title: HIF-1α activation results in actin cytoskeleton reorganization and modulation of Rac-1 signaling in endothelial cells.
abstract: BACKGROUND
  Hypoxia is a major driving force in vascularization and vascular remodeling. Pharmacological inhibition of prolyl hydroxylases (PHDs) leads to an oxygen-independent and long-lasting activation of hypoxia-inducible factors (HIFs). Whereas effects of HIF-stabilization on transcriptional responses have been thoroughly investigated in endothelial cells, the molecular details of cytoskeletal changes elicited by PHD-inhibition remain largely unknown. To investigate this important aspect of PHD-inhibition, we used a spheroid-on-matrix cell culture model.
  
  
  RESULTS
  Microvascular endothelial cells (glEND.2) were organized into spheroids. Migration of cells from the spheroids was quantified and analyzed by immunocytochemistry. The PHD inhibitor dimethyloxalyl glycine (DMOG) induced F-actin stress fiber formation in migrating cells, but only weakly affected microvascular endothelial cells firmly attached in a monolayer. Compared to control spheroids, the residual spheroids were larger upon PHD inhibition and contained more cells with tight VE-cadherin positive cell-cell contacts. Morphological alterations were dependent on stabilization of HIF-1α and not HIF-2α as shown in cells with stable knockdown of HIF-α isoforms. DMOG-treated endothelial cells exhibited a reduction of immunoreactive Rac-1 at the migrating front, concomitant with a diminished Rac-1 activity, whereas total Rac-1 protein remained unchanged. Two chemically distinct Rac-1 inhibitors mimicked the effects of DMOG in terms of F-actin fiber formation and orientation, as well as stabilization of residual spheroids. Furthermore, phosphorylation of p21-activated kinase PAK downstream of Rac-1 was reduced by DMOG in a HIF-1α-dependent manner. Stabilization of cell-cell contacts associated with decreased Rac-1 activity was also confirmed in human umbilical vein endothelial cells.
  
  
  CONCLUSIONS
  Our data demonstrates that PHD inhibition induces HIF-1α-dependent cytoskeletal remodeling in endothelial cells, which is mediated essentially by a reduction in Rac-1 signaling.
authors.forename: Alexander
authors.initials: A
authors.lastname: Weidemann
authors.forename: Johannes
authors.initials: J
authors.lastname: Breyer
authors.forename: Margot
authors.initials: M
authors.lastname: Rehm
authors.forename: Kai-Uwe
authors.initials: KU
authors.lastname: Eckardt
authors.forename: Christoph
authors.initials: C
authors.lastname: Daniel
authors.forename: Iwona
authors.initials: I
authors.lastname: Cicha
authors.forename: Klaudia
authors.initials: K
authors.lastname: Giehl
authors.affiliation: Department of Nephrology and Hypertension, Universitätsklinikum Erlangen, Universität Erlangen-Nürnberg, Loschgestrasse 8, 91054 Erlangen, Germany. margarete.goppelt-struebe@uk-erlangen.de.
authors.forename: Margarete
authors.initials: M
authors.lastname: Goppelt-Struebe
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D002465:Cell Movement; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D055806:Microvessels / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D064800:Prolyl-Hydroxylase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction; D018874:Spheroids, Cellular / Q000166:cytology / Q000502:physiology; D054462:p21-Activated Kinases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D000199:Actins; D000600:Amino Acids, Dicarboxylic; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D064800:Prolyl-Hydroxylase Inhibitors; C107953:RAC1 protein, human; D054462:p21-Activated Kinases; D054460:rho-Associated Kinases; D020830:rac1 GTP-Binding Protein; C040947:oxalylglycine