reactive oxygen species-caspase-3 relationship in mediating blood-brain barrier endothelial cell...

Reactive Oxygen Species-Caspase-3 Relationship inMediating Blood–Brain Barrier Endothelial CellHyperpermeability Following Oxygen–GlucoseDeprivation and Reoxygenation

HIMAKARNIKA ALLURI, HAYDEN W. STAGG, RICKESHA L. WILSON, ROBERT P. CLAYTON, DEVENDRA A.

SAWANT, MADHAVI KONERU, MADHAVA R. BEERAM, MATTHEW L. DAVIS, AND BINU THARAKAN

Departments of Surgery and Pediatrics, Texas A&M University Health Science Center College of Medicine and Scott & White Healthcare, Temple,

Texas, USA

Address for correspondence: Binu Tharakan, Ph.D., F.A.H.A, Department of Surgery, Scott and White Healthcare & Texas A&M University Health

Science Center College of Medicine, 702 S.W. H.K. Dodgen Loop, Temple, TX 76504, USA. E-mail: [email protected]

Received 30 August 2013; accepted 21 December 2013.

ABSTRACT

Objective: Microvascular hyperpermeability that occurs due to

breakdown of the BBB is a major contributor of brain vasogenic

edema, following IR injury. In microvascular endothelial cells,

increased ROS formation leads to caspase-3 activation following IR

injury. The specific mechanisms, by which ROS mediates micro-

vascular hyperpermeability following IR, are not clearly known. We

utilized an OGD-R in vitro model of IR injury to study this.

Methods: RBMEC were subjected to OGD-R in presence of a

caspase-3 inhibitor Z-DEVD, caspase-3 siRNA or an ROS inhibitor

L-AA. Cytochrome c levels were measured by ELISA and caspase-3

activity was measured fluorometrically. TJ integrity and cytoskeletal

assembly were studied using ZO-1 immunofluorescence and rho-

damine phalloidin staining for f-actin, respectively.

Results: OGD-R significantly increased monolayer permeability,

ROS formation, cytochrome c levels, and caspase-3 activity

(p < 0.05) and induced TJ disruption and actin stress fiber

formation. Z-DEVD, L-AA and caspase-3 siRNA significantly

attenuated OGD-R-induced hyperpermeability (p < 0.05) while

only L-AA decreased cytochrome c levels. Z-DEVD and L-AA

protected TJ integrity and actin cytoskeletal assembly.

Conclusions: These results suggest that OGD-R-induced hyper-

permeability is ROS and caspase-3 dependent and can be regulated

by their inhibitors.

KEY WORDS: vascular hyperpermeability, blood–brain barrier, endo-

thelial cells, ischemia reperfusion

Abbreviations used: AFC, 7-amino-4-trifluoromethylcoumarin;

BBB, blood–brain barrier; DCFDA, 2′,7′-dichlorofluorescein diac-

etate; EDTA, ethylenediaminetetraacetic acid; FITC, fluorescein

isothiocyanate; IR, ischemia reperfusion; L-AA, L-ascorbic acid;

MMP, matrix metalloproteinases; MPT, mitochondrial permeabil-

ity transition; OGD-R, oxygen–glucose deprivation and reoxygen-

ation; PBS, phosphate-buffered saline; RBMEC, rat brain

microvascular endothelial cells; ROS, reactive oxygen species;

SOD, superoxide dismutase; TJ, tight junction; TJP, tight junction

proteins; Z-DEVD, Z-D(OMe)-E(OMe)-V-D(OMe)-FMK; ZO-1,

zonula occludens-1.

Please cite this paper as: Alluri H, Stagg HW, Wilson RL, Clayton RP, Sawant DA, Koneru M, Beeram MR, Davis ML, Tharakan B. Reactive oxygen species-

caspase-3 relationship in mediating blood–brain barrier endothelial cell hyperpermeability following oxygen–glucose deprivation and reoxygenation.

