title: Cell adhesion molecule expression in cultured human iris endothelial cells.
abstract: OBJECTIVE
  To develop a method to isolate human iris microvascular endothelial cells (HIECs) for exploring their constitutive and inflammatory agent-modulated expression of intercellular adhesion molecules (ICAM)-1 and -2, vascular cell adhesion molecule (VCAM)-1, and E-selectin.
  
  
  METHODS
  Endothelial cells from collagenase-digested irises were isolated on the basis of their expression of platelet endothelial cell adhesion molecule (PECAM)-1, using antibody-coupled magnetic beads. Cells were characterized as endothelial based on morphologic criteria, their expression of PECAM-1 and von Willebrand factor, their uptake of acetylated low-density lipoprotein, and their ability to form capillary-like networks on a synthetic basement membrane. Constitutive and inflammatory agent-modulated expression of ICAM-1 and -2, VCAM-1, and E-selectin was evaluated by the reverse transcription-polymerase chain reaction, enzyme-linked immunocellular assays (ELICAs), Western blot analysis, and functional studies of leukocyte adhesion to HIEC monolayers.
  
  
  RESULTS
  HIECs constitutively expressed mRNA and protein for ICAM-1 and -2, but only low to nondetectable levels of VCAM-1 or E-selectin. When stimulated with endotoxin- or tumor necrosis factor (TNF)-alpha, ICAM-1, VCAM-1, and E-selectin were potently and time- and dose-dependently upregulated at both the message and protein levels. By contrast, ICAM-2 message and protein were slowly downregulated by inflammatory agents over time, but nonetheless remained present and functional. Overall, cytokine- or endotoxin-activation of HIECs resulted in enhanced adhesiveness for leukocytes.
  
  
  CONCLUSIONS
  ICAM-1, VCAM-1, and E-selectin have been previously implicated in mediating anterior ocular inflammation. This is a report of the selective isolation of HIECs, with a demonstration of differential expression and regulation of these adhesion molecules in them. In addition, this is the first demonstration of the regulated expression of ICAM-2 in any ocular microvascular cells.
authors.affiliation: Department of Ophthalmology, Casey Eye Institute, Portland, OR, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Silverman
authors.forename: D O
authors.initials: DO
authors.lastname: Zamora
authors.forename: Y
authors.initials: Y
authors.lastname: Pan
authors.forename: P V
authors.initials: PV
authors.lastname: Texeira
authors.forename: S R
authors.initials: SR
authors.lastname: Planck
authors.forename: J T
authors.initials: JT
authors.lastname: Rosenbaum
mesh_terms: D000293:Adolescent; D000328:Adult; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000235:genetics; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D019040:E-Selectin / Q000096:biosynthesis / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004731:Endotoxins / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics; D007498:Iris / Q000098:blood supply*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics
chemical_list: D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D019040:E-Selectin; D004731:Endotoxins; C065289:ICAM2 protein, human; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1