title: EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells.
abstract: Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders.
authors.affiliation: Department of Haematology, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Research Institute of Internal Medicine, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway. Electronic address: benedicte.stavik@rr-research.no.
authors.forename: Benedicte
authors.initials: B
authors.lastname: Stavik
authors.affiliation: Department of Haematology, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Research Institute of Internal Medicine, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Institute of Basic Medical Sciences, University of Oslo, Postboks 1072 Blindern, 0316 Oslo, Norway. Electronic address: s.e.serrano@medisin.uio.no.
authors.forename: Sandra
authors.initials: S
authors.lastname: Espada
authors.affiliation: Department of Haematology, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Research Institute of Internal Medicine, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Postboks 1072 Blindern, 0316 Oslo, Norway. Electronic address: x.y.cui@medisin.uio.no.
authors.forename: Xue Yan
authors.initials: XY
authors.lastname: Cui
authors.affiliation: Department of Medical Genetics, Oslo University Hospital and University of Oslo, Postboks 4950 Nydalen, 0424 Oslo, Norway. Electronic address: nina.iversen@medisin.uio.no.
authors.forename: Nina
authors.initials: N
authors.lastname: Iversen
authors.affiliation: Research Institute of Internal Medicine, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Hospital for Rheumatic Diseases, Margrethe Grundtvigsvei 6, 2609 Lillehammer, Norway. Electronic address: sverre.holm@rr-research.no.
authors.forename: Sverre
authors.initials: S
authors.lastname: Holm
authors.affiliation: Department of Haematology, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Research Institute of Internal Medicine, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway. Electronic address: uxmaow@ous-hf.no.
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Mowinkel
authors.affiliation: Research Institute of Internal Medicine, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Postboks 1072 Blindern, 0316 Oslo, Norway. Electronic address: bente.halvorsen@rr-research.no.
authors.forename: Bente
authors.initials: B
authors.lastname: Halvorsen
authors.affiliation: Department of Haematology, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Research Institute of Internal Medicine, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway. Electronic address: grethe.skretting@medisin.uio.no.
authors.forename: Grethe
authors.initials: G
authors.lastname: Skretting
authors.affiliation: Department of Haematology, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Research Institute of Internal Medicine, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Postboks 1072 Blindern, 0316 Oslo, Norway. Electronic address: p.m.sandset@medisin.uio.no.
authors.forename: Per Morten
authors.initials: PM
authors.lastname: Sandset
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia; D002460:Cell Line; D015536:Down-Regulation; D015951:Factor Xa / Q000096:biosynthesis / Q000235:genetics; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics; D013925:Thromboplastin / Q000096:biosynthesis / Q000235:genetics; D013927:Thrombosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D015951:Factor Xa; D008074:Lipoproteins; D013925:Thromboplastin; D000099162:Endothelial PAS Domain-Containing Protein 1; C051928:lipoprotein-associated coagulation inhibitor
keywords: Atherosclerosis; HIF-2α; Hypoxia; Tissue factor pathway inhibitor