized to the RTK the Grb2/SOS complex is able to activate membrane-localized Ras, bridging TGFβ receptor activation with the MAPK signaling pathway. In its activated, GTP-bound state, Ras is able to phosphorylate and activate the Raf-MEK-Erk MAPK cascade [19, 20].

Erk, through its functioning as a serine/threonine kinase, not only regulates intracellular mitogen signaling in the cytoplasm but also translocates to the nucleus where it regulates the activity of various transcriptional regulators [36]. Through its modulation of gene transcription, Erk mediates TGFβ-induced disassembly of adherens junctions and enhanced migration, two key events in TGFβ-induced EMT [34]. As such, Erk activation is required, however insufficient (since it must cooperate with Smad signaling), for TGFβ-induced EMT [37].

JNK and p38 MAPK signaling pathways are also activated in response to TGFβ signaling. This activation is dependent on the scaffolding protein TRAF6, which associates with activated TGFβRII through its C-terminal TRAF domain. TGFβRII-­bound TRAF6 undergoes auto-polyubiquitination, leading to its association with the MAP3K, TAK1 [38]. TAK1 is required for activation of both the JNK and p38 MAPK pathways via activation of MKK4-JNK and MKK3/6-p38 cascades [39]. In fact, TAK1 is indispensable for JNK and p38 MAPK activation, and embryos deficient in TAK1 suffer from vascular defects whose phenotype is similar to ALK1 and endoglin mutants [40]. The activation of TGFβ-TAK1-JNK/p38 MAPK pathways is independent of Smad-mediated transcription; however, these signaling pathways cooperate with Smad signaling in order to regulate TGFβ-induced cellular functions such as apoptosis [41] and EMT [42]. Recent studies revealed that different isoforms of p38 MAPK are responsible for the differential effects of VEGF and TGFβ on endothelial cells. In particular, TGFβ is able to shift VEGF signaling from pro-survival to pro-apoptotic isoforms of p38. Thus in the absence of TGFβ, VEGF supports endothelial proliferation, but when TGFβ is also present, endothelial cell death can occur [43].

2.2.2 Rho-GTPase

TGFβ also rapidly activates RhoA signaling in a Smad-independent manner [44]. However, TGFβ signaling has also been shown to lead to a downregulation of RhoA protein in response to TGFβ activation of the Par6 polarity pathway [45]. Par6 is a scaffolding protein that complexes with TGFβRI at tight junctions. Following ligand binding to TGFβRII, it travels to the tight junction where it complexes with TGFβRI and phosphorylates Par6 at serine 345. Activated Par6 recruits the E3 ubiquitin ligase, Smurf1, to tight junctions where it ubiquitinates and targets RhoA for degradation, leading to localized RhoA downregulation at tight junctions [45]. This results in a loss of polarity and enhanced cellular motility. This localized degradation is responsible for the dissolution of tight junctions, reorganization of the actin cytoskeleton, and extension of filopodia [46], all of which are essential for EMT [45]. The potential role of the Par6 pathway in vascular biology has been recently highlighted by in vitro studies on endothelial-mesenchymal transition (EndoMT), a process induced by TGFβ (discussed in more detail in later sections), which is essential for heart valve formation in the developing embryo. It was observed that blockade of Par6 activation abrogated EndoMT in response to TGFβ2, and this was dependent on the presence of both ALK-5 and type 3 TGFβ receptor betaglycan [47]. We have recently demonstrated Par6 activation in response to TGFβ1 in bovine aortic endothelial cells, particularly at low (0.5 ng/mL and lower) TGFβ concentrations (Viloria-Petit, unpublished observations). Since low TGFβ concentrations have been previously observed to be pro-­angiogenic [48], our results suggest that Par6 activation might mediate angiogenesis in response to TGFβ.

2.2.3 PI3K (AKT)

The Akt signaling pathway is also activated downstream of TGFβ through TGFβRI phosphorylation of PI3K, an upstream kinase of Akt. PI3K interacts with TGFβRII independent of receptor activation and upon ligand stimulation is brought in contact with TGFβRI, where it is phosphorylated [49]. Downstream activation of the Akt signaling pathway is required for TGFβ-induced EMT and does so by two proposed mechanisms: first, through its ability to mediate TGFβ-induced actin filament reorganization and enhanced cellular migration and secondly, through Akt’s activation of downstream mTOR [50]. The mTOR signaling pathway regulates cellular translation levels, and Akt-mTOR activation is believed to facilitate Smad-mediated transcriptional programs.

