**4. Synopsis of VEGF-induced endothelial responses and their role in tumor angiogenesis**

VEGF/VEGFR-2 signal transduction leads to six major endothelial responses: cell survival, invasion and migration into the surrounding tissue, proliferation, vascular permeability and vascular inflammation. These endothelial responses are tightly integrated to allow tumor angiogenesis to progress successfully.

Vascular permeability—crucial for normal tissue homeostasis—is a prerequisite for VEGFinduced angiogenesis. Endothelial permeability is mediated by the so-called transcellular and paracellular pathways, that is, solutes and cells can pass through (transcellular) or between (paracellular) endothelial cells. Transcellular passage requires cell fenestration and/or a complex system of transport vesicles that includes organelles called vesiculo-vacuolar organelles, while the paracellular pathway depends on the coordinated opening and closure of endothelial cell-to-cell junctions, combined with cell retraction. VEGF is involved in both transcellular and paracellular permeability. VEGF/VEGFR-2 axis induces increased endothelial permeability mainly through SRC-mediated signaling of VE-cadherin internalization (see Sections 3.4 and 3.5) that disrupts the endothelial barrier function, and by activation of PLCγ that mediates an increase in intracellular Ca2+ resulting in Ca2+-calmodulin-dependent regulation of eNOS (see Section 3.2). Activation of eNOS—also achieved by AKT-mediated phosphorylation—promotes an increase in vascular permeability by NO production that is followed by vasodilatation. VEGF-induced vascular permeability contributes to the dissemination of extracellular proteases and the deposition of a fibrin gel provisional stroma that changes the extracellular matrix of normal tissues from anti- to pro-angiogenic and stromagenic, favoring and supporting inward migration of endothelial cells and the growth of new endothelial sprouts [111]. In addition, the reduced vessel integrity may promote leukocyte extravasation and facilitate exit of metastasis from the primary tumors.

VEGF-induced vascular permeability goes in parallel with vascular inflammation. Although VEGF is not an inflammatory cytokine, VEGF-induced gene transcription—in particular through NFAT activation—includes a conspicuous inflammatory component (see Section 3.2). This could conceivably promote attraction of inflammatory cells that may contribute to the angiogenic response. Indeed, it is well established that tumor angiogenesis is linked to inflammation. On the one hand, tumor cells can be killed by the immune system; on the other hand, tumor can use leukocytes to supports its expansion.

Endothelial cell invasion into the surrounding tissue is made possible by means of the release of MMPs, which degrade the basal membrane and the extracellular matrix and allow the migration of endothelial cells to form capillary sprouts. Endothelial cells express different MMPs, and it has been reported that their expression is induced by VEGF and regulated by Ets transcription factors [112, 113]. Signaling pathways, such as MAP kinase cascade, PI3K/ AKT axis, and Ca2+-specific signals, converge on the Ets transcription factors, controlling their activity.

VEGF-induced endothelial cell migration and proliferation are tightly regulated and coordinated spatio-temporal behaviors, which—in parallel with sustained cell survival—enable angiogenic sprouting and capillary lumen formation, necessary to create the new vessels devoted to support tumor growth and metastasis spread. VEGF-induced endothelial survival and proliferation are stimulated primarily via PI3K/AKT and PLCγ/ERK pathways, respectively, involving—as above described—several signaling intermediates. VEGF-induced endothelial cell migration appears to be regulated by a larger number of pathways, including the involvement of PLCγ, PI3K, RHO GTPases, SRC and FAK activities.

## **5. Conclusions**

In this chapter, we attempted in particular to describe the best characterized signal transduction events downstream to VEGF/VEGFR-2 axis involved in tumor angiogenesis. Multiple VEGF-induced signaling pathways take part in the promotion of different biological responses in endothelial cells. Although it is possible to recognize distinct patterns along VEGF-induced signaling, they are intricate, characterized by the involvement of a number of enzymes and adaptor/scaffold proteins—whose activity converges and branches at many point—and by the presence of VEGFR-2 molecular partners influencing endothelial cell sensitivity to VEGF and receptor signal output. This depicts a complex signal network induced by VEGF, where the apparent redundancy in operating signaling pathways is likely to reflect a need for a fine-tuning and a differential control of the biological effects in response to VEGF [25]. Although past decade has seen an important advancement in our understanding of VEGF signaling, there is still a lack of insight in many aspects of VEGF/VEGFR-2 signal transduction, in particular for what concerns its fine regulation. A further elucidation of the multifaced VEGF signaling network in the context of endothelial biology is crucial for developing new potential anti-angiogenic therapies. In parallel with current therapies that directly target VEGF and VEGFR-2, agents able to influence key molecular player—proximal, median or distal to VEGFR-2—could be of clinical interest.

## **Acknowledgement**

Publication costs have been supported by "Associazione Italiana per la Ricerca sul Cancro" (AIRC), project code 12182.
