Cell adhesion molecule expression in cultured human iris endothelial cells

M. D. Silverman, D. O. Zamora, Y. Pan, P. V. Texeira, Stephen Planck, James (Jim) Rosenbaum

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

PURPOSE. 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)-α, 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.

Original languageEnglish (US)
Pages (from-to)2861-2866
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume42
Issue number12
StatePublished - 2001

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Cell Adhesion Molecules
Iris
E-Selectin
Vascular Cell Adhesion Molecule-1
Intercellular Adhesion Molecule-1
Endothelial Cells
CD31 Antigens
Endotoxins
Leukocytes
Adhesiveness
Proteins
von Willebrand Factor
Collagenases
Basement Membrane
Reverse Transcription
Down-Regulation
Tumor Necrosis Factor-alpha
Western Blotting
Cytokines
Inflammation

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Cell adhesion molecule expression in cultured human iris endothelial cells. / Silverman, M. D.; Zamora, D. O.; Pan, Y.; Texeira, P. V.; Planck, Stephen; Rosenbaum, James (Jim).

In: Investigative Ophthalmology and Visual Science, Vol. 42, No. 12, 2001, p. 2861-2866.

Research output: Contribution to journalArticle

Silverman, M. D. ; Zamora, D. O. ; Pan, Y. ; Texeira, P. V. ; Planck, Stephen ; Rosenbaum, James (Jim). / Cell adhesion molecule expression in cultured human iris endothelial cells. In: Investigative Ophthalmology and Visual Science. 2001 ; Vol. 42, No. 12. pp. 2861-2866.
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T1 - Cell adhesion molecule expression in cultured human iris endothelial cells

AU - Silverman, M. D.

AU - Zamora, D. O.

AU - Pan, Y.

AU - Texeira, P. V.

AU - Planck, Stephen

AU - Rosenbaum, James (Jim)

PY - 2001

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N2 - PURPOSE. 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)-α, 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.

AB - PURPOSE. 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)-α, 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.

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