Cerebral hypoxia and reoxygenation (H/R) is a component of various diseases including traumatic brain injury, cardiac arrest, and ischemic stroke [
]. Blood–brain barrier (BBB) integrity is modulated by production of reactive oxygen species (ROS) and subsequent oxidative stress in the setting of H/R [
]. For example, studies using bovine brain microvessel endothelial cells subjected to H/R stress reported discrete changes in tight junction protein localization that correlated with increased paracellular permeability to sucrose, a vascular marker that does not cross the intact BBB [
]. Similar observations have been reported in vivo where H/R induced disassembly of occludin oligomers in rat brain microvessels [
]. Furthermore, studies in the same model system showed increased CNS accumulation of sucrose [
] and dextrans [
], evidence indicating BBB dysfunction in response to H/R. Indeed, vascular changes induced by H/R can have deleterious consequences. Enhanced BBB permeabilization can lead to vasogenic edema and cause clinically significant increases in brain volume and intracranial pressure [
]. Additionally, substances that are typically contained within the systemic circulation, including drugs, can leak into brain parenchyma and potentially cause neurotoxicity. Clearly, there is a critical need to preserve BBB integrity in diseases with an H/R component.
In order to develop therapeutic approaches that can confer BBB protection, it is essential to identify specific biological mechanisms that contribute to oxidative stress-induced damage of the brain microvasculature. Indeed, furthering our understanding of the endothelial cell antioxidant defense system will enable advancement of such pharmacological strategies. The endogenous antioxidant glutathione (GSH) is a vital component of this antioxidant defense system. In vivo studies have demonstrated that cerebral GSH levels are significantly decreased in response to reperfusion injury [
] and GSH depletion is associated with increased BBB permeability to both sucrose and sodium fluorescein [
]. Although this does not reflect large-scale BBB disruption, this leak is clinically significant by permitting increased paracellular transport of potentially toxic small molecules. Decreased GSH levels in response to H/R may involve membrane transport processes mediated by multidrug resistance proteins (Mrps). Mrps are members of the ATP-binding cassette (ABC) superfamily of efflux transporters, primarily transport organic anions and conjugated metabolites, and are encoded by genes from ABC subfamily C (i.e.,
]. Both GSH and glutathione disulfide (GSSG) are known transport substrates for Mrp1, Mrp2, and Mrp4. For example, studies in primary cultures of rat astrocytes showed that GSH transport could be blocked using MK571, an established inhibitor of Mrp1 and Mrp2 [
]. Similarly, Mrp4 is also believed to be involved in transport of GSH in the brain [
]. Indeed, these observations point towards endogenous transporters that can be targeted to preserve endothelial GSH levels and provide BBB protection in the setting of H/R.
Effective targeting of Mrps to reduce GSH efflux and confer BBB protection requires identification and characterization of regulatory pathways that control expression of these transporters. One such pathway is signaling mediated by nuclear factor E2-related factor (Nrf2). Nrf2 is normally inactive in the cytoplasm and rapidly degraded when associated with Kelch-like ECH-associated protein 1 (Keap1). Under conditions of oxidative stress, Keap1 dissociates and allows Nrf2 to translocate to the nucleus and initiate transcription of genes containing an antioxidant response element (ARE) [
]. Nrf2 has been shown to induce expression of Mrp1, Mrp2, and Mrp4 and the genes that encodes these proteins (i.e.,
Abcc1, Abcc2, Abcc4
) at the BBB as well as in other tissues [
]. At present, involvement of Nrf2 in regulating Mrp transporter expression at the BBB has not been evaluated under pathophysiological conditions.
In the present study, we show increased expression of
Abcc1, Abcc2, and Abcc4 mRNA transcripts in brain microvessels via Nrf2 signaling in the setting of H/R. Specifically, we show for the first time that H/R activates Nrf2 signaling at the BBB and that Nrf2 binds to an antioxidant response element in the promoter of all three genes that encode GSH transporting Mrp isoforms. These data provide critical information that can inform future studies aimed at targeting Mrp transporters to confer BBB protection in diseases with an H/R component.
