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Formation of new vascular networks, whether in health or disease, is based on the ability of the constituent cells to participate in the web of intercellular communications. The mediators of these interactions can be broadly divided into several categories, including (a) specialized ("professional") signaling effectors required for vascular homeostasis, (b) pleiotropic effectors endowed with angiogenesis-regulating activities (stimulators and inhibitors), along with other functions, and (c) other regulators that link angiogenesis to processes, such as hemostasis, bone marrow stimulation, neuronal growth, or immunity (Carmeliet and Jain, 2011). Although this molecular network contains redundancies, feedbacks and complex response patterns, at the heart of it are individual key molecules that can serve as potential targets for the anti-angiogenic therapy (Table 11–1 and Figs. 11–5, 11–6, 11–7, 11–8, 11–9, 11–10). Key elements of this molecular circuitry are shown in Figure 11–5 and are discussed below.
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11.4.1 Vascular Endothelial Growth Factor Family
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VEGF-A, also known as vascular permeability factor (VPF), is the key member of a larger family of related polypeptides, which also includes VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placenta growth factor (PlGF), all related to platelet-derived growth factor (PDGF) (Dvorak et al, 1995; Ferrara, 2005). These ligands form functional homo- and heterodimers and interact with at least 4 different receptors (VEGFRs), of which VEGFR1/FLT-1, VEGFR2/KDR/FLK-1), and VEGFR3/FLT-4 are signaling tyrosine kinases (RTKs), while Neuropilin 1 (NRP1) is a coreceptor (Ferrara, 2005). The relatively selective (albeit not exclusive) expression of VEGFRs by ECs (of blood vessels, lymphatics, and in tumors) allows VEGFs to control vascular processes in a potent and combinatorial manner (see Fig. 11–5). Thus, VEGFR1 binds VEGF-A, VEGF-B and PlGF, while VEGFR2 interacts mainly with VEGF-A (and VEGF-E), and VEGFR3 interacts preferentially with VEGF-C and VEGF-D. NRP1, the semaphorin receptor involved in neuronal guidance, is also expressed by ECs, where it acts as a coreceptor for VEGFR2. NRP1 binds only certain VEGF-A splice isoforms (eg, VEGF165) (Ferrara, 2005).
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Germline deletion of each of the VEGF receptors leads to early embryonic lethality amidst vascular defects (Carmeliet and Jain, 2011), suggesting their essential role in vascular development and angiogenesis. However, the angiogenic responses elicited by exposure to the naturally occurring VEGF-A homodimers are mainly mediated mainly by VEGFR2. This involves robust phosphorylation and recruitment of intracellular signaling targets (see Fig. 11–5). Although VEGFR1 also binds VEGF-A with high affinity (10-fold greater than VEGFR2), the phosphorylation of this receptor in ECs is weak, and deletion of its kinase domain is relatively inconsequential for vascular development (Carmeliet and Jain, 2011). Indeed, VEGFR1 is often expressed as a splice variant composed of the soluble extracellular domain (sFlt-1), which acts as a natural VEGF antagonist (VEGF "sink") (Carmeliet and Jain, 2011). However, VEGFR1 does possess an important regulatory function for macrophages, and certain cancer cells, in which it mediates migratory responses in response to VEGF (Carmeliet and Jain, 2011).
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The crucial role of VEGF/VEGF-A is underscored by the unprecedented embryonic lethality and profound vascular defects in mouse embryos lacking even a single vegf allele (haploinsufficiency) (Carmeliet and Jain, 2011; Ferrara, 2005). Indeed, VEGF-A acts as a potent mitogen, motility, and survival factor for ECs, and a chemoattractant for their progenitors (Ferrara, 2005). These powerful influences are regulated by the organ- and context-specific alternative splicing of the VEGF-A transcript resulting in several protein isoforms. The main species generated during this process are designated according to the number of their constituent amino acids and include VEGF121 (121 amino acids), VEGF145, VEGF165, VEGF189, and VEGF206 (Ferrara, 2005). This splicing removes various sequences from within the region encoded by exons 6 and 7, while leaving the sequences corresponding to exons 1 to 5 and 8 intact.
