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282:1485-1494, 2002. doi:10.1152/ajpheart.00645.2001Am J Physiol Heart Circ PhysiolKaren S. Mark and Thomas P. Davis

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Cerebral microvascular changes in permeability andtight junctions induced by hypoxia-reoxygenation

KAREN S. MARK AND THOMAS P. DAVISDepartment of Pharmacology, University of Arizona College of Medicine,

Tucson, Arizona 85724-5050

Received 23 July 2001; accepted in final form 26 November 2001

Mark, Karen S., and Thomas P. Davis. Cerebral micro-vascular changes in permeability and tight junctions inducedby hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol282: H1485 H1494, 2002; 10.1152/ajpheart.00645.2001.Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are criticalfor maintaining brain homeostasis and low permeability.Both integral (claudin-1 and occludin) and membrane-associ-ated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins com-

bine to form these TJ complexes that are anchored to thecytoskeletal architecture (actin). Disruptions of the BBB havebeen attributed to hypoxic conditions that occur with ischemicstroke, pathologies of decreased perfusion, and high-altitudeexposure. The effects of hypoxia and posthypoxic reoxygen-ation in cerebral microvasculature and corresponding cellularmechanisms involved in disrupting the BBB remain unclear.This study examined hypoxia and posthypoxic reoxygenationeffects on paracellular permeability and changes in actin andTJ proteins using primary bovine brain microvessel endothe-lial cells (BBMEC). Hypoxia induced a 2.6-fold increase in[14C]sucrose, a marker of paracellular permeability. Thiseffect was significantly reduced (58%) with posthypoxicreoxygenation. After hypoxia and posthypoxic reoxygenation,actin expression was increased (1.4- and 2.3-fold, respec-

tively). Whereas little change was observed in TJ proteinexpression immediately after hypoxia, a twofold increase inexpression was seen with posthypoxic reoxygenation. Fur-thermore, immunofluorescence studies showed alterations inoccludin, ZO-1, and ZO-2 protein localization during hypoxiaand posthypoxic reoxygenation that correlate with the ob-served changes in BBMEC permeability. The results of thisstudy show hypoxia-induced changes in paracellular perme-ability may be due to perturbation of TJ complexes and thatposthypoxic reoxygenation reverses these effects.

endothelial cells; cytoarchitecture; blood-brain barrier; im-munofluorescence

WHEREAS RESEARCH INVOLVING hypoxic stress has tradi-tionally focused on identifying and treating risk factorsassociated with ischemic stroke (17, 18, 43), hypoxia asa result of high-altitude exposure has also been asso-ciated with impairment in neurological function (26,37). Early intervention after ischemic stroke has beenshown to reduce tissue damage associated with in-creased cerebrovascular permeability (7, 39). Recently,

research has expanded into examining cellular effectsof cerebral ischemia-reperfusion. Ischemic stroke is areduction or cessation of blood flow to the brain, whichdeprives the cerebral tissue of important nutrients andoxygen as well as removal of metabolic waste products.Whereas this decrease in oxygen supply to the brain(i.e., hypoxia) has been shown to increase cerebrovas-cular permeability (1, 19), there are also reports (42,66) of increased permeability in peripheral and cere-bral endothelial cells during posthypoxic reperfusion(i.e., reoxygenation). Little is known about cellularresponse of cerebral endothelial cells during high-alti-tude hypoxia. It remains unclear whether the hypoxicinsult or the posthypoxic reoxygenation induces func-tional changes in the blood-brain barrier (BBB). Fur-thermore, the molecular alterations that occur duringthese changes in paracellular permeability require ex-amination.

Brain microvessel endothelial cells (BMEC) form ametabolic and physical barrier separating the periph-ery from the brain to maintain cerebral homeostasis (5,28). The lack of fenestrations and presence of tight

junctional (TJ) complexes differentiate BMECs fromperipheral microvascular endothelium. Whereas adhe-rens junctions and other junctional proteins contributeto cell-to-cell contacts in the paracellular cleft, TJ com-plexes are critical for restricting paracellular diffusionin the cerebral microvasculature (4, 13, 55).

TJs are complexes of plasma membrane proteinsthat connect to the cytoarchitecture via membrane-associated accessory proteins (49). Claudins and occlu-din are integral transmembrane proteins, which inter-act with plasma membranes of adjacent cells formingthe TJ barrier (63, 65). Cytoplasmic TJ accessory proteins[e.g., zonula occluden (ZO)-1, ZO-3, and cingulin] connectTJs to the cytoskeleton (i.e., actin) (8, 33). ZO-1 and ZO-2

are membrane-associated guanylate kinase proteins crit-ical in forming and stabilizing TJs by binding occludin tothe cytoarchitecture (33, 36). Figure 1 is a schematic ofBMEC TJ complexes. Although TJ complexes have beenidentified, little is known about alterations in these pro-teins under pathological insult, i.e., hypoxia and reoxy-genation conditions.