The mammalian Mrp family belongs to the ABCC group of proteins, which contains 13 members including one ion channel (i.e., CFTR), two surface receptors (i.e., SUR1 and 2) and a truncated protein that does not mediate transport (i.e., ABCC13) [
]. Several functionally characterized Mrp isoforms have been localized to the mammalian BBB. These include Mrp1, Mrp2, Mrp4, Mrp5 and Mrp6 [
]. The presence of multiple Mrp isoforms at the BBB is a critical determinant in controlling delivery of therapeutic agents to the brain. Additionally, the ability of Mrp isoforms to actively efflux the endogenous antioxidant glutathione (GSH) has significant implications for diseases with an H/R component. GSH is responsible for maintenance of cellular redox balance and antioxidant defense in the brain. It has been previously demonstrated that various Mrps are upregulated in response to oxidative stress conditions, which leads to enhanced cellular efflux of GSH [
]. Increased functional expression of Mrp isoforms at the BBB could cause reduced endothelial cell concentrations of GSH, an alteration in cellular redox status, and increased potential for cell injury and death. Therefore, biological mechanisms that can modulate Mrp expression at the BBB in response to oxidative stress require further investigation.
A thorough understanding of signaling pathways involved in Mrp regulation in the setting of H/R will enable development of pharmacological approaches to target Mrp-mediated efflux (i.e., GSH transport) for the purpose of preventing BBB dysfunction. One intriguing pathway is signaling mediated by Nrf2, a sensor of oxidative stress [
]. In the presence of ROS, the cytosolic Nrf2 repressor Keap1 undergoes structural alterations that cause dissociation from the Nrf2-Keap1 complex. This enables Nrf2 to translocate to the nucleus and induce transcription of genes that possess an antioxidant response element at their promoter [
]. It has been demonstrated that activation of Nrf2 signaling induces expression of Mrp1, Mrp2, and Mrp4 [
]. Our data expands upon these previous studies by showing Nrf2-mediated increases in mRNA transcript expression for
in rat brain microvessels. We also show increased Nrf2 nuclear translocation in the setting of H/R and that Nrf2 binds to the ARE in the respective promoter for
ur findings are novel and highly significant because we have shown, for the first time, that H/R-induced activation of Nrf2 leads to increased expression of mRNA transcripts for transporters endogenously expressed at the BBB. This is a rapid response, which may indicate that genes involved in the H/R stress response may be available for immediate activation in an effort to protect the vasculature from dysfunction and subsequent leak of circulating solutes. It is also intriguing that changes in
mRNA transcript expression occur following H/R but are not apparent in the setting of hypoxia despite activation of Nrf2 signaling under both conditions. Such changes may be reflective of the dramatic increase in ROS production following H/R. For example, Fabian and Kent demonstrated increased production of superoxide anions by neutrophils following reperfusion, an event that can greatly exacerbate BBB dysfunction [
]. Such dramatic increases in ROS production can certainly induce cellular changes independent of signaling pathways that are activated in response to hypoxia [
Recent evidence has shown that Nrf2 is a component of a complex signaling pathway, which involves additional factors for promoter activation and subsequent modulation of transport mechanisms at the BBB. For example, sulforaphane-induced increases in ABC transporter functional expression at the BBB can be abolished using pifithrin, an inhibitor of p53 signaling, or in p53 null mice [
]. In contrast, nutlin-3, a p53 activator, increased P-gp transport activity in mouse brain capillaries [
]. Of particular note, this study also demonstrated that pharmacological inhibitors of p38 MAPK signaling (i.e., SB203580) and nuclear factor-κB (NF-κB) signaling (i.e.,
4-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine, SN50) blocked effects of sulforaphane and nutlin-3 on P-gp activity [
]. Taken together, the work of Wang and colleagues suggests that effects of Nrf2 signaling on ABC transporters at the BBB requires involvement of p53, p38 MAPK, and NF-κB signaling.