3 TGFb and Endothelial Sprouting, Proliferation, and Permeability

Extensive evidence suggests that TGFβ plays a role during the activation phase of angiogenic sprouting by promoting vascular permeability, proliferation, and migration of endothelial cells. TGFβ also mediates the reverse events that occur during the resolution phase of angiogenesis, including inhibition of endothelial cell migration and proliferation and decreased permeability, which are necessary for vessel stabilization [51].

3.1 Endothelial Sprouting

Endothelial sprouting involves two distinct endothelial cell phenotypes: the tip cells, which lead the newly forming vessel sprout, and the stalk cells, which proliferate and form the lumen of the new vessel [52]. These cells are initially part of a mature vessel. An increase in permeability and migratory characteristics allows these cells to delaminate from the endothelium and become involved in the newly forming vessel sprout. Proliferation must be suppressed in the tip cells and enhanced in the stalk cells to ensure their respective functions. Finally, when the new vessel is in place, there must be a reversion back to characteristics of cells in a quiescent endothelium, which includes a decrease in permeability, proliferation, and migratory capacity [52].

One of the key regulators of sprouting during angiogenesis is VEGF-A since tip cell migration largely depends on the gradient of VEGF-A, which binds to VEGF receptor 2 on endothelial cells [53]. Studies in mice and zebra fish have contributed to our understanding of the role of VEGF and Notch signaling in vessel sprouting. VEGF binding to VEGFR2 on a tip cell activates VEGFR signaling leading to increased expression of the Notch ligand Dll4, which in turn binds to Notch1 receptor on adjacent stalk cells. Notch signaling in the latter reduces VEGR2 and VEGR3 expression, making cells insensitive to VEGF stimulation, thereby suppressing the tip cell phenotype [54].

TGFβ effects on in vitro endothelial cell sprouting are variable, including induction, repression, or no effect depending on the concentration of TGFβ, the type of endothelial cells employed, and the source of signaling activation, i.e. whether constitutively activated receptors or exogenously added ligand was used [55]. The nature of the angiogenic response to TGFβ depends on the balance of ALK1 vs. ALK5 signaling input, with ALK1 predominantly promoting sprouting and ALK5 favoring the resolution/stabilization phase of angiogenesis [55]. The inhibitory effect of an ALK1 antibody on endothelial cell sprouting in vitro and on angiogenesis in two different tumor models supports this concept [56]. However, recent studies on developmental angiogenesis in mice suggest that, rather than promoting sprouting, ALK1 signaling cooperates with Notch signaling to repress VEGF responsiveness, tip cell formation, and sprouting [57]. Whether these discrepancies represent differences in developmental vs. pathologic angiogenesis remains to be determined. Interestingly, we have recently found TGFβ to decrease endothelial VEGFR2 [58] expression via ALK5 signaling. Thus, ALK5 signaling may potentially contribute to endothelial cell insensitivity to VEGF stimulation that might be necessary for both maintenance of the stalk cell fate during sprouting and the resolution phase of angiogenesis.

Finally, EndoMT has been hypothesized to be one of the mechanisms mediating angiogenic sprouting in response to TGFβ. EndoMT is the process whereby cells from a quiescent, stable endothelium delaminate from this cell layer and take on a fibroblastoid phenotype. During this process, endothelial cells experience loss of adherens and tight junctions and their associated markers including vascular endothelial (VE)-cadherin, zonula occludens (ZO)-1, and claudin-5. The cells’ transition towards a mesenchymal phenotype is associated with the gain of mesenchymal markers such as α-smooth muscle actin and fibroblast-specific protein-1, as well as motility [59, 60]. As previously mentioned, EndoMT mediates cardiac development and is also responsible for pathologic tissue fibrosis; however, its role in promoting angiogenesis is still unknown [60]. It is possible that a partial, reversible form of EndoMT facilitates angiogenic sprouting since loss of cell–cell junctions is required for endothelial cells to delaminate from the existing vessel. An EndoMT of the tip cells could similarly promote the invasive/migratory characteristics that are necessary for them to guide the vessel sprout.

