Transendothelial permeability changes induced by free radicals in an in vitro model of the blood-brain barrier

doi:10.1016/S0891-5849(99)00112-4

Free Radical Biology and Medicine

Volume 27, Issues 5–6, September 1999, Pages 667-672

https://doi.org/10.1016/S0891-5849(99)00112-4 Get rights and content

Abstract

In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 μM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.

Introduction

The mammalian brain is protected from chemical insult by the blood-brain barrier (BBB), which prevents the entry of most polar xenobiotics into the brain [1]. The BBB is formed by capillary endothelial cells that show tight junctional structures, allowing the strict regulation of ion and substance exchange between blood and brain. In addition to a physical barrier, there is a metabolic barrier, formed by the enzymes of the endothelial cytosol, which metabolize some of the permeable molecules that are recognized as substrates [2]. These enzymes include monoamine oxidase and xanthine oxidase, which react with oxygen to produce free radicals [3], as well as NADPH-cytochrome P450 reductase, which is able to transfer two electrons using a large variety of substrates of both endogenous and exogenous origin [4], [5]. This electron transfer may occur in two steps for molecules with strong redox potentials, generating a free radical intermediate which may undergo further reduction under hypoxic conditions. In aerobic conditions, this intermediate can combine with molecular oxygen to form superoxide radicals and regenerate the parent compound, giving rise to futile redox cycling [6]. This redox cycling has been demonstrated in vitro using rat brain preparations [7]. Oxygen free radicals can then target protein, polyunsaturated lipids, and nucleic acids, and may lead to cell damage, and ultimately, to cell death [8].

The oxygen demand of the brain is high compared to other tissues, and both arteries and capillaries are areas of high oxygen tension. In addition, brain microvessels are rich in polyunsaturated fatty acids and have high levels of activity of a number of enzymes [9]. The activity of NADPH-cytochrome P450 reductase has been found to be 2-fold higher in brain microvessels compared to brain homogenates [10]. The BBB may therefore constitute a potential target for the toxic action of compounds undergoing futile redox cycling.

Previous experiments have shown the metabolism of quinones, bipyridinium ions, and nitroheterocyclic compounds by isolated brain microvessels, which may lead to the production of superoxide radicals [7]. Although brain endothelial cells possess high intracellular levels of antioxidative defense mechanisms, such as glutathione (GSH), GSH peroxidase, superoxide dismutase (SOD), and catalase [3], [9], aging and/or chronic exposure to environmental toxins and drugs may render the BBB susceptible to toxic damage and alter specific BBB functions, which may result in loss of cerebral homeostasis [1].

In the present study, we studied the effects of superoxide radical production on BBB permeability. As an in vitro BBB model, we used an immortalized endothelial cell line from rat brain capillaries, RBE4 [11]. The cells were grown to confluence on collagen-coated semi-permeable inserts in co-culture with C6 glioma cells to preserve the inductive effect of astrocytic factors on BBB properties in the endothelial cells [12]. We investigated the ability of a number of different xenobiotics, which have been shown to increase superoxide production in isolated capillaries, to increase the permeability of an endothelial monolayer as a model of BBB opening in conditions of oxygen free radical toxicity.

Section snippets

Culture medium components

DMEM F-12, α-MEM, fetal calf serum (FCS), basic fibroblast growth factor (bFGF), glutamine, penicillin, streptomycin, and geneticin (G418) were purchased from Sigma (Poole, Dorset, UK).

Chemicals and solvents

Benzylviologen duroquinone, nitrofurazone, methylviologen, menadione, SOD, catalase, dimethyl-sulfoxide (DMSO) and other routine chemicals were supplied by Sigma.

Radioisotopes

[³H]sucrose (specific activity 10 Ci/mmol) and [¹⁴C]sucrose (specific activity 500 mCi/mmol) were purchased from Amersham International (Little

Effect of menadione, nitrofurazone, and methylviologen on endothelial permeability

The permeability coefficient of control RBE4 monocellular layers (Pe) was 4.11 ± 0.29 × 10⁻³ cm·min⁻¹ (mean ± SEM, n = 21 in 8 experiments). This value is similar to values previously reported for RBE4 cells in co-culture with primary astrocytes [13]. Both benzylviologen and 100 μM menadione significantly increased sucrose permeability over control values, whereas nitrofurazone, methylviologen, or 10 μM menadione did not significantly increase sucrose clearance (Table 1). At the concentration

Discussion

Previous studies on isolated brain microsomes and microvessels have shown that menadione undergoes a NADPH-dependent metabolism, resulting in the formation of large quantities of superoxide radicals [7]. This production occurs through a redox cycling corresponding to a monoelectronic transfer from NADPH to the chemical, with a subsequent formation of superoxide from molecular oxygen [4]. Other studies on primary cultures of rat brain endothelial cerebrovascular cells also demonstrated that

Acknowledgements

The authors gratefully acknowledge the financial help of the Biomed II program (BMH1-CT92-1193).

