**3. Vasculogenesis**

Likewise, TGF-β acts as a cytostatic factor by decreasing c-Myc expression and downregulating the inhibitor of DNA-binding protein (ID) 1 and ID3 transcription factors. ID1 and ID3 are involved in differentiation, cell cycle progression and self-renewal of stem cells [21, 22]. TGF-β elicits c- Flk-1myc repression by promoting SMAD3 binding to a repressing Smadbinding element (RSBE) at the c-myc promoter [23]. c-Myc can be recruited to the promoters of *CDKN1A* and *CDKN2B* by the Myc-interacting zinc-finger (MIZ-1). This blocks CDK expression and results in apoptosis [24]. Additionally, in endothelial cells (ECs), TGF-β can target a second receptor type 1, ALK-1, which signals through Smad1/5/8 and stimulates

**Figure 2.** TGF-β canonical pathway. **(a)**In normal cells and early stages of cancer TGF-β promotes cell cycle arrest. Repressors of the pathway are shown in red. Blue dots represent protein phosphorylation and **(b)**in endothelial cells, an

6 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Several proteins are known to antagonize canonical TGF-β signaling. For example, (i) PI3K activates AKT which phosphorylates the SMADs-FOXO complex and inhibits its translocation to the nucleus [21], (ii) foxhead box G1 (FOXG1) inhibits the SMADs-FOXO complex [21], (iii) SMAD7 can trigger TβRI for proteosomal degradation by recruiting SMAD-specific E3 ubiquitin protein ligase (SMURF1) and SMURF2 [26], (iv) SMAD6 blocks SMAD1 through SMAD4 binding, (v) Erk proteins phosphorylate SMADs and inhibit their nuclear translocation, (vi) BAMBI, a pseudoreceptor, dimerizes with TβRI leading to its inactivation, (vii) FKBP12 binds to TβRI and impedes its phosphorylation, activation and signaling [27] and (viii) protein arginine N-methyltransferase 1 (PRMT1) methylates SMAD6 and allows BMP

angiogenic factors, such as interleukin 1 receptor-like 1 and ID1 (**Figure 2**) [25].

signaling through SMADs1/5 [28–30].

alternative pathway promotes cell proliferation.

During embryogenesis, the development of the vascular system is divided into three stages, vasculogenesis, angiogenesis and arteriogenesis. Vasculogenesis occurs in embryonic organs as well as extraembryonic tissues such as the placenta, yolk sac and allantois [31]. The earliest discernible structures in vasculogenesis, the blood islands, are formed in the mouse yolk sac by embryonic day (E) 6.5–7. This structure contains precursor cells or hemangioblasts, which differentiate to EC and hematopoietic cells [32]. At E8.5, cells located toward the periphery of the blood island, or angioblasts, differentiate into EC, while cells located toward the central region give rise to hematopoietic precursor cells. Next, lumenization takes place; tight junctions and basement membranes develop, and pericytes are recruited to blood vessels and promote maturation [33].

Several growth factors have been identified to regulate vasculogenesis, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), the hedgehog family, neuropilins, integrins, fibronectin and TGF-βs. FGF-2 has been reported to participate in the generation of the angioblast in quail/chick chimeras and in vessel formation [34].

Hedgehog signaling has been shown to be crucial in the initial steps of vasculogenesis. It promotes differentiation of the primitive endoderm into both, endothelial and hematopoietic lineage [35]. For instance, blocking Indian hedgehog (Ihh) causes signaling repression from the visceral ectoderm and consequently abrogation of vasculogenesis and hematopoiesis in anterior epiblast [35]. Deletion of *Ihh* in mouse caused 50% lethality at midgestation with the remaining 50% dying at birth. Defects in blood vessel formation have been proposed as the cause for the lethality, which has been supported by experiments showing: (i) deletion of Sonic hedgehog (*Shh*) in mice resulted in a reduction in vascularization in lung [36], (ii) overexpression of *Shh* resulted in an increase in vascularization in neuroectoderm [37], (iii) depletion of *Shh* in zebrafish caused defective vasculature [38] and (iv) depletion of *Ihh* from stem cell-derived embroid bodies inhibited blood island differentiation [39].

VEGF signaling is crucial in vasculogenesis. Genetic studies have shown that deletion of *Flt-1* (VEGFR1), *Flk-1/KDR* (VEGFR2) and one or both alleles of *VEGF* cause embryonic lethality. VEGFR1 mutants exhibit aberrant central localization of the angioblasts in the blood island, instead of their normal localization toward the periphery [40]. These results implied that the growth of ECs was not inhibited in this region and led to the idea that VEGR1 hampers signaling from VEGF by ligand sequestration [33]. In addition, VEGFR2 mutants die around E9. In these embryos, both vasculogenesis and hematopoiesis do not initiate which was explained by faulty blood island in which cell migration was abrogated [41, 42]. Similarly, mutant heterozygous for VEGF die by E11 and showed impaired vasculogenesis and angiogenesis. These embryos showed severe abnormalities, such as underdeveloped brain and heart, decreased number of nucleated red blood cells in blood islands and aberrant vasculature in nervous system and placenta [43].