Address for reprint requests and other correspondence: T. P.Davis, Univ. of Arizona, 1501 N. Campbell, PO Box 245050, Tucson,AZ 85724-5050 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

Am J Physiol Heart Circ Physiol 282: H1485H1494, 2002;10.1152/ajpheart.00645.2001.

0363-6135/02 $5.00 Copyright 2002 the American Physiological Societyhttp://www.ajpheart.org H1485
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Hypoxia and reoxygenation alter actin distribution

(12). Cellular mechanisms induced by these changes inoxygen levels leading to increased permeability,changes in TJ complexes, and cytoarchitecture need tobe investigated. During ischemic stroke with reducedperfusion, hypoglycemia and hypoxia result in energydepletion (62), ion imbalance, and release of cellularmediators (38, 53) that may lead to cell injury or death(38). Recent attention has been given to investigatingcellular mechanisms affected by ischemia (1, 44).Whereas hypoxia-aglycemia has been shown to induceincreased BMEC permeability via calcium-dependentpathways (1), the effects on TJ complexes are not welldefined. Additionally, reoxygenation effects on intra-cellular mechanisms involved in altering BMEC per-

meability remain largely unknown.This study used a well-characterized model of the

BBB, bovine BMEC (BBMEC), to examine the relation-ship between functional and molecular alterations incerebral microvasculature induced by hypoxia andposthypoxic reoxygenation.

MATERIALS AND METHODS

Materials. Transwell polyester membrane inserts, mini-mal essential medium, Hams F-12 medium, and Permanoxtissue culture slide chambers were purchased from Fisher(St. Louis, MO). Bovine fibronectin, bovine serum albumin,3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide(MTT), and equine serum were purchased from Sigma (St.

Louis, MO). Rat tail collagen (type I) was purchased fromCollaborative Biomedical Products (Medford, MA). Bicincho-ninic acid protein assay kit was purchased from Pierce (In-dianapolis, IN). All other nutrients, salts, and antibioticsused in culture media or assay buffers were of cell culturequality from Sigma. Epithelial voltohmmeter and STX-2chopstick electrodes were purchased from World PrecisionInstruments (Sarasota, FL).

Antibodies. Rabbit polyclonal anti-ZO-1, anti-ZO-2, anti-occludin, and anti-claudin-1 antibodies documented to reactwith human, mouse, rat, and dog were purchased fromZymed Laboratories (San Francisco, CA). Anti-ZO-1 is di-rected against amino acids 463 1109 of human ZO-1 cDNA.

Anti-ZO-2 recognizes the central portion of the protein and

has some cross-reactivity with unidentified proteins in the50- to 85-kDa region. Anti-occludin is directed against theCOOH-terminal 150 amino acids of human occludin. Theanti-claudin-1 antibody recognizes the COOH-terminus re-gion of the protein. Mouse monoclonal antibody (Sigma) di-rected against the COOH-terminus of actin (clone AC-40)reacts with human, bovine, chicken, dog, rat, and mousespecies. Anti-mouse IgG and anti-rabbit IgG horseradishperoxidase-conjugated secondary antibodies were purchasedfrom Amersham (Buckinghamshire, UK).

Isolation and culturing of BBMECs. Fresh bovine brainswere obtained from the University of Arizona Animal Ser-

vices for use as an in vitro BBB model. Primary BBMECswere collected from gray matter of bovine cerebral cortexeswith the use of enzymatic digestion and centrifugal separa-

tion methods previously described (48). BBMECs wereseeded (50,000 cells/cm2) on collagen-coated, fibronectin-treated 75-cm2 tissue culture flasks or 12-well Transwellpolyester membrane inserts (0.4 m pore/12 mm diameter).Culture media was composed of 45% minimum essentialmedium, 45% Hams F-12 nutrient mix, 10 mM HEPES, 13mM NaHCO3, 50 g/ml gentamicin, 10% equine serum, 2.5g/ml amphotericin B, and 100 g/ml heparin sodium. Cellcultures were grown in a humidified 37C incubator with95% room air-5% CO2, and culture medium was replacedevery other day until the BBMEC monolayers reached con-fluency (1113 days).

Hypoxia-reoxygenation treatment. Several studies (19, 61)that examined the effects of hypoxia have used 95% N2-5%

CO2 mixtures to reduce atmospheric O2, whereas others haveused 100% N2 (12, 31). In this study, a hypoxic workstation(Coy Laboratories; Grass Lake, MI) was infused with 1%O2-99% N2 to treat confluent BBMEC monolayers for 24 h at37C. After 24-h hypoxia treatment, monolayers were used inpermeability experiments or immunofluorescence studies, orprotein was harvested for Western blot analyses. Alterna-tively, after exposure to hypoxic conditions, BBMEC mono-layers were reoxygenated in 95% room air-5% CO2 at 37Cfor 2 h, followed by permeability, Western blot, or immuno-fluorescent microscopy experiments. Changes in permeabil-ity, protein expression, or localization patterns after thesetreatment protocols were compared with control monolayers

Fig. 1. Schematic drawing of proposed junctional archi-tecture of cerebromicrovessel endothelial cells. Tightjunctions (TJ) consisting of the integral membrane pro-teins occludin and claudin are located towards the api-

cal side of endothelial cells with their correspondingcytoplasmic accessory proteins [i.e., zonula occluden(ZO)-1, ZO-2, ZO-3, and cingulin] connecting TJs to thecytoskeleton (i.e., actin; not shown). Adherens junctions(AJ), located toward the basolateral side of endothelialcells, are composed of the integral membrane boundcadherins and cytoplasmic accessory proteins (i.e., -and -catenin).