An emerging concept is that Nrf2 acts as a double-edged sword [
]. Activation of Nrf2 signaling at the BBB is generally considered to be protective owing to its activation of cytoprotective pathways; pre- and post-treatment administration of Nrf2 activators confer BBB protection in animal models of stroke and traumatic brain injury [
]. A subset of Nrf2 target genes are involved in the synthesis and metabolism of GSH, including GCLC and GCLM (subunits of glutamate–cysteine ligase), glutathione peroxidase, and glutathione reductase [
]. Indeed, increased expression of GSH synthetic genes can lead to increased cellular production of this critical antioxidant. However, oxidative stress increases the functional expression of Mrp1 [
], and oxidative stress induced by metals or H
has been previously shown to increase Mrp1-mediated export of GSH and GSSG [
]. Upregulation of Mrp isoforms in glial cells may have neuroprotective effects in the setting of oxidative stress through release of GSH into brain parenchyma where it can be readily accessed by neurons [
]. However, an alteration in the balance of Mrp isoforms via activation of Nrf2 signaling may have considerably different effects than in brain parenchyma. Indeed, efflux of GSH by Mrp isoforms expressed at the abluminal membrane of the BBB may provide some neuroprotection; however, increased GSH efflux due to enhanced Mrp-mediated transport can adversely affect redox balance and antioxidant defense at the brain microvascular endothelium and contribute to barrier dysfunction in the setting of H/R. This indicates that studies designed to develop pharmacological approaches based on targeting Mrp isoforms at the BBB must consider both neuroprotective and vascular protective effects associated with these transporters. Additionally, increased functional expression of Mrp isoforms at the BBB can negatively affect endothelial cell inflammation and repair pathways. Endogenous mediators involved in such pathways include leukotriene C4, a known Mrp1/Mrp2 substrate [
], and prostaglandin E
In order to fully comprehend the implications of Nrf2-mediated upregulation of
gene expression at the BBB, future studies must be undertaken to assess Mrp localization in the brain microvasculature. Expression and localization of Mrp isoforms at the BBB is species-dependent and remains highly controversial [
]. Localization of Mrp1 is thought to be at the abluminal plasma membrane in brain microvascular endothelial cells in rodents, but at the luminal membrane in humans [
]. Mrp4 has been detected on the luminal surface of the BBB in rat; however, abluminal expression has not been confirmed [
]. Based on qPCR and proteomic analysis, Mrp4 is the most abundant of the three GSH-transporting isoforms in human brain microvessels [
]. Mrp2 is likely localized to the luminal aspect of the BBB, but several studies have failed to detect Mrp2 at the protein level [
]. This may be due to low basal expression of Mrp2, which may be increased in response to cellular stressors such as oxidative stress [
]. In mice, there are notable differences in Mrp expression between strains and between vessels of different diameters. For example, FVB mice appear to lack Mrp2 in brain vessels, but it is present in C57BL/6 and Swiss mice [
]. This same study also showed that Mrp1is most abundant in vessels 20–50 μm in diameter [
]. Rigorous assessment of Mrp isoform localization will undoubtedly inform the development of therapeutic strategies to protect the BBB in diseases with an H/R component. Furthermore, these studies should include both male and female experimental animals in order to determine differences in Mrp localization based on sex.
KI reviewed experimental data and prepared the manuscript; JY assisted with experimental work. PTR participated in experimental design, performed experiments, performed data analysis, assisted with preparation of the manuscript, and obtained funding via R01-NS084941. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
All animal experiments were approved by the University of Arizona Institutional Animal Care and Use Committee and are clearly outlined via protocol #11-252.
This work was supported by a grant from the National Institute of Neurological Disease and Stroke (NINDS), National Institutes of Health (NIH) (Grant #R01-NS084941) to PTR.