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* Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands;
Weill Medical College, Cornell University, New York, USA;
Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes, UK;
Institut Cochin, Departement de Biologie Cellulaire, Paris, France;
|| Institute of Molecular Pharmacology, Berlin, Germany; and
¶ Solvay Pharmaceuticals Research Laboratories, Weesp, The Netherlands
2Correspondence: Department of Molecular Cell Biology and Immunology, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands, E-mail: he.devries{at}vumc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: blood-brain barrier • claudin-5 • occludin • protein kinase B
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
TJs are continuous membrane strands located at the apical site between brain ECs, which consist of transmembrane and cytoplasmic proteins that are associated with the actin cytoskeleton. The transmembrane proteins occludin and the claudins mediate cellular interaction between brain ECs and play a major role in TJ functioning. Occludin is a phosphoprotein that spans the plasma membrane four times with intracellular location of both the amino and the carboxyl termini (2
, 3)
. Occludin expression is associated with increased electrical resistance and decreased paracellular transport (4)
. Claudins comprise a multigene family consisting of more than 20 members and contain 2 extracellular loops and 4 transmembrane domains and interact in both a homophilic and heterophilic way with claudins of adjacent cells (5
, 6)
. Claudin-5 is a critical component of the BBB as it closes the BBB for small molecules up to 800 Da (7)
. The carboxyterminal parts of both occludin and claudins interact with membrane-associated recruiting proteins of the zona occludens (ZO) protein family (8
, 9)
. ZO proteins are reported to link transmembrane proteins to the actin cytoskeleton and have signaling potential (10
, 11)
. Through its interaction with TJ molecules, the actin cytoskeleton plays an active role in maintaining TJ integrity and BBB function (12)
. Several cytoplasmic signaling molecules, such as Rho, PI3 kinase, protein kinase C (PKC), Ca2+, heterotrimeric G proteins, cyclic adenosine monophosphate (cAMP), and phospholipase C have been localized to TJ complexes and may regulate their assembly and disassembly [for review see (13
, 14)
].
A defective function of the BBB is reported in neuroinflammatory diseases like multiple sclerosis (MS) (15
, 16)
, HIV-associated dementia (17)
and encephalitis (18
, 19)
, stroke, and brain trauma (20)
. Pathological events that may occur at the BBB include structural and spatial alterations of the TJ, enhanced permeability for blood-derived components, and infiltration of inflammatory cells into the CNS. In these processes, proinflammatory mediators like chemokines (21)
and cytokines (22
, 23)
play an important role. Previously we showed that reactive oxygen species (ROS), which are highly reactive molecules, are produced during monocyte migration and contribute to BBB injury and subsequent inflammation in the brain (24
, 25)
. Indeed, scavenging extracellular ROS by lipoic acid and luteolin prevented the development of clinical signs in animal models for MS, acute and chronic experimental allergic encephalomyelitis (24
, 26)
. Currently, specific ROS signaling pathways within brain ECs that target the TJ to disengage are unclear, although previous studies from our group revealed that ROS induced enhanced Ca2+ mobilization and inositol (1
, 4
, 5)
-trisphosphate (IP3) formation in brain ECs via phospholipase C (25)
. Furthermore, ROS may activate protein tyrosine kinases in brain endothelial cells, which in turn may lead to tyrosine phosphorylation of TJ proteins (27)
.