H1486 HYPOXIA-INDUCED CHANGES IN BRAIN ENDOTHELIAL CELLS

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incubated for 24 h at 37C in 95% room air-5% CO2 (normoxicconditions).

Gas analysis of cell culture medium. Gas values [pH, PCO2(mmHg), PO2 (mmHg), and bicarbonate (mmol/l)] were mea-sured in cell culture medium after exposure of BBMECmonolayers to normoxic (control), hypoxic, or posthypoxicreoxygenation conditions using a blood gas analyzer (model

ABL-505 Radiometer; Copenhagen, Denmark).Cytotoxicity study of BBMECs after hypoxia-reoxygenation.

Viability of BBMECs after exposure to normoxia, hypoxia, orhypoxia with reoxygenation was assessed using the MTTcytotoxicity assay (27). Confluent BBMEC monolayers wereexposed to normoxic, hypoxic, or hypoxic with reoxygenationconditions. The culture medium was removed and cells wererinsed with phosphate-buffered saline (PBS). The cells wereincubated with 5 mg/ml of MTT for 2 h at 37C. Excess MTTwas removed and cells were rinsed with PBS before solubi-lizing in a 50:50 mixture of dimethyl formamide and 20%(wt/vol) sodium dodecyl sulfate (pH 4.7). Absorbance read-ings were taken at 550 nm using a Labsystems MicroplateReader (Fisher; Tustin, CA). Viability was expressed as apercentage of control cells exposed to normoxic conditions.

BBMEC monolayer permeability experiments. BBMEC

monolayer integrity was assessed by transendothelial elec-trical resistance (TEER) measurements. Those monolayerswith resistance values 120 cm2 were used to assess thepermeability effects of hypoxia and reoxygenation onBBMEC monolayers. The passage of [14C]sucrose (a paracel-lular marker) across the BBMEC monolayer was employedbecause this method is more sensitive for detecting smallchanges in permeability compared with TEER measure-ments (20).

After treatment, confluent BBMEC monolayers were incu-bated with assay buffer composed of (in mM) 122 NaCl, 3KCl, 1.4 CaCl2, 1.2 MgSO4, 25 NaHCO3, 10 HEPES, 10glucose, and 0.5 K2HPO4 for 30 min at 37C under the samegas conditions. Permeability across BBMEC monolayers wasdetermined by adding [14C]sucrose (0.5 Ci) to the lumenal

side (upper compartment of Transwell). Samples (50 l) wereremoved from the ablumenal side (lower chamber of Trans-well) at 0, 60, and 120 min and replaced with fresh assaybuffer. The concentration of [14C]sucrose applied to the lu-menal side was determined by removing samples (50 l) attime zero and replacing with fresh assay buffer and [14C]su-crose. The amount of radioactivity in the samples from per-meability studies was determined by using a BeckmanLS5000 TD beta counter. Permeability coefficients (PC) for[14C]sucrose were expressed in the following equation aspreviously described (15)

PC (cm/min) V

SA Cd

Cr

T

where V is volume in receiver chamber (1.5 cm3

), SA issurface area of cell monolayer (1 cm2), Cd is the concentrationof marker in the donor chamber at time 0, and Cr is theconcentration of marker in the receiver at sample time T.

Western blot protein analysis. After being treated withhypoxia, hypoxia-reoxygenation, or normoxic (control) condi-tions, protein was isolated from confluent BBMEC monolay-ers using the TRI reagent protocol (Sigma). Briefly, proteinwas separated from RNA and DNA by chloroform and etha-nol extraction and then precipitated using isopropanol, fol-lowed by washing with guanidinium chloride-95% ethanolbefore dissolving in 1% sodium dodecyl sulfate. Protein wasquantified using the bicinchoninic acid method.

Protein samples (20 g) were separated using an electro-phoretic field on Novex 412% Tris-glycine gels at 125 V for7590 min. The proteins were transferred to polyvinylidenefluoride membranes with 240 mA at 4C for 30 min. Themembranes were blocked using 5% nonfat milk-Tris-bufferedsaline (20 mM Tris base-137 mM NaCl, pH 7.6) with 0.1%Tween 20 and incubated overnight at 4C with primaryantibodies (1:1,0001:2,000 dilution) in PBS-0.5% BSA. Themembranes were washed with 5% nonfat milk-Tris-buffered

saline buffer before incubation with the respective secondaryantibody at a 1:2,000 dilution (in PBS-0.5% BSA) for 30 minat room temperature. Membranes were developed using theenhanced chemiluminescence method (ECL; Amersham)and protein bands were visualized on X-ray film. Semiquanti-tation of the protein was done with the use of imaging software(Scion) and the results are reported as a percentage of control.