Neuropilins are co-receptors for VEGF receptors. Nrp1 is found in ECs of arteries, while neuropilin 2 (Nrp2) is found at the endothelium of lymphatic vessels and veins. Deletion of *Nrp1* in mice affects severely the central and peripheral nervous systems, as well as the yolk sac vasculature [44]. In contrast, depletion of *Nrp2* has no effects in the vasculature of arteries or veins, but it does affect angiogenesis of the lymphatic vasculature [45, 46]. In addition, mice harboring deletions in both neuropilins have shown obstruction in vasculogenesis in the yolk sac and in the formation of the primary vascular plexus [47].

## **4. Developmental angiogenesis**

Angiogenesis is the formation of new blood vessels from existing vasculature. It occurs by mechanisms including sprouting angiogenesis and intussusceptive angiogenesis (**Figure 3**). Sprouting angiogenesis initiates with the selection of endothelial tip cells at the vessel wall. These cells react toward extracellular stimuli and secrete proteolytic enzymes to digest the surrounding ECM. Tip cells are connected to endothelial stalk cells to direct the vascular sprout [33, 48, 49]. Once the new tube is formed and a lumen is established, the vessel is stabilized by the recruitment of pericytes to capillaries or vascular smooth muscle cells (vSMC) to arteries and veins [6, 33], leading to re-establishment of mature blood vessels.

Intussusceptive angiogenesis, also known as splitting angiogenesis, results in the formation of intermediate intracapillary pillars. This mechanism is more efficient than sprouting angiogenesis since it does not require cell proliferation. Instead, it needs the reorganization of existing ECs. In this process, (i) ECs from opposite sides of the blood vessel make contacts, (ii) ECs from both ends reorganize and cause a splitting in the vessel wall, (iii) an interstitial pillar core is generated and (iv) myofibroblasts, pericytes and finally collagen invade the pillar and a basement membrane is formed [33, 49, 50].

**Figure 3.** Mechanisms of angiogenesis. **(a)**Sprouting and **(b)**intussusceptive angiogenesis.

## **5. TGF-β in vasculogenesis and angiogenesis**

#### **5.1. Mutant phenotypes in mice lacking components of the TGF-B pathway**

TGF-β mRNA was initially detected by PCR in preimplantation stages and in situ expression was present as early as E7.5, suggesting important roles in early development [51, 52]. In the embryo, proper TGF-β was detected in angioblast progenitors within the primitive heart mesoderm. Likewise, its expression was detected in extraembryonic tissues, including in the allantois mesoderm, and within blood islands of the yolk sac [53].

Deletion of the TGF-β1 gene in mice resulted in 50% lethality in utero, with the remaining 50% of mutant mice surviving up to three weeks postnatally. Histopathology analyses showed multifocal inflammatory cell infiltration and necrosis in several organs, especially the heart and stomach [53, 54]. It was subsequently shown that maternal contributions of *TGFB1* RNA and other genetic and epigenetic factors contributed to 50% postnatal survival [55].

The 50% of TGF-β mutants showed lethality and resorption by E10.5. Analysis of E8.5 embryos did not show significant morphological defects. However, analysis of E9.5 and E10.5 embryos resulted in a range of phenotypic defects within the yolk sac. While in some cases, vasculogenesis was delayed; in other cases, a dramatic reduction in size was observed and it was accompanied by weak and disorganized primary vessels, with some areas displaying complete vessel depletion. Analysis of the yolk sac vasculature indicated that the defects occurred during differentiation of ECs and hematopoietic cells. In contrast, the initial differentiation of mesodermal cells into ECs was not affected [52].

Genetic ablation of TβRII resulted in very similar phenotypes as in TGF-β1 mutants, with alterations in yolk sac vasculature and embryonic lethality by E10.5- E11.5 [56]. Mutants in endoglin also showed defects in vascular vessels within and outside the embryo. Embryo lethality was observed at E11.5, with mutants developing focal hemorrhage [57, 58]. Similarly, engineered mutations in mice that abrogate the expression of *ALK1, ALK5, SMAD1* or *SMAD5* resulted in defects in cardiovascular development [57, 59, 60].