Microcirculation 21: 187–195, 2014.

INTRODUCTION

Microvascular hyperpermeability, the excessive leakage of

fluid and proteins from small blood vessels into the

extravascular space is one of the major causes of vasogenic

brain edema and occurs in a variety of disease and clinical

conditions, the most important of which is IR injury [8]. IR

injury occurs as an early secondary injury leading to a

nonspecific, inflammatory response triggering mitochondrial

permeabilization and is associated with the production of

various free radicals also known as ROS [14,18,21,23,26]. IR

injury is one of the frequent causes of various debilitating

symptoms associated with stroke, cardiac diseases, peripheral

vascular disease, and traumatic brain and spinal cord injuries

[15,22,30,34,40]. A hallmark of IR injury is the rapid inflow

of blood following an acute/chronic occlusion of blood

vessels, triggering the formation of free radicals [9,13,29].

Compared to the peripheral blood vessels, the cerebral vessels

DOI:10.1111/micc.12110

Original Article

ª 2013 John Wiley & Sons Ltd 187

are more sensitive to blood flow changes, being composed of

high amount of unsaturated lipids and need high rate of

metabolism to occur, which renders them most vulnerable to

the reperfusion injury [25,33]. IR injury has been extensively

studied in various disease conditions but the intracellular

mechanisms that lead to IR-induced vascular hyperperme-

ability and cerebral edema are not clearly known. In this

study, using an established in vitro system of IR injury, that

created an environment of anoxia reperfusion, we studied

how oxygen and nutrient deprivation followed by reoxygen-

ation induces microvascular endothelial hyperpermeability

mediated by ROS and caspase-3.

Free radicals or ROS are highly reactive molecules with

short half-lives and the presence of the unpaired electrons

in their orbitals make them chemically unstable [6,31].

ROS includes superoxide, hydroxyl, nitric oxide, hydrogen

peroxide, and peroxynitrite radicals, formed mainly at the

level of the mitochondrial electron transport chain [8].

They trigger a variety of secondary injuries in the body,

such as lipid peroxidation of membranes (including the

mitochondrial membranes), inactivation of various pro-

teins, excitotoxicity, depolymerization of polysaccharides,

and nucleic acid disruption and inflammation [6,10,19]. In

the cell, mitochondria plays an important role in buffering

the free oxygen radicals by converting them into water in

the final step of the electron transport chain of oxidative

phosphorylation [12]. Pathogenic insult or IR trigger

increased ROS production, which causes redox imbalance

in the cell, outstripping the endogenous antioxidant

mechanisms like ascorbic acid, SOD, catalase, etc.

[16,21,24,34]. There is increasing evidence that ROS are

key mediators of endothelial barrier dysfunctions in the

body including in the BBB [7,27,31,43] but the mechanisms

by which they control microvascular permeability are not

clearly known.

BBB endothelial cell tight junctions are critical for main-

taining the homeostasis of brain and for regulating micro-

vascular permeability [2]. The tight junctions are composed

of transmembrane proteins such as claudins, occludins, and

junctional adhesion molecules and are connected to the actin

cytoskeleton through a scaffolding protein ZO-1 on the

cytoplasmic side [2–4,16,25]. The tight junctions are also one

of the primary targets for ROS and proteolytic enzymes such

as caspases and MMPs [5,9,15,16]. In this study, we

hypothesized that ROS formation following oxygen–glucosedeprivation/reoxygenation is a major inducer of mitochon-

drial cytochrome c release and subsequent caspase-3 mediated

BBB tight junction breakdown and hyperpermeability.

We mainly addressed the following questions in this

study: (i) Is there any involvement of mitochondrial

mechanisms that mediate caspase-3 induced breakdown of

the BBB resulting in microvascular hyperpermeability

following OGD-R? (ii) Is there ROS mediation in inducing

caspase-3 mediated breakdown of the BBB and associated

hyperpermeability? (iii) Is there an involvement of actin

cytoskeletal assembly in IR-induced barrier dysfunction and

permeability? (iv) Are endogenous antioxidants effective

against ROS mediated barrier dysfunction and hyperper-

meability?