Immunofluorescence of BBMECs. Confluent BBMECsmonolayers grown on Permanox chamber slides (Fisher)were exposed to 24-h hypoxia, hypoxia with 2-h reoxygen-ation, or normoxic conditions before immunofluorescencestaining. After treatment, culture medium was removed andmonolayers were rinsed with prewarmed PBS. Cells werefixed with 3.7% formaldehyde-PBS for 10 min at room tem-perature (RT) and permeabilized using 1% Triton-X-PBS at

RT for 10 min. After fixing and permeabilization, the mono-layers were blocked with 1% BSA-PBS for 1 h at RT.

Confluent BBMEC monolayers from each treatment group(normoxia, hypoxia, and posthypoxic reoxygenation) wereincubated with anti-occludin (10 g/ml), anti-claudin-1 (10g/ml), anti-ZO-2 (5 g/ml), or anti-ZO-1 (5 g/ml) primaryantibody at RT for 30 min. The cells were rinsed with 1%BSA/PBS, followed by incubation with a fluorescein-conju-gated secondary antibody (4 g/ml; Alexafluor 488 in 1%BSA-PBS; Molecular Probes) for 30 min at RT in the dark.

Confluent BBMEC monolayers from each treatment groupwere incubated with Alexafluor 488-conjugated phalloidin,which binds specifically to actin filaments. The phalloidinstain was reconstituted with 1% BSA-PBS (5 U/ml) applied tothe luminal side of each monolayer and incubated for 30 min

at RT in the dark. The fluorescent-stained cells were rinsedthree times with PBS before being mounted on a coverslipwith 50% glycerol-PBS and sealed.

Photographs were taken with the use of a 60 oil immer-sion objective on a Nikon TE300 fluorescent microscope witha fluorescein filter.

Data analysis. Statistical analysis of the data was donewith the use of one-way analysis of variance with Newman-Keuls multirange post hoc comparison of the means (10).

RESULTS

Gas analysis of treated BBMECs. Table 1 shows thechanges in pH, PCO2 (mmHg), PO2 (mmHg), and bicar-

Table 1. Gas analysis of cell culture medium

Treatment Group pHPCO2,

mmHgPO2,

mmHgHCO3

,mmol

Normoxic 7.240.08 25.17.0 1 39.32.3 8.41.5Hypoxic 8.060.09 1.70.1 46.57.3 6.51.6Posthypoxic

reoxygenation 7.370.03 10.81.7* 129.23.6 6.10.8

Data are means SE; n 5. Measurements were performed inculture medium. *P 0.05 and P 0.01 compared with normoxicconditions using one-way ANOVA and Newman-Keuls post hoc anal-ysis.

H1487HYPOXIA-INDUCED CHANGES IN BRAIN ENDOTHELIAL CELLS

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bonate (HCO3; mmol/l) levels in the BBMEC cell cul-

ture medium. As expected, PO2 levels dropped signifi-cantly (P value 0.01) during the hypoxic (1% O2-99%N2) phase with a rapid return (within 5 min) to controllevels during reoxygenation. Similarly, PCO2 levels de-creased during hypoxia with a subsequent increasewhen O2 was reintroduced to the system. In concertwith the declining PCO2, the pH increased significantlyin the culture medium of BBMECs treated to 24-hhypoxia. There was no change in bicarbonate (HCO3

)

levels in any of the treatment groups.Cytotoxicity of hypoxia-treated BBMECs. There was

no significant difference in cell viability of theBBMECs exposed to 24-h hypoxia (1% O2-99% N2) orhypoxia with reoxygenation compared with normoxiccontrols using the MTT assay (112% and 100%, respec-tively, compared with control; Table 2). This indicatesthat the decreased PO2 or increased pH levels wereinsufficient to cause cell death.

Permeability of [14C]sucrose in BBMECs. Table 2shows that, whereas there was no difference inBBMEC monolayer TEER measurements in any of thetreatments (109123 cm2), there was a significantincrease (P 0.01) in permeability of [14C] sucrose inthe hypoxia-treated group compared with controlBBMEC monolayers (12.57 vs. 4.80 cm/min 104,respectively). Reoxygenation for 2 h attenuated thehypoxia-induced permeability increase by 58% (8.04cm/min 104).

