Brain Genomic Response following Hypoxia and Re-oxygenation inthe Neonatal RatIDENTIFICATION OF GENES THAT MIGHT CONTRIBUTE TO HYPOXIA-INDUCED ISCHEMIC TOLERANCE*

Received for publication, May 13, 2002, and in revised form, July 25, 2002Published, JBC Papers in Press, July 26, 2002, DOI 10.1074/jbc.M204619200

Myriam Bernaudin‡§, Yang Tang‡, Melinda Reilly‡, Edwige Petit¶, and Frank R. Sharp‡

From the ‡Department of Neurology and Neuroscience Program, University of Cincinnati, Cincinnati, Ohio 45267 and¶UMR 6551-CNRS, Universite de Caen, IFR 47, Caen, France

Hypoxic preconditioning (8% O2, 3 h) produces toler-ance 24 h after hypoxic-ischemic brain injury in neona-tal rats. To better understand the ischemic tolerancemechanisms induced by hypoxia, we used oligonucleo-tide microarrays to examine genomic responses in neo-natal rat brain following 3 h of hypoxia (8% O2) andeither 0, 6, 18, or 24 h of re-oxygenation. The resultsshowed that hypoxia-inducible factor (HIF)-1- but notHIF-2-mediated gene expression may be involved inbrain hypoxia-induced tolerance. Among the genes reg-ulated by hypoxia, 12 genes were confirmed by real timereverse transcriptase-PCR as follows: VEGF, EPO,GLUT-1, adrenomedullin, propyl 4-hydroxylase �, MT-1,MKP-1, CELF, 12-lipoxygenase, t-PA, CAR-1, and an ex-pressed sequence tag. Some genes, for example GLUT-1,MT-1, CELF, MKP-1, and t-PA did not show any hypoxicregulation in either astrocytes or neurons, suggestingthat other cells are responsible for the up-regulation ofthese genes in the hypoxic brain. These genes were ex-pressed in normal and hypoxic brain, heart, kidney,liver, and lung, with adrenomedullin, MT-1, and VEGFbeing prominently induced in brain by hypoxia. Theseresults suggest that a number of endogenous molecularmechanisms may explain how hypoxic preconditioningprotects against subsequent ischemia, and may providenovel therapeutic targets for treatment of cerebralischemia.

Impaired oxygen (hypoxia) or reduced blood flow (ischemia)to the brain is a major cause of morbidity and mortality in theperinatal period, often resulting in cognitive impairment, sei-zures, and other neurological disabilities. Although hypoxia-ischemia animal models have increased our understanding ofthe processes leading to cell death, there are still no pharma-cological treatments available to reduce cell death in ischemicneonatal brain.

Interestingly, cells can be protected when a non-injurioushypoxic stress is performed several hours or days before alethal hypoxic-ischemic stress (preconditioning). This phenom-enon is called tolerance. Ischemic tolerance can be achieved inbrain by several preconditioning sublethal stresses such ashypoxia (1–3), ischemia itself (4), hypothermia (5), hyperther-

mia (6), hyperbaric oxygenation (7), metabolic inhibitors (8),spreading depression (9), as well as cytokines (10, 11).

As hypoxic preconditioning is non-invasive and reproducible,this model has been used to study the mechanisms protectingthe brain against hypoxia-ischemia particularly in newbornrats (3, 12–14). In addition, hypoxic preconditioning alsoinduces tolerance against focal transient (15) and permanent(16) cerebral ischemia in adult mice. These studies suggestedthat hypoxia-inducible factor-1 (HIF-1)1 could be an impor-tant mediator of hypoxia-induced tolerance to ischemia (13,14, 16). Indeed, hypoxic preconditioning induces expressionof HIF-1� and its target genes in neonatal (14) and adultbrain (16). In addition, desferrioxamine and cobalt chloride,two agents that activate HIF-1 (17), also induce toleranceagainst hypoxia-ischemia in neonatal rat brain (13). HIF-1 isan important transcription factor regulating gene expressionin response to hypoxia. Moreover, HIF-1 target genes such aserythropoietin (EPO) and vascular endothelial growth factor(VEGF) protect the brain against ischemia (11, 18–20). Thissuggests that HIF-1 might be involved in the establishmentof ischemic tolerance in brain. However, it is possible thatthere are other adaptive mechanisms underlying thisprotection.

Understanding changes in gene expression in brain followingexposure to hypoxia could reveal new mechanisms of ischemictolerance. Accordingly, we exploited DNA microarray technol-ogy to investigate the brain genomic response of neonatal rat tohypoxia (3 h, 8% O2). Previous studies have analyzed the geneexpression after hypoxia in fish (21), PC12 pheochromocytomacells (22, 23), glioblastoma cells (24), and Hep3B hepatocarci-noma cells (25). Therefore, this is the first in vivo study of thegenomic response to hypoxia in neonatal rat brain and invertebrates in general.

EXPERIMENTAL PROCEDURES

Animals—All animal experiments were performed in strict accord-ance with National Institutes of Health guidelines, and animal proto-cols were approved by the University of Cincinnati Animal ResearchCommittee. Sprague-Dawley rats (Harlan, Indianapolis, IN) were ac-climated to the animal quarters at least 3 days prior to study. Theanimals, maintained on a 12-h light/dark cycle, were given food andwater ad libitum. In addition, all experimental and control animalswere housed in the same room prior to and at the conclusion of thestudy.

