Dataset Information

Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood-brain barrier model.

ABSTRACT:

Objective

Nuclear receptors and cytochrome P450 (CYP) regulate hepatic metabolism of several drugs. Nuclear receptors are expressed at the neurovascular unit of patients with drug-resistant epilepsy. We studied whether glucocorticoid receptor (GR) silencing or inhibition in human epileptic brain endothelial cells (EPI-ECs) functionally impacts drug bioavailability across an in vitro model of the blood-brain barrier (BBB) by CYP-multidrug transporter (multidrug resistance protein 1, MDR1) mechanisms.

Methods

Surgically resected brain specimens from patients with drug-resistant epilepsy, primary EPI-ECs, and control human brain microvascular endothelial cells (HBMECs) were used. Expression of GR, pregnane X receptor, CYP3A4, and MDR1 was analyzed pre- and post-GR silencing in EPI-ECs. Endothelial cells were co-cultured with astrocytes and seeded in an in vitro flow-based BBB model (DIV-BBB). Alternatively, the GR inhibitor mifepristone was added to the EPI-EC DIV-BBB. Integrity of the BBB was monitored by measuring transendothelial electrical resistance. Cell viability was assessed by glucose-lactate levels. Permeability of [³ H]sucrose and [¹⁴ C]phenytoin was quantified. CYP function was determined by measuring resorufin formation and oxcarbazepine (OXC) metabolism.

Results

Silencing and inhibition of GR in EPI-ECs resulted in decreased pregnane X receptor, CYP3A4, and MDR1 expression. GR silencing or inhibition did not affect BBB properties in vitro, as transendothelial electrical resistance and P_sucrose were unaltered, and glucose metabolism was maintained. GR EPI-EC silencing or inhibition led to (1) increased P_phenytoin BBB permeability as compared to control; (2) decreased CYP function, indirectly evaluated by resorufin formation; (3) improved OXC bioavailability with increased abluminal (brain-side) OXC levels as compared to control.

Significance

Our results suggest that modulating GR expression in EPI-ECs at the BBB modifies drug metabolism and penetration by a mechanism encompassing P450 and efflux transporters. The latter could be exploited for future drug design and to overcome pharmacoresistance.

SUBMITTER: Ghosh C

PROVIDER: S-EPMC6282717 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Publications

Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood-brain barrier model.

Ghosh Chaitali C Hossain Mohammed M Mishra Saurabh S Khan Sameena S Gonzalez-Martinez Jorge J Marchi Nicola N Janigro Damir D Bingaman William W Najm Imad I

Epilepsia 20180928 11

<h4>Objective</h4>Nuclear receptors and cytochrome P450 (CYP) regulate hepatic metabolism of several drugs. Nuclear receptors are expressed at the neurovascular unit of patients with drug-resistant epilepsy. We studied whether glucocorticoid receptor (GR) silencing or inhibition in human epileptic brain endothelial cells (EPI-ECs) functionally impacts drug bioavailability across an in vitro model of the blood-brain barrier (BBB) by CYP-multidrug transporter (multidrug resistance protein 1, MDR1) ...[more]

PMID: 30264400

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Project description:Hypoglycemia impairs blood-brain barrier (BBB) endothelial function; a major hallmark in the pathogenesis of various CNS disorders. Previously, we have demonstrated that prolonged hypoglycemic exposure down-regulated BBB endothelial NF-E2 related factor-2 (Nrf2) expression; a redox-sensitive transcriptional factor that regulates endothelial function. Here, we sought to determine the functional role of Nrf2 in preserving BBB integrity and molecular mechanisms underlying hypoglycemia-induced Nrf2 down-regulation in vitro using human cerebral microvascular endothelial cell line (hCMEC/D3). Cell monolayers were exposed to normal or hypoglycemic (5.5 or 2.2mM D-glucose) media for 3-24h. Pharmacological or gene manipulation (by silencing RNA) approaches were used to investigate specific molecular pathways implicated in hypoglycemia-induced Nrf2 degradation. BBB integrity was assessed by paracellular permeability to labeled dextrans of increasing molecular sizes (4-70kDa). Silencing Nrf2 expression in hCMEC/D3 cells abrogated the expression of claudin-5 and VE-cadherin, while ZO-1 was up-regulated. These effects were paralleled by a decrease in electrical resistance of hCMEC/D3 monolayers and potential increase in permeability to all labeled dextrans. Hypoglycemic exposure (3-24h) led to progressive and sustained down-regulation of Nrf2 (without affecting mRNA) and its target, NQO-1, with a concomitant increase in the cytosolic pool of E3 ubiquitin ligase, Siah2 (but not Keap1). Pretreatment with protease inhibitor MG132, or selective knock-down of Siah2 (but not Keap1) significantly attenuated hypoglycemia-induced Nrf2 destabilization. While hypoglycemic exposure triggered a significant increase in BBB permeability to dextrans, silencing Siah2 gene abrogated the effects of hypoglycemia and restored BBB integrity. In summary, our data indicate a potential role for Nrf2 signaling in regulating tight junction integrity and maintaining BBB function. Nrf2 suppression by increased Siah2-driven proteasomal degradation mediates hypoglycemia-evoked endothelial dysfunction and loss of BBB integrity. Overall, this study suggests that sustained activation of endothelial Nrf2 signaling could have therapeutic potential to prevent hypoglycemia-induced cerebrovascular dysfunction.

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Dataset Information

Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood-brain barrier model.

Objective

Methods

Results

Significance

Publications

Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood-brain barrier model.

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