MATERIALS AND METHODS

RBMECPrimary cultures of RBMECs derived from adult Sprague

Dawley rats were obtained from Cell Applications (San

Diego, CA, USA). RBMECs were initially grown on 0.05%

fibronectin-coated cell culture dishes, using rat brain endo-

thelial cell complete media in a cell culture incubator (95%

O2, 5% CO2 at 37°C). RBMECs were treated with 0.25%

trypsin-EDTA for cell detachment and were grown on

fibronectin-coated Transwell inserts, chamber slides or

regular dishes for experimental purposes. RBMEC passages

6–8 were chosen for all the experiments.

Chemicals and ReagentsRBMEC Culture Medium was purchased from Cell Appli-

cations, Inc. Dulbecco’s Modified Eagle Medium without

glucose was obtained from Life Technologies (Grand Island,

NY, USA). Transwell-24 well plates were obtained from

Corning, USA. 5% Fibronectin from bovine plasma, L-AA,

Nunc Lab Tek II-CC, 8-well glass chamber slides and FITC-

Dextran-10 were purchased from Sigma Aldrich (St. Louis,

MO, USA). Rabbit anti- ZO-1 and rhodamine phalloidin

were purchased from Life Technologies. QIA70 Caspase-3

activity fluorometric assay kit was bought from EMD

Millipore/Calbiochem (Billerica, MA, USA). DCFDA cellular

ROS detection assay kit was bought from Abcam, USA.

Caspase-3 siRNA and control siRNA were obtained from

Dharmacon/Thermoscientific (Pittsburgh, PA, USA). Pierce

BCA protein assay kit was bought from Thermo Scientific

(Waltham, MA, USA). Cytochrome c ELISA kit and Z-

DEVD-FMK (Z-D(OMe)-E(OMe)-V-D(OMe)-FMK) were

obtained from R&D Systems (Minneapolis, MN, USA).

OGD-R In Vitro ModelOGD-R was performed as described previously [40,42,43].

Briefly, RBMECs were grown in RBMEC complete growth

media as monolayers on Transwell inserts, chamber slides or

regular dishes in a cell culture incubator (95% O2, 5% CO2 at

37°C) as described above. Later, the cells were exposed to no

glucose DMEM (Life Technologies) and placed in a hypoxic/

anoxic chamber (95% N2 and 5% CO2 at 37°C) in a cell

culture incubator. This was followed by a reperfusion face by

exposure to normal incubation conditions (95% O2, 5% CO2

at 37°C) and regular RBMEC growth medium for one hour

and three hours, respectively.

H. Alluri et al.

188 ª 2013 John Wiley & Sons Ltd

Effect of OGD-R on Brain MicrovascularEndothelial Cell Monolayer PermeabilityRBMEC monolayers were grown on fibronectin-coated

Transwell inserts for 96 hours, followed by treatment with

Z-DEVD (10 lM) or L-AA (10 mM). Cells were later

exposed to oxygen–glucose deprivation (two hours) followed

by reoxygenation (one hour and three hours) as described

above. The following treatment groups were maintained:

Control (untreated group), OGD-R one hour, OGD-R three

hours, Z-DEVD + OGD-R one hour, Z-DEVD + OGD-R

three hours, L-AA + OGD-R one hour, L-AA + OGD-R

three hours, Z-DEVD, L-AA. FITC-dextran-10 (1 mg/mL)

probe was applied to the upper reservoir of the monolayer

plate for 30 minutes. Samples were collected from the lower

compartment and fluorescent intensity was measured (485-

and 520-nm, excitation and emission, respectively).The

change in permeability was calculated as a percentage of

the untreated control values. Each experimental group

consisted of five replicates.

