ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression

Cardiovasc Res. 2012 Jan 1;93(1):111-9. doi: 10.1093/cvr/cvr265. Epub 2011 Oct 13.

Abstract

Aims: The vascular endothelial growth factor (VEGF) stimulates angiogenesis by induction of vessel permeability, proliferation, and migration of endothelial cells, an important process in ischaemic diseases. ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) (cytohesin-2) is a guanine exchange factor important for cellular signalling through ARF GTPases. However, a role for ARNO in VEGF-dependent endothelial processes has so far not been documented. Therefore, we investigated whether ARNO has a role in VEGF-dependent activation of endothelial cells and thus vessel permeability.

Methods and results: ARNO expression was observed in endothelial cells in vitro by RT-PCR, western blotting, and immunofluorescence as well as ex vivo by immunohistochemical staining of mouse aorta. Treatment with the cytohesin inhibitor SecinH3 or with an ARNO siRNA prevented VEGF-dependent Akt activation, assessed by detection of phosphorylated Akt, and proliferation of endothelial cells in vitro, measured by methylthiazoletetrazolium (MTT) reduction. In addition, ARNO suppression reduced VEGF-induced permeability in vessels of the mouse (C57BL/6) cremaster muscle in vivo, as measured by extravasation of fluorescein isothiocyanate (FITC)-dextran. Moreover, ARNO knock-down accelerated ligand-induced reduction in vascular endothelial growth factor receptor-2 (VEGFR-2) surface expression, internalization, and degradation, as assessed by flow cytometry and western blotting, respectively.

Conclusion: Our findings indicate an important and novel role for endothelial ARNO in VEGF-dependent initiation of angiogenesis by regulation of VEGFR-2 internalization in endothelial cells, resulting in the activation of the Akt pathway, vessel permeability, and ultimately endothelial proliferation. Thus, ARNO may be a new essential player in endothelial signalling and angiogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Capillary Permeability / physiology
  • Cell Proliferation
  • Cells, Cultured
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism
  • GTPase-Activating Proteins / antagonists & inhibitors
  • GTPase-Activating Proteins / genetics
  • GTPase-Activating Proteins / metabolism*
  • Gene Knockdown Techniques
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Mice
  • Neovascularization, Physiologic
  • Phosphorylation
  • Proto-Oncogene Proteins c-akt / metabolism
  • RNA, Small Interfering / genetics
  • Signal Transduction
  • Vascular Endothelial Growth Factor A / metabolism*
  • Vascular Endothelial Growth Factor Receptor-2 / metabolism*

Substances

  • GTPase-Activating Proteins
  • RNA, Small Interfering
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • cytohesin-2
  • vascular endothelial growth factor A, mouse
  • Vascular Endothelial Growth Factor Receptor-2
  • Proto-Oncogene Proteins c-akt