Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Irina V. Balyasnikova*, Sergei M. Danilov, Vladimir R. Muzykantov, Aron B. Fisher

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

Previous work has suggested that not all immunoreactive angiotensin- converting enzyme (ACE) in tissues or cells is in a biologically active State. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31- positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.

Original languageEnglish (US)
Pages (from-to)545-554
Number of pages10
JournalIn Vitro Cellular and Developmental Biology - Animal
Volume34
Issue number7
DOIs
StatePublished - 1998

Keywords

  • Angiotensin-converting enzyme
  • Cell culture
  • Human umbilical vein endothelial cells
  • Immunocytochemistry
  • Phorbol ester

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

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