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Originally published online as doi:10.1189/jlb.0605340 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1223-1232.)
© 2005 by Society for Leukocyte Biology

Alcohol-induced oxidative stress in brain endothelial cells causes blood-brain barrier dysfunction

J. Haorah*, B. Knipe*, J. Leibhart*, A. Ghorpade* and Y. Persidsky*,{dagger},1

* Departments of Pharmacology and Experimental Neuroscience, Center for Neurovirology and Neurodegenerative Disorders, and
{dagger} Pathology/Microbiology, University of Nebraska Medical Center, Omaha

1 Correspondence: Center for Neurovirology and Neurodegenerative Disorders, Departments of Pathology and Microbiology and Pharmacology and Experimental Neuroscience, 985215 Nebraska Medical Center, Omaha, NE 68198-5215. E-mail: ypersids{at}unmc.edu

ABSTRACT

Brain microvascular endothelial cells (BMVEC) connected by tight junctions (TJ) form a tight monolayer at the blood-brain barrier (BBB). We investigated the idea that BBB dysfunction seen in alcohol abuse is associated with oxidative stress stemming from ethanol (EtOH) metabolism in BMVEC. Exposure to EtOH induced catalytic activity/expression of EtOH-metabolizing enzymes, which paralleled enhanced generation of reactive oxygen species (ROS). EtOH-mediated oxidative stress led to activation of myosin light chain (MLC) kinase, phosphorylation of MLC and TJ proteins, decreased BBB integrity, and enhanced monocyte migration across BBB. Acetaldehyde or ROS donors mimicked changes induced by EtOH in BMVEC. Thus, oxidative stress resulting from alcohol metabolism in BMVEC can lead to BBB breakdown in alcohol abuse, serving as an aggravating factor in neuroinflammatory disorders.

Key Words: tight junctions • monocyte migration

INTRODUCTION

Blood-brain barrier (BBB) composed of brain microvascular endothelial cells (BMVEC) serves as a protective shield between blood and the central nervous system (CNS). BMVEC possess unique barrier functional properties as compared with microvascular endothelium of other organs and are connected by tight junctions (TJ), which are composed of transmembrane proteins occludin/claudin-5 and intracellular zonula occludens (ZO-1, -2, and -3). TJ ensure structural integrity and low permeability of monolayer [1 ]. Alcohol abuse and neuroinflammatory conditions [such as CNS human immunodeficiency virus type 1 (HIV-1) infection] are associated with BBB impairment. Diffusion-imaging studies in patients with chronic alcoholism suggest that white matter abnormalities are related to BBB dysfunction [2 ]. Similarly, neuroimaging indicated existence of a BBB leak in white matter during HIV-1 encephalopathy, which was reversed by suppression of virus replication and correlated with cognitive improvement after initiation of antiretroviral therapy [3 , 4 ]. Monocytes/macrophages egress across BBB seen in HIV-1 encephalitis (HIVE), the neuropathologic correlate of HIV-1 encephalopathy, paralleled TJ impairment of BMVEC documented by diminished immunostaining for occludin and ZO-1 [5 , 6 ]. It has been shown that the CNS suffers the additive effects of alcohol abuse and HIV-1 in infected patients [7 ] by imaging studies.

Previous studies have clearly indicated that long-term alcohol abuse leads to profound functional and morphological changes in the CNS, regardless of nutritional status. Neuropathologic examination of brain tissue from chronic alcoholics indicated the presence of neurodegeneration, ranging from minor dendritic and synaptic changes to neuronal cell death [8 ]. Thus, multiple lines of evidence suggest that chronic and excessive ethanol (EtOH) consumption may enhance oxidative injury of neural cells. By mechanisms not well understood, EtOH induces activity of the EtOH-metabolizing enzyme cytochrome P450-2E1 (CYP2E1) and enhances reactive oxygen species (ROS) generation in the brain (for review, see ref. [9 ]). It is interesting that expression of CYP2E1, the enzyme that catalyzes EtOH to ROS and acetaldehyde (AA), was prominent in the white matter of animals chronically exposed to alcohol [10 ]. Oxidative stress, associated with inflammatory factors secreted by activated macrophages or viral proteins, was suggested as an underlying cause of BBB disruption in HIV-1 CNS infection [11 ]. Overall, little is known about the mechanisms involved in BBB compromise during HIV-1 CNS infection and excessive alcohol consumption, conditions promoting neuroinflammation and oxidative stress [6 , 9 , 12 13 14 ].

Using our previously developed BBB models and primary BMVEC, we demonstrated that EtOH or its metabolite AA in nontoxic concentrations decreased BBB tightness via activation of myosin light chain (MLC) kinase (MLCK), leading to phosphorylation of MLC/TJ proteins and promoted monocyte migration across BBB models [15 ]. Antioxidant treatment or suppression of EtOH metabolism in BMVEC prevented these phenomena, suggesting that alcohol-associated BBB impairment is mediated, in part, by oxidative stress [15 ]. The current study addresses the role of oxidative stress in BBB impairment caused by alcohol exposure and whether these mechanisms exacerbate BBB dysfunction associated with HIVE. Here, we provide evidence that EtOH metabolites AA and ROS activate MLCK, leading to phosporylation of MLC/TJ proteins and resulting in decreased BBB integrity and enhanced monocyte migration across the BBB. All of these effects were reproduced in the BBB by mediators of oxidative stress. Our data demonstrate a clear link amongst BMVEC alcohol metabolism, oxidative stress, and BBB impairment, with potential implications in neuroinflammation and alcohol abuse.

MATERIALS AND METHODS

Cell isolation and culture
Primary human BMVEC were isolated from the temporal cortex of brain tissue obtained during surgical removal of epileptogenic foci in adult patients by the procedure described previously [16 ] and supplied by Dr. Marlys Witte (University of Arizona, Tucson). Routine evaluation for von Willebrand factor, Ulex europeus lectin, and CD31 demonstrated that cells were >99% pure. Primary bovine BMVEC were isolated from the gray matter of fresh bovine cerebral cortices through enzymatic digestion and subsequent gradient density centrifugation [17 ]. As described before [15 ], freshly isolated cells were cultured in 96-well plates, 24-well tissue-culture inserts (Cyclopore® polyethylene terephthalate membrane, pore diameter 0.4 µm or 3 µm, Collaborative Biochemical Products, Becton Dickinson, San Jose, CA), or in 75 cm2 flasks. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) activity as described previously [18 ]. The following concentrations were used in our experiments: 50 mM EtOH (corresponding to 0.23%); 1 mM 4-methylpyrazole (4-MP), an inhibitor of EtOH metabolizing enzymes CYP2E1 and alcohol dehydrogenase (ADH); 50 µM uric acid (UA) antioxidant; 100 µM AA; 10 µM ML-7 (a specific inhibitor of MLCK); 100 µM H2O2, superoxide donor; 50 µM S-nitroso-N-acetyl-penicillamine (SNAP) nitric oxide (NO) donor; and 50 µM 3-morpholinylsydnoneimine (SIN-1; peroxynitrite donor). All compounds and inhibitors were used in nontoxic concentrations determined by cell viability assay, MTT (96 h exposure).