Western blot analysis of cytoskeletal and TJ proteins.Alterations in expression of proteins that form TJs andcytoskeletal architecture were examined after hypoxia(24 h), hypoxia-reoxygenation (24 h/2 h), or normoxic(control) conditions. Table 3 summarizes the Westernblot analyses of hypoxia and posthypoxic reoxygen-ated-treated BBMECs compared with controls. An in-creased expression (1.4-fold) of the major cytoskeletal

protein (actin) occurred after hypoxia and continued toincrease (2.3-fold) on reoxygenation (Fig. 2). In con-trast, hypoxia alone had little effect on the expression

of TJ proteins (i.e., claudin-1, occludin, ZO-1, or ZO-2;Fig. 2). Whereas there was little change in claudin-1expression, a significant increase was observed in ex-pression of occludin, ZO-1, and ZO-2 (1.4-, 1.8-, and1.9-fold; respectively) with posthypoxia reoxygenation.

Immunofluorescence of hypoxia-treated BBMEC mono-layers. BBMEC monolayers exposed to hypoxia for 24 hshowed changes in actin localization, with increasedstaining of the filaments and development of stresstangles (arrowheads in Fig. 3). More of these actin

stress tangles became apparent after reoxygenation. As with the protein expression of claudin-1, littlechange in the distribution pattern of this protein wasseen after hypoxia or posthypoxic reoxygenation. Incontrast, significant disruptions were seen in localiza-tion of occludin and ZO-2 after hypoxia (arrows in Fig.3). These changes were less apparent in posthypoxicreoxygenation correlating to the increased protein ex-pression (Fig. 2). The localization pattern of ZO-1 ap-pears more diffuse throughout the cells after 24 hhypoxia. The distribution of ZO-1 and ZO-2 in posthy-poxic reoxygenation BBMECs resembles that of controlmonolayers (Fig. 3); however, there is more intensestaining at cell-to-cell contact points.

DISCUSSION

Endothelial cells of the cerebral microvasculatureserve as a frontline defense, protecting neurons andglial cells from harmful insult. This study and othersfocus on understanding how BMECs respond tochanges in O2 content of blood seen in ischemic orhigh-altitude hypoxia. Under normal conditions ofroom air (21% O2), arterial PO2 (PaO

2) levels range from

80100 mmHg and interstitial PO2 (PIO2) values range

from 1525 mmHg or 25% of PaO2

(24). Because ofthe diffusion of O2 across membranes, decreases in

PaO2 will result in similar reductions in PIO2. Withhigh-altitude exposure or a narrowing of cerebral cap-illaries such as in ischemic hypoxia, the available O2

Table 2. BBMEC permeability and cell viability

Treatment GroupPermeability Coefficient of

[14C]sucrose (cm/min 104) TEER, cm2Cell Viability,

%Control

Normoxic 4.800.46 1232.3 100.05.6Hypoxic 12.571.31 1122.0 112.42.2Posthypoxic reoxygenation 8.041.10* 1091.0 100.36.1n 9 6 6

Data are means SE; n, no. of cell cultures. BBMEC, bovine brain microvessel endothelial cells; TEER, transendothelial electrialresistance. *P 0.05 and P 0.01 compared with normoxic conditions; P 0.01 compared with hypoxic conditions using one-way ANOVAand Newman-Keuls post hoc analysis.

Table 3. Effects of treatment on BBMEC protein expression

Treatment Group Actin, % Claudin-1, % Occludin, % ZO-1, % ZO-2, %

Normoxic 1002 1001 1002 1003 1001Hypoxic 14111* 995 975 1024 976Posthypoxic reoxygenat ion 23216 1035 13811 17116 17822

Data are percentage of control with means SE. ZO, zona occludens. *P 0.05 and P 0.01 compared with normoxic conditions usingone-way ANOVA and Newman-Keuls post hoc analysis.


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decreases with a subsequent reduction in PaO2

thatvaries depending on the severity of exposure. In fact,

the severity and length of decreased O2 available to thecerebral microvessel endothelial cells will impact thedegree of brain tissue damage.

Hypoxia as an element of ischemic stroke or high-altitude exposure has been examined using both invivo and in vitro models. In vivo models for ischemicstroke have shown decreased PO2 values and increasedPCO2 with corresponding decrease in pH (41, 47),whereas models for high-altitude hypoxia have showndecreased PO2 and PCO2 with a rise in pH (6, 51).Typically, O2 concentrations in the range of 612%have been used to study hypoxic effects in vivo with PO2

values of 3547 mmHg (16, 25, 50). In Wistar rats, acombination of middle cerebral artery occlusion andhypoxia (PaO

2of 47 mmHg) resulted in increased brain

tissue damage compared with controls (50).Although middle cerebral artery occlusion provides a

model for examining tissue damage (primarily neuro-nal) that occurs during focal ischemia, there is nodocumentation of PO2 values for the injury site. Fur-

thermore, it does not allow for examination of paracel-lular permeability changes across the capillary endo-thelium nor protein localization changes that mayoccur during a hypoxic insult. Such changes are bestexamined with in vitro cell culture models. A variety ofapproaches for inducing a hypoxic environment havebeen used in peripheral and cerebral cell culture mod-els, including O2 chelating agents (gas-paks) and an-aerobic chambers infused with various gas mixtures(100% N2, 95% N2-5% CO2, or a mix of 85% N2-10%H2-5% CO2) to maintain an anoxic or hypoxic environ-ment for periods ranging from 20 min to 72 h. To date,few studies have reported PO2, PCO2, and pH values

from cell culture models of hypoxic insult (3, 54, 56,60). In rat mesangial cells, Archer et al. (3) examinedthe effects of graded hypoxia on inducible nitric oxide(NO) synthase function using atmospheric O2 levels of0, 2.5, 10, and 21%, resulting in culture medium PO2levels of 32, 46, 85, and 140 mmHg, respectively (3).

