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Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells

Academic Article
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Overview

authors

  • Schleef, R. R.
  • Loskutoff, David J.
  • Podor, T. J.

publication date

  • June 1991

journal

  • Journal of Cell Biology  Journal

abstract

  • Immunogold EM was employed to compare the distribution of type 1 plasminogen activator inhibitor (PAI-1) on the surface of agonist-activated human umbilical vein endothelial cells (HUVECs) with that of control, unactivated cells. As previously observed, (Schleef, R.R., T.J. Podor, E. Dunne, J. Mimuro, and D.J. Loskutoff. J. Cell Biol. 110:155-163), analysis of cross-sections of nonpermeabilized control HUVEC monolayers stained first with affinity-purified rabbit antibodies to PAI-1 and then with gold-conjugated goat anti-rabbit IgG, revealed the presence of relatively few gold particles (less than 1-2% of the total) on the apical cell surface. The majority of gold particles were detected primarily in the extracellular matrix between the culture substratum and the cell membrane. In contrast, treatment of HUVECs with tumor necrosis factor alpha (TNF alpha; 200 U/ml, 24 h) or with lipopolysaccharide (LPS; 10 micrograms/ml, 24 h) resulted in an increased staining of PAI-1 not only in the extracellular matrix, but also on the apical cell surface (10-fold increase). Immunoabsorption of the rabbit anti-PAI-1 with purified PAI-1, or treatment of HUVECs with tissue-type plasminogen activator (2.5 micrograms/ml, 2 h, 4 degrees C) reduced the amount of staining both on the apical surface and in the extracellular matrix of agonist-activated HUVECs by 80-95%. The topographical location of PAI-1 on the cell surface was examined further by coupling immunogold staining with high resolution surface replication. Transmission EM of surface replicas from TNF alpha- or LPS-activated HUVECs revealed a general increase in PAI-1 staining both on planar regions and within indentations of the apical cell surface. Nonactivated HUVECs revealed PAI-1-specific immunogold particles only in areas of exposed extracellular matrix between the cells and occasionally at regions of cell-cell contacts. Analysis of activated bovine aortic endothelial cells by immuno-electron microscopy, immunologic assays, and flow cytometry revealed similar increases in surface PAI-1. These increases in surface PAI-1 could be detected by 3 h and continued over a 24-h period. The expression of PAI-1 on the luminal surface of endothelial cells during immune or inflammatory reactions could reduce endothelial fibrinolytic activity, thus, promoting the localized, pathologic formation of intravascular thrombi.

subject areas

  • Cells, Cultured
  • Endothelium, Vascular
  • Flow Cytometry
  • Humans
  • Lipopolysaccharides
  • Microscopy, Immunoelectron
  • Plasminogen Inactivators
  • Tumor Necrosis Factor-alpha
  • Umbilical Veins
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Identity

PubMed Central ID

  • PMC2289026

International Standard Serial Number (ISSN)

  • 0021-9525

Digital Object Identifier (DOI)

  • 10.1083/jcb.113.6.1413

PubMed ID

  • 2045419
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Additional Document Info

start page

  • 1413

end page

  • 1423

volume

  • 113

issue

  • 6

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