Dichlorofluorescein-diacetate (DCF-DA) assay
DCF-DA is converted to highly fluorescent DCF by ROS. The fluorescence was detected at excitation 488 nm and at 525 nm emission spectra using a fluorescence plate reader as described previously [19 ]. Results were expressed as specific mean fluorescence intensity (MFI) per mg protein.

CYP2E1 and ADH activity
For ADH and CYP2E1 activity assays, extracted protein lysates were centrifuged at 35,000 rpm (105,000 g) for 1 h at 4°C. High-speed cytosolic fractions (supernatants) were used for ADH activity assay, whereas the microsomes (pellets) were resuspended in 1x phosphate-buffered saline for CYP2E1 activity assay. CYP2E1 activity was determined by hydroxylation of p-nitrophenol to 4-nitrocatechol mediated by CYP2E1 as described before [19 ]. ADH catalytic activity was assayed by formation of reduced nicotinamide adenine dinucleotide (NADH) from NAD oxidation following the method described previously [20 ]. Briefly, 50 µl high-speed cytosolic samples were added to 430 µl reaction mixture consisting of 0.5 M Tris-HCl, 0.01 M dithiothreitol, and 0.5 M EtOH in 1.5 ml disposable cuvette, prewarmed for 5 min, followed by addition of 20 µl 90 mM NAD, mixed well. After 30 s, the change in optical density was monitored at 340 nm using the time-drive kinetics for 10 min at 30 s intervals, and the activity was expressed as nmoles/min/mg total cellular protein.

Transendothelial electrical resistance (TEER) and monocyte migration
The BBB models were constructed [16 ] by placing the BMVEC (200 µl containing 20,000 cells) in the upper chamber of 24-well tissue-culture inserts (Cyclopore® polyethylene terephthalate membrane, pore diameter 0.4 µm for TEER or 3 µm for monocyte migration). The cells were cultured for at least 4 days before use. Using this in vitro BBB model [16 ], the TEER and monocyte migration across the BBB of endothelial cell monolayers were studied as described previously [15 ]. Electrical resistance of the blank inserts (150 {Omega} cm2) with media alone was subtracted from the TEER readings obtained from inserts with confluent endothelial cell monolayers. The resulting TEER values (200–250 {Omega} cm2) represented the resistance ("tightness") of the endothelial cell monolayers. For migration studies across the BBB constructs, peripheral blood monocytes obtained from HIV- and hepatitis B-seronegative donors by leukopheresis were purified by counter-current centrifugal elutriation [21 ] and applied to upper chambers of BBB models (105 cells/insert). Migrated monocytes were counted on the lower chamber after immunostaining with the CD68 antibody (macrophage marker at 1:100 dilution, Dako, Carpenteria, CA).

Immunoconjugation, immunoprecipitation, and Western blot
Immunoconjugation, immunoprecipitation, and Western blot analyses were performed as described [15 ]. Antibodies to MLC, MLCK (Sigma Chemical Co., St. Louis, MO), phospho-MLC-2 (serine 19/threonine18; Cell Signaling, Inc., Beverly, MA), occludin, ZO-1, claudin-5, and antiphosphoserine (Zymed, San Francisco, CA) were used for immunoconjugation, immunoprecipitation, and Western blot analyses. Results were expressed as ratio of relative intensity of target protein to that of the internal standard, {alpha}-actin.

Statistical analysis
Results were expressed as mean values (±SEM), and a value of P < 0.05 was considered significant. The statistical significance effects of EtOH or inhibitor effects on TEER, monocyte migration, and expression of proteins were assessed by two-way ANOVA analyses with Newman-Keuls post-test for multiple comparisons.

RESULTS

EtOH induces ROS generation and ROS-nitrated protein accumulation in BMVEC
Our previous experiments demonstrated that EtOH-induced activation of MLCK led to phosphorylation of MLC and TJ proteins. As a result, BMVEC monolayers demonstrated redistributed TJ staining, gap formation, decreased BBB integrity, and enhanced monocyte migration [15 ]. AA mimicked EtOH-induced effects, and inhibition of EtOH metabolism in endothelial cells reversed these changes. We hypothesized that oxidative stress could be the cause of BBB impairment. To explore this idea, we first examined whether EtOH could lead to oxidative stress in endothelial cells using the DCF-DA assay. H2O2 (100 µM), a ROS-donating agent, and SNAP (50 µM), a NO donor, were used as positive controls. EtOH-exposed BMVEC showed a 38% increase in reactive species levels compared with control (P<0.01), and this increase in ROS formation was abrogated by the metabolic EtOH inhibitor (4-MP) or antioxidant (UA; Fig. 1A ). As expected, treatment of BMVEC with H2O2 or SNAP, individually or in combination, showed significant increases in reactive species production as compared with control (38%, 34%, and 42%, respectively; P<0.01; Fig. 1A ), and antioxidant treatment reverted ROS production to control levels (P>0.05).



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  		Figure 1. EtOH metabolism in BMVEC leads to oxidative stress, which is reversible by antioxidant. BMVEC were treated with EtOH (50 mM) or Ros donors for 2 h, with or without inhibitor of EtOH metabolism (4-MP) or antioxidant (50 µM UA). (A) DCF fluorescence was measured as specific MFI per milligram protein. (B) Representative immunoreactive bands for 210 kDa nitrated proteins and {alpha}-actin. (C) Changes in nitrotyrosine (210 kDa) content, expressed as relative intensity ratio of 210 kDa nitrated protein to that of {alpha}-actin. Results were expressed as mean values (±SEM; n=4) and represented by the bar graph. *, Statistical differences (P<0.01) as compared with untreated control cells.

