Regulation of ABCB1 expression and activity in porcine brain endothelial cells

Abstract

The blood-brain barrier (BBB) is a neurovascular structure that acts as a physical and metabolic barrier, which restricts the transport of xenobiotics and endogenous molecules to the brain. Brain endothelial cells of the BBB express transmembrane ATP-binding cassette (ABC) transporters capable of pumping out compounds to the bloodstream, and these contribute to barrier function. ABCB1 (P-glycoprotein) is one of the most clinically relevant ABC transporter expressed at the BBB. Its expression and activity can be regulated through genomic and physical mechanisms, and in vitro models have proved invaluable for study of these mechanisms under several conditions. In this thesis, an in vitro BBB model, comprising primary porcine brain endothelial cells (PBECs) was shown to possess phenotypic features comparable with BBB endothelial cells. In vitro PBECs possessed spindle-like morphology and expressed functional alkaline phosphatase and γ-glutamyl transpeptidase. Verapamil-sensitive ABCB1 activity and Ko 143-sensitive ABCG2 activity were also observed. The characterised PBEC model was subjected to treatments with drugs and other compounds known for being ligands of members of the nuclear receptor family, including glucocorticoid receptor (GR), pregnane X receptor (PXR), constitutive androstane receptor (CAR) and retinoid X receptor (RXR). The model was also tested to assess the effect of compounds involved in the progression of inflammatory conditions including pro-inflammatory mediators, neuroactive metabolites and dietary omega-3 long-chain polyunsaturated fatty acids (PUFAs). Activation of GR by the ligands dexamethasone and hydrocortisone significantly up-regulated ABCB1 expression and activity, which was abrogated under a co-treatment with the GR antagonist mifepristone. The human PXR ligand rifampicin and the CAR ligand CITCO both significantly decreased ABCB1 activity, however this decrease was mediated through direct interaction of the compounds with the transporter and not via interaction with the nuclear receptors themselves. The pro-inflammatory cytokine interleukin-1β up-regulated ABCB1 expression and activity, which was counteracted by the interleukin-1 receptor antagonist. Treatment with the long-chain PUFAs arachidonic acid (AA), docosahexaenoic acid (DHA) and eicosapentenoic acid (EPA) significantly down-regulated ABCB1 activity without modifying its expression. Use of N-methyl-D-aspartate (NMDA) failed to induce expression of ABCB1 via activation of the NMDA receptor, probably due to absence of the NR1 subunit or expression of a non-functional configuration. Use of inhibitors of the nuclear transcription factor kappaB (NF-kappaB) did not revert the IL-1β-induced ABCB1 activity. However, long-chain PUFAs counteracted the glucocorticoid and IL-1β-mediated increase in ABCB1 activity without influencing the already up-regulated protein expression. In summary, the findings of this thesis project will help to better understand the factors that govern the activity of ABCB1 expressed in a relevant to human brain endothelial cell model, in terms of response against drugs, pro-inflammatory and anti-inflammatory mediators involved in the progression of chronic diseases.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jeffrey Penny (Supervisor) & Costas Demonacos (Supervisor)