The ATP-binding cassette transporters ABCB1 and ABCC1 are not regulated by hypoxia in immortalised human brain microvascular endothelial cells

Pauline Patak,Eric Metzen,Simon T Schäfer,Dirk M Hermann,Fengyan Jin

doi:10.1186/2040-7378-3-12

Abstract

BackgroundATP-binding cassette transporters at the blood-brain barrier are actively regulated upon ischemic stroke in a way that impedes the access of pharmacological compounds to the brain tissue. The luminal endothelial transporter ABCB1 was recently shown to be increased, whereas the abluminal transporter ABCC1 was decreased on ischemic brain capillaries. In vitro studies using epithelial cells suggested that ABCB1 is regulated during hypoxia in a hypoxia-inducible factor (HIF)-1α-dependent way.MethodsIn order to investigate whether hypoxia might be responsible for the expression changes of ABCB1 and ABCC1 in the ischemic brain, the immortalised human brain microvascular endothelial cell line hCMEC/D3 was exposed to hypoxia (1%) or anoxia (0%). Cell lysates were analysed by Western blot to detect the protein expression of ABCB1, ABCC1, HIF-1α and HIF-2α.ResultsDuring hypoxia, an accumulation of HIF-1α and HIF-2α was noticed in hCMEC/D3 cells that followed different time kinetics. Both HIF-1α and HIF-2α abundance increased within 4 h of hypoxia. HIF-1α levels decreased to below detection levels within 16 h of hypoxia, whereas HIF-2α remained elevated even after 48 h. No changes of ABCB1 and ABCC1 expression were detected, neither on the mRNA nor protein level.ConclusionOur data suggests that other factors than hypoxia may be responsible for the expression changes of ATP-binding cassette transporters in the ischemic brain.

Highlights

ATP-binding cassette transporters at the blood-brain barrier are actively regulated upon ischemic stroke in a way that impedes the access of pharmacological compounds to the brain tissue
hypoxia-inducible factor (HIF)-1a and HIF-2a expression in hCMEC/D3 cell line during normoxia and hypoxia To eludicate the impact of cell confluency on hypoxiainducible expression of HIF-1a and HIF-2a, cells seeded at different densities were exposed to 1% oxygen for 6 hours and compared with control cells that were simultaneously kept under normoxia with or without the addition of 0.1 mM deferoxamine mesylate (DFO)
Kinetics of HIF-1a and HIF-2a expression in hCMEC/D3 during hypoxia To evaluate the kinetics of hypoxic induction of HIF-1a and HIF-2a, hCMEC/D3 cells were exposed to different durations of hypoxia

Summary

Introduction

ATP-binding cassette transporters at the blood-brain barrier are actively regulated upon ischemic stroke in a way that impedes the access of pharmacological compounds to the brain tissue. ABC transporters are regulated on brain capillary endothelial cells in a coordinated way that impedes the delivery of drugs into the brain. Since hypoxia is a major factor contributing to ischemic injury, we were intrigued by the question whether HIF1a-dependent signaling is responsible for the ischemia-induced expression changes of ABC transporters in endothelial cells. To elucidate this issue, we exposed the cells of the immortalised human brain microvascular endothelial cell line hCMEC/D3 to conditions of ambient hypoxia

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