The blockade of cyclooxygenases-1 and -2 reduces the effects of hypoxia on endothelial cells

M.A Gloria,A Pacheco-Silva,N.O.S Câmara,M.A Cenedeze

doi:10.1590/s0100-879x2006000900006

Abstract

Hypoxia activates endothelial cells by the action of reactive oxygen species generated in part by cyclooxygenases (COX) production enhancing leukocyte transmigration. We investigated the effect of specific COX inhibition on the function of endothelial cells exposed to hypoxia. Mouse immortalized endothelial cells were subjected to 30 min of oxygen deprivation by gas exchange. Acridine orange/ethidium bromide dyes and lactate dehydrogenase activity were used to monitor cell viability. The mRNA of COX-1 and -2 was amplified and semi-quantified before and after hypoxia in cells treated or not with indomethacin, a non-selective COX inhibitor. Expression of RANTES (regulated upon activation, normal T cell expressed and secreted) protein and the protective role of heme oxygenase-1 (HO-1) were also investigated by PCR. Gas exchange decreased partial oxygen pressure (PaO2) by 45.12 +/- 5.85% (from 162 +/- 10 to 73 +/- 7.4 mmHg). Thirty minutes of hypoxia decreased cell viability and enhanced lactate dehydrogenase levels compared to control (73.1 +/- 2.7 vs 91.2 +/- 0.9%, P < 0.02; 35.96 +/- 11.64 vs 22.19 +/- 9.65%, P = 0.002, respectively). COX-2 and HO-1 mRNA were up-regulated after hypoxia. Indomethacin (300 microM) decreased COX-2, HO-1, hypoxia-inducible factor-1alpha and RANTES mRNA and increased cell viability after hypoxia. We conclude that blockade of COX up-regulation can ameliorate endothelial injury, resulting in reduced production of chemokines.

Highlights

Acute renal failure, a clinical feature of ischemia and reperfusion injury (IRI), remains a major cause of morbidity and mortality in hospitalized patients [1]
Normal T cell expressed and secreted (RANTES) protein is a key molecule involved in these events which is secreted by endothelial cells and activated leukocytes [6,7]
Endothelial cells subjected to gaseous hypoxia showed a higher level of lactate dehydrogenase (LDH) release (35.96 ± 11.64 vs 22.19 ± 9.65%, P = 0.002) and low viability compared to those that remained in a non-hypoxic state

Summary

Introduction

A clinical feature of ischemia and reperfusion injury (IRI), remains a major cause of morbidity and mortality in hospitalized patients [1]. The cellular mechanisms involved in IRI include ATP depletion, impaired calcium regulation, generation of reactive oxygen species (ROS), and the production of proinflammatory cytokines/chemokines. All these mediators contribute to activation of endothelial cells [4,5]. Endothelial cell activation is associated with pro-coagulant and pro-inflammatory phenotypes. The up-regulation of class II molecules of the major histocompatibility complex, together with secretion of chemokines, promotes leukocyte adhesion and further transmigration of leukocytes through the endothelial barrier into the tissue. Normal T cell expressed and secreted (RANTES) protein is a key molecule involved in these events which is secreted by endothelial cells and activated leukocytes [6,7]

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Results

Conclusion