1. Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells.
- Author
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Fearnley GW, Smith GA, Odell AF, Latham AM, Wheatcroft SB, Harrison MA, Tomlinson DC, and Ponnambalam S
- Subjects
- Cell Physiological Phenomena, Cell Separation, Cells, Cultured, Flow Cytometry, Gene Expression, Gene Knockdown Techniques, Humans, Microscopy, Fluorescence, Primary Cell Culture, Protein Transport, Proteolysis, RNA Interference, Receptors, Vascular Endothelial Growth Factor metabolism, Reverse Genetics, Umbilical Cord cytology, rab GTP-Binding Proteins genetics, rab GTP-Binding Proteins metabolism, Endosomes metabolism, Human Umbilical Vein Endothelial Cells metabolism, Signal Transduction, Vascular Endothelial Growth Factor A physiology
- Abstract
The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function., (© 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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