title: Hypoxia regulated gene transcription in human optic nerve lamina cribrosa cells in culture.
abstract: OBJECTIVE
  Vascular hypoperfusion, extracellular matrix remodeling and axon loss are pathological characteristics of the glaucomatous optic nerve head. We report a novel study demonstrating transcriptional responses in optic nerve lamina cribrosa (LC) cells exposed to in vitro hypoxic stress.
  
  
  METHODS
  Primary cultures of human glial fibrillary acid protein (GFAP) negative LC cells were generated from four donors. Cells were exposed to 24 hours of hypoxic stress (1% O2) or normoxia (21% O2). Hypoxia responsive genes were identified using Affymetrix HG-U133A microarrays (n = 3) and validated with real time PCR (n = 3). Secreted protein was measured by ELISA (n = 4) and cellular protein by Western blot (n = 4). Expression data were annotated with NIH DAVID software and putative transcription factor sites in hypoxia-responsive gene promoters were identified using Core_TF software.
  
  
  RESULTS
  Hypoxia-sensitive genes included those involved in apoptosis (e.g., BNIP3), neurogenesis (e.g., STC1), extracellular matrix (e.g., MIF, DDR1/TrkE, and IGFR2), mitochondrion (e.g., CYP1B1) and angiogenesis (e.g., VEGF). Real time PCR for selected genes supported the expression changes identified by microarray. ELISA and Western blot validated corresponding changes in protein production. Promoter sequence interrogation revealed putative conserved transcription factor binding sites (e.g., HIF and CREB) in the promoters of the hypoxia responsive genes.
  
  
  CONCLUSIONS
  Our data show that LC cell gene expression is sensitive to reduced oxygen levels in vitro and provides bioinformatic evidence of the potential transcriptional regulators of this response.
authors.affiliation: Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland. ruaidhri.kirwan@ucd.ie
authors.forename: Ruaidhrí P
authors.initials: RP
authors.lastname: Kirwan
authors.forename: Luca
authors.initials: L
authors.lastname: Felice
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
authors.forename: Colm J
authors.initials: CJ
authors.lastname: O'Brien
authors.forename: Martin O
authors.initials: MO
authors.lastname: Leonard
mesh_terms: D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D000860:Hypoxia / Q000235:genetics*; D008928:Mitochondria / Q000235:genetics; D055495:Neurogenesis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D009898:Optic Disk / Q000166:cytology / Q000378:metabolism; D009900:Optic Nerve / Q000166:cytology / Q000378:metabolism*; D018384:Oxidative Stress; D060888:Real-Time Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics
chemical_list: D016326:Extracellular Matrix Proteins; D014157:Transcription Factors