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Consequently, VEGF-A splice isoforms exhibit reduced heparin binding, increased solubility and diminished association with both cellular membranes and the ECM, depending on their size. Thus, the shortest VEGF121 isoform is highly soluble, while VEGF189 is mostly cell bound, and VEGF165 expresses intermediate properties, and the greatest angiogenic activity. The latter is a function of the more stable angiogenic gradient that VEGF165 (VEGF164 in the mouse) can form within the interstitial space in tissues. This gradient favors directional responses of tip cells and robust sprouting. In addition, unlike VEGF121, VEGF165 interacts with both VEGFR2 and NRP1, which is thought to contribute to more pronounced endothelial responses (Ferrara, 2005). Notably, the VEGF-A splicing process also results in expression of alternative ligands endowed with antagonistic (antiangiogenic) activities. For instance, one such variant, VEGF165b, interferes with angiogenesis, and exerts a modulating influence on responses of blood vessels to angiogenic growth factors involved in tissue remodeling and inflammation (Nowak et al, 2008).
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The remaining members of the VEGF family play more restricted roles in vascular processes, and germline deletions of the respective genes do not have haploinsufficient consequences. VEGF-C/D control lymphangiogenesis, while VEGF-B provides additional survival protection to ECs. VEGF-E is a VEGF-like gene found in the genome of the Orf virus (Ferrara, 2005). In some instances, the effects of these factors become prominent only under pathological conditions (eg, in the case of PLGF). Finally, although VEGF-related factors were originally viewed as having mainly paracrine effects, VEGF-A was recently shown to be expressed at low, but functionally meaningful, levels by ECs themselves, which contributes to vascular homeostasis (Lee et al, 2007).
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11.4.2 Platelet-Derived Growth Factor Family
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This family of VEGF-related growth factors consists of 4 members: PDGF-A, PDGF-B, PDGF-C, and PDGF-D, the homo- or heterodimers of which interact preferentially with 1 of 2 main cellular receptor tyrosine kinases, namely, PDGFRα and PDGFRβ, (Andrae et al, 2008). While PDGFs play multiple roles in development, disease, and cancer, they are also central to vascular growth, especially blood vessel maturation. Thus, PDGF-BB homodimers are produced at high levels in endothelial tip cells and in phalanx cells of arteriogenic vessels, whereby they attract mural cells harboring PDGFRβ (Andrae et al, 2008). Other members of this family may also contribute to various angiogenic events indirectly, for example, by influencing the expression of angiogenesis-related genes (eg, VEGF) in cancer cells and fibroblasts (Dong et al, 2004). Indeed, recent studies suggest that these mechanisms may bypass the requirement for VEGF and contribute to tumor resistance to VEGF-directed therapies (Shojaei et al, 2009).
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This family of VEGF-unrelated growth factors has been found to induce VEGF-like effects in ECs, especially in endocrine organs. This includes growth, migration, permeability, fenestration (formation of trans-endothelial openings), and angiogenesis (LeCouter et al, 2002). These mediators consist of the endocrine gland VEGF/prokineticin 1 (EG-VEGF/PK1) and Bombina variegata-secreted protein 8/prokineticin 2 (Bv8/PK2), which both interact with their respective G-protein–coupled receptors (PK-R1 and PK-R2) (Ferrara, 2010). Although prokineticins appear to have a role in homeostasis of the microcirculation in endocrine glands, they are also found in cancer, where they may be produced by myeloid cells and contribute to VEGF-independent angiogenesis (Shojaei et al, 2009).