Aim of current study was to elucidate the ROS-dependent signaling pathways regulating TJ dynamics in brain ECs. We provide evidence that ROS selectively activate signaling cascades involving RhoA, PI3 kinase, and protein kinase B (PKB/Akt) leading to rearrangements of the actin cytoskeleton and spatial redistribution and disappearance of occludin and claudin-5, inducing altered BBB integrity. Selective inhibitors of identified signaling pathways and antioxidants reversed observed alterations and prevented ROS-induced monocyte migration across an in vitro model of the BBB. Molecular understanding of the regulation of the function and integrity of the BBB is essential to identify agents that can prevent BBB dysfunction in neuroinflammatory diseases thus limiting neurological deficits.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of brain cell line overexpressing claudin-5-YFP
The DNA sequence of mouse claudin-5 fused to the N terminus of YFP was subcloned from the pEYFP-N1 vector (28)
into the modified retroviral vector pLZRS-IRES-zeocin. The resulting construct, pLZRS-Claudin-5-YFP-IRES-zeocin, was transfected into amphotropic Phoenix retrovirus producer cells. Subsequently, virus containing supernatant was used to transduce immortalized rat GP8/3.9 brain endothelial cells (ECs) as described (29)
. Expression and localization of claudin-5-YFP were determined by confocal laser scanning microscopy (CLSM) at room temperature (Leica TCS SP2 AOBS microscope, HCX PL APO 63x/1.30 lens; Leica Microsystems B.V., Rijswijk, The Netherlands). The cells were grown on collagen-coated glass coverslips and fixed with 4% paraformaldehyde. Claudin-5-YFP was labeled with polyclonal rabbit anticlaudin-5 followed by Alexa 594-conjugated secondary antibodies. Mounted coverslips were analyzed by sequential excitation at 514 /594 nm.
Cell culture
The Lewis immortalized rat brain endothelial cell line GP8/3.9 (30)
, the GFP-occludin- (29)
, and the claudin-5-YFP expressing GP8/3.9 cell lines were cultured as described (31)
. The human immortalized brain endothelial cell line hCMEC/D3 was cultured as described (32)
. Primary rat brain ECs were isolated and cultured as described (33)
ROS generating system
A mixture of 0.02 U/ml xanthine oxidase and 100 µM hypoxanthine was used to generate ROS as described previously (25)
. This mixture is known to produce constant levels of predominantly superoxide and to a lesser extent hydrogen peroxide and hydroxyl radicals (34)
.
Live cell analysis of TJ rearrangements
The GFP-occludin and claudin-5-YFP expressing immortalized rat GP8/3.9 cells were cultured to confluence on collagen-coated 42 mm diameter glass cover slips in Ham’s F12 medium supplemented with 2.5% fetal calf serum. ROS was generated as described above. CLSM was performed at 37°C
Electrical cell substrate impedance sensing (ECIS) assay
ECISTM Model 1600R (Applied BioPhysics, Troy, NY, USA) was used to measure TEER in confluent monolayers of immortalized human hCMEC/D3 cells. In short, 250 µl of cell suspension (8x105 cells per ml) was seeded to each well of an 8W10 ECIS array equilibrated with EC growth medium without serum and coated with collagen. When monolayers reached maximum resistance (
850 
) xanthine oxidase (0.08 U/ml) and 100 µM hypoxanthine were added to generate ROS and the endothelial integrity was measured in real-time as described (35)
.
Permeability of the BBB in vitro
Immortalized human hCMEC/D3 cells were cultured onto collagen (upper side, Sigma) coated Costar Transwell filters (pore-size 0.4 µm; Corning Incorporated, Corning, NY, USA), and rat primary ECs were cultured onto collagen en fibronectin coated Costar Transwell filters in endothelial cell growth medium 1:1 mixed with astrocyte-conditioned medium (36)
. Permeability for FITC-dextran (150 kDa) was assayed as described before (37)
, and the influence of ROS on the permeability was tested. At various time-points after addition of ROS samples were collected from the acceptor chambers for measurement of fluorescence intensity using a FLUOstar Galaxy microplate reader (BMG Labtechnologies, Offenburg, Germany), excitation 485 nm, emission 520 nm.
Western blot of tight junction proteins and PKB phosphorylation
GFP-occludin- or claudin-5-YFP expressing immortalized rat GP8/3.9 cells were grown to confluence and incubated with inhibitors for Rho GTPases (toxin B, 5 ng/ml), PI3 kinase (wortmannin, 2 µM) or PKB (triciribine, 12.5 µM) 2 h prior to ROS treatment. Immortalized rat GP8/3.9 cells were grown to confluence and maintained for 48 h in serum-free cell culture medium and incubated with wortmannin (2 µM), toxin B (5 ng/ml), superoxide dismutase (5000 U/ml), catalase (5000 U/ml), mannitol (50 mM), lipoic acid (300 µM), or luteolin (50 µM) 2 h prior to ROS treatment. After ROS treatment cells were lysed and proteins were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Membranes were blocked with 5% lowfat milk powder and incubated with primary antibodies. Immunoreactive proteins were detected with horseradish peroxidase-conjugated secondary antibodies. Supersignal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA) was used for detection of immunoreactive proteins. Protein band intensity was quantified using ImageQuant TL software (Amersham Biosciences, Piscataway, NJ, USA) and presented as percentage of control.
