ATP-binding cassette transporters in immortalised human brain microvascular endothelial cells in normal and hypoxic conditions.

Abstract

Background: Rapid reperfusion following ischemia is the most effective therapy in stroke therapy. However, the success may be compromised by ischemia & reperfusion (I/R) injury and at the human blood-brain barrier (BBB), therefore the effects on transendothelial transport are of special interest. Current studies suggest the ATP-binding cassette (ABC) transporters to be regulated upon ischemic stroke in a way that impedes the effects of drug therapy. The immortalised human brain microvascular endothelial cell line hCMEC/D3 provides most of the unique properties of the BBB with respect to transport and might be a reliable in vitro model to study transendothelial transport after I/R.

Methods: We exposed hCMEC/D3 cells to 24 hours of hypoxia alone and to hypoxia followed by 60 min of reoxygenisation as an in vitro model for I/R. Western blot showed mild upregulation of hypoxia inducible factor (HIF-1α) after hypoxia alone and RNA lysates were analysed with a well-established real-time RT-PCR-based TaqMan low-density array detecting 47 of 48 known human ABC transporters.

Results: No significant increases of ABC mRNA expression levels were detected neither in hypoxic nor in I/R samples. However, slight decrease of ABCC1 in hypoxic and I/R samples and of ABCA10 and ABCD3 in I/R samples was observed.

Conclusion: Our data suggests that hCMEC/D3 cell line and - at the moment - in vitro models in general are a poor basis for stroke research but may be enhanced by co-culturing more cells of the neurovascular unit inducing an overall ischemic response at the BBB.