References (20)

M.H Livertoux et al.
Superoxide production mediated by the redox cycling of xenobiotics in rat brain microsomesdependence on the reduction potentials
Brain Res.
(1996)
J.F Ghersi-Egea et al.
Enzyme-mediated superoxide formation initiated by exogenous molecules in rat brain
Toxicol. Appl. Pharmacol.
(1991)
P Lagrange et al.
Superoxide anion production during monoelectronic reduction of xenobiotics by preparations of rat brain cortex, microvessels and choroid plexus
Free Radic. Biol. Med.
(1994)
C.P LeBel et al.
Oxygen radicalscommon mediators of neurotoxicity
Neurotoxicol. Teratol.
(1991)
J.F Ghersi-Egea et al.
A new aspect of the protective functions of the blood-brain barrieractivities of four drug-metabolizing enzymes in isolated brain microvessels
Life Sci.
(1988)
R.J Rist et al.
F-actin cytoskeleton and sucrose permeability of primary cultured and immortalized brain microvascular endothelial cell monolayerseffects of cAMP and astrocytic factors
Brain Res.
(1997)
R.A Kozar et al.
Free radical induced alterations in endothelial cell function
J. Surg. Res.
(1994)
S.P Olesen
Rapid increase in blood-brain barrier permeability during severe hypoxia and metabolic inhibition
Brain Res.
(1986)
M.M Murphy et al.
ReviewPeroxynitrite: a biologically significant oxidant
Gen. Pharmacol.
(1998)
S Imaizumi et al.
The influence of oxygen free radicals on the permeability of the monolayer of cultured brain endothelial cells
Neurochem. Int.
(1996)

There are more references available in the full text version of this article.

Cited by (82)