Rho-activation assay
Rho-activation was assayed using a RhoA G-LISA kit (Cytoskeleton, Denver, CO, USA). Immortalized rat GP8/3.9 cells were grown to 60% confluence and maintained for 48 h in serum-free cell culture medium. Cells were treated with ROS for 30 min, washed with ice-cold PBS, and scraped in ice-cold lysisbuffer. The RhoA G-LISA assay was performed according to manufacturer’s prescription.
AMAXA transfection of GP8/3.9 cells
Immortalized rat GP8/3.9 cells (1.0 x106) were transfected with 3 µg of DNA constructs (empty vector, dominant-negative Rho A N19, or constitutively active RhoA V14 (38)
, kind gift from Alan Hall (MRC Laboratory for Molecular Cell Biology, University College London, London, UK) using the Basic Nucleofector Kit for Primary Mammalian Endothelial CellsTM (Amaxa Biosystems, Gaithersburg, MD, USA).
Monocyte migration
Monocyte migration of primary human monocytes across confluent monolayers of immortalized human hCMEC/D3 cells was studied with time-lapse video microscopy as described previously (25)
. Freshly isolated monocytes (7.5 x105/ml) were added to confluent monolayers of hCMEC/D3 cells, and the number of migrated monocytes was assessed after 4 h. The migration assay was conducted in the absence and presence of ROS and inhibitors for Rho GTPases (toxin B, 5 ng/ml), PI3 kinase (wortmannin, 2 µM), or PKB (triciribine, 12.5 µM).
Statistical analysis
Data were analyzed statistically by means of analysis of variance (ANOVA) and Student-Newman-Keuls test. Statistical significance was defined as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. ROS time-dependently reduced TEER across a monolayer of immortalized human hCMEC/D3 brain ECs (Fig. 1
A; maximal effect after 20 min (35±3.9% decrease, P<0.05) or a monolayer of primary rat brain ECs (Fig. 1B
; maximal effect after 45 min (28±2.5% decrease, P<0.05). We next determined the paracellular permeability toward large hydrophilic molecules. Addition of ROS to a human brain endothelial monolayer on Transwell filters enhanced leakage of FITC-dextran (150 kDa; 34±12.5% increase after 45 min, rising to 100±32.6% increase after 180 min, P<0.05, Fig. 1C
). Similarly, ROS enhanced leakage of FITC-dextran across a monolayer of primary rat brain ECs (maximal effect after 60 min, 27±4.4% increase, P<0.05, Fig. 1D
). ROS did not affect brain EC viability, which was routinely checked by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assay and 7-amino-actinomycin D (7AAD) exclusion by flow cytometry within this time frame (data not shown).
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BBB integrity is dynamically regulated by the actin cytoskeleton and dependent on the presence of tight junctions. Therefore, cytoskeletal alterations induced by ROS may be responsible for increased BBB permeability. To gain insight into the effect of ROS on actin cytoskeleton dynamics in brain endothelium, we determined the formation of F-actin bundles (stress-fibers) on exposure to ROS as described previously (24)
. ROS induced a significant increase of stress fiber formation at 60 min on the addition of ROS (31±6.1% increase, P<0.001, Fig. 2
A). In addition, hydrogen peroxide (100 µM) solely similarly increased the number of F-actin fibers (26±8.6%, P<0.05, Fig. 2B
). Hydrogen peroxide did not affect brain EC viability as was assayed by MTT and 7AAD (data not shown).