 
Level of nitrotyrosine was our next indicator of oxidative stress, as ROS leads to formation of nitrated proteins. Using antinitrotyrosine antibody, we detected three nitrotyrosine proteins (molecular weights of 210, 97, and 68 kDa). The 210-kDa protein was the major nitrated protein as compared with 97 kDa or 68 kDa nitrated proteins. Exposure of BMVEC to EtOH, ROS donors, or with inhibitor/scavenger revealed similar effects on levels of each nitrated protein. Figure 1B and 1C , demonstrates data for the 210-kDa nitrotyrosine-positive bands. Similar to ROS levels, EtOH exposure increased content of 210 kDa nitrated protein (by 26%), which was reversed to control level by 4-MP or antioxidant treatment. These results further supported the idea that production of ROS and subsequent nitrotyrosine formation were associated with EtOH metabolism/oxidative stress. Similarly, BMVEC exposure to superoxide (H2O2) or NO (SNAP) resulted in a 37% increase of 210 kDa nitrated protein content as compared with control, untreated cells (Fig. 1C) . However, the combination of H2O2 and SNAP did not further increase the nitrotyrosine level in BMVEC. Peroxynitrite treatment of BMVEC using SIN-1 also enhanced the nitrotyrosine expression (data not shown). Treatment with antioxidants, which would reduce the generation of reactive species, prevented enhanced nitrotyrosine formation, indicating a role for reactive species in formation of nitrotyrosine. Our results suggest that EtOH metabolism by endothelial cells can form reactive species and lead to nitrotyrosine modifications on proteins, indicative of involvement of oxidative stress as one of the mechanisms for effects of EtOH on BBB.

EtOH metabolism in endothelial cells
As EtOH metabolism inhibitor blocked ROS and nitrotyrosine formation in EtOH-treated BMVEC, we next investigated functional activity and expression of two putative EtOH-metabolizing enzymes, CYP2E1 and ADH, in BMVEC. ADH metabolizes EtOH to AA, and CYP2E1-mediated EtOH metabolism leads to production of AA and ROS.

We detected high levels of CYP2E1 catalytic activity (129 nmoles/mg protein) and protein expression in BMVEC. Exposure of BMVEC to EtOH (50 mM) resulted in a 1.6-fold increase of CYP2E1 catalytic activity (up to 200 nmoles/hr/mg protein; P<0.01) and augmented protein level (49%; P<0.01) when compared with the control, unexposed cells (Fig. 2A 2C and 2D ). EtOH-induced increase in CYP2E1 activity and protein content were diminished to control level either by 4-MP or by withdrawal of EtOH from the culture media. Similar results were obtained for ADH activity and protein content in brain endothelial cells. EtOH (50 mM) treatment of BMVEC resulted in a 1.6-fold increase in ADH catalytic activity (from 28 to 44 nmoles/min/mg total cellular protein; P<0.01) and a 43% increase in ADH protein level (P<0.01) as compared with unexposed controls. Addition of 4-MP (ADH inhibitor) or withdrawal of EtOH decreased ADH activity and protein content to basal levels. Using liver microsomal or cytosolic fractions as positive controls (generous gift of Dr. Terrence Donohue, Alcohol Research Center, VA Medical Center, Omaha, NE), derived from control or EtOH pair-fed rats for 5 weeks on a liquid diet, we assayed CYP2E1 and ADH activity in human BMVEC (50 mM EtOH) and monocyte-derived macrophages (MDM; 25 mM EtOH) after 2 h EtOH exposure. Our results indicate that CYP2E1 levels following EtOH exposure increased from 43 to 168 nmoles/hr/mg protein in liver microsomes, from 126 to 164 nmoles/hr/mg protein in human BMVEC, and from 61 to 109 nmoles/hr/mg protein in human MDM. Similarly, ADH activity increased from 41 to 59 nmoles/min/mg protein in liver microsomes, from 20 to 37 nmoles/min/mg protein in human BMVEC, and from 27 to 42 nmoles/min/mg protein. Our data indicate that ADH and CYP2E1 are highly expressed and are functionally active in bovine as well as in human BMVEC, resulting in EtOH metabolism to AA and reactive species.



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  		Figure 2. EtOH effects on CYP2E1 and ADH activity and expression in BMVEC. (A) CYP2E1 activity (nmole/hr/mg protein) and (B) ADH activity (nmole/min/mg protein) in primary bovine BMVEC after 2 h EtOH (50 mM) treatment with or without 4-MP or 2 h after EtOH withdrawal. (C) represents immunoreactive bands for CYP2E1, ADH, and internal standard {alpha}-actin. (D) CYP2E1 and (E) ADH contents were expressed as ratio of densitometric intensity of them to {alpha}-actin. Results were expressed as mean values ± SEM (n=4). *, Statistical differences (P<0.01) as compared with untreated control cells.

 
Oxidative stress activates MLCK and leads to MLC phosphorylation
We reported previously that EtOH metabolism in BMVEC results in MLCK activation and MLC phosphorylation [15 ]. To verify whether EtOH metabolites mediate MLCK activation, we analyzed MLCK expression and MLC phosphorylation in BMVEC after exposure to EtOH, AA, or ROS donors using immunoprecipitation and Western blot. Consistent with previously published results, EtOH-treated BMVEC demonstrated a significant increase in phosphorylated MLC (p-MLC) and MLCK content as compared with untreated controls (Fig. 3B and 3D ). Addition of ethanol metabolic inhibitor or antioxident reversed the EtOH-mediated changes. To clarify contribution of AA and oxidative stress in MLCK activation, we treated BMVEC with exogenous AA or ROS/NO donors. BMVEC exposed to AA (100 µM) showed an increase in the phoshorylated form of MLC and increased MLCK expression (49% and 33%, respectively; P<0.01) with a minor decrease in total MLC protein expression. The magnitude of these changes was similar to ones induced by EtOH (Fig. 3A 3B 3C) . BMVEC treatment with superoxide (H2O2), NO (SNAP), or peroxynitrite (SIN-1) showed a significant increase in the content of p-MLC (37%, 38%, and 45%, respectively; P<0.01) and augmented MLCK protein expression (27%, 28%, and 33%, respectively; P<0.01; Fig. 3A 3B 3D ). Treatment with the peroxynitrite donor significantly reduced expression of total MLC (26%; P<0.01); however, superoxide and NO donors had minimal effects on MLC expression. Antioxidant or specific inhibitor MLCK (ML-7) prevented peroxynitrite-, superoxide-, and NO-mediated MLCK activation, confirming a role of oxidative stress in this process. Overall, our results suggest that EtOH metabolism and resulting oxidative stress could lead to MLCK activation and MLC phosphorylation.



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  		Figure 3. EtOH and oxidative stress induce activation of MLCK and MLC phosphorylation. BMVEC were treated for 2 h with EtOH, AA, H2O2, SNAP, and SIN-1, or cells were preincubated with 4-MP, ML-7, or UA for 0.5 h prior to application of test compounds. Immunoprecipitation and Western blot analyses were performed to determine the expression of p-MLC, MLC, and MLCK. (A) Representative immunoreactive bands of MLC, MLCK, p-MLC, and {alpha}-actin. Relative intensity was expressed as the ratio of the target protein to that of {alpha}-actin. Results were expressed as mean values (±SEM; n=4) and were represented by the bar graph for (B) p-MLC, (C) MLC, and (D) MLCK. Antibodies to antiphosphoserine-MLC were used to measure p-MLC. *, Statistical differences (P<0.01) as compared with untreated control cells.