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11.4.4 Angiopoietins and TIE Receptors
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At least 3 related ligands known as angiopoietins (ANG1, -2, and -4) play diverse roles in endothelial cell survival, vascular development and maturation, as well as angiogenesis and lymphangiogenesis in humans (Ang3 is a mouse ortholog of ANG4) (Yancopoulos et al, 2000; Jones et al, 2001). Angiopoietins interact with TIE2/TEK receptor tyrosine kinase expressed preferentially (but not exclusively) on ECs, which also harbor a related orphan receptor known as TIE1 with poorly understood function. Nonetheless, both TIE1 and TIE2 receptors are essential for vascular development (Augustin et al, 2009). Their activity is controlled by the vascular endothelial receptor tyrosine phosphatase (VE-PTP) (Li et al, 2009). As mentioned earlier, the best understood function of the angiopoietin/TIE2 circuitry is in the regulation of EC survival, vascular permeability, and recruitment of mural cells. In this setting, ANG1 emanates from perivascular tissues and acts as the TIE2 agonist. In contrast, ANG2 is produced largely by ECs exposed to VEGF, and it blocks ANG1/TIE2 interaction, thereby destabilizing endothelial-PC contacts (see Fig. 11–5). Thus, in the presence of VEGF, the exposure to ANG2 promotes vascular sprouting, but when VEGF levels are low, ANG2 promotes vascular regression (Augustin et al, 2009 for review).
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Cell-cell contact-dependent regulatory interactions often involve Notch receptors (NOTCH 1 to 4) and their 5 cell-associated ligands, including Jagged (JAG) 1, JAG 2, Delta-like 1, 3, and 4 (DLL1, DLL3, and DLL4) (Dufraine et al, 2008). NOTCH 1 and 4 are expressed in ECs (NOTCH 4 preferentially) and, along with DLL4, are required for proper vascular development. DLL4 is expressed in angiogenic tip cells and plays a role in maintaining their identity, whereas JAG 1 may be involved in modulating these effects and recruitment of additional tip cells. JAG 1 may also mediate direct interactions between endothelial and mural cells, or between tumor cells and the vasculature (Dufraine et al, 2008). Another Notch ligand, DLL1, is involved in vascular remodeling and arteriogenesis. Interactions with these ligands trigger proteolytic release of the intracellular domain of NOTCH (ICN), which is responsible for modulation of gene expression and cellular effects (Dufraine et al, 2008).
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11.4.6 Ephrins and EPH Receptors
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The definition of arterial and venous identity in the developing vascular system is largely attributed to the unique bidirectional signaling mechanism mediated by transmembrane ligands of the ephrin family, notably Ephrin B2 (on the arterial side) and their EPHB4 receptors (on the venous side; see Fig. 11–5). The expression of other Ephrins (eg, A1, A2, B1 and B3) and additional EPH receptors (EPHB2, EPHB3) is also observed in ECs, including in tumors. These Ephrins are implicated in various angiogenesis-related processes, such as endothelial–PC interactions, interactions of blood vessels with tumor cells, and cooperation with other angiogenic factors. The latter is exemplified by Ephrin B2-dependent regulation of VEGFR2 endocytosis and signaling (Wang et al, 2010).
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11.4.7 Vascular Integrins, Cadherins, and Adhesion Molecules
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Adhesion molecules connect ECs with their extraluminal, intercellular, and intraluminal surroundings (see Fig. 11–7). Thus, direct interaction between the abluminal surfaces of ECs and the extravascular ECM (basement membrane) is essential for survival, homeostasis, and angiogenic activity of these cells. Quiescent ECs are anchored to the permanent basement membrane, which is composed of laminin and collagen type IV. In contrast, angiogenic ECs are surrounded by provisional ECM containing fibrin, vitronectin, fibronectin, and partially proteolyzed collagens. These various interactions are mediated by a family of heterodimeric, transmembrane ECM receptors, known as integrins (each composed of α and β subunits), which recognize specific motifs within their target ECM proteins (eg, RGD peptides).