3.2 Endothelial Permeability

Signaling via the ALK5 TGFβ receptor has been shown to promote and inhibit vascular permeability, depending on cell context. TGFβ can induce permeability in pulmonary endothelial cell monolayers, which is attenuated by treatment with SB-431542, an ALK5 kinase inhibitor [61]. Specifically, SB-431542 upregulates the expression of the endothelial-specific tight junction component, claudin-5 [62]. In contrast, in vivo blockade of TGFβ signaling in mouse retinal endothelial cells leads to increased permeability and decreases vessel barrier function. Both in vivo and in vitro analyses demonstrated that TGFβ signaling blockade resulted in increased endothelial permeability characterized by decreased interaction between the tight junction proteins occludin and ZO-1 [63].

As mentioned above, TGFβ-mediated EndoMT may contribute to an increase in endothelial permeability that is necessary for angiogenic sprouting. During EndoMT, there is a decrease in expression of the adherens junction protein VE-cadherin, as well as tight junction proteins ZO-1 and claudin-5. TGFβ has been shown to downregulate claudin-5 at the transcriptional level, and VE-cadherin has been observed to upregulate expression of claudin-5 [64, 65]. Thus, TGFβ-­mediated downregulation of VE-cadherin during EndoMT can indirectly decrease expression of claudin-5, resulting in the loss of both adherens junctions and tight junctions with a concomitant increase in endothelial permeability.

Along with TGFβ, VEGF has also been shown to be an important mediator of endothelial permeability during angiogenesis [66]. VEGF has a demonstrated function in modulating VE-cadherin at the adherens junctions through tyrosine phosphorylation, which leads to an increase in permeability [67]. Since TGFβ induces VEGF expression in vascular endothelial cells, this relationship may provide an alternative mechanism whereby TGFβ can modulate VE-cadherin expression and therefore increase endothelial cell permeability [68]. TGFβ’s ability to downregulate VEGF receptor 2 expression can also provide an additional means by which TGFβ can regulate and perhaps reverse vascular permeability during the resolution phase of angiogenesis.

3.3 Endothelial Proliferation and Migration

TGFβ can enhance cell proliferation at low doses and suppress proliferation at high doses. The presence of both type I receptors ALK1 and ALK5 may provide a means by which TGFβ’s dual role in proliferation is regulated [69]. Activation of ALK1 has been primarily shown to stimulate proliferation and migration of endothelial cells during the activation phase of angiogenesis [69, 70]. The downstream effector of ALK1 responsible for this process is Id1, an inhibitor of differentiation that is required for proliferation and migration. When ALK1 is active, both endothelial cells and fibroblasts are induced to express Id1 [69, 71]. It is interesting to note that ALK1 in combination with ALK5 is a potential negative regulator of endothelial cell migration and proliferation, which may play a role in the resolution phase of angiogenesis [72–74]. Possible mediators of the inhibitory effects of ALK1 signaling are JNK and ERK, but the process is not fully characterized in endothelial cells [72].

In contrast to ALK1, ALK5 seems to have more defined anti-proliferative roles during both the activation and the resolution phases of angiogenesis [69]. It is believed that activated Smad2/3 proteins cooperate with nuclear corepressors to repress the transcription of c-myc and cyclin-dependent kinase (CDK) genes and with nuclear co-activators to activate transcription of p15 and p21, two major inhibitors of the cell cycle, collectively inhibiting proliferation [75, 76]. ALK5 has been specifically shown to prevent proliferation and migration in endothelial cell spheroid assays and embryonic stem cell-derived endothelial cells, whereas the ALK5 kinase inhibitor, SB-431542, has opposite effects [62, 77]. Furthermore, in vitro studies have found that ALK5-induced blood vessel maturation is mediated by the induction of plasminogen activator inhibitor (PAI)-1 in endothelial cells. PAI-1 prevents degradation of the provisional ECM that surrounds the nascent vessel, hence promoting vessel maturation during the resolution phase [69]. Thus, ALK5 likely plays roles in both inhibiting proliferation of the tip cells during activation and in both tip and stalk cells during resolution of angiogenesis. While a balance between ALK1 and ALK5 may be important to mediate the effects of TGFβ on the endothelium, their actions are not mutually exclusive, and they may serve as regulators of one another.