 
Oxidative stress leads to phosphorylation of TJ proteins
Since MLCK activation results in impaired TJ integrity [15 ], we next examined effects of EtOH metabolites on expression of TJ proteins including occludin, claudin-5, and ZO-1 and their phosphorylation, which is associated with the loosening of BBB. Similar to our previously published findings, EtOH-mediated MLCK activation in BMVEC increased phosphorylation of occludin and claudin-5 (Fig. 4A 4B 4C 4D ) and diminished contents of total occludin and claudin-5 as compared with their respective controls by 32% and 37%, respectively (P<0.01, data not shown). In addition, we found that EtOH also enhanced ZO-1 phosphorylation at serine residues by 43% (P<0.01) without affecting ZO-1 content (Fig. 4E 4F) . Similar to MLC phosphorylation, 4-MP or antioxidant inhibited the EtOH-induced increase in TJ protein phosphorylation. BMVEC exposure to AA resulted in enhanced phosphorylation of serine residues on occludin (by 60%), claudin-5 (by 61%), and ZO-1 (by 50%; P<0.01; Fig. 4B 4D 4F) , paralleling EtOH-mediated effects and MLCK activation. Likewise, BMVEC treatment with donors of superoxide, NO, or peroxynitrite enhanced the phosphorylation of occludin (by 45–67%), claudin-5 (by 40–59%), and ZO-1 (by 44–67%; P<0.01) when compared with their respective controls (Fig. 4B 4D 4F) . Exposure of BMVEC to peroxynitrite donor significantly decreased the contents of TJ proteins (41–45%; P<0.01). While superoxide or NO donors reduced the content of total occludin and ZO-1 (by 26–38%) without affecting claudin-5 content (data not shown). Oxidative stress-mediated augmentation of TJ protein phosphorylation and the decrease of TJ content were prevented by antioxidant. MLCK inhibitor blocked phosphorylation of TJ proteins; however, it did not prevent the oxidative stress-induced decrease in TJ protein contents. Thus, AA and ROS donors reproduced changes induced in BMVEC by EtOH. In toto, these data further support the notion that oxidative stress-mediated phosphorylation of TJ proteins via MLCK activation contributes to impairment of TJ assembly and BBB dysfunction.



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  		Figure 4. EtOH exposure and oxidative stress lead to phosphorylation of TJ proteins. BMVEC were treated for 2 h treatment with EtOH, AA, H2O2, SNAP, and SIN-1, or cells were pre-incubated with 4-MP, ML-7, or UA for 0.5 h prior to application of test compounds. Immunoprecipitation and Western blot analyses were performed to determine the expression of TJ proteins. Results were expressed as mean values (±SEM; n=4). (A) Representative immunoreactive bands of phosphorylated and total occludin, (B) ratio of phosphorylated/total occludin, (C) representative immunoreactive bands of phosphorylated and total claudin-5, (D) ratio of phosphorylated/total claudin-5, (E) representative immunoreactive bands of phosphorylated and total ZO-1, (F) ratio of phosphorylated/total ZO-1. Phosphorylated forms of TJ proteins were determined by probing the total immunoprecipitated proteins with antiphosphoserine antibody. *, Statistical differences (P<0.01) as compared with untreated control cells.

 
EtOH-induced oxidative stress affects TEER and monocyte migration across the BBB
Finally, we determined whether EtOH-induced oxidative stress alters BBB integrity (measured by TEER) and pattern of monocyte migration across the BBB. Application of AA (100 µM) for 2 h reduced TEER by 26% in BBB models, and treatment with donors of superoxide, NO, or peroxynitrite resulted in a 19% to 26% decrease of TEER, which was prevented by antioxidant pretreatment (Fig. 5A ). Consistent with our previous findings, EtOH exposure (50 mM, 2 h) led to a significant decrease in TEER, which was reversed by 4-MP or antioxidant treatment (Fig. 5A) , indicating involvement of reactive species in the loss of BBB integrity. These functional changes paralleled enhanced monocyte migration across the BBB constructs after application of EtOH, AA, or exogenous reactive species donors (Fig. 5B) . Similar to TEER results, all of these treatments for 2 h significantly increased monocyte migration across the BBB (by 222–286%; P<0.01; Fig. 5B ). Effects of EtOH on monocyte migration were prevented by 4-MP or reversed to almost control level by antioxidant (only 46% higher than control as compared with 160% EtOH treated without UA). Antioxidant reduced monocyte passage augmented by peroxynitrite (Fig. 5B , last bar on right) or H2O2 and NO (data not shown) to control levels. All results shown for bovine BMVEC (three different donors) were reproduced with human BMVEC (two independent donors, data not shown). In summary, EtOH metabolism in BMVEC resulted in oxidative stress leading to modification of cytoskeleton organization and TJ assembly. These functional changes led to loss of BBB integrity and increased leukocyte migration across the BBB.



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  		Figure 5. Effects of EtOH, AA, and oxidative stress on TEER and monocyte migration across BBB. (A) EtOH, AA, H2O2, SNAP, and SIN-1 significantly reduced TEER, and TEER measurement across the BMVEC monolayer assessed the BBB tightness. TEER values recorded in ohms ({Omega})/insert area were corrected to {Omega}/cm2 from three experiments in triplicate. Results were then expressed as mean percent of controls at initial time-point (±SEM; n=3). (B) Monolayer treatment for 2 h with EtOH, AA, or exogenous reactive species donors significantly increased monocyte (Mo) migration across the BBB. 4-MP and UA inhibited monocyte migration across the BBB enhanced by EtOH or SIN-1 exposure. *, Statistical differences (P<0.01) as compared with untreated control cells.

 
DISCUSSION

The present study demonstrates that EtOH-metabolizing enzymes ADH and CYP2E1 are expressed in BMVEC, and EtOH metabolism in BMVEC leads to oxidative stress, resulting in MLCK activation (Fig. 6 ). Activated MLCK phosphorylates MLC and TJ proteins, leading to cytoskeletal rearrangement and impairment of TJ integrity. These events cause BBB dysfunction and enhanced leukocyte migration across BBB. It is important that our data indicate that oxidative stress per se could result in similar BBB alterations via the same pathways.



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  		Figure 6. Schematic representation: effects of EtOH and oxidative stress on BBB. EtOH metabolism in brain endothelial cells results in production of AA and reactive species. These, in turn, activate MLCK, leading to phosphorylation of MLC and TJ proteins, functional alterations of TJ, and cytoskeletal rearrangement. Ultimately, these changes result in BBB compromise and enhanced leukocyte migration across BBB.