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Furthermore, we have investigated the effect of ROS on the behavior of the TJ proteins claudin-5 and occludin. To study real-time dynamics of TJ proteins, we used a immortalized rat GP8/3.9 brain EC line overexpressing GFP-occludin, which was previously used in our group to study occludin dynamics under inflammatory conditions (29)
. In addition, we generated a immortalized rat brain EC line overexpressing fluorescently labeled claudin-5, in which claudin-5-YFP was localized at the plasma membrane, recognized by anticlaudin-5 antibodies and colocalized with endogenous ZO-1 (supplementary Fig. 1A–F). Further characterization of this cell line is shown in supplementary Fig. 1G–I. Live cell microscopy demonstrated that ROS, within 30 min, induced ruffling of cellular junctions (indicated by white arrows), which coincided with redistribution of occludin (Fig. 3
A) and claudin-5 (Fig. 3C
) from the cellular junctions. Western blot analysis demonstrated that occludin expression decreased within a similar time frame, indicating that loss of membrane-associated occludin is directly followed by loss of protein expression (Fig. 3B
). In addition, ROS-induced claudin-5 disappearance was detected after 60 min (Fig. 3D
). Cell viability was assayed by MTT and 7AAD and remained unaffected (data not shown).
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ROS specifically and transiently activates PKB in brain endothelial cells
A number of signal transduction pathways, including the PI3 kinase pathway (39
, 40)
, have been suggested to be involved in the regulation of TJs and the cytoskeleton and may regulate ROS-induced signals. Therefore, we hypothesize that its downstream target PKB could also be implicated in these processes. To study the effect of ROS on PKB activation in immortalized rat GP8/3.9 brain ECs, we determined the phosphorylation state of PKB by Western blotting. Addition of ROS significantly induced transient PKB phosphorylation at Ser-473, which is essential for PKB activation (41)
, without affecting total PKB levels. PKB phosphorylation was maximal at 30 min after the addition of ROS (52±7.1% increase, P<0.001, Fig. 4
A), which was more potent than hydrogen peroxide alone (32±7.7% increase, P<0.01, Fig. 4B
). Incubation with wortmannin, a selective inhibitor of PI3 kinase, revealed that ROS-induced PKB phosphorylation was dependent on PI3 kinase (69±11.3% decrease compared to ROS alone, P<0.01, Fig. 4A
). Another selective inhibitor of PI3 kinase, LY294002, equally inhibited ROS-induced PKB phosphorylation (data not shown). ROS can be scavenged by broad-spectrum antioxidants like lipoic acid and luteolin, or by more specific scavengers like catalase, superoxide dismutase (SOD) and mannitol. Superoxide can be converted by SOD to hydrogen peroxide. In turn, hydrogen peroxide can be decomposed by catalase into water and oxygen. Alternatively, hydrogen peroxide can be used to generate hydroxyl radicals, which can be scavenged by mannitol. All these antioxidants completely inhibited ROS-induced PKB phosphorylation, demonstrating that superoxide, and to a certain extent hydrogen peroxide and hydroxyl radical, contribute to the ROS-induced PKB phosphorylation (Fig. 4C
).
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Rho is an upstream mediator of PI3 kinase signaling
To identify upstream mediators of ROS-induced PKB phosphorylation, we examined the effects of specific inhibitors of candidate signal transduction molecules in this pathway. Inhibition of Rho GTPase-mediated signaling by toxin B (5 ng/ml) completely blocked ROS-induced PKB phosphorylation (P<0.001), indicating that activation of Rho family GTPases is a prerequisite of ROS-induced PKB activation in brain ECs (Fig. 5
A). In addition, our results showed that ROS induced RhoA activation (Fig. 5B
). To test whether RhoA activity was required for ROS-induced activation of PKB, we overexpressed a dominant-negative mutant of RhoA (RhoA N19) or a constitutively active mutant of RhoA (RhoA V14) (38)
in immortalized rat GP8/3.9 brain EC. The expression of constitutively active RhoA coincided with increased baseline PKB phosphorylation levels, which slightly increased after ROS addition, whereas the presence of dominant-negative RhoA resulted into decreased baseline PKB phosphorylation levels and blocked ROS-induced PKB phosphorylation (Fig. 5C
). These results indicate that ROS induces PKB phosphorylation through RhoA-activation.
