Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells

Methods Enzymol. 2014;535:265-92. doi: 10.1016/B978-0-12-397925-4.00016-X.

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.

Keywords: Endosome; Endothelial; GTPase; Migration; Proliferation; Signaling; Trafficking; Tubulogenesis; VEGF-A; VEGFR2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Physiological Phenomena
  • Cell Separation
  • Cells, Cultured
  • Endosomes / metabolism*
  • Flow Cytometry
  • Gene Expression
  • Gene Knockdown Techniques
  • Human Umbilical Vein Endothelial Cells / metabolism*
  • Humans
  • Microscopy, Fluorescence
  • Primary Cell Culture
  • Protein Transport
  • Proteolysis
  • RNA Interference
  • Receptors, Vascular Endothelial Growth Factor / metabolism
  • Reverse Genetics
  • Signal Transduction*
  • Umbilical Cord / cytology
  • Vascular Endothelial Growth Factor A / physiology*
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism

Substances

  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Receptors, Vascular Endothelial Growth Factor
  • rab GTP-Binding Proteins