 
Here, we present evidence that EtOH-metabolizing enzymes ADH and CYP2E1 are functionally active in BMVEC, and EtOH exposure significantly increases the levels and activities of both enzymes. Metabolism of EtOH by ADH generates toxic AA, while metabolism of EtOH by CYP2E1 produces AA and ROS [22 , 23 ]. Consistent with our results, enhanced CYP2E1 activity was shown to correlate with increased oxidative stress in liver tissue from EtOH-fed animals [24 25 26 ] and rat fetal astrocytes [27 ]. We reported similar results for human macrophages where an EtOH-induced increase in CYP2E1 activity and content correlated with augmented ROS production [19 ], suggesting that CYP2E1 may be the common pathway of EtOH metabolism in cells of mesodermal origin (macrophages and endothelium). Previously, CYP2E1 expression was documented in rat brain tissues [28 , 29 ] and endothelial cells of different sources (human coronary artery [30 , 31 ], human umbilical vein [32 , 33 ], porcine, or bovine endothelial cells [33 ]). Martinez and colleagues [34 ] reported that rat brain endothelial cells express high levels of ADH in vivo. However, the current study is the first report demonstrating high levels of ADH/CYP2E1 activity and expression in bovine and human BMVEC, their further increases by EtOH exposure, and links these BMVEC EtOH metabolism related changes to oxidative stress. According to our results, the basal level of CYP2E1 catalytic activity in bovine/human BMVEC was similar to those of rat/mouse liver microsomes [35 ]. However, the degree of increase in CYP2E1 activity induced by EtOH was much higher in rat/mouse liver microsomes (4-fold) as compared with bovine or human BMVEC (1.3- to 1.6-fold). Furthermore, bovine/human BMVEC and human macrophages exhibited ADH activities comparable with that of rat liver cytosolic proteins. As AA converts xanthine dehydrogenase to xanthine oxidase, leading to enhanced ROS production [36 ], ADH activity (metabolizing EtOH to AA) may also contribute to BBB injury associated with oxidative stress. This may explain our data indicating the AA ability to mimic effects of EtOH/oxidative stress on cytoskeletal and TJ protein changes in BMVEC.

Organ dysfunction stemming from alcohol abuse has significant clinical implications [37 ]. Oxidative stress associated with alcohol abuse exacerbated endothelial and epithelial cell injury in lungs seen during acute inflammation in trauma patients [38 ]. These pathologic changes were ameliorated by antioxidant treatment in an animal model with chronic alcohol exposure [39 ]. ROS-related damage of brain tissue was documented in alcoholics including cerebral edema, neuronal loss, and damage of BBB [40 ]. Other pathophysiologic conditions associated with oxidative stress may decrease BBB integrity and enhance monocyte migration. Increased expression of adhesion molecules on endothelial cells and their respective ligands on monocytes-macrophages occur shortly after exposure to oxidative stress, or lipid oxidation products can be modulated by antioxidants [41 42 43 ]. Leukocyte-endothelial cell interactions via adhesion molecules lead to ROS generation and disruption of junctional complexes [44 ]. ROS scavenging inhibits these effects on junctions and transendothelial leukocyte migration [45 ]. Leukocyte adhesion to endothelium induces ROS production, which changes the functional state of endothelium, facilitating leukocyte passage [46 ], and our data suggest that the EtOH metabolism in BMVEC can activate similar pathways, increasing monocyte migration.

By a yet-unknown mechanism, oxidative stress activated MLCK in BMVEC, which phosphorylated cytoskeletal (MLC) and TJ proteins, diminishing the integrity of the BMVEC monolayer. Exogenously supplied AA, peroxide, peroxynitrite, and NO mimicked EtOH-metabolic effects in BMVEC, leading to reduced TEER and increased monocyte migration across the BBB, further supporting the role of oxidative stress in BBB dysfunction. Similar to our findings, Zhao and Davis [47 ] reported the peroxide-induced activation of MLCK and subsequent MLC phosphorylation in primary pulmonary endothelial cells.

We demonstrated that MLC, claudin-5, and occludin were predominantly phosphorylated at serine and minimally at threonine residues after BMVEC treatment with EtOH [15 ]. Here, we present evidence that EtOH-induced oxidative stress via MLCK activation led to phosphorylation of MLC and TJ proteins. Mediators of oxidative stress (peroxide, peroxynitrite, and NO) elicited the same response in BMVEC. Phosphorylation of MLC changes BBB integrity, likely through altered actin-myosin interactions. In endothelial cells, MLC phosphorylation at serine/threonine residues is associated with cell contraction, cytoskeletal rearrangement, or loss of barrier integrity, and the MLCK inhibitor can prevent these changes [15 , 48 , 49 ].

Although occludin phosphorylation at serine/threonine residues altered the actin-cytoskeletal interactions [50 , 51 ], occludin phosphorylation at tyrosine residues attenuated the interactions with ZO-1, -2, and -3 [52 ]. Occludin phosphorylation at these sites also increased the permeability of endothelial monolayers [53 , 54 ], and dephosphorylation restored barrier tightness [55 ]. Diminished occludin expression, enhanced occludin phosphorylation at serine residues, and increased permeability were observed in endothelial cells exposed to H2O2 [56 ]. These studies concur with our findings of enhanced occludin phosphorylation and decreased content in BMVEC caused by oxidative stress.

Claudin-5 is believed to be a vital component of TJ in the BBB [57 , 58 ]. Thus, decreased claudin-5 content mediated by oxidative stress/EtOH metabolism observed here could be one of the important contributing factors for BBB breakdown. Increased ZO-1 phosphorylation at serine/threonine residues by EtOH, AA, or ROS donors correlated with augmented monocyte migration across the BBB, and these findings are supported by those of others that phosphorylation of ZO-1 at these sites was associated with an increase in paracellular permeability in BMVEC after hypoxia [59 ]. ZO-1 phosphorylation at tyrosine residues resulted in augmented permeability of endothelial monolayers [53 ]. Exposure of endothelial cells to H2O2 [60 ] resulted in decreased of TEER and ZO-1/occludin redistribution, similar to effects of EtOH in BMVEC [15 ].

A number of studies demonstrated inhibitory effects of EtOH on cytokine and chemokine production and reduction of leukocyte migration. In vivo, these effects are usually observed after acute short-term alcohol exposure [61 ], and more chronic studies pointed to existence of vascular injury promoting leakiness and leukocyte adhesion [62 ]. Similarly, chronic exposure to EtOH in vitro led to increased cytokine and NO production in glial cells [63 ]. Diffusion-imaging studies in patients with chronic alcoholism suggest that white matter abnormalities may be related to BBB dysfunction [2 ]. In our system, alcohol-induced leakiness of BBB and enhanced monocyte migration were highly reproducible phenomena. Differences between in vitro and in vivo systems and length of EtOH exposure in vivo could explain seemingly contradictory observations.