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PKB phosphorylation and Rho-activation mediate the effects of ROS on brain EC integrity
Next, we examined whether Rho, PI3 kinase, and PKB are involved in cytoskeleton and TJ alterations induced by ROS. Western blot analysis revealed that the Rho-inhibitor toxin B, the PI3 kinase inhibitor wortmannin, and the PKB inhibitor triciribine prevented ROS-induced disappearance of GFP-occludin (Fig. 6
A) and claudin-5-YFP (Fig. 6B
). Moreover, these inhibitors also reduced ROS-induced formation of actin stress fibers (Fig. 6C
). Together, these results suggest that signaling pathways involving Rho, PI3 kinases, and PKB mediate ROS-induced cytoskeleton and TJ reorganization.
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Specific inhibitors for Rho, PI3 kinase, and PKB can prevent ROS-induced transendothelial monocyte migration
Under pathological conditions, ROS significantly contribute to BBB dysfunction and inflammation in the brain by enhancing cellular migration. To study the role of Rho, PI3 kinase, and PKB in ROS-induced monocyte migration, we used an in vitro model of the BBB as described previously (25)
. ROS induced the migration of primary human monocytes across immortalized human hCMEC/D3 brain ECs (38±3.8% increase, Fig. 7
). Specific inhibitors of Rho (42±3.5% decrease), PI3 kinase (40±3.5% decrease), and PKB (41±3.3% decrease) prevented this ROS-induced migration (Fig. 7
, ***P<0.001). In addition, the same inhibitors also prevented ROS-induced migration of primary rat monocytes across immortalized rat GP8/3.9 brain ECs (data not shown) and a specific RhoA inhibitor (C3 transferase) prevented ROS-induced migration of primary human monocytes across immortalized human hCMEC/D3 brain ECs (Supplementary Fig. 2). Furthermore, basal monocyte migration across untreated human brain ECs (##P<0.01) was also inhibited by the same specific inhibitors for Rho (16±2.4% decrease), PI3 kinase (17±2.3% decrease), and PKB (14±1.6% decrease). Together, these results indicate that these signaling pathways mediate ROS-induced monocyte migration across an in vitro model of the BBB.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Here, we demonstrate for the first time that extracellular ROS modulate BBB integrity by transient activation of the PI3 kinase and PKB pathway via RhoA, which acutely disrupts the integrity of the TJs, allowing paracellular transport to occur.
ROS altered brain EC integrity within minutes by decreasing TEER and increasing brain EC permeability. ROS also induced cytoskeletal rearrangements, redistribution, and disappearance of both occludin and claudin-5 in a time-dependent manner. As claudin-5 is thought to be a critical component of TJ in the BBB (7)
, ROS-induced redistribution and disappearance of claudin-5 may be an important mechanism underlying enhanced BBB permeability. Recently, it was shown that hydrogen peroxide exposure enhances BBB permeability and reduces TEER (27
, 42
, 43)
as well as facilitate the formation of actin stress fibers in bovine brain endothelium (43)
. Our data confirm and extend these results as superoxide, which is the most predominant ROS produced in our ROS generating system, has the capacity to do this as well. The organization of TJ proteins occludin and ZO-1 is altered by exogenous ROS in both epithelial cells (44
45
46)
and brain ECs (43
, 47)
. However, we are the first to demonstrate that ROS affected claudin-5 expression at the cellular junction.
In this study we established that the PKB pathway is an important mediator of ROS-induced alterations in brain ECs. Exogenous superoxide and hydrogen peroxide activated PI3 kinase and its downstream target PKB in a transient manner in brain ECs. Moreover, inhibition of PI3-kinase or PKB activity prevented the formation of F-actin stress-fibers as well as the disappearance of occludin and claudin-5, indicating that the PI3 kinase and PKB signaling pathway have a pivotal role in ROS-induced loss of BBB integrity. Thus far, knowledge about the role of ROS-induced signal transfer in the regulation of claudin-5 in brain ECs is limited. It has been reported that claudin-5 can be phosphorylated after exposure of brain ECs to ROS (27
, 48)
. Furthermore, evidence suggests that the phosphorylation state of occludin is important in the regulation of TJ assembly and disassembly (49)
. Occludin can be phosphorylated on Ser, Thr, and Tyr residues (50)
and in epithelial cells extracellular ROS induced tyrosine phosphorylation and dissociation of occludin from intercellular junctions (44
, 45)
. In addition, the ROS-induced claudin-5 and occludin disappearance may also be explained by protein degradation. However, no degradation products were detected using the current antibodies (data not shown). Previously (29)
, we postulated that the ubiquitin-proteasome pathway might affect tight junction stability, since in vivo evidence suggests that occludin is ubiquitinated and its turnover is retarded after treatment with a proteasome inhibitor (51)
. Whether such a pathway is involved in occludin and claudin-5 degradation remains to be determined. Together, our data provide conclusive evidence that the PI3 kinase and PKB pathway mediates ROS-induced dysregulation of occludin and claudin-5 and suggest that PKB is the putative kinase that may regulate occludin and claudin-5 dynamics.
