Chapter 080. Cancer Cell Biology and Angiogenesis (Part 17) VEGF and its receptors are required for vasculogenesis (the de novo formation of blood vessels from differentiating endothelial cells, as occurs during embryonic development) and angiogenesis under normal (wound healing, corpus luteum formation) and pathologic processes (tumor angiogenesis, inflammatory conditions such as rheumatoid arthritis). VEGF-A is a heparin-binding glycoprotein with at least four isoforms (splice variants) that regulates blood vessel formation by binding to the RTKs VEGFR1 and VEGFR2, which are expressed on all ECs in addition to a subset of hematopoietic cells (Fig. 80-8). VEGFR2 regulates EC proliferation, migration, and survival, while VEGFR1 may act as an antagonist of R1 in ECs but is probably also important for angioblast differentiation during embryogenesis. Tumor vessels appear to be more dependent on VEGFR signaling for growth and survival than normal ECs. While VEGF signaling is a critical initiator of angiogenesis, this is a complex process regulated by additional signaling pathways (Fig. 80-9). The angiopoietin, Ang1, produced by stromal cells, binds to the EC RTK Tie-2 and promotes the interaction of ECs with the ECM and perivascular cells, such as pericytes and smooth-muscle cells, to form tight, non-leaky vessels. PDGF and basic fibroblast growth factor (bFGF) help to recruit these perivascular cells. Ang1 is required for maintaining the quiescence and stability of mature blood vessels and prevents the vascular permeability normally induced by VEGF and inflammatory cytokines. For tumor cell–derived VEGF to initiate sprouting from host vessels, the stability conferred by the Ang1/Tie2 pathway must be perturbed; this occurs by the secretion of Ang2 by ECs that are undergoing active remodeling. Ang2 binds to Tie2 and is a competitive inhibitor of Ang1 action: under the influence of Ang2, preexisting blood vessels become more responsive to remodeling signals, with less adherence of ECs to stroma and associated perivascular cells and more responsiveness to VEGF. Therefore, Ang2 is required at early stages of tumor angiogenesis for destabilizing the vasculature by making host ECs more sensitive to angiogenic signals. Since tumor ECs are blocked by Ang2, there is no stabilization by the Ang1/Tie2 interaction, and tumor blood vessels are leaky, hemorrhagic, and have poor association of ECs with underlying stroma. Sprouting tumor ECs express high levels of the transmembrane protein ephrin-B2 and its receptor, the RTK EPH whose signaling appears to work with the angiopoietins during vessel remodeling. During embryogenesis, EPH receptors are expressed on the endothelium of primordial venous vessels while the transmembrane ligand ephrin-B2 is expressed by cells of primordial arteries; the reciprocal expression may regulate differentiation and patterning of the vasculature. A number of ubiquitously expressed host molecules play critical roles in normal and pathologic angiogenesis. Proangiogenic cytokines, chemokines, and growth factors secreted by stromal cells or inflammatory cells make important contributions to neovascularization, including bFGF, transforming growth factor-α (TGF-α), TNF-α, and IL-8. In contrast to normal endothelium, angiogenic endothelium overexpresses specific members of the integrin family of ECM- binding proteins that mediate EC adhesion, migration, and survival. Specifically, expression of integrins α v β 3 , α v β 5 , and α 5 β 1 mediate spreading and migration of ECs and are required for angiogenesis induced by VEGF and bFGF, which in turn can upregulate EC integrin expression. The α v β 3 integrin physically associates with VEGFR2 in the plasma membrane and promotes signal transduction from each receptor to promote EC proliferation (via focal adhesion kinase, src, PI3K, and other pathways) and survival (by inhibition of p53 and increasing the Bcl- 2/Bax expression ratio). In addition, α v β 3 forms cell surface complexes with matrix metalloproteinases (MMPs), zinc-requiring proteases that cleave ECM proteins, leading to enhanced EC migration and the release of heparin-binding growth factors including VEGF and bFGF. EC adhesion molecules can be upregulated (i.e., by VEGF, TNF-α) or downregulated (by TGF-β); this, together with chaotic blood flow explains poor leukocyte-endothelial interactions in tumor blood vessels and may help tumor cells avoid immune surveillance. Cells derived from hematopoietic progenitors in the host bone marrow contribute to tumor angiogenesis in a process linked to the secretion of VEGF and PlGF (placenta-derived growth factor) by tumor cells and their surrounding stroma. VEGF promotes the mobilization and recruitment of circulating endothelial cell precursors (CEPs) and hematopoietic stem cells (HSCs) to tumors where they co-localize and appear to cooperate in neovessel formation. CEPs express VEGFR2, while HSCs express VEGFR1, a receptor for VEGF and PlGF. Both CEPs and HSCs are derived from a common precursor, the hemangioblast. CEPs are thought to differentiate into ECs, whereas the role of HSC-derived cells (such as tumor-associated macrophages) may be to secrete angiogenic factors required for sprouting and stabilization of ECs (VEGF, bFGF, angiopoietins) and to activate MMPs, resulting in ECM remodeling and growth factor release. In mouse tumor models and in human cancers, increased numbers of CEPs and subsets of VEGFR-expressing HSCs can be detected in the circulation, which may correlate with increased levels of serum VEGF. It is not yet known whether levels of these cells have prognostic value or if changes during treatment correlate with inhibition of tumor angiogenesis. Whether CEPs and VEGFR1-expressing HSCs are required to maintain the long-term integrity of established tumor vessels is also unknown. Lymphatic vessels also exist within tumors. Development of tumor lymphatics is associated with expression of VEGFR3 and its ligands VEGF-C and VEGF-D. The role of these vessels in tumor cell metastasis to regional lymph nodes remains to be determined, since, as discussed above, interstitial pressures within tumors are high and most lymphatic vessels may exit in a collapsed and nonfunctional state. However, VEGF-C levels correlate significantly with metastasis to regional lymph nodes in lung, prostate, and colorectal cancers, . Chapter 080. Cancer Cell Biology and Angiogenesis (Part 17) VEGF and its receptors are required for vasculogenesis (the de novo. cells, binds to the EC RTK Tie-2 and promotes the interaction of ECs with the ECM and perivascular cells, such as pericytes and smooth-muscle cells, to form tight, non-leaky vessels. PDGF and. VEGF and PlGF (placenta-derived growth factor) by tumor cells and their surrounding stroma. VEGF promotes the mobilization and recruitment of circulating endothelial cell precursors (CEPs) and