title: Human microvascular endothelial cell prostaglandin E1 synthesis during in vitro ischemia-reperfusion.
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 PGE(1) 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% O(2)), ischemia (1.5% O(2)), and reperfusion (20% O(2)). Prostaglandin E(2) 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. PGE(1) release increased significantly during the initial 30 min of ischemia, but rapidly fell below normoxic levels by 90 and 120 min. During reperfusion, PGE(1) 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 PGE(1) 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 PGE(2) secretion, despite relative decreases in PGHS-2 mRNA.
authors.affiliation: Department of Surgery, Massachusetts General Hospital Harvard Medical, Boston, Massachusetts 02131, USA. mtwatkins@partners.org
authors.forename: Michael T
authors.initials: MT
authors.lastname: Watkins
authors.forename: Hassan
authors.initials: H
authors.lastname: Al-Badawi
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Russo
authors.forename: Hiram
authors.initials: H
authors.lastname: Soler
authors.forename: Brian
authors.initials: B
authors.lastname: Peterson
authors.forename: George M
authors.initials: GM
authors.lastname: Patton
mesh_terms: D000527:Alprostadil / Q000096:biosynthesis*; D002460:Cell Line; D002470:Cell Survival; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000032:analysis / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D019344:Lactic Acid / Q000096:biosynthesis; D008565:Membrane Proteins; D008833:Microcirculation; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D012867:Skin / Q000098:blood supply
chemical_list: D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D019344:Lactic Acid; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D000527:Alprostadil; D015232:Dinoprostone