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Angiogenesis relies on the spatial and temporal coordination of endothelial migration and proliferation to form new blood vessels. This occurs through synchronous activation of multiple downstream pathways which facilitate vascular development. Proangiogenic growth factors and supporting extracellular matrix allow the formation of capillary-like tubules, reminiscent of microvascular beds, in vitro. In this chapter, we describe a methodology for the establishment of vascular networks by co-culture of endothelial cells and fibroblasts to facilitate the study of tubulogenic and angiogenic potential. We detail the use of siRNA mediated knockdown to deplete target genes of interest, in either the endothelial or fibroblast cells, to allow the assessment of their role in angiogenesis. Finally, we detail how these vascular networks may be stained using immunofluorescence to allow quantification of angiogenic potential in vitro., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lymphatic vessels constitute a specialized vasculature that is involved in development, cancer, obesity, and immune regulation. The migration of lymphatic endothelial cells (LECs) is critical for vessel growth (lymphangiogenesis) and vessel remodeling, processes that modify the lymphatic network in response to developmental or pathological demands. Using the publicly accessible results of our genome-wide siRNA screen, we characterized the migratome of primary human LECs and identified individual genes and signaling pathways that regulate LEC migration. We compared our data set with mRNA differential expression data from endothelial and stromal cells derived from two in vivo models of lymphatic vessel remodeling, viral infection and contact hypersensitivity-induced inflammation, which identified genes selectively involved in regulating LEC migration and remodeling. We also characterized the top candidates in the LEC migratome in primary blood vascular endothelial cells to identify genes with functions common to lymphatic and blood vascular endothelium. On the basis of these analyses, we showed that LGALS1 , which encodes the glycan-binding protein Galectin-1, promoted lymphatic vascular growth in vitro and in vivo and contributed to maintenance of the lymphatic endothelial phenotype. Our results provide insight into the signaling networks that control lymphangiogenesis and lymphatic remodeling and potentially identify therapeutic targets and biomarkers in disease specific to lymphatic or blood vessels., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.

Objective: Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor tyrosine kinase that regulates vascular physiology. However, mechanism(s) by which VEGFR2 signaling and trafficking is coordinated are not clear. Here, we have tested endocytic Rab GTPases for regulation of VEGFR2 trafficking and signaling linked to endothelial cell migration., Methods and Results: Quiescent VEGFR2 displays endosomal localization and colocalization with the Rab5a GTPase, an early endosome fusion regulator. Expression of GTP or GDP-bound Rab5a mutants block activated VEGFR2 trafficking and degradation. Manipulation of Rab7a GTPase activity associated with late endosomes using overexpression of wild-type or mutant proteins blocks activated VEGFR2 trafficking and degradation. Depletion of Rab7a decreased VEGFR2 Y1175 phosphorylation but increased p42/44 (pERK1/2) MAPK phosphorylation. Endothelial cell migration was increased by Rab5a depletion but decreased by Rab7a depletion., Conclusions: Rab5a and Rab7a regulate VEGFR2 trafficking toward early and late endosomes. Our data suggest that VEGFR2-mediated regulation of endothelial function is dependent on different but specific Rab-mediated GTP hydrolysis activity required for endosomal trafficking.

The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A(2)-alpha (cPLA(2)alpha) cleaves phospholipids in a Ca(2+)-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca(2+)-independent association of cPLA(2)alpha with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA(2)alpha and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA(2)alpha is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA(2)alpha and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA(2)alpha activity and endothelial cell prostaglandin generation.