#### **5.2. Roles of TGF-β in angiogenesis**

vasculature [44]. In contrast, depletion of *Nrp2* has no effects in the vasculature of arteries or veins, but it does affect angiogenesis of the lymphatic vasculature [45, 46]. In addition, mice harboring deletions in both neuropilins have shown obstruction in vasculogenesis in the yolk

Angiogenesis is the formation of new blood vessels from existing vasculature. It occurs by mechanisms including sprouting angiogenesis and intussusceptive angiogenesis (**Figure 3**). Sprouting angiogenesis initiates with the selection of endothelial tip cells at the vessel wall. These cells react toward extracellular stimuli and secrete proteolytic enzymes to digest the surrounding ECM. Tip cells are connected to endothelial stalk cells to direct the vascular sprout [33, 48, 49]. Once the new tube is formed and a lumen is established, the vessel is stabilized by the recruitment of pericytes to capillaries or vascular smooth muscle cells (vSMC) to arteries and veins [6, 33], leading to re-establishment

Intussusceptive angiogenesis, also known as splitting angiogenesis, results in the formation of intermediate intracapillary pillars. This mechanism is more efficient than sprouting angiogenesis since it does not require cell proliferation. Instead, it needs the reorganization of existing ECs. In this process, (i) ECs from opposite sides of the blood vessel make contacts, (ii) ECs from both ends reorganize and cause a splitting in the vessel wall, (iii) an interstitial pillar core is generated and (iv) myofibroblasts, pericytes and finally collagen invade the pillar and

sac and in the formation of the primary vascular plexus [47].

8 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**4. Developmental angiogenesis**

a basement membrane is formed [33, 49, 50].

**5. TGF-β in vasculogenesis and angiogenesis**

**5.1. Mutant phenotypes in mice lacking components of the TGF-B pathway**

**Figure 3.** Mechanisms of angiogenesis. **(a)**Sprouting and **(b)**intussusceptive angiogenesis.

allantois mesoderm, and within blood islands of the yolk sac [53].

TGF-β mRNA was initially detected by PCR in preimplantation stages and in situ expression was present as early as E7.5, suggesting important roles in early development [51, 52]. In the embryo, proper TGF-β was detected in angioblast progenitors within the primitive heart mesoderm. Likewise, its expression was detected in extraembryonic tissues, including in the

of mature blood vessels.

Early work to determine the roles for TGF-βs in ECs was contradictory. TGF-β signaling was initially found to inhibit cell migration and proliferation [61, 62], yet later studies indicated that it promotes cell proliferation [63–66]. The relative levels of expression of TGF-β seem to partially explain these discrepancies, with low doses promoting angiogenesis and higher levels resulting in growth inhibition of ECs and maturation of blood vessels [66, 67]. For instance, during blood vessel coverage by smooth muscle cells, TGF-β paracrine signaling from ECs to mesenchymal cells results in vascular smooth muscle cell and pericyte differentiation [6].

TGF-β also plays a role in the angiogenic process of hypoxic tissue. For instance, during infarction (stroke), neovascularization occurs primarily at the ischemic penumbra (periphery of the infarct), which correlates with high levels of both mRNA and active TGF-β protein [68]. Similarly, during organ transplant VEGF and TGF-β1, levels are increased in devascularized hypoxic tissue. TGF-β3 was also upregulated in hypoxic tissues, but to a lesser degree [69].

TGF-β regulates angiogenesis by different mechanisms; for example, it is involved in vessel proliferation and maturation by alternating two signaling cascades with opposite effects (ALK1 and ALK5). Likewise, TGF-β can promote its own expression, and it upregulates the expression of other angiogenic factors such as, platelet-derived growth factor (PDGF), interleukine-1, basic fibroblast growth factor (bFGF), tumor necrosis factor alpha and transforming growth factor alpha [70]. TGF-β can change the functions of other factors, such as VEGF, from prosurvival to pro-apoptotic [71]. Similarly, *in vitro* work has shown that in ECs TGF-β upregulates the expression of endothelin (*EDN1*), *PDGFA* and *PDGFB*, nitric oxide synthase 3 (*NOS3*), actin, alpha 2, smooth muscle, aorta (*ACTA2*), secreted protein acidic and cysteine rich (*SPARC*), *TSP-1*, fibronectin (*FN1*), collagens (*COL1A1, COL4A1*, and *COL5A1*), plasminogen activator (*PLAU*), serpin family E member 1 (*SERPINE1*) and integrins (*ITGB1, ITGB3, ITGAV, ITGA2* and *ITGA5*). It can also downregulate several genes, such as selectin-E (*SELE*), *KDR*, von Willebrand factor (*VWR*), thrombomodulin (*THBD*), monocyte chemo-attractant protein (*MCP1*), C-X-C motif chemokine ligand 1 (*CXCL1*), integrins (*ITGB1, ITGB3, ITGA5* and *ITGA6*), *TIMP1* and *PLAU* [70].