The current study used primary BBMECs as an invitro BBB model to investigate paracellular permeabil-ity changes induced by hypoxia and posthypoxic reoxy-genation. Changes in expression and localization ofcytoskeletal and TJ proteins were also examined underthe same conditions. Whereas the benefits of astrocytecoculture conditions (astrocyte, glial cells, or condi-tioned media) have been debated, BBMECs alone were

used in this study for three reasons. First, this modelallows direct assessment of hypoxia-reoxygenationeffects in cerebral microvascular endothelial cells with-out confounding their response by variations in astro-cyte culturing techniques. Second, this allows a consis-tent model for evaluating the functional and molecularchanges in brain microvessel endothelial cells (perme-ability, protein expression, and protein localization).Third, immunocytochemistry observations are bestwhen the cells are grown on slides, which do not allowablumenal access where astrocytes would exert theirinfluence.

The current study used a decreased O2 level to sim-

ulate the reduced oxygen available to cerebral mi-crovessel endothelial cells during hypoxic insults suchas ischemic stroke or high-altitude exposure. The mod-erate hypoxic conditions (1% 0.5% O2 throughout theexperiment) resulted in cell culture medium PO2 mea-surements of 47 7 mmHg compared with normoxiccontrols of 139 2 mmHg. These PO2 values are inagreement with the PO2values used in both in vivo andin vitro studies of hypoxic exposure (2, 3, 50). Althoughthe PCO2 declined with a subsequent increase in pH,there was no change in HCO3

levels (Table 1). In arelated study, BBMECs exposed to 1% O2-94% N2-5%

Fig. 2. Hypoxia-reoxygenation effects on protein expression of bo-vine brain microvessel endothelial cells (BBMECs). Confluent mono-layers were exposed to normoxia (C), 24-h hypoxia (H) or hypoxiawith 2-h reoxygenation (HR). Representative Western blots for actin(42 kDa), claudin-1 (44 kDa dimer), occludin (65 kDa), ZO-1 (220kDa), and ZO-2 (160 kDa) are shown (n 4).


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pheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/http://ajpheart.physiology.org/

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CO2 had a mean pH value of 7.12, PCO2 of 33 mmHg,and PO2 value of 55 mmHg with similar permeabilityresults (data not shown). Changes in pH, PCO2, and PO2

did not affect cell viability as demonstrated by the MTTcytotoxicity assay.

Whereas TEER measurements showed little changeunder the conditions in this study, radiolabeled sucrose(346 mol wt) proved to be a more sensitive marker formeasuring changes in paracellular permeability (Table2). Differences in permeability of [14C]sucrose wereobserved in hypoxia-treated BBMEC monolayers com-pared with normoxic (controls) and posthypoxic reoxy-genated monolayers. BBMEC monolayers exposed tohypoxia for 24 h showed a significant (2.6-fold) increasein [14C]sucrose permeability compared with control

monolayers. These findings are consistent with previ-ous reports (1, 19, 23) showing increased permeabilityin brain capillary endothelial cells treated with hyp-

oxia for periods ranging from 2 to 48 h.Permeability changes shown in the current study

correlate with alterations in TJ proteins and actin, asshown in Fig. 1. Increased expression of actin and theemergence of stress fiber tangles seen in Fig. 3 corre-late with increased paracellular permeability suggest-ing hypoxia-induced alterations in cell morphology.This is supported by previous studies showing alter-ations in the cytoskeleton of 4 h hypoxia-treated bovineaortic endothelial cells (12) and actin filament rear-rangement in brain endothelial cells after hypoxia for2448 h (53). This change in cell morphology and

Fig. 3. Immunofluorescence showingprotein localization in BBMECs. Con-fluent monolayers were exposed to nor-moxic (Control), 24-h hypoxia (Hypoxic),or hypoxia with 2-h reoxygenation (Re-oxygenated) conditions. After treatment,the monolayers were incubated with pri-mary antibodies directed against actin,

claudin-1, occludin, ZO-1, and ZO-2,followed by Alexafluor 488-conjugatedsecondary antibodies. Representativepictures for each treatment group andprotein are shown (n 3). Arrowheads,actin stress tangles; arrows, disrup-tions in the expression of occludin andZO-2. *Increased cytosolic staining ofZO-1 and ZO-2.