Hypertension & dementia: Pathophysiology & potential utility of antihypertensives in reducing disease burden
2024, Pharmacology and Therapeutics
Dementia is a common cause of disability and dependency among the elderly due to its progressive neurodegenerative nature. As there is currently no curative therapy, it is of major importance to identify new ways to reduce its prevalence. Hypertension is recognised as a modifiable risk factor for dementia, particularly for the two most common subtypes; vascular dementia (VaD) and Alzheimer's disease (AD). From the current literature, identified through a comprehensive literature search of PubMed and Cochrane Library, this review aims to establish the stage in adulthood when hypertension becomes a risk for cognitive decline and dementia, and whether antihypertensive treatment is effective as a preventative therapy.
Observational studies generally found hypertension in mid-life (age 45-64) to be correlated with an increased risk of cognitive decline and dementia incidence, including both VaD and AD. Hypertension manifesting in late life (age $\geq$ 65) was demonstrated to be less of a risk, to the extent that incidences of high blood pressure (BP) in the very elderly (age $\geq$ 75) may even be related to reduced incidence of dementias. Despite the evidence linking hypertension to dementia, there were conflicting findings as to whether the use of antihypertensives was beneficial for its prevention and this conflicting evidence and inconsistent results could be due to the methodological differences between the reviewed observational and randomised controlled trials. Furthermore, dihydropyridine calcium channel blockers and potassium-sparing diuretics were proposed to have neuroprotective properties in addition to BP lowering. Overall, if antihypertensives are confirmed to be beneficial by larger-scale homogenous trials with longer follow-up durations, treatment of hypertension, particularly in mid-life, could be an effective strategy to considerably lower the prevalence of dementia. Furthermore, greater clarification of the neuroprotective properties that some antihypertensives possess will allow for better clinical practice guidance on the choice of antihypertensive class for both BP lowering and dementia prevention.
Anti-neuroinflammatory effect of 6,8,1′-tri-O-methylaverantin, a metabolite from a marine-derived fungal strain Aspergillus sp., via upregulation of heme oxygenase-1 in lipopolysaccharide-activated microglia
2018, Neurochemistry International
In the course of searching for anti-neuroinflammatory metabolites from marine-derived fungi, three fungal metabolites, 6,8,1′-tri-O-methylaverantin, 6,8-di-O-methylaverufin, and 5-methoxysterigmatocystin were isolated from a marine-derived fungal strain Aspergillus sp. SF-6796. Among these, 6,8,1′-tri-O-methylaverantin induced the expression of heme oxygenase (HO)-1 protein in BV2 microglial cells. The induction of HO-1 protein was mediated by the activation of nuclear transcription factor erythroid-2 related factor 2 (Nrf2), and was regulated by the p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/protein kinase B signaling pathways. Furthermore, 6,8,1′-tri-O-methylaverantin suppressed the overproduction of pro-inflammatory mediators, such as nitric oxide, prostaglandin E₂, inducible nitric oxide synthase, and cyclooxygenase-2 in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. These anti-neuroinflammatory effects were mediated through the negative regulation of the nuclear factor kappa B pathway, repressing the phosphorylation and degradation of inhibitor kappa B-α, translocation into the nucleus of p65/p50 heterodimer, and DNA-binding activity of p65 subunit. The anti-neuroinflammatory effect of 6,8,1′-tri-O-methylaverantin was partially blocked by a selective HO-1 inhibitor, suggesting that its anti-neuroinflammatory effect is at least partly mediated by HO-1 induction. In this study, 6,8,1′-tri-O-methylaverantin also induced HO-1 protein expression in primary microglial cells, and this correlated with anti-neuroinflammatory effects observed in LPS-stimulated primary microglial cells. In conclusion, 6,8,1′-tri-O-methylaverantin represents a potential candidate for use in the development of therapeutic agents for the regulation of neuroinflammation in neurodegenerative diseases.
Increases in intracellular calcium perturb blood-brain barrier via protein kinase C-alpha and apoptosis
2016, Biochimica et Biophysica Acta - Molecular Basis of Disease
Citation Excerpt :
Another element of BBB hyperpermeability during ischaemic stroke is the increased damage observed during reperfusion which can lead to vasogenic brain oedema. During reperfusion there is a substantial increase in oxidative stress which contributes to BBB impairment [26,27]. Many studies suggest that increases in ROS levels disrupt endothelial tight junctions, resulting in increased BBB permeability [28–30].
An increase in intracellular calcium represents one of the early events during an ischaemic stroke. It triggers many downstream processes which promote the formation of brain oedema, the leading cause of death after an ischaemic stroke. As impairment of blood–brain barrier (BBB) accounts for much of oedema formation, the current study explored the impact of intracellular calcium on barrier integrity in relation to protein kinase C, caspase-3/7, plasminogen activators and the pro-oxidant enzyme NADPH oxidase. Human brain microvascular endothelial cells alone or in co-culture with human astrocytes were subjected to 4 h of oxygen–glucose deprivation alone or followed by 20 h of reperfusion (OGD ± R) in the absence or presence of inhibitors for urokinase plasminogen activator (amiloride), NADPH oxidase (apocynin), intracellular calcium (BAPTA-AM) and protein kinase C-α (RO-32-0432). Endothelial cells with protein kinase C-α knockdown, achieved by siRNA, were also exposed to the above conditions. BBB permeability was measured by transendothelial electrical resistance and Evan's blue-albumin and sodium fluorescein flux. Intracellular calcium and total superoxide anion levels, caspase-3/7, NADPH oxidase, plasminogen activator and protein kinase C activities, stress fibre formation, the rate of apoptosis and BBB permeability were increased by OGD ± R. Treatment with the specific inhibitors or knockdown of protein kinase C-α attenuated them. This study reveals successive increases in intracellular calcium levels and protein kinase C-α activity are key mechanisms in OGD ± R-mediated impairment of BBB. Furthermore inhibition of protein kinase C-α may be therapeutic in restoring BBB function by reducing the rate of cytoskeletal reorganisation, oxidative stress and apoptosis.
