Endothelial junction formation and neutrophil transmigration in culture

A. R. Burns, P. C. Walker, E. S. Brown, T. T.thurmnn, S. I. Simon, C. W. Smith

Research output: Contribution to journalArticle

Abstract

Smeets et al. showed that glutaraldehyde-crosslinked gelatin is a superior substrate for long term (>20 days) culturing of human umbilical vein endothelial cell (HUVEC) monolayers (Biotech. Histochem. 67:241, 1992). Although HUVEC borders stain with silver and express PECAM-1, the characterization of junctional complexes (tight junctions or zonula occludens (ZO), gap junctions (GJ), and zonula adherens (ZA)) remains to be determined. Using transmission electron microscopy we show that ZO, although present between HUVEC in vivo, were not detected in primary or first pass (P1) cultures. At 2 days post-confluence, GJ and ZA were found in 28% and 75% of the P1 endothelial borders, respectively. Borders (17%) lacking GJ or ZA were designated as open contacts (OC). The frequency of GJ, ZA, and OC was not statistically different at 10 days post-confluence, however the increased CV associated with ZA (11% at 2 days; 70% at 10 days) and OC (22% at 2 days; 101% at 10 days) suggests a decline in junctional integrity. Consistent with this observation is a 40% decline hi electrical resistance between 2 and 10 day old monolayers. At 2 days, immunofluorescent pan-cadherin staining was observed along P1 endothelial borders but was absent and discontinuous at the tri-cellular corner or "Y" junction between three adjacent endothelial cells. We show that the maioritv of neutroohils mierate across IL-1-stimulated (10 U/ml, 4h) 2 day old monolayers at "Y" junctions.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number6
StatePublished - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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