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Integrins are viewed as functional hubs that localize and regulate the activities of other angiogenic effectors, including VEGFR2, other receptors, MMPs, angiopoietins, and intracellular signal transducing kinases (PKB/AKT, FAK, ILK, SRC). Growth factors upregulate the expression of several Integrins on the surface of ECs in blood vessels (αvβ3, αvβ5, α1β1, α2β1, α4β1, α5β1) and lymphatics (α1β1, α2β1, α4β1, α9β1) (Avraamides et al, 2008). Although acute pharmacological disruption of the adhesive function of certain integrins (αvβ3) resulted in antiangiogenic effects (Cheresh and Stupack, 2002), the corresponding gene targeting studies suggested a more complex involvement. This is illustrated by the enhanced, rather than reduced, tumor angiogenesis in β3/β5-deficient mice (Reynolds et al, 2002). Indeed, other mutations of vascular integrins may either reduce (β3 mutant, α1β1), or increase (α2β1) adult angiogenesis (Avraamides et al, 2008).
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In contrast to the outward-oriented adhesion that is mediated by endothelial integrins, vascular endothelial cadherin (VE-cadherin/CD144) mediates formation of intercellular junctions between ECs within the vascular tube. This contact contributes to the unique properties of the endothelial lining (barrier function, restricted permeability, homotypic adhesion) (Nyqvist et al, 2008). VE-cadherin is selectively expressed by the cells of endothelial lineage and serves as their genetic marker. Importantly, gene targeting studies revealed that VE-cadherin is essential for vascular development, and for the functionality of some of its key regulators, such as VEGFR2 (Carmeliet and Jain, 2011). N-cadherin is also expressed by ECs, albeit not as selectively, and is required for vascular integrity and developmental angiogenesis. Cadherins bind to other cadherins on the adjacent cells and orchestrate formation of physical contacts, referred to as adherens junctions. These structures along with tight junctions (involving claudins) influence the functional integration of the endothelial lining.
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Interactions between the endothelium and circulating immune, myeloid, inflammatory, and progenitor cells, and platelets are mediated by a diverse class of intraluminal adhesion molecules, including selectins (eg, E-selectin), integrins (α4β1/VLA4), and members of the immunoglobulin family of cell adhesion molecules (CAMs, eg, ICAM-1/2 and VCAM1), all of which play different roles in angiogenesis (Francavilla et al, 2009).
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11.4.8 Angiogenesis-Regulating Proteases
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During blood vessel formation, the dissolution of the endothelial basement membrane, liberation of progenitor cells from the bone marrow, release of VEGF and other growth factors from their ECM stores, and the activation/modulation of the coagulation system, all involve various classes of proteases and their endogenous inhibitors (Kalluri and Zeisberg, 2006; van Hinsbergh et al, 2006). Of particular note are matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs 1 to 4), often expressed in the tumor microenvironment (see Fig. 11–6). These molecules participate in ECM breakdown, generation of angiogenesis-regulating protein fragments, regulation of tumor and endothelial cell invasion, and may also act as ligands of cellular receptors (Kessenbrock et al, 2010). The main MMPs involved in tumor angiogenesis are MMP-1, MMP-2, MMP-9, and MMP-14. Their role is illustrated by the impairment of tumor neovascularization in mice with disrupted MMP9 gene expression (Kessenbrock et al, 2010). Other proteases involved in angiogenesis include cathepsins, coagulation factor VIIa, thrombin (IIa), urokinase-type plasminogen activator (uPA), as well as members of the disintegrin and metalloproteinase domain (ADAM) and thrombospondin motif-containing (ADAMTS) families of proteases (van Hinsbergh et al, 2006).