The variation in roles played by ALK1 and ALK5 as well as the balance between these two type I TGFβ receptors is likely dependent on cellular context, with the cross talk between them providing a mechanism whereby TGFβ can strategically regulate proliferation of the tip and stalk cells during angiogenesis. It should also be noted that VEGF has also been shown to play a role in proliferation of endothelial cells during angiogenesis which is dependent on its concentration [53]. Since VEGF is a positive regulator of proliferation and TGFβ has been shown to be an inducer of VEGF expression in endothelial cells, this interaction provides yet another regulatory mechanism for TGFβ to control proliferation [53, 68].

4 TGFb Co-receptors in Angiogenesis

The human type III TGFβ co-receptors endoglin and betaglycan are type I integral membrane glycoproteins [78, 79]. Betaglycan is universally expressed on nearly all cell types and is the most highly expressed of the TGFβ superfamily receptors [80]. However, the expression of betaglycan in some cell types, specifically vascular endothelial cells with the exception of those forming the endocardium [81], appears to be weak or absent, and instead, endothelial cells predominantly express the related TGFβ co-receptor, endoglin [80]. Both endoglin and betaglycan are generally expressed on the cell surface as homodimers, with endoglin homodimers being linked by disulfide bridges; however, endoglin and betaglycan are capable of forming heteromeric complexes in microvascular endothelial cells [79, 80]. Both type III co-receptors also exist as soluble forms. Betaglycan shedding is mediated in part by membrane-type matrix metalloproteinase1 (MT1-MMP) and plasmin [82], while soluble endoglin is produced by cleavage of the membrane-­bound endoglin at close proximity to the transmembrane domain by matrix metalloproteinase 14 (MMP14) [83].

Endoglin expression is potently stimulated by hypoxia, BMP9, and TGFβ via ALK1, while TNFα exerts an inhibitory effect on endoglin expression in endothelial cells [3, 84, 85]. Both betaglycan and endoglin cytoplasmic domains can be phosphorylated by serine/threonine kinases [86, 87]. ALK5 is responsible for the phosphorylation of endoglin’s cytoplasmic tail, which has been shown to be necessary for the activation of TGFβ-dependent ALK1 signaling [88]. Thus, ALK5 is indirectly responsible for ALK1 activation via endoglin, which in turn is necessary for endothelial cell proliferation. The phosphorylation of endoglin has been shown to influence its subcellular localization, probably by modulating its interaction with adhesive proteins such as zyxin and zyxin-related protein 1 (ZRP-1), hence modifying the adhesive properties of endoglin-expressing cells [86].

It is not fully understood how endoglin regulates TGFβ-dependent responses. Endothelial cells that lack endoglin experience decreased proliferation due to diminished ALK1 activity and increased ALK5 activity [89]. The increase in ALK5 and subsequent TGFβ-induced growth inhibition, even at low concentrations of TGFβ, which normally promote proliferation [69], may also be due in part to decreased inhibition of ALK5 by ALK1 [89]. ALK1 has been shown to interrupt ALK5 signaling, likely acting downstream of Smad2/3 phosphorylation [70]. Thus ALK1 may be involved in a negative regulatory mechanism that is able to mediate the anti-proliferative effects of ALK5 in endothelial cells. However, endoglin association with TβRII results in alteration of its phosphorylated status, thus ensuing loss of ALK5 from the TGFβ receptor complex, possibly explaining endoglin’s inhibitory effect on ALK5 signaling [90]. Furthermore, studies conducted on human umbilical vein endothelial cells demonstrate that ALK1-dependent inhibition of cell adhesion is counteracted by endoglin phosphorylation [90, 91]. These results suggest that endoglin interaction with TGFβ signaling receptors via both its extracellular and cytoplasmic domains might affect TGFβ cell responses.

4.1 Regulation of TGFb Ligand Access to Co-receptors

Betaglycan binds multiple members of the TGFβ family, including TGFβ1, TGFβ2, TGFβ3, activin A, BMP2, BMP4, and BMP7 [92–94]. Betaglycan also plays a role in presenting the ligand to TβRII, leading to either enhanced or inhibited signaling [95]. Unlike betaglycan, endoglin binds TGFβ1 and TGFβ3 but not TGFβ2 [96]. Other endoglin ligands include activins and BMPs, and endoglin can also interact with activin type II receptors [97]. Therefore, functional differences and similarities found between betaglycan and endoglin could be due to differences between these two proteins’ ligand binding profiles.