Inhibition of Rho activation prevented ROS-induced cytoskeleton rearrangements and disappearance of occludin and claudin-5. These data indicate that ROS activate Rho, which subsequently may affect the actin cytoskeleton via PI3 kinase and PKB. It has been reported that cytoskeleton depolymerization causes redistribution of TJ molecules (52
, 53)
. Hence, signaling molecules that control the organization of the actin cytoskeleton may indirectly be involved in the regulation of TJs. The family of Ras-related small GTP-binding proteins RhoA, Rac1, and Cdc42 are such regulators of the actin cytoskeleton, and it has been described that RhoA activation leads to phosphorylation of occludin and claudin-5 and TJ reorganization(18
, 54
, 55)
. The broad-spectrum Rho-GTPase inhibitor toxin B blocked ROS-induced PKB phosphorylation. However, a more specific inhibitor of RhoA (C3 transferase) prevented ROS-induced cytoskeleton rearrangements. Overexpression of constitutively active RhoA in our brain ECs coincided with increased baseline PKB phosphorylation levels, whereas the presence of dominant-negative RhoA resulted into decreased baseline PKB phosphorylation levels and blocked ROS-induced PKB phosphorylation. We therefore conclude that RhoA is an upstream activator of the PI3 kinase pathway, which is in line with previous studies (56
, 57)
. It should be noted that the relationship between PKB and the Rho family of GTPases is not unambiguous and may depend on stimuli and cell type. A number of reports suggest that Rho-activation is a downstream event of PI3 kinase signaling (58
, 59)
. In our brain EC model, however, Rho acts upstream of PI3 kinase and ensuing activation of PKB allows direct interaction with the cytoskeleton (60)
.
Specific inhibitors for Rho, PI3 kinase, and PKB mediate ROS-induced monocyte migration across an in vitro model of the human BBB. Infiltration of leukocytes into the CNS is a crucial event during neuroinflammatory diseases. We have previously shown that PI3 kinase plays an important role during ROS-induced monocyte transmigration across rat brain ECs (25)
. Furthermore, inhibition of RhoA in brain endothelium resulted in decreased cellular migration in vitro and in vivo (61
, 62)
. Our data confirm and extend these results as a specific inhibitor of PKB can prevent ROS-induced monocyte migration across an in vitro model of the BBB.
In summary, our data provide evidence that ROS specifically activate the PKB signaling pathway via RhoA in brain ECs. We have conclusively shown that activation of the PKB pathway, either directly or indirectly via the actin cytoskeleton, causes dissociation and subsequent disappearance of the TJ proteins claudin-5 and occludin. Furthermore, inhibiting PKB activity prevented ROS-induced monocyte migration across an in vitro model of the BBB. Our results may support the imagination of an immunomodulatory role of ROS in TJ dynamics. By down-regulating TJ assembly, ROS contribute to altered BBB integrity, thereby facilitating the influx of leukocytes and thus supporting an inflammatory response. Furthermore, PKB phosphorylation and RhoA activation may be early markers of BBB dysfunction. Agents that selectively inhibit this effect, like antioxidants or signaling inhibitors for RhoA, PI3 kinase, or PKB, may be used therapeutically to modulate neuroinflammatory diseases complicated by BBB dysfunction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication February 9, 2007. Accepted for publication May 17, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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