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11.4.9 Angiogenesis Stimulators and Inhibitors
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The mechanisms described above are required for the execution of one or more of the angiogenic programs, but they are not necessarily the only or initial triggers of tumor angiogenesis. In cancer and other angiogenesis-dependent diseases, vascular growth may be initiated by a global shift in expression of several molecules, and the related cumulative change in the regulatory environment, the constituents of which are broadly classified as angiogenesis stimulators and inhibitors (see Table 11–1). It is believed that the onset of angiogenesis ("angiogenic switch," see Fig. 11–8) is triggered when the balance between these opposing influences is tilted beyond a certain discrete threshold, and in favor of stimulators (Folkman and Kalluri, 2003). Although this transition may appear as a binary event, the biological mechanisms involved can be complex, discontinuous, incremental, or oscillatory. The angiogenic threshold and the magnitude of endothelial cell responses may also differ depending on the genetic background and several other factors (Rohan et al, 2000). Furthermore, the composition of angiogenic factors may change over time during tumor progression (see Fig. 11–8), leading to a series of transitions rather than a single switch (Folkman and Kalluri, 2003).
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Stimulators of angiogenesis include factors acting directly on ECs, such as VEGF, PlGF, FGF1/2, HGF, or interleukin 8 (IL-8). Similar effects can also result from indirect actions of their potent inducers, for example, transforming growth factors alpha or beta (TGFα, TGFβ) and several other cytokines and chemokines (eg, pleiotrophin). Some of these factors may act by recruitment of inflammatory cells (eg, IL-6), or bone marrow progenitor cells (eg, VEGF or stromal derived factor 1 [SDF1]), or through concomitant stimulation of both endothelial and inflammatory cells (eg, IL-8) (Kerbel, 2008).
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The endogenous angiogenesis inhibitors are thought to maintain blood vessels in their quiescent state, or limit the magnitude of angiogenic responses, both locally and systemically (Folkman and Kalluri, 2003). Angiogenesis inhibitors belong to different classes of molecules, including certain ECM proteins (thrombospondins 1 and 2) (Bouck et al, 1996), or their proteolytic fragments (endostatin, tumstatin, arresten), fragments of enzymes and zymogens (angiostatin, PEX domain), fragments of coagulation-related chemokines (platelet factor 4 [PF4]), and of hormones (prolactin 16-kDa fragment), along with certain cytokines (interferons α, β, and γ) (Folkman and Kalluri, 2003; see Table 11–1).
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11.4.10 Coagulation System and Platelets as Regulators of Tumor Angiogenesis
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The discovery of VEGF in 1983 (Senger et al, 1983), and its role as a vascular permeability factor (VPF), originally linked angiogenesis to the coagulation system, especially through the observation of the associated extravascular leakage of plasma and formation of proangiogenic fibrin matrix (Dvorak et al, 1995). Although tumor angiogenesis was found to occur also in the absence of fibrinogen and fibrin, proangiogenic activities can be ascribed to several effectors of the coagulation system, such as TF/thromboplastin, thrombin and several others (Rickles et al, 2003). These factors may act directly on ECs, or stimulate the expression of VEGF, IL-8, and other angiogenic proteins by cancer cells and stroma. An important role is attributed to circulating blood platelets (Pinedo et al, 1998), which may serve as reservoirs and carriers of angiogenic factors (eg, plasma VEGF) and inhibitors (PF4, TSP-1). Indeed, platelets appear to take up, accumulate and segregate such factors in their granules and may selectively release them at sites of angiogenesis (Klement et al, 2009).
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In addition to simple molecular signals (eg, soluble VEGF), ECs and other elements of the tumor microenvironment may receive and emit more complex (multimolecular) messages, encapsulated in cell membrane-derived structures known as microvesicles, microparticles, or exosomes. Microvesicles may deliver angiogenic growth factors (VEGF, FGF), inflammatory cytokines (IL-1), enzymes (MMPs), enzyme inducers (EMMPRIN), and other mediators, including messenger RNA (mRNA), microRNA, and DNA. Interestingly, microvesicles derived from cancer cells may also contain active and phosphorylated oncoproteins that may enter and reprogram ECs (eg, trigger autocrine production of VEGF) thereby possibly contributing to tumor angiogenesis (Al-Nedawi et al, 2009).