Effect of Caspase-3 Knockdown and OGD-R onBrain Microvascular Endothelial Cell MonolayerPermeabilityRBMECs were grown on fibronectin-coated Transwell

membranes as monolayers for 96 hours. They were treated

with control siRNA or caspase-3 siRNA for 48 hours. This

was followed by exposure of caspase-3 siRNA group to OGD-

R (one hour and three hours) conditions. The following

treatment groups were maintained: Control (untreated),

OGD-R one hour, OGD-R three hours, Caspase-3 siR-

NA + OGD-R one hour, and Caspase-3 siRNA + OGD-R

three hours. FITC-dextran-10 (1 mg/mL) was applied in the

upper chamber for 30 minutes and the fluorescent intensity

was measured as described above. Each experimental group

consisted of five replicates.

Effect of OGD-R on BBB Tight Junction Integrityand Cytoskeletal AssemblyRBMECs were grown on chamber slides and pretreated with

Z-DEVD (10 lM; one hour) or L-AA (10 mM; one hour)

followed by exposure to OGD-R (one hour) as described

above. To study tight junction integrity, the cells were

processed for immunofluorescence localization of ZO-1. Cells

were fixed in 4% paraformaldehyde in PBS for 15 minutes and

permeabilized with 0.5% Triton X-100 in PBS for 15 minutes.

After blocking with 2% bovine serum albumin in PBS, cells

were incubated overnight with an anti-rabbit primary anti-

body against ZO-1(1:150), followed by incubation with an

FITC tagged anti-rabbit secondary antibody for one hour at

room temperature. The cells were washed and the slides

mounted using Vectashield antifade reagent containing DAPI

(Vector Laboratories, Burlingame, CA, USA). To study the

actin cytoskeleton, f-actin fibers were stained with rhodamine

phalloidin. For this purpose, cells were fixed and permeablized

as described above followed by exposure to rhodamine

Phalloidin (1:50) for 20 minutes, prior to mounting as above.

The cells were visualized and they were scanned at a single

optical plane with an Olympus Fluoview 300 confocal

microscope (Center Valley, PA, USA). Each experimental

group consisted of two replicates.

Effect of OGD-R on ROS FormationRBMECs were grown on 96 well plates for 96 hours and

pretreated with L-AA (10 mM; one hour) followed by

OGD-R (one hour and three hours) as described above. An

untreated control group and an L-AA treated group were

also maintained. ROS levels were determined using a

membrane-permeable dye DCFDA. DCFDA was applied to

the cells at 37°C for 30 minutes. Each experimental group

consisted of four replicates. ROS in the cells oxidize DCFDA,

yielding the fluorescent product -(-6)-chloromethyl-29,79-

dichlorodihydrofluorescein. The fluorescence intensity was

measured at an excitation of 485 nm and an emission of

535 nm.

Effect of OGD-R on Mitochondrial Release ofCytochrome cRBMECs were grown in culture dishes and treated with L-AA

(10 mM; one hour), followed by OGD-R (one hour and

three hours) as described above. An untreated group served

as the control group. Each experimental group consisted of

five replicates. Cell lysates were obtained by scraping the cells

in the presence of a lysis buffer (provided in the ELISA kit)

followed by centrifugation at 10,000 g for 60 minutes at 4°C.Supernatant was collected, protein concentration was esti-

mated using BCA method (Pierce, Rockford, IL, USA) and

were used to perform an ELISA (R&D Systems). Briefly, the

samples were added to a 96 well plate and treated with a

conjugate regent, transferred to a cytochrome c antibody-

coated microwell plate, and incubated at room temperature

for 60 minutes. The wells were washed and treated with a

substrate reagent and incubated for 30 minutes, followed by

addition of a stop solution. The optical density was read at

450 nm using a colorimetric plate reader. The concentration

of cytochrome c was calibrated from a standard curve.