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increased paracellular permeability suggests disrup-tions at TJ protein complexes.

Although no significant changes in protein expres-sion of TJ proteins (claudin, occludin, ZO-1, and ZO-2)were observed in BBMECs after a 24-h hypoxic insult,there were changes in protein localization that corre-late with functional changes (i.e., increased permeabil-ity) seen in this study. Claudin-1, which is considered

the critical protein in forming TJ complexes, is embed-ded in the plasma membrane with short cytoplasmicsections that bind to occludin. Whereas the protein isimportant to the TJ complex, previous studies (11, 22,67) examining claudin-1 have shown variations in im-munofluorescence patterns of claudin-1 protein includ-ing staining at cell boundaries and diffuse punctatedistribution. In this study, little change in the proteinexpression or localization pattern of claudin-1 was ob-served with hypoxia or posthypoxic reoxygenation com-pared with normoxic controls (Figs. 2 and 3).

Occludin, another integral TJ protein, has two extra-cellular loops and long intracellular NH2-terminal and

COOH-terminal regions suggesting that it connectsclaudins with TJ accessory proteins (i.e, ZO-1 andZO-2) and the plasma membranes of adjacent cells.Figure 3 shows that under normoxic conditions, occlu-din has a continuous distribution pattern at the cellboundaries. Similarly, staining of the TJ accessoryproteins ZO-1 and ZO-2 were localized along theplasma membrane at cell-to-cell contacts under controlconditions. Exposure to hypoxia resulted in perturba-tions of the plasma membrane pattern of occludin andZO-2 proteins compared with controls. In addition,both ZO-1 and ZO-2 show a diffuse pattern throughouthypoxic-treated BBMECs with discontinuous stainingat the cell borders compared with control monolayers

(Fig. 3). This is supported by other studies examiningthe hypoxic effects on localization of ZO-1 in BMECs(20). These disruptions in TJ proteins at cell-to-cellcontact sites correlate with increased paracellular per-meability and changes seen in actin distribution pat-terns (i.e., stress tangles). Together, these results in-dicate that hypoxic insult to brain endothelial cellsinduces restructuring of the TJ and cytoarchitecture(i.e., changes in protein localization) that correlatewith observed functional changes (i.e., increased para-cellular permeability) with little change in protein ex-pression. This exposure to a hypoxic environment maytrigger relocation of some TJ proteins from the plasma

membrane to the cytoplasm. It is well known thatprotein phosphorylation regulation is a means of intra-cellular signaling, as cells use phosphorylation of en-zymes and proteins to regulate cellular functions (pro-tein degradation and enzyme activation) (58, 63). Thepossibility of TJ proteins relocating to the cytoplasmmay be due to changes in phosphorylation states thatoccur with changes in phosphatase or kinase activity.The examination of the phosphorylation states of theseTJ and cytoskeletal proteins during hypoxic insult willclarify whether this cellular mechanism is involved inprotein localization and BBB permeability changes.

Whereas this study and others have shown pertur-bations of BBB permeability in various ischemia-hypoxia models (1, 19, 23, 52), others (12, 32, 42) havealso reported increased paracellular permeability anddisruptions in actin during posthypoxic reoxygenation.The present study showed that reoxygenation attenu-ated the hypoxia-induced increase in permeability by58% (Table 2). This recovery with reoxygenation corre-

lates with changes in protein expression and localiza-tion of actin and TJ proteins. Increasing TJ proteinsynthesis to enhance weakened cell-to-cell contactsmay be an endothelial cell response to cytoarchitec-tural alterations and increased paracellular perme-ability. Whereas a modest rise (1.4-fold) in expressionof actin protein was seen after hypoxic insult, thesecells showed a 2.3-fold increase in protein expressionwith 2-h posthypoxic reoxygenation compared withnormoxic monolayers. This increase in protein expres-sion coincides with an increased number of stress fibertangles (Fig. 3) and a reduction in permeability. Al-though no change in actin expression was observedwith hypoxic exposure of bovine aortic endothelialcells, a 50% increase in expression was seen withposthypoxic reoxygenation (12). Because actin is theprimary protein forming the cytoskeletal structure,these increases in actin protein and stress tangles mayprovide additional scaffolding for TJ proteins to rein-force cell-to-cell contacts that become weakened byhypoxic insult. Whereas studies (21, 29, 49) have dem-onstrated that claudin is vital to forming the tightjunction seal, this study shows little change in proteinexpression or localization of claudin-1 after hypoxia orposthypoxic reoxygenation. This suggests that claudinis not involved in the hypoxia-induced paracellularpermeability changes. In contrast, the increased ex-

pression of occludin, ZO-1, and ZO-2 after reoxygen-ation indicates alterations of TJ complexes and theirconnection to the cytoarchitecture during the reoxy-genation stage of an ischemic event. This is furthersupported by the distribution patterns seen in these TJproteins after a hypoxic insult when gaps are apparentand during posthypoxic reoxygenation when smallerand fewer gaps are seen. In addition, increased expres-sion of the TJ proteins and actin with posthypoxicreoxygenation correlates well with a reduction in para-cellular permeability of BBMEC monolayers comparedwith monolayers treated to hypoxia alone (Table 2).This suggests that O2 reintroduced to the system stim-ulates cellular mechanisms to reinforce cell-to-cell con-

tact (TJ complexes), resulting in attenuation of para-cellular permeability induced during hypoxic insult.Whether a sufficient reoxygenation period can inducecellular changes to reduce paracellular permeability tocontrol levels requires further investigation.