Levels of ALK1 and ALK5 determine TGF-β mitogenic or mitostatic responses in ECs. ALK5- Smad2/3 signaling stimulates transcription of ECM proteins such as fibronectin and plasminogen activator inhibitor type 1, which promote the resolution of angiogenesis by inducing vessel maturation. In contrast, signaling via the ALK1-Smad1/5/8 pathway generates antiangiogenic responses [25, 51]. This requires a TGF-β accessory receptor, endoglin, which enhances ALK1 signaling and inhibits ALK5 cytostatic phenotype [72]. More recent work has shown that in the mouse eye retina the leucine-rich alpha-2-glycoprotein (Lrg1), which binds endoglin, promotes angiogenesis through Alk1-Smad1/5/8 in the presence of TGF-β [73].

#### *5.2.1. TGF-β signaling in CNS development*

In the mammalian CNS, neurons, astrocytes, pericytes and ECs closely interact to form a multicellular neurovascular unit [11]. During embryonic brain development, TGF-β is critical for sprouting angiogenesis of the CNS. In particular, TGF-β has been shown to work in conjunction with αν integrins to regulate paracrine signaling between neuroepithelial cells and ECs within neurovascular units. In mouse, embryos deletion of αν integrin showed vascular defects that were restricted mainly to the brain. This phenotype was recapitulated in β8 integrin mutant embryos. In contrast, deletion of β3 and β5 integrins did not cause brain vasculature abnormalities [74, 75]. Cell type-specific deletion of αν and β8 integrins in nervous system glial cells resulted in developmental intracerebral hemorrhage as well as postnatal motor dysfunction and seizures. Of note, the brain hemorrhage observed in embryos was absence in adult mice, suggesting that a compensatory mechanism that repairs hemorrhage occurs after birth [75, 76]. Interestingly, αν ablation in vascular ECs did not show a phenotype [75]. In later work, in which an outbred background was used to overcome the effects of β8−/− embryonic lethality, it was shown that adult mice lacking β8 integrin displayed neurovascular pathologies [77]. Most notably, adult β8 integrin mutants displayed a reduction in olfactory bulb size and abnormalities at the subventricular zone and rostral migratory stream. Neuroblasts generated in the subventricular zone utilize blood vessels as guides to migrate within the rostral migratory stream and differentiated to neurons within the olfactory bulbs. The size-reduced olfactory bulbs in adult β8−/− mice revealed essential roles for this integrin in promoting neuroblast migration along blood vessels. These defects correlated with a reduction in TGF-β signaling in neurospheres dissected from β8−/− mice [77].

The brain vascular defects observed in *Itgb8* null mutants are also shared by *Tgfb1* and *Tgfb3* loss of function mutants. In addition, mutating the integrin-binding RGD binding site in *Tgfb1* leads to early embryonic lethality [78]. Similarly, mice lacking both β6 and β8 integrins showed similar phenotypes as null mutants for *Tgfb1* and *Tgfb3* [78].

More recently, integrin β8 and Nrp1 have been shown to mediate neuroepithelial-endothelial cell interactions. β8 integrin in the neuroepithelium activates TGF-β signaling in ECs, while Nrp1 suppresses canonical TGF-β signaling, thus controlling normal sprouting angiogenesis [79]. Disruption of TGF-β signaling by targeting β8 integrin or Nrp1 results in excessive vessels sprouting and branching and formation of dysplastic glomeruloid-like vessels that are hemorrhagic [80].

The ανβ8-TGF-β connection in developmental angiogenesis in the brain also regulated neovascularization in the developing retina, where β8 integrin is expressed in astrocytes and Muller glial cells, a neuroepithelial cell type specifically found in the retina [81]. β8−/− retinas display abnormalities in the formation of the secondary vascular plexus, including impaired sprouting and formation of blood vessels with glomeruloid-like tufts. In addition, intraretinal hemorrhage was detected [81]. Furthermore, ablation of αν or β8 integrins but not of *Tgfbr2* in astrocytes resulted in defects in angiogenesis, and blocking TGF-β1 with neutralizing antibodies affected paracrine signaling to ECs [81]. This work was confirmed later in a study showing that *Tgfbr2* deletion in ECs of neonatal mice caused bleeding in the brain and vascular abnormalities, hemorrhage and deficiency in the formation of the deeper vascular network in the retina [82, 83]. Similarly, reduced Smad2 phosphorylation was observed in ECs from retina of *Tgfbr2* knockout mice [82].