Activin and NADPH-oxidase in preeclampsia: Insights from in vitro and murine studies
2015, American Journal of Obstetrics and Gynecology
Clinical management of preeclampsia has remained unchanged for almost 5 decades. We now understand that maternal endothelial dysfunction likely arises because of placenta-derived vasoactive factors. Activin A is one such antiangiogenic factor that is released by the placenta and that is elevated in maternal serum in women with preeclampsia. Whether activin has a role in the pathogenesis of preeclampsia is not known.
To assess the effects of activin on endothelial cell function, we cultured human umbilical vein endothelial cells in the presence of activin or serum from normal pregnant women or pregnant women with preeclampsia, with or without follistatin, a functional activin antagonist or apocynin, a NADPH oxidase (Nox2) inhibitor. We also administered activin to pregnant C57Bl6 mice, with or without apocynin, and studied maternal and fetal outcomes. Last, we assessed endothelial cell Nox2 and nitric oxide synthase expression in normal pregnant women and pregnant women with preeclampsia.
Activin and preeclamptic serum induced endothelial cell oxidative stress by Nox2 up-regulation and endothelial cell dysfunction, which are effects that are mitigated by either follistatin or apocynin. The administration of activin to pregnant mice induced endothelial oxidative stress, hypertension, proteinuria, fetal growth restriction, and preterm littering. Apocynin prevented all of these effects. Compared with normal pregnant women, women with preeclampsia had increased endothelial Nox2 expression.
An activin-Nox2 pathway is a likely link between an injured placenta, endothelial dysfunction, and preeclampsia. This offers opportunities that are not novel therapeutic approaches to preeclampsia.
Effect of oxidative stress on UDP-glucuronosyltransferases in rat astrocytes
2012, Toxicology Letters
Citation Excerpt :
Menadione was dissolved in DMSO; benzyl viologen and paraquat were dissolved in Hank's Balanced Saline Solution (HBSS) and added directly in the culture medium, to reach the final concentrations of 10 μM (menadione), 60 μM (benzyl viologen) and 400 μM (paraquat). Menadione, benzyl viologen and paraquat were used at concentrations corresponding to the apparent Km of their NADPH-dependent reduction, for the maximal superoxide production measured in rat brain microsomes and brain cultured cells (Lagrange et al., 1999). As antioxidant treatment, astrocytes were incubated with 500 μM melatonin, added in the culture medium at the same time with the xenobiotic compounds.
The present work reports data regarding effects of an induced oxidative stress on the mainly expressed isoforms of UDP-glucuronosyltransferases (UGTs) in the brain. UGT1A6 and UGT1A7 expression and enzymatic activities toward the 1-naphthol were analyzed in rat cultured astrocytes following the exposure for 48 h to redox-cycling xenobiotic compounds such as quinones and bipyridinium ions. The expression of NADPH:cytochrome P450 reductase and NAD(P)H:quinone oxidoreductase 1 (NQO1) was also investigated. Oxidative stress induced significant deleterious changes in astrocyte morphology, decreased cell viability and inhibited catalytic function of UGTs as a result of protein oxidation. Alternatively, in the surviving impaired astrocytes, oxidative conditions induced a significant overactivity and overexpression of xenobiotic detoxification enzymes, as adaptive response. These effects were significantly prevented by the presence of melatonin, suggesting its direct antioxidant action on reactive oxygen species, reflected further on the glucuronidation activity and transcriptional regulation of both UGT1A6 and UGT1A7. Results show that both catalytic properties of UGTs and the expression of UGT1A6, UGT1A7, NQO1 and NADPH:cytochrome P450 reductase in rat astrocytes are greatly influenced by the pro-oxidative environment. In conclusion, an experimental increase in oxidative cellular status could have both immediate and long term consequences on detoxification enzymatic system activity and expression.
Vitamin K3-2,3-epoxide induction of apoptosis with activation of ROS-dependent ERK and JNK protein phosphorylation in human glioma cells
2011, Chemico-Biological Interactions
2-Methyl-1,4-naphthoquinone (menadione or vitamin K3; EPO) and K3-2,3-epoxide (EPO1), but not vitamin K3-3-OH (EPO2), exhibited cytotoxicity that caused DNA fragmentation and chromatin condensation in U87 and C6 cells. EPO1 showed more-potent cytotoxicity than EPO, and the IC₅₀ values of EPO and EPO1 in U87 cells were 37.5 and 15.7 μM, respectively. Activation of caspase 3 enzyme activity with cleavage of caspase 3 protein was detected in EPO1-treated U87 and C6 cells, and the addition of the caspase 3 peptidyl inhibitor, DEVD-FMK, reduced the cytotoxic effect of EPO1. An increase in the intracellular ROS level by EPO1 was observed in the DCHF-DA analysis, and EPO1-induced apoptosis and caspase 3 protein cleavage were prevented by adding the antioxidant, N-acetyl-cysteine (NAC), with decreased ROS production elicited by EPO1. Activation of ERK and JNK, but not p38, via phosphorylation induction was identified in EPO1- but not EPO- or EPO2-treated U87 and C6 cells, and this was blocked by adding NAC. However, the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125, showed no effect on EPO1-induced cytotoxicity in either cell type. Our findings demonstrate that 2,3-epoxide substitution significantly potentiates the apoptotic effect of vitamin K3 via stimulating ROS production, which may be useful in the chemotherapy of glioblastoma cells.

View all citing articles on Scopus

View full text

Original ContributionsTransendothelial permeability changes induced by free radicals in an in vitro model of the blood-brain barrier

Abstract

Introduction

Section snippets

Culture medium components

Chemicals and solvents

Radioisotopes

Effect of menadione, nitrofurazone, and methylviologen on endothelial permeability

Discussion

Acknowledgements

Brain Res.

Toxicol. Appl. Pharmacol.

Free Radic. Biol. Med.

Neurotoxicol. Teratol.

Life Sci.

Brain Res.

J. Surg. Res.

Brain Res.

Gen. Pharmacol.

Neurochem. Int.

Original Contributions
Transendothelial permeability changes induced by free radicals in an in vitro model of the blood-brain barrier