In the case of the type III co-receptor betaglycan, its function as a co-receptor to specific members of the TGFβ superfamily is carried out through its ectodomain, which consists of two independent ligand-binding domains. The residual carboxy-terminal half of the protein is necessary for protein anchoring to the cell membrane [94]. Comparative studies between endoglin and betaglycan intracellular responses to TGFβ signaling found a distinctive role for the extracellular domains [98]. Exchanging the extracellular domain between these two co-­receptors did not alter endoglin ligand binding potential; however, in contrast to betaglycan, TβRII is essential for endoglin binding of TGFβ1, activin A, BMP2, and BMP7 [97, 98]. The soluble form of endoglin reduced binding of TGFβ1 by interfering with its interaction with TβRII, and soluble endoglin suppressed TGFβ1 signaling in endothelial cells [99].

As previously mentioned, TGFβ can signal in endothelial cells through either ALK1 or ALK5, resulting in the stimulation of endothelial cell proliferation and migration (ALK1) or inhibition of these responses (ALK5) [89]. Forced expression of endoglin led to inhibition of TGFβ/ALK5 signaling, and subsequent blockade of TGFβ induced growth inhibitory effect on endothelial cells [89, 98, 100, 101]. Moreover, endoglin can block apoptosis in response to hypoxia and TGFβ. When endoglin-expressing and endoglin-deficient endothelial cells were both exposed to TGFβ1 under hypoxic stress, the presence of endoglin was sufficient to block the synergistic pro-apoptotic effect of TGFβ1 and hypoxia [84]. Additionally, in endothelial cells the endoglin cytoplasmic tail interacts with β-arrestin2, leading to endoglin-mediated inhibitory effects on TGFβ-induced ERK activation and migration [102]. Finally, endoglin is able to inhibit cell migration through its interaction with LIM domain containing focal adhesion proteins such as zyxin, possible in a TGFβ-independent fashion [103].

During embryogenesis, inflammation, and wound healing, modifications in vascular structure occur, and endoglin expression is elevated during these modifications [104, 105]. The importance of endoglin function in maintaining normal vascular structure is underlined by the relationship between mutations in the endoglin gene and HHT, which is a disorder characterized by the formation of small dilated blood vessels and arteriovenous malformations (AVMs) in the vasculature of lung, liver, and brain [106, 107]. Studies done to elucidate the role endoglin plays in the enhancement of the TGFβ/ALK1 signaling pathway suggest that endothelial cell response to TGFβ is critically dependent on endoglin functional association with ALK1 [90]. The results from these studies agree with what is seen in cases of HHT where the predominant mutations are in either human endoglin (ENG) or ALK1 (ACVRL1) genes [108, 109].

5 TGFb and Vascular Mural Cells

The structure of microvessels varies between different tissue beds, and one of the major alterations is in the nature and prevalence of mural cells. Pericytes are found in capillaries, venules, and small arterioles, while true vascular smooth muscle cells are associated with larger arterioles and the macrocirculation [110]. In addition, there are significant differences in pericyte coverage and phenotype between vascular beds [111], and the ratio of pericytes to endothelium can vary from an almost 1:2 ratio in retina [112] to less than one pericyte for every ten endothelial cells. Pericytes can also be additionally specialized for tissue-specific vascular function, becoming glomerular mesangial cells (kidney) or Ito/stellate cells (liver), for example [110].

Mural cells play significant roles in the stabilization, functionality, and phenotype determination of the microcirculation, and recruitment of these cells is an essential part of the so-called resolution stage of sprouting angiogenesis [113]. During development, platelet-derived growth factors (PDGFs) act as potent chemoattractants for mural cell precursors and are produced by endothelial cells during vasculogenesis in the embryo and during sprouting angiogenesis in adult tissue. There is evidence from in vitro studies that PDGF-B can induce TGFβ production via the MAPK/ERK pathway and angiopoietin 1 (Ang-1) production via PKC and PI3K pathways during vascular smooth muscle differentiation of 10T1/2 cells [114]. Furthermore, TGFβ can downregulate this PDGF-B induction of Ang-1, and both TGFβ and Ang-1 synergistically reduce PDGF production by vascular endothelial cells, suggesting that cross talk between endothelium and mural cell precursors is essential for maturation of the microvascular bed [114]. There is also evidence that monocyte chemoattractant protein 1 (MCP-1) is also chemoattractant for vascular smooth muscle and 10T1/2 cells [115]. MCP-1 is upregulated in ischemic regions of brain associated with endoglin positive microcirculation and in human brain microvessel endothelial cells exposed to ischemia in vitro, highlighting the potential role of TGFβ-mediated pathways in angiogenic recovery of reperfused brain after stroke [116].

