title: ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Walter-Brendel Centre for Experimental Medicine, Munich, Germany.
authors: Hanna K
authors: HK
authors: Mannell
authors: Joachim
authors: J
authors: Pircher
authors: Daniel I
authors: DI
authors: Chaudhry
authors: Stefan K C
authors: SK
authors: Alig
authors: Elisabeth G
authors: EG
authors: Koch
authors: Ramona
authors: R
authors: Mettler
authors: Ulrich
authors: U
authors: Pohl
authors: Florian
authors: F
authors: Krötz
mesh_terms: D000818:Animals; D002199:Capillary Permeability / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051379:Mice; D018919:Neovascularization, Physiologic; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C102474:cytohesin-2; C467480:vascular endothelial growth factor A, mouse; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt