title: HIF-2α Expression Regulates Sprout Formation into 3D Fibrin Matrices in Prolonged Hypoxia in Human Microvascular Endothelial Cells.
abstract: BACKGROUND
  During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis.
  
  
  OBJECTIVE
  We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling.
  
  
  RESULTS
  Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction.
  
  
  CONCLUSIONS
  Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, De Boelelaan 1118 1081 HV, Amsterdam, The Netherlands.|A-Skin Nederland BV, De Boelelaan 1117, Amsterdam 1007 MB, The Netherlands.
authors.forename: Tessa D
authors.initials: TD
authors.lastname: Nauta
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, De Boelelaan 1118 1081 HV, Amsterdam, The Netherlands.
authors.forename: Monique C A
authors.initials: MC
authors.lastname: Duyndam
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, De Boelelaan 1118 1081 HV, Amsterdam, The Netherlands.|A-Skin Nederland BV, De Boelelaan 1117, Amsterdam 1007 MB, The Netherlands.
authors.forename: Ester M
authors.initials: EM
authors.lastname: Weijers
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, De Boelelaan 1118 1081 HV, Amsterdam, The Netherlands.
authors.forename: Victor M W
authors.initials: VM
authors.lastname: van Hinsbergh
authors.affiliation: Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, De Boelelaan 1118 1081 HV, Amsterdam, The Netherlands.
authors.forename: Pieter
authors.initials: P
authors.lastname: Koolwijk
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques; D015687:Cell Hypoxia; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005337:Fibrin / Q000737:chemistry*; D006801:Humans; D055806:Microvessels / Q000166:cytology; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051880:Receptors, Notch / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D005337:Fibrin; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051880:Receptors, Notch; D014409:Tumor Necrosis Factor-alpha; D014568:Urokinase-Type Plasminogen Activator; D042461:Vascular Endothelial Growth Factor A; D000099162:Endothelial PAS Domain-Containing Protein 1