Effect of OGD-R on Caspase-3 ActivityRBMECs were grown in culture dishes and treated with L-AA

(10 mM; one hour) alone or L-AA, followed by OGD-R (one

hour and three hours) as described above. An untreated

group served as the control group. Caspase-3 activity was

measured using a caspase-3 activity assay kit (EMD

Millipore/Calbiochem). The Z-DEVD substrate provided in

the kit was labeled with a fluorescent molecule, AFC. The cell

Blood-Brain Barrier and Hyperpermeability


lysates were obtained as described above using lysis buffer

provided in the assay kit. This was followed by protein

estimation and treatment with the substrate conjugate. Each

experimental group consisted of four replicates. The resulting

fluorescent intensity was measured in a fluorescent plate

reader capable of measuring excitation at 400 nm and

emission at 505 nm.

Statistical AnalysisData are expressed as the mean � SEM (%). Statistical

differences among groups were determined by one-way

ANOVA followed by Bonferroni post hoc test to determine

significant differences between specific groups. These proce-

dures were followed for analysis of results from monolayer

permeability, ROS, cytochrome c and caspase-3 studies. A

value of p < 0.05 was considered a statistically significant

difference.

RESULTS

OGD-R Induces BBB Endothelial Cell MonolayerHyperpermeabilityOGD-R (one hour and three hours) induced significant

increase in permeability of the monolayer, evidenced by

increased FITC-dextran-10 leakage to the lower chamber

(p < 0.05; Figure 1). Pretreatment with caspase-3 inhibitor,

Z-DEVD (10 lM; one hour) or L-AA (10 mM; one hour)

attenuated this effect (p < 0.05; Figure 1). This shows that

caspase-3 and ROS play important roles in promoting BBB

hyperpermeability. Caspase-3 siRNA-transfected monolayers

showed significant decrease in permeability following expo-

sure to OGD-R compared to OGD-R alone group (p < 0.05;

Figure 2).

OGD-R Induces Disruption of BBB Endothelial CellTight JunctionsThe immunofluorescence study demonstrated junctional

continuity of ZO-1, whereas cells exposed to OGD-R showed

discontinuity of junctional staining demonstrating the loss of

barrier integrity. Pretreatment with Z-DEVD (10 lM; one

hour) and L-AA (10 mM; one hour) decreased OGD-R-

induced tight junction disruption (Figure 3A). This study

demonstrates that ROS and caspase-3 promotes monolayer

permeability by targeting the tight junctions.

Rhodamine phalloidin staining for f-actin in normal control

cells demonstrated clear cytoskeletal assembly with no stress

fiber formation. Cells exposed to OGD-R showed an increase

in stress fiber formation. Pretreatment ofOGD-R exposed cells

with Z-DEVD (10 lM; one hour) or L-AA (10 mM; one hour)

showed decrease in f-actin stress fiber formation (Figure 3B).

OGD-R Induces ROS Formation in BBB EndothelialCellsRBMECs exposed to OGD-R (one hour and three hours)

resulted in a significant increase in ROS formation evidenced

by increased fluorescent intensity (p < 0.05). Pretreatment of

cells exposed to OGD-R with Z-DEVD showed no significant

change in ROS formation compared to OGD-R alone group.

Treatment of cells with L-AA (10 mM; one hour) prior to

OGD-R resulted in a significant decrease in ROS formation

compared to OGD-R alone group (Figure 4).

OGD-R Induces Mitochondrial Release ofCytochrome c in BBB Endothelial CellsOGD-R (one hour and three hours) induced significant

increase in mitochondrial release of cytochrome c evidenced

by significant increase in its cytosolic levels (p < 0.05).