Culture of 10T1/2 cells with vascular endothelial cells leads to activation of latent TGFβ (similar to what is seen with EC/smooth muscle cell coculture [117]) and subsequent TGFβ-driven 10T1/2 cell differentiation into pericyte-like cells [118]. Endothelial-mural cell precursor contact is required for this TGFβ activation, and coculture of endothelial cells with mesenchymal precursors from mutant mouse embryos demonstrates that cell coupling via gap junction protein connexin 43 is essential for this activity [119]. Coculture of endothelial cells and 10T1/2 cells enhances the survival of both cell types; ECs require active ALK5 signaling for this, while 10T1/2 cells in coculture employ other pathways for survival [63]. This TGFβ-mediated reciprocal interaction between vascular components is relevant for both vascular functionality (such as permeability/barrier function) and neural retinal cell survival in adult mice [63]. Proper pericyte/endothelial cell interactions are also essential for maintaining blood–brain barrier characteristics in cerebral vessels. This is mediated via endothelial cell Smad4 signaling, which in cooperation with Notch signaling leads to increased N-cadherin expression and stable endothelial-mural cell adhesion [120]. In mesenchymal stem cells, TGFβ induces production of the Notch ligand Jagged1 and subsequent vascular smooth muscle cell-specific gene expression via Smad3 and Rho kinase pathways [121].

TGFβ signaling via endoglin or ALK1 is able to reduce endothelial activation via TGFβ/ALK5 and therefore tends to promote vessel destabilization and proliferation/sprouting [89]. Endoglin in cooperation with αv integrin leads to TGFβ activation and also signals for subsequent reduced pericyte migration. The matricellular protein “secreted protein acidic and rich in cysteine” (SPARC) is able to interfere with TGFβ-mediated inhibition of pericyte migration via its ability to prevent endoglin from incorporating into pericyte focal contacts and associating αv integrin [122]. Interestingly, endoglin is able to associate with αv integrin independent of the formation of focal adhesions, and endoglin may interfere with pericyte focal adhesion formation or maturation, partially accounting for its ability to reduce mural cell migration upon TGFβ stimulation [122]. Rivera and Brekken propose a model whereby, as pericytes come into contact with endothelial cells, SPARC is degraded or removed from the integrin complex, leading to TGFβRII/αv integrin/TGFβ interactions and subsequent signaling [122].

Mice null for endothelial expression of the tumor suppressor LKB1 display early embryonic death associated with defective yolk sac vessel recruitment of mesenchymal precursors of vascular smooth muscle cells, similar to what is seen in endoglin knockout murine embryos [123, 124]. LKB1 null endothelial cells were defective in TGFβ production, implicating this kinase in regulation of TGFβ production, via as yet unclear mechanisms [123].

6 Pathological Angiogenesis

Angiogenesis plays key roles in reproduction, development, growth, and wound healing and can drive the so-called angiogenesis-dependent diseases such as diabetic retinopathy, chronic inflammation, and cancer [125, 126]. There is growing evidence that, despite underlying fundamental similarities the angiogenesis occurring under such pathological settings displays significant alterations in pathways and processes. While such differences complicate our understanding of the angiogenic process, they can also provide opportunities for therapeutic intervention specifically targeting pathological neovascularization [126–128]. In this section, we describe in more detail some examples of “pathological angiogenesis” where TGFβ plays a significant role.

6.1 HHT

HHT is an autosomal dominant syndrome associated with epistaxis, AVMs in multiple organs, and dilated regions of high capillary density or telangiectases [106]. There are two commonly identified genetic defects in this condition accounting for two types of HHTs; HHT1 arises due to mutation of the TGFβ type III receptor endoglin and HHT2 from mutation in the TGFβ type I receptor ALK1 [106]. Additionally, Smad4 mutations has also been identified in patients with a syndrome characterized by both juvenile polyposis and HHT [129]. Pulmonary circulation is especially affected in all HHT cases, leading to potential for life-threatening hemorrhage. Studies have found a general loss of pulmonary capillaries and gain of AVM with primarily venous identity endothelium, perhaps due to excessive endothelial proliferation. Thus, loss of TGFβ regulation of endothelial quiescence and endothelial differentiation may be an underlying molecular defect in these individuals [130]. This is highlighted by the newly identified mutation in PTPN14 associated with HHT, especially the pulmonary manifestations [131]. PTPN14 codes for a protein tyrosine phosphatase; its expression is modulated by both ALK1 and EphrinB2, and PTPN14 knockdown leads to increased angiogenesis in vitro due to enhanced ­number of tip cells [131].