Cytokines, chemokines, other secretory inflammatory products and viral proteins, such as gp120 or Tat produced by virus-infected and/or immune-activated macrophages, are thought to be the ultimate cause of HAD [6 ]. Oxidative stress plays an important role in HIV-1 CNS infection [12 ]. It has been shown that HIV-1 Tat protein-induced oxidative stress altered ZO-1 protein in mouse brain [64 ] and claudin-5 protein in rat brain endothelial cells [11 ], leading to disruption of BBB. Thus, apart from proinflammatory products or viral proteins, oxidative stress could be the significant contributing factor in HIV-1-infected patients with a history of alcohol abuse, further worsening BBB dysfunction. Indeed, it has been shown that CNS suffers the additive effects of alcohol abuse and HIV-1 [7 ]. Additional studies will be necessary to address relevance of our in vitro findings and potential synergy amongst multiple deleterious factors such as viral proteins, proinflammatory cytokines, and oxidative stress and their net effects on BBB during HIV-1 CNS infection in the setting of alcohol abuse. This will be accomplished using an animal model for HIVE [65 ].

ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health Grants R21 AA013846 and R01 MH65151 (to Y. P.). The authors of this manuscript appreciate excellent administrative support from Ms. Robin Taylor and Ms. Julie Ditter. We thank Dr. Mark Thomas for critical reading of the manuscript. Liver microsomal and cytosolic fractions, which served as positive controls, were provided Dr. Terrence Donohue (Alcohol Research Center, VA Medical Center, Omaha, NE).

Received June 23, 2005; revised August 1, 2005; accepted August 8, 2005.

