title: Hypoxia and hypoxia mimetics inhibit TNF-dependent VCAM1 induction in the 5A32 endothelial cell line via a hypoxia inducible factor dependent mechanism.
abstract: BACKGROUND
  We previously reported that iron chelators inhibit TNFα-mediated induction of VCAM-1 in human dermal microvascular endothelial cells. We hypothesized that iron chelators mediate inhibition of VCAM-1 via inhibition of iron-dependent enzymes such as those involved with oxygen sensing and that similar inhibition may be observed with agents which simulate hypoxia.
  
  
  OBJECTIVE
  We proposed to examine whether non-metal binding hypoxia mimetics inhibit TNFα-mediated VCAM-1 induction and define the mechanisms by which they mediate their effects on VCAM-1 expression.
  
  
  METHODS
  These studies were undertaken in vitro using immortalized dermal endothelial cells, Western blot analysis, ELISA, immunofluorescence microscopy, quantitative real-time PCR, and chromatin immunoprecipitation.
  
  
  RESULTS
  Hypoxia and the non-iron binding hypoxia mimetic dimethyl oxallyl glycine (DMOG) inhibited TNFα-mediated induction of VCAM-1. DMOG inhibition of VCAM-1 was dose-dependent, targeted VCAM-1 gene transcription independent of NF-κB nuclear translocation, and blocked TNFα-mediated chromatin modifications of relevant elements of the VCAM-1 promoter. Combined gene silencing of both HIF-1α and HIF-2α using siRNA led to a partial rescue of VCAM expression in hypoxia mimetic-treated cells.
  
  
  CONCLUSIONS
  Iron chelators, non-metal binding hypoxia mimetics, and hypoxia all inhibit TNFα-mediated VCAM-1 expression. Inhibition is mediated independent of nuclear translocation of NF-κB, appears to target TNFα-mediated chromatin modifications, and is at least partially dependent upon HIF expression. The absence of complete VCAM-1 expression rescue with HIF silencing implies an important regulatory role for an Fe(II)/α-ketoglutarate dioxygenase distinct from the prolyl and asparagyl hydroxylases that control HIF function. Identification of this dioxygenase may provide a valuable target for modulating inflammation in human tissues.
authors.affiliation: Department of Dermatology, Atlanta Veterans Affairs Medical Center, Atlanta, GA, USA.
authors.forename: Todd V
authors.initials: TV
authors.lastname: Cartee
authors.forename: Kellie J
authors.initials: KJ
authors.lastname: White
authors.forename: Marvin
authors.initials: M
authors.lastname: Newton-West
authors.forename: Robert A
authors.initials: RA
authors.lastname: Swerlick
mesh_terms: D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology*; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D015687:Cell Hypoxia; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D000600:Amino Acids, Dicarboxylic; D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human; C040947:oxalylglycine
references.citation: Cell. 1990 Feb 23;60(4):577-84
references.pmid: 1689216
references.citation: Immunity. 1999 Feb;10(2):143-51
references.pmid: 10072067
references.citation: Cell. 2006 May 5;125(3):483-95
references.pmid: 16603237
references.citation: J Exp Med. 2005 Jan 3;201(1):105-15
references.pmid: 15630139
references.citation: Nature. 2006 Jul 20;442(7100):307-11
references.pmid: 16732293
references.citation: Nat Immunol. 2001 Jul;2(7):652-7
references.pmid: 11429551
references.citation: Arthritis Res Ther. 2009;11(1):215
references.pmid: 19291263
references.citation: Antioxid Redox Signal. 2007 Jul;9(7):979-84
references.pmid: 17508919
references.citation: J Clin Invest. 1993 Oct;92(4):1866-74
references.pmid: 7691889
references.citation: Phytochemistry. 2004 Jan;65(1):7-17
references.pmid: 14697267
references.citation: Mol Cell Biol. 1995 May;15(5):2558-69
references.pmid: 7537851
references.citation: Springer Semin Immunopathol. 1995;16(4):427-36
references.pmid: 7570293
references.citation: Blood. 1996 Jan 1;87(1):211-7
references.pmid: 8547644
references.citation: J Mol Med (Berl). 2011 Jul;89(7):657-65
references.pmid: 21336851
references.citation: Acta Derm Venereol Suppl (Stockh). 1994;186:21-2
references.pmid: 7521104
references.citation: J Clin Invest. 2011 Jun;121(6):2155-7
references.pmid: 21606603
references.citation: J Immunol. 2011 Jan 15;186(2):1091-6
references.pmid: 21149600
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 3;103(40):14767-72
references.pmid: 17003112
references.citation: N Engl J Med. 2003 Jan 2;348(1):15-23
references.pmid: 12510038
references.citation: J Immunol. 1992 Jan 1;148(1):78-83
references.pmid: 1370173
references.citation: J Invest Dermatol. 2003 May;120(5):871-9
references.pmid: 12713595
references.citation: Cell Metab. 2009 Jan 7;9(1):11-22
references.pmid: 19117543
references.citation: Cell. 2001 Oct 5;107(1):43-54
references.pmid: 11595184
references.citation: Crit Rev Biochem Mol Biol. 2004 Jan-Feb;39(1):21-68
references.pmid: 15121720
references.citation: N Engl J Med. 2006 Mar 2;354(9):899-910
references.pmid: 16510744
references.citation: Mutat Res. 2008 Apr 2;640(1-2):174-9
references.pmid: 18294659
references.citation: J Invest Dermatol. 2009 Mar;129(3):723-9
references.pmid: 18787531
references.citation: J Immunol. 2009 May 1;182(9):5469-76
references.pmid: 19380795
references.citation: J Immunol. 2006 Oct 15;177(8):4962-5
references.pmid: 17015677
references.citation: J Cell Biochem. 2010 Jun 1;110(3):671-86
references.pmid: 20512927
references.citation: J Invest Dermatol. 1991 Aug;97(2):190-6
references.pmid: 1906507
references.citation: Cochrane Database Syst Rev. 2006 Jul 19;(3):CD006097
references.pmid: 16856112
references.citation: J Allergy Clin Immunol. 1996 Dec;98(6 Pt 2):S270-7
references.pmid: 8977536
references.citation: Ann N Y Acad Sci. 2009 Oct;1177:19-29
references.pmid: 19845603
references.citation: Cell. 2003 Mar 7;112(5):645-57
references.pmid: 12628185
references.citation: Annu Rev Cell Dev Biol. 1999;15:551-78
references.pmid: 10611972
references.citation: Blood. 2008 Aug 15;112(4):1482-92
references.pmid: 18451309
references.citation: Am J Physiol Heart Circ Physiol. 2004 Mar;286(3):H878-83
references.pmid: 14630627
references.citation: Proc Natl Acad Sci U S A. 2006 Nov 28;103(48):18154-9
references.pmid: 17114296
references.citation: Genes Dev. 2008 May 1;22(9):1115-40
references.pmid: 18451103
references.citation: J Biol Chem. 2006 Aug 11;281(32):22575-85
references.pmid: 16760477