The results from this study demonstrate that cere-bral microvessel endothelial cells undergo molecularand functional changes during times of hypoxic-reper-fusion stress, but the cellular signaling systems thatconnect the stress to these changes are not well char-acterized. In recent reviews by del Zoppo et al. (14) andStanimirovic et al. (59), various mediators, including


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the proinflammatory cytokines tumor necrosis factor(TNF)- and interleukin (IL)-1, NO, and prostaglan-dins, are discussed in relation to their involvementwith disruptions in BBB integrity during ischemicstroke. Sharkey et al. (56) showed an increased expres-sion of the angiogenic cytokine vascular endothelialgrowth factor in endometrial cells exposed to decreasedO2. In addition, hypoxia-induced increases in perme-

ability of porcine brain endothelial cells have beencorrelated to such a rise in vascular endothelial growthfactor (19). Temporal expression of cytokines (TNF-,IL-6, and transforming growth factor-) has been re-ported in focal ischemic model of stroke (30). In addi-tion to release of these cytokines, the loss of O2 has alsobeen shown to increase intracellular calcium levels,decrease levels of ATP, and the release of excitatoryamino acids and NO (35, 38, 46). Abbruscato et al. (1)showed that calcium channel inhibitors (nifedipine andSKF-96365) prevented increased permeability inducedby hypoxia-aglycemia (1). Posthypoxic reoxygenationhas been shown to decrease the influx of calcium in

bovine brain endothelial cells (34). This suggests thatthe recovery in permeability seen in the present studymay be due to calcium mobilization by the endothelialcells. NO has been shown to increase permeability incerebral microvasculature (45). Because NO is pro-duced constitutively by calcium-dependent endothelialNO synthase and by calcium-independent inducibleNO synthase, alterations in calcium levels may impactNO formation and other calcium-dependent cellularmechanisms (i.e., kinase activation). Decreased levelsof ATP during hypoxic exposure have also been asso-ciated with increased permeability and actin rear-rangement in brain endothelial cells (53). Changes inATP levels are likely to impact phosphorylation states

of cellular proteins. Whereas some proteins such asoccludin are phosphorylated and tagged for degrada-tion, phosphorylation is also a form of intracellularsignaling (9, 57, 64). Recent studies (9, 57, 63, 64) haveshown a correlation between increased phosphoryla-tion of TJ proteins (ZO-1, ZO-2, and occludin) andincreased permeability in both kidney and brain cellculture models. Indeed, it has been suggested thatchanges in phosphorylation of these cellular proteins isresponsible for perturbations in cell-to-cell adhesion(40, 63). Whereas a 2.3-fold increase in tyrosine phos-phorylation was seen in aortic endothelial cells exposedto 4-h hypoxia with 30-s posthypoxic reoxygenation

(12), it is not clear which proteins are phosphorylated.Therefore, further work is required to determine whichof these intracellular signaling systems connect thehypoxic insult and subsequent reoxygenation with themolecular and functional alterations demonstrated inthe present study.

In summary, we report that hypoxia induces in-creased paracellular permeability that is significantlyreduced on reoxygenation and these functionalchanges are associated with alterations in actin and TJprotein distribution. Whereas hypoxia-induced alter-ations in ZO-1 have been demonstrated previously (20),

this is the first report examining the effects of hypoxiaand posthypoxic reoxygenation on protein expressionand localization of actin and TJ proteins in the cerebralmicrovasculature. Understanding cellular mechanismsinduced by hypoxia and posthypoxic reoxygenationthat alter BBB permeability will contribute to devel-oping pharmacotherapies for treatment or preventionof decreased O2 conditions as seen with high-altitudeexposure and ischemic stroke.

This study was supported by National Institute of NeurologicalDisorders and Stroke Grant R01-NS-39592 and University of Ari-zona Foundation Office of the Vice President for Research andGraduate Studies Grant 210777.

REFERENCES

1. Abbruscato TJ and Davis TP. Combination of hypoxia/agly-cemia compromises in vitro blood-brain barrier integrity.J Phar-macol Exp Ther 289: 668675, 1999.

2. Al-Haboubi HA and Ward BJ. Microvascular permeability ofthe isolated rat heart to various solutes in well-oxygenated andhypoxic conditions. Int J Microcirc Clin Exp 16: 291301, 1996.

3. Archer SL, Freude KA, and Shultz PJ. Effect of gradedhypoxia

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