6.2 Organ Fibrosis

Due to the known association between TGFβ signaling and fibrosis in many ­systems, it is perhaps not surprising that vascular manifestations of this situation arise. Both the mural cell and endothelial cell components of the microcirculation are documented targets of TFGβ-mediated fibrosis in several organs. For instance, during the development of liver cirrhosis, hepatic stellate cells or Ito cells (the vascular sinusoidal mural cells) express excessive collagen upon TGFβ signaling and in a neuropilin-1-dependent fashion [132]. In the kidney, the renal glomerulus is prone to mesangial cell proliferative glomerulonephritis. These cells are modified and specialized pericytes of the glomerular filtration capillaries, and their proliferation is driven in part by excessive TGFβ production [133]. Interestingly, the bioactive lipid mediator sphingosine-1 phosphate1 (S1P1) is able to cross activate TGFβ signaling in renal mesangial cells via Smad1, 2, and 3 [133], indicating possible transactivation of TGFβ signaling pathways.

In addition to targeting vascular mural cells to promote fibrosis, TGFβ can also induce endothelial-to-mesenchymal transition (EndoMT), a process by which endothelial cells transform into mesenchymal cells, such as fibroblasts and bone cells. This phenomenon is well documented in heart and kidney fibrosis and is mediated by the snail family of transcriptional repressors. Both canonical and noncanonical TGFβ signaling, including Smad, MEK, PI3K, p38 MAPK, c-Abl, and PKC-δ signaling, have been reported to mediate an EndoMT response to TGFβ (reviewed in [59]).

6.3 Cancer

Perhaps the best-studied example of pathological angiogenesis where TGFβ plays a significant role is in the neovascularization of solid tumors. TGFβ orchestrates a switch from vascular inhibition to pro-angiogenic activity, likely indirectly via stimulation of cancer cell production of pro-inflammatory and immune suppressive gene products (as reviewed by Tian et al. [113]). Proteomic comparison of angiogenesis in glioblastoma to physiological angiogenesis (endometrial tissue) found numerous TGFβ target genes overexpressed in glioma vessels compared to endometrium, in particular TGFβ-induced protein ig-h3, periostin, integrin-αv, and tenascin C [134]. We found that TGFβ was able to downregulate the expression of VEGFR2 in colorectal tumor vasculature in an Alk5/Smad2-­dependent fashion [58]. VEGFR2 expression on glioma blood vessels increased with tumor progression, and the proportion of p-Smad2-positive endothelial cells was significantly higher in tumor vessels compared to normal brain vasculature [135]. There is evidence that ALK1 signaling in cancer angiogenesis may modulate cross talk between EC and pericytes, and inhibition of ALK1 may be especially effective in VEGF refractory tumors [128]. Our results support the possibility that ALK5 activation in endothelial cells may be, at least in part, responsible for development of tumor vessel refractoriness to VEGF inhibition by modulating the levels of VEGFR2 and likely VEGF dependence on endothelial cells.

Finally, endoglin, an essential modulator of TGFβ signaling in endothelial cells, has been shown to be significantly upregulated in tumor-associated endothelium and its expression correlated with poor prognosis in patients with various tumor types including breast, lung, colorectal, prostate, gastric, endometrial, hepatocellular, ovarian, cervical, and head and neck cancers, as well as glioblastoma (reviewed in [136]). Tumor growth and vascularization is reduced in Eng− heterozygous mice [137], and both endoglin-neutralizing antibodies [136] and soluble endoglin [138] target the tumor vasculature and inhibit tumor growth in experimental models, suggesting endoglin as another potential therapeutic target in cancer.

7 Conclusion

In summary, this review highlights the central actions of TGFβ on the vascular components undergoing angiogenesis, which are broad ranging and context-­dependent. In particular, the pleomorphic responses to TGFβ occurring in pathological vs. physiological angiogenesis provide avenues for improved understanding and therapeutic control of these events.