0

50

100

150

200

FIT

C-d

extr

an fl

uore

scen

t int

ensi

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con

trol

)

Control OGD-R 1 hour OGD-R 3 hours Z-DEVD+OGD-R1 hour

Z-DEVD+OGD-R3 hours

L-AA+OGD-R1 hour

L-AA+OGD-R3 hours

*a

*c*b*b

*a

*c

Figure 1. Caspase-3 inhibitor (Z-DEVD) and L-AA attenuates OGD-R-induced RBMEC monolayer hyperpermeability. Monolayer permeability is expressed

as a percentage of the change in FITC-dextran-10 fluorescent intensity (30 minutes accumulation). ‘*’ indicates statistical significance (p < 0.05; n = 5);

‘a’ indicates significant change compared to control group; ‘b’ indicates significant change compared to OGD-R one hour; ‘c’ indicates significant change

compared to OGD-R three hours.

H. Alluri et al.


Pretreatment with L-AA (10 mM; one hour) attenuated

OGD-R-induced increase in cytosolic cytochrome c levels

significantly (p < 0.05; Figure 5A). Caspase-3 siRNA-

transfected monolayers showed significant decrease in

permeability following OGD-R compared to OGD-R alone

group (p < 0.05; Figure 2).

OGD-R Induces Caspase-3 Activity in BBBEndothelial CellsOGD-R (one hour and three hours) induced significant

increase in caspase-3 activity (p < 0.05). Pretreatment with

L-AA (10 mM; one hour) attenuated OGD-R-induced increase

in caspase-3 activity significantly (p < 0.05; Figure 5B).

0

50

100

150

200

FIT

C-d

extr

an fl

uore

scen

t int

ensi

ty(%

con

trol

)

Control OGD-R 1 hour OGD-R 3 hours si RNA+OGD-R1 hour

siRNA+OGD-R3 hours

*a*a

*c*b

Figure 2. OGD-R induced hyperpermeability is significantly decreased by caspase-3 siRNA in RBMEC monolayers. ‘*’ indicates statistical significance

(p < 0.05; n = 5); ‘a’ indicates significant change compared to control group; ‘b’ indicates significant change compared to OGD-R one hour; ‘c’ indicates

significant change compared to OGD-R three hours. ‘Control group’ indicates an untreated group.

Control OGD-R 1 hour Z-DEVD + OGD-R Z-DEVD L-AA+OGD-R

Control OGD-R Z-DEVD + OGD-R Z-DEVD L-AA+OGD-R

A

B

Figure 3. (A) Immunofluorescence localization of tight junction protein ZO-1 demonstrating disruption of the tight junctions following OGD-R in

RBMECs. Z-DEVD and L-AA provides protection against OGD-R-induced disruption of the tight junctions. Arrows indicate tight junction disruption. (B)

Rhodamine phalloidin staining for f-actin stress fiber formation demonstrating changes in cytoskeletal assembly following OGD-R in RBMECs. OGD-R

increased the formation of f-actin stress fibers. Z-DEVD and L-AA decreased the formation of f-actin stress fibers. Arrows indicate actin stress fiber

formation. Each experimental group consisted of two replicates.



0

Contro

l

OGD-R 1

hour

OGD-R 3

hours

L-AA+OGD-R

-

1 hou

r

L-AA+OGD-R

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rsL-A

A

Z-DEVD

Z-DEVD+OGD-R

1 hou

r

Z-DEVD+OGD-R

3 hou

rs

20406080

100120140160180

RO

S Fo

rmat

ion

(% C

ontr

ol) *a *a

*b

*a *a

*c

Figure 4. L-AA attenuates OGD-R-induced ROS formation in RBMECs whereas Z-DEVD showed no significant effect. ‘*’ indicates statistical significance(p < 0.05; n = 4); ‘a’ indicates significant change compared to control group; ‘b’ indicates significant change compared to OGD-R one hour; ‘c’ indicates

significant change compared to OGD-R three hours.