REFERENCES

  1. Engelhardt, B. (2003) Development of the blood-brain barrier Cell Tissue Res. 314,119-129[CrossRef][Medline]
  2. Mann, K., Agartz, I., Harper, C., Shoaf, S., Rawlings, R. R., Momenan, R., Hommer, D. W., Pfefferbaum, A., Sullivan, E. V., Anton, R. F., Drobes, D. J., George, M. S., Bares, R., Machulla, H. J., Mundle, G., Reimold, M., Heinz, A. (2001) Neuroimaging in alcoholism: ethanol and brain damage Alcohol. Clin. Exp. Res. 25,104S-109S[Medline]
  3. Gendelman, H. E., Zheng, J., Coulter, C. L., Ghorpade, A., Che, M., Thylin, M., Rubocki, R., Persidsky, Y., Hahn, F., Reinhard, J., Jr, Swindells, S. (1998) Suppression of inflammatory neurotoxins by highly active antiretroviral therapy in human immunodeficiency virus-associated dementia J. Infect. Dis. 178,1000-1007[Medline]
  4. Avison, M. J., Nath, A., Greene-Avison, R., Schmitt, F. A., Greenberg, R. N., Berger, J. R. (2004) Neuroimaging correlates of HIV-associated BBB compromise J. Neuroimmunol. 157,140-146[CrossRef][Medline]
  5. Dallasta, L. M., Pisarov, L. A., Esplen, J. E., Werley, J. V., Moses, A. V., Nelson, J. A., Achim, C. L. (1999) Blood-brain barrier tight junction disruption in human immunodeficiency virus-1 encephalitis Am. J. Pathol. 155,1915-1927[Abstract/Free Full Text]
  6. Persidsky, Y., Gendelman, H. E. (2003) Mononuclear phagocyte immunity and the neuropathogenesis of HIV-1 infection J. Leukoc. Biol. 74,691-701[Abstract/Free Full Text]
  7. Fein, G., Fletcher, D. J., Di Sclafani, V. (1998) Effect of chronic alcohol abuse on the CNS morbidity of HIV disease Alcohol. Clin. Exp. Res. 22,196S-200S[CrossRef][Medline]
  8. Harper, C. (1998) The neuropathology of alcohol-specific brain damage, or does alcohol damage the brain? J. Neuropathol. Exp. Neurol. 57,101-110[Medline]
  9. Sun, A. Y., Sun, G. Y. (2001) Ethanol and oxidative mechanisms in the brain J. Biomed. Sci. 8,37-43[CrossRef][Medline]
  10. Hansson, T., Tindberg, N., Ingelman-Sundberg, M., Kohler, C. (1990) Regional distribution of ethanol-inducible cytochrome P450 IIE1 in the rat central nervous system Neuroscience 34,451-463[CrossRef][Medline]
  11. Andras, I. E., Pu, H., Tian, J., Deli, M. A., Nath, A., Hennig, B., Toborek, M. (2005) Signaling mechanisms of HIV-1 Tat-induced alterations of claudin-5 expression in brain endothelial cells J. Cereb. Blood Flow Metab. Epub ahead of print.
  12. Boven, L. A., Gomes, L., Hery, C., Gray, F., Verhoef, J., Portegies, P., Tardieu, M., Nottet, H. S. (1999) Increased peroxynitrite activity in AIDS dementia complex: implications for the neuropathogenesis of HIV-1 infection J. Immunol. 162,4319-4327[Abstract/Free Full Text]
  13. Flora, G., Pu, H., Lee, Y. W., Ravikumar, R., Nath, A., Hennig, B., Toborek, M. (2005) Proinflammatory synergism of ethanol and HIV-1 Tat protein in brain tissue Exp. Neurol. 191,2-12[CrossRef][Medline]
  14. Belmadani, A., Neafsey, E. J., Collins, M. A. (2003) Human immunodeficiency virus type 1 gp120 and ethanol coexposure in rat organotypic brain slice cultures: curtailment of gp120-induced neurotoxicity and neurotoxic mediators by moderate but not high ethanol concentrations J. Neurovirol. 9,45-54[Medline]
  15. Haorah, J., Heilman, D., Knipe, B., Chrastil, J., Ghorpade, A., Miller, D. W., Persidsky, Y. (2005) Ethanol-induced activation of myosin light chain kinase leads to dysfunction of tight junctions and blood-brain barrier compromise Alcohol. Clin. Exp. Res. 29,999-1009[CrossRef][Medline]
  16. Persidsky, Y., Stins, M., Way, D., Witte, M. H., Weinand, M., Kim, K. S., Bock, P., Gendelman, H. E., Fiala, M. (1997) A model for monocyte migration through the blood-brain barrier during HIV-1 encephalitis J. Immunol. 158,3499-3510[Abstract]
  17. Andus, K. L., Borchardt, R. T. (1987) Bovine brain microvessel endothelial cell monolayers as a model for the blood-brain barrier Ann NY Acad. Sci. 507,9-18[CrossRef]
  18. Manthorpe, M., Fagnani, R., Skaper, S. D., Varon, S. (1986) An automated colorimetric microassay for neuronotrophic factors Brain Res. 390,191-198[CrossRef][Medline]
  19. Haorah, J., Heilman, D., Diekmann, C., Osna, N., Donohue, T. M., Jr, Ghorpade, A., Persidsky, Y. (2004) Alcohol and HIV decrease proteasome and immunoproteasome function in macrophages: implications for impaired immune function during disease Cell. Immunol. 229,139-148[CrossRef][Medline]
  20. Clemens, D. L., Halgard, C. M., Miles, R. R., Sorrell, M. F., Tuma, D. J. (1995) Establishment of a recombinant hepatic cell line stably expressing alcohol dehydrogenase Arch. Biochem. Biophys. 321,311-318[CrossRef][Medline]
  21. Gendelman, H. E., Orenstein, J. M., Martin, M. A., Ferrua, C., Mitra, R., Phipps, T., Wahl, L. A., Lane, H. C., Fauci, A. S., Burke, D. S., et al (1988) Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes J. Exp. Med. 167,1428-1441[Abstract/Free Full Text]
  22. Persson, J. O., Terelius, Y., Ingelman-Sundberg, M. (1990) Cytochrome P-450-dependent formation of reactive oxygen radicals: isozyme-specific inhibition of P-450-mediated reduction of oxygen and carbon tetrachloride Xenobiotica 20,887-900[Medline]
  23. Ingelman-Sundberg, M., Johansson, I., Yin, H., Terelius, Y., Eliasson, E., Clot, P., Albano, E. (1993) Ethanol-inducible cytochrome P4502E1: genetic polymorphism, regulation, and possible role in the etiology of alcohol-induced liver disease Alcohol 10,447-452[CrossRef][Medline]
  24. French, S. W., Wong, K., Jui, L., Albano, E., Hagbjork, A. L., Ingelman-Sundberg, M. (1993) Effect of ethanol on cytochrome P450 2E1 (CYP2E1), lipid peroxidation, and serum protein adduct formation in relation to liver pathology pathogenesis Exp. Mol. Pathol. 58,61-75[CrossRef][Medline]
  25. Wu, D., Cederbaum, A. I. (2003) Alcohol, oxidative stress, and free radical damage Alcohol Res. Health 27,277-284[Medline]
  26. Jimenez-Lopez, J. M., Cederbaum, A. I. (2005) Protein kinase C signaling as a survival pathway against CYP2E1-derived oxidative stress and toxicity in HepG2 cells J. Pharmacol. Exp. Ther. 312,998-1006[Abstract/Free Full Text]
  27. Montoliu, C., Sancho-Tello, M., Azorin, I., Burgal, M., Valles, S., Renau-Piqueras, J., Guerri, C. (1995) Ethanol increases cytochrome P4502E1 and induces oxidative stress in astrocytes J. Neurochem. 65,2561-2570[Medline]
  28. Anandatheerthavarada, H. K., Shankar, S. K., Bhamre, S., Boyd, M. R., Song, B. J., Ravindranath, V. (1993) Induction of brain cytochrome P-450IIE1 by chronic ethanol treatment Brain Res. 601,279-285[CrossRef][Medline]
  29. Warner, M., Gustafsson, J. A. (1994) Effect of ethanol on cytochrome P450 in the rat brain Proc. Natl. Acad. Sci. USA 91,1019-1023[Abstract/Free Full Text]
  30. Borlak, J., Walles, M., Levsen, K., Thum, T. (2003) Verapamil: metabolism in cultures of primary human coronary arterial endothelial cells Drug Metab. Dispos. 31,888-891[Abstract/Free Full Text]
  31. Kapucuoglu, N., Coban, T., Raunio, H., Pelkonen, O., Edwards, R. J., Boobis, A. R., Iscan, M. (2003) Immunohistochemical demonstration of the expression of CYP2E1 in human breast tumor and non-tumor tissues Cancer Lett. 196,153-159[CrossRef][Medline]
  32. Hoebel, B. G., Steyrer, E., Graier, W. F. (1998) Origin and function of epoxyeicosatrienoic acids in vascular endothelial cells: more than just endothelium-derived hyperpolarizing factor? Clin. Exp. Pharmacol. Physiol. 25,826-830[Medline]
