Human microvascular endothelial cell prostaglandin E1 synthesis during in vitro ischemia-reperfusion

Michael T. Watkins, Hassan Al-Badawi, Andrea L. Russo, Hiram Soler, Brian Peterson, George M. Patton

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Ischemia-reperfusion injury is a microvascular event documented in numerous in vivo animal models. In animal models, prostaglandin and prostaglandin analogues have been found to ameliorate reperfusion injury. These studies were undertaken to evaluate human microvascular endothelial PGE1 synthesis during in vitro ischemia followed by reperfusion. Human (neonatal) microvascular endothelial cell (MEC) cultures (n = 6) were subjected to sequential 2 h periods of normoxia (20% O2), ischemia (1.5% O 2), and reperfusion (20% O2). Prostaglandin E2 synthesis in conditioned media was determined by ELISA. Steady state levels of MEC prostaglandin H synthase (PGHS)-1 and -2 mRNA were assessed at the end of each 2-h period using RT-PCR and a quantitative mRNA ELISA. MEC PGHS protein levels were analyzed using an ELISA. PGE1 release increased significantly during the initial 30 min of ischemia, but rapidly fell below normoxic levels by 90 and 120 min. During reperfusion, PGE1 release returned to normoxic levels at 30, 60, and 90 min, and exceeded normoxic levels at 120 min. PGHS-1 mRNA levels were undetectable during all experimental conditions. PGHS-2 mRNA levels were unchanged by ischemia, but were decreased by reperfusion. In contrast, PGHS-2 protein levels increased 3-fold during ischemia, and remained elevated during reperfusion. Human MEC do not express PGHS-1 mRNA in vitro. Prolonged ischemia decreases MEC PGE1 synthesis, and stimulates increased PGHS-2 protein levels without altering the steady state levels of COX-2 mRNA. During reperfusion, increased PGHS-2 protein levels persist and are associated with stimulated PGE2 secretion, despite relative decreases in PGHS-2 mRNA.

Original languageEnglish (US)
Pages (from-to)472-480
Number of pages9
JournalJournal of cellular biochemistry
Volume92
Issue number3
DOIs
StatePublished - Dec 1 2004

Keywords

  • Endothelium
  • Ischemia
  • Prostaglandin H synthase
  • Reperfusion

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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