0

10

20

30

40

Cyt

ochr

ome

c le

vels

(ng/

mL

)

*a

*c*b

*a

Control OGD-R1 hour

OGD-R3 hours

Z-DEVD+OGD-R1 hour

L-AA+OGD-R3 hours

0

50

100

150

200

*a

Cas

pase

-3 a

ctiv

ity (%

con

trol

)

Control OGD-R1 hour

OGD-R3 hours

L-AA+OGD-R1 hour

L-AA+OGD-R3 hours

L-AA

*b*a

*c

A

B

Figure 5. OGD-R induced mitochondrial release of cytochrome c and caspase-3 activation is decreased by L-AA in RBMECs. (A) OGD-R induced

significant increase in cytosolic cytochrome c levels and this effect was decreased by L-AA. (B) OGD-R induces significant increase in caspase-3 activity.

This effect was significantly reduced by L-AA pretreatment. ‘*’ indicates statistical significance (p < 0.05; n = 5); ‘a’ indicates significant change compared

to control group; ‘b’ indicates significant change compared to OGD-R one hour; ‘c’ indicates significant change compared to OGD-R three hours.

H. Alluri et al.


DISCUSSION

In this study, our primary focus was to investigate the

mechanistic relationship between ROS and caspase-3 and

their downstream effect on BBB integrity and microvas-

cular permeability following oxygen–glucose deprivation

and reoxygenation, an in vitro model of IR Injury. The

important findings of the study are (i) brain microvascular

endothelial cell monolayer hyperpermeability that occurs

following OGD-R, is promoted by mitochondria-mediated

activation of caspase-3; (ii) mitochondria-mediated regu-

lation of microvascular hyperpermeability is associated

with tight junction disruption and cytoskeletal disorgani-

zation; (iii) ROS plays an important role in promoting

microvascular permeability via caspase-3 induced tight

junction disruption and cytoskeletal disorganization;

(iv) L-AA, an endogenous antioxidant has protective

functions against OGD-R-induced brain microvascular

hyperpermeability.

Previous studies have shown that mitochondrial oxidative

stress caused by ROS, after reperfusion injury promotes

mitochondrial release of cytochrome c [6]. Cytochrome c is a

highly conserved heme protein loosely attached to the inner

mitochondrial membrane and its release occurs depending

on the changes in MPT pore that is a key event in the

activation of caspase-3. MPT is known to alter in response to

oxidative stress induced by mitochondrial ROS [41]. The

exogenous ROS also induce MPT pore opening and

cytochrome c release in isolated mitochondria, which could

be blocked by drugs that protect mitochondrial transmem-

brane potential [36,37,41]. In this study, an increase in ROS

formation and mitochondrial release of cytochrome c and

their relationship to endothelial cell monolayer permeability

following OGD-R was observed. In addition to ROS

production by mitochondrial respiratory chain, ROS gener-

ation by cytoplasmic enzymes also has been reported in

different cell types [28,33]. However, this study does not

clearly discriminate between the two different sources of

ROS. Cytochrome c release and activation of caspase-3 are

the key events in the apoptotic signaling pathway. Produc-

tion of ROS during apoptosis can also be caspase dependent

[36]. Activation of caspase-3 as well as neuroprotection

following caspase-3 inhibition has been reported in cerebral

ischemia [11,29]. In this study OGD-R induced ROS

formation, mitochondrial release of cytochrome c, and

caspase-3 activation followed by BBB endothelial cell hyp-

erpermeability. L-AA a powerful antioxidant attenuated all

these effects. Thus, our studies demonstrate ROS formation

and activation of caspase-3 as major mechanisms that control

endothelial cell barrier dysfunction and hyperpermeability

following OGD-R.

OGD-R as an in vitromodel of ischemia reperfusion injury

has been well established [1,17,43]. However, the specific

effects of various combinations of oxygen and glucose levels

are not clearly known. We have used an anoxia-reperfusion

strategy using an oxygen–glucose deprivation and reoxygen-

ation system with complete deprivation of oxygen and

glucose followed by reoxygenation. For this purpose, we used

95% nitrogen/5% CO2 and a glucose-free medium as

described previously [1,17]. Anoxia reperfusion has been

previously known to generate ROS formation in vitro [1,43].