  33. Farin, F. M., Pohlman, T. H., Omiecinski, C. J. (1994) Expression of cytochrome P450s and microsomal epoxide hydrolase in primary cultures of human umbilical vein endothelial cells Toxicol. Appl. Pharmacol. 124,1-9[CrossRef][Medline]
  34. Martinez, S. E., Vaglenova, J., Sabria, J., Martinez, M. C., Farres, J., Pares, X. (2001) Distribution of alcohol dehydrogenase mRNA in the rat central nervous system. Consequences for brain ethanol and retinoid metabolism Eur. J. Biochem. 268,5045-5056[Medline]
  35. Bray, B. J., Perry, N. B., Menkes, D. B., Rosengren, R. J. (2002) St. John’s wort extract induces CYP3A and CYP2E1 in the Swiss Webster mouse Toxicol. Sci. 66,27-33[Abstract/Free Full Text]
  36. Sultatos, L. G. (1988) Effects of acute ethanol administration on the hepatic xanthine dehydrogenase/oxidase system in the rat J. Pharmacol. Exp. Ther. 246,946-949[Abstract/Free Full Text]
  37. Zima, T., Fialova, L., Mestek, O., Janebova, M., Crkovska, J., Malbohan, I., Stipek, S., Mikulikova, L., Popov, P. (2001) Oxidative stress, metabolism of ethanol and alcohol-related diseases J. Biomed. Sci. 8,59-70[Medline]
  38. Burnham, E. L., Brown, L. A., Halls, L., Moss, M. (2003) Effects of chronic alcohol abuse on alveolar epithelial barrier function and glutathione homeostasis Alcohol. Clin. Exp. Res. 27,1167-1172[CrossRef][Medline]
  39. Velasquez, A., Bechara, R. I., Lewis, J. F., Malloy, J., McCaig, L., Brown, L. A., Guidot, D. M. (2002) Glutathione replacement preserves the functional surfactant phospholipid pool size and decreases sepsis-mediated lung dysfunction in ethanol-fed rats Alcohol. Clin. Exp. Res. 26,1245-1251[CrossRef][Medline]
  40. Pratt, O. E., Rooprai, H. K., Shaw, G. K., Thomson, A. D. (1990) The genesis of alcoholic brain tissue injury Alcohol Alcohol. 25,217-230[Abstract/Free Full Text]
  41. Blouin, E., Halbwachs-Mecarelli, L., Rieu, P. (1999) Redox regulation of ß2-integrin CD11b/CD18 activation Eur. J. Immunol. 29,3419-3431[CrossRef][Medline]
  42. Kunsch, C., Luchoomun, J., Grey, J. Y., Olliff, L. K., Saint, L. B., Arrendale, R. F., Wasserman, M. A., Saxena, U., Medford, R. M. (2004) Selective inhibition of endothelial and monocyte redox-sensitive genes by AGI-1067: a novel antioxidant and anti-inflammatory agent J. Pharmacol. Exp. Ther. 308,820-829[Abstract/Free Full Text]
  43. Pratico, D. (2005) Antioxidants and endothelium protection Atherosclerosis 181,215-224[CrossRef][Medline]
  44. Cook-Mills, J. M. (2002) VCAM-1 signals during lymphocyte migration: role of reactive oxygen species Mol. Immunol. 39,499-508[CrossRef][Medline]
  45. Matheny, H. E., Deem, T. L., Cook-Mills, J. M. (2000) Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase J. Immunol. 164,6550-6559[Abstract/Free Full Text]
  46. van Buul, J. D., Hordijk, P. L. (2004) Signaling in leukocyte transendothelial migration Arterioscler. Thromb. Vasc. Biol. 24,824-833[Abstract/Free Full Text]
  47. Zhao, Y., Davis, H. W. (1998) Hydrogen peroxide-induced cytoskeletal rearrangement in cultured pulmonary endothelial cells J. Cell. Physiol. 174,370-379[CrossRef][Medline]
  48. Yuan, S. Y., Wu, M. H., Ustinova, E. E., Guo, M., Tinsley, J. H., De Lanerolle, P., Xu, W. (2002) Myosin light chain phosphorylation in neutrophil-stimulated coronary microvascular leakage Circ. Res. 90,1214-1221[Abstract/Free Full Text]
  49. Kawkitinarong, K., Linz-McGillem, L., Birukov, K. G., Garcia, J. G. (2004) Differential regulation of human lung epithelial and endothelial barrier function by thrombin Am. J. Respir. Cell Mol. Biol. 31,517-527[Abstract/Free Full Text]
  50. Sakakibara, A., Furuse, M., Saitou, M., Ando-Akatsuka, Y., Tsukita, S. (1997) Possible involvement of phosphorylation of occludin in tight junction formation J. Cell Biol. 137,1393-1401[Abstract/Free Full Text]
  51. Wong, V. (1997) Phosphorylation of occludin correlates with occludin localization and function at the tight junction Am. J. Physiol. 273,C1859-C1867
  52. Kale, G., Naren, A. P., Sheth, P., Rao, R. K. (2003) Tyrosine phosphorylation of occludin attenuates its interactions with ZO-1, ZO-2, and ZO-3 Biochem. Biophys. Res. Commun. 302,324-329[CrossRef][Medline]
  53. Antonetti, D. A., Barber, A. J., Hollinger, L. A., Wolpert, E. B., Gardner, T. W. (1999) Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors J. Biol. Chem. 274,23463-23467[Abstract/Free Full Text]
  54. Hirase, T., Kawashima, S., Wong, E., Uemada, T., Rikitake, Y., Tsukita, S., Yokoyama, M., Staddon, J. (2001) Regulation of tight junction permeability and occludin phosphorylation byRhoA-p160ROCK-dependent and independent mechanism J. Biol. Chem. 276,10423-10431[Abstract/Free Full Text]
  55. Antonetti, D. A., Wolpert, E. B., DeMaio, L., Harhaj, N. S., Scaduto, R. C., Jr (2002) Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin J. Neurochem. 80,667-677[CrossRef][Medline]
  56. Kevil, C. G., Oshima, T., Alexander, B., Coe, L. L., Alexander, J. S. (2000) H(2)O(2)-mediated permeability: role of MAPK and occludin Am. J. Physiol. Cell Physiol. 279,C21-C30[Abstract/Free Full Text]
  57. Furuse, M., Hata, M., Furuse, K., Yoshida, Y., Haratake, A., Sugitani, Y., Noda, T., Kubo, A., Tsukita, S. (2002) Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice J. Cell Biol. 156,1099-1111[Abstract/Free Full Text]
  58. Nitta, T., Hata, M., Gotoh, S., Seo, Y., Sasaki, H., Hashimoto, N., Furuse, M., Tsukita, S. (2003) Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice J. Cell Biol. 161,653-660[Abstract/Free Full Text]
  59. Fischer, S., Wobben, M., Marti, H. H., Renz, D., Schaper, W. (2002) Hypoxia-induced hyperpermeability in brain microvessel endothelial cells involves VEGF-mediated changes in the expression of zonula occludens-1 Microvasc. Res. 63,70-80[CrossRef][Medline]
  60. Lee, H. S., Namkoong, K., Kim, D. H., Kim, K. J., Cheong, Y. H., Kim, S. S., Lee, W. B., Kim, K. Y. (2004) Hydrogen peroxide-induced alterations of tight junction proteins in bovine brain microvascular endothelial cells Microvasc. Res. 68,231-238[CrossRef][Medline]
  61. Saeed, R. W., Varma, S., Peng, T., Tracey, K. J., Sherry, B., Metz, C. N. (2004) Ethanol blocks leukocyte recruitment and endothelial cell activation in vivo and in vitro J. Immunol. 173,6376-6383[Abstract/Free Full Text]
  62. Han, J. Y., Miura, S., Akiba, Y., Higuchi, H., Kato, S., Suzuki, H., Yokoyama, H., Ishii, H. (2001) Chronic ethanol consumption exacerbates microcirculatory damage in rat mesentery after reperfusion Am. J. Physiol. Gastrointest. Liver Physiol. 280,G939-G948[Abstract/Free Full Text]
  63. Davis, R. L., Syapin, P. J. (2004) Acute ethanol exposure modulates expression of inducible nitric-oxide synthase in human astroglia: evidence for a transcriptional mechanism Alcohol 32,195-202[CrossRef][Medline]
  64. Pu, H., Tian, J., Andras, I. E., Hayashi, K., Flora, G., Hennig, B., Toborek, M. (2005) HIV-1 Tat protein-induced alterations of ZO-1 expression are mediated by redox-regulated ERK1/2 activation J. Cereb. Blood Flow Metab. Epub ahead of print.
  65. Potula, R., Poluektova, L., Knipe, B., Chrastil, J., Heilman, D., Dou, H., Takikawa, O., Munn, D. H., Gendelman, H. E., Persidsky, Y. (2005) Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in animal model of HIV-1 encephalitis Blood 106,2382-2390[Abstract/Free Full Text]



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