Also, the glucose-free medium used in this study does not

contain L-AA as a basic component. However, L-AA may be

present as a constituent of the fetal bovine serum. This model

may not be a true replication of human hypoxia in vivo, but

is a highly reproducible anoxia-reperfusion model to study

various signaling pathways involved in ischemia/anoxia-

reperfusion conditions.

To evaluate the integrity of BBB endothelial cells, we used

ZO-1 protein localization and visualization of actin cyto-

skeleton based on f-actin staining. Our studies demonstrated

tight junction (TJ) disruption evidenced by discontinuous

localization of ZO-1 and increased formation of f-actin stress

fibers. ZO-1 is a scaffolding protein that plays a major role in

determining the integrity of the BBB, as its carboxyl terminal

binds to the actin cytoskeleton, while its N-terminal binds to

the TJPs such as claudin-5 and occludin [16,20]. Further-

more, recent studies suggest that, ZO-1 and ZO-2 are

decreased following ischemic brain injury [7]. Our evalua-

tion of results from the immunofluorescence studies are

focused on tight junction integrity based on ZO-1 localiza-

tion along the cell-cell junctions. It is not clear if the

cytoplasmic staining observed in some of the groups (Z-

DEVD, Z-DEVD + OGD-R one hour, and L-AA + OGD-R

one hour) is definitely related to ZO-1 redistribution or due

to any nonspecific background staining. The precise regula-

tion of the structure and dynamics of the actin cytoskeleton

is essential for many developmental and physiological

processes including cell-cell adhesion. Actin stress fibers are

contractile actomyosin bundles found in many cultured

nonmuscle cells, where they play a major role in cell adhesion

and morphogenesis [38]. In several occasions, in endothelial

cells, an increase in actin stress fiber formation has been

associated with barrier dysfunction and hyperpermeability

[35,37].

L-AA, commonly known as vitamin C, is an endogenously

occurring, small molecular weight, water soluble vitamin

[39]. Our studies demonstrated that L-AA effectively inhib-

ited the release of mitochondrial cytochrome c, a major step

to the regulation of caspase-3 activation. It has been shown

that vitamins such as vitamin E, C and A possess antioxidant

properties and play an important role in inhibiting ROS

production [22]. While our studies support the antioxidant

properties of L-AA, our results also demonstrate how L-AA

regulates barrier integrity and permeability by targeting

mitochondrial release of cytochrome c and subsequent



caspase-3 activation. We did not study the effect of caspase-3

inhibition using Z-DEVD on cytochrome c levels, as caspase-

3 activation is a downstream event to mitochondrial

cytochrome c release. This was further supported by the

observation that Z-DEVD had no effect on OGD-R induced

ROS formation.

In conclusion, the present findings suggest that OGD-R

induces ROS formation and cytochrome c release leading to

caspase-3-mediated disruption of BBB tight junctions and

microvascular hyperpermeability. Furthermore, our studies

demonstrate that the endogenous antioxidant L-AA has

protective benefits against barrier disruption and hyperper-

meability by preserving tight junction integrity as well as the

actin cytoskeletal assembly. Our results thus provide a

mechanistic relationship between ROS formation, caspase-3

activation, and BBB endothelial hyperpermeability following

OGD-R.

PERSPECTIVE

Blood-Brain Barrier (BBB) dysfunction is a major contri-

butor to the vasogenic brain edema that occurs, following

ischemia-reperfusion (IR) injury. The present findings

suggest that OGD-R, an in vitro model of IR injury induces

ROS formation and cytochrome c release leading to caspase-

3 mediated disruption of BBB tight junctions leading to

microvascular hyperpermeability. Inhibition of this pathway

may have therapeutic benefit against IR injury.

ACKNOWLEDGMENTS

We acknowledge Scott and White Hospital Research Grants

Program for financial support and Texas A&M Health

Science Center College of Medicine Integrated Imaging

Laboratory for the use of the confocal laser microscope.

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