* This work was supported by National Institutes of Health GrantsNS28167, NS38743, NS43252, NS42774, and AG19561 and an Ameri-can Heart Association Bugher award (to F. R. S.). The costs of publica-tion of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: UMR-CNRS 6551, Bd.Henri Becquerel, BP5229, F-14074 Caen Cedex, France. Tel.: 33-2-31-56-60-45; Fax: 33-2-31-56-61-99; E-mail: m_bernaudin@hotmail.com.

1 The abbreviations used are: HIF-1, hypoxia-inducible factor-1;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Q-RT, quantita-tive one-step reverse transcriptase; RT, reverse transcriptase; VEGF,vascular endothelial growth factor; EPO, erythropoietin; EST, ex-pressed sequence tag; t-PA, tissue-type plasminogen activator; JNK,c-Jun N-terminal kinase; MAP, mitogen-activated protein; MT-1, met-allothionein-1; MKP-1, MAP kinase phosphatase-1; GLUT-1, glucosetransporter-1; CAR-1, cell adhesion regulator-1.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 42, Issue of October 18, pp. 39728–39738, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Primary Neuron and Astrocyte Cultures and Hypoxia Induction—Neocortical cultures of neurons from rat embryos at 14–15 days ofembryonic development (Sprague-Dawley rats, CERJ, Le Genest Saint-Isle, France) were prepared as described (26). Cultures were allowed togrow in a serum-free, chemically defined medium: Dulbecco’s modifiedEagle’s medium plus N-2 (Invitrogen) for 3 days in a humidified 5% CO2

incubator at 37 °C before use.Neocortical cultures of astrocytes were prepared from neonatal (1–

3-day-old) rats (Sprague-Dawley CERJ) as described (26). Cultureswere allowed to grow in a humidified 5% CO2 incubator at 37 °C toconfluency (15–20 days) before use in Dulbecco’s modified Eagle’s me-dium supplemented with 10% fetal calf serum, 50 units/ml penicillin,and 50 �g/ml streptomycin (Invitrogen).

Hypoxia was induced by placing neuron and astrocyte cultures in amodular incubator chamber (Anaerobic Chamber, Plas Labs, Lansing,MI) gassed with a mixture consisting of 4.5% CO2 and 95.5% N2. Thegrowth medium was replaced by the same medium (without serum forastrocytes) that had been equilibrated with a gas mixture consisting of4.5% CO2 and 95.5% N2 for 30 min before bathing the cells. The hypoxiawas maintained for 3 h, i.e. the duration known to induce tolerance invivo (1–3, 12). Immediately after hypoxia, total RNA was prepared fromcultured cells as described below.

Hypoxia Preconditioning Procedures in Animals—Male and femalerats at postnatal day 6 (P6) (Sprague-Dawley Harlan) were used. Thehypoxia chamber (Reming Bioinstruments, Redfield, NY) consists offour identical Plexiglas chambers, each 16 liters in size, that are ar-ranged adjacent to one another. The inlet O2, CO2, and N2 are fixedusing gas tanks of appropriate concentrations and valves to monitorinlet gas concentrations. The exiting gas is continuously monitored forO2, CO2, and N2 as well. These data are monitored and stored for eachexperiment. Rat pups were placed in cages inside these large Plexiglaschambers through which 8% O2 and 92% N2 were circulated for a periodof 3 h. This degree and duration of hypoxia is not known to produce anyinjury in brain and has been shown to protect the newborn brain fromsubsequent hypoxic-ischemic injury (1–3, 12). Control animals from thesame littermates were placed in one of the four chambers and exposedto ambient oxygen (approximately 21% O2) for the same duration. After3 h of hypoxia or normoxia, all animals were returned to their dams for0, 6, 18, or 24 h (re-oxygenation period). After the different re-oxygen-ation periods, rat pups (at P6 or P7) were anesthetized with ketamine(100 mg/kg) and xylazine (20 mg/kg), and their brains were removed forRNA or protein isolation (Fig. 1). In addition, total RNA was alsoprepared from different organs (heart, kidney, liver, and lung) after 3 hof hypoxia and from controls.

RNA Isolation—After 3 h of hypoxia or normoxia followed by differ-ent periods of re-oxygenation (0, 6, 18, or 24 h), rat pups were anesthe-tized with ketamine (100 mg/kg) and xylazine (20 mg/kg), and theirbrain (whole brain hemisphere), heart, kidney, liver, and lung wereremoved as rapidly as possible for RNA isolation. Total RNA wasisolated with TRIzol Reagent (Invitrogen) according to the manufactur-er’s protocol. Briefly, the tissue pellets were homogenized in TRIzolReagent using a syringe. After extraction with chloroform (an addi-tional step of phenol/chloroform extraction is added to the manufactur-er’s protocol), RNA was precipitated by isopropyl alcohol and subjectedto further purification using an RNeasy mini kit (Qiagen, Valencia, CA).

The quality and quantity of extracted total RNA samples were exam-ined by loading 5 �g of each sample on a denaturing agarose gel.

Microarray Analysis—Microarray expression analysis was per-formed according to the Affymetrix expression analysis technical man-ual (Affymetrix GeneChip® Expression Analysis Manual, P/N 700217–700222, Affymetrix, Santa Clara, CA). Double-stranded cDNA issynthesized from total RNA. To synthesize the first cDNA strand, ahigh pressure liquid chromatography-purified oligo(dT) primer was an-nealed to the RNA, and extension by reverse transcriptase was per-formed in the presence of deoxyoligonucleotides. The second strand wassynthesized using DNA polymerase I. Double-stranded cDNA was pu-rified using a modified phenol/chloroform extraction procedure (Eppen-dorf, 5 Prime 3 3 Prime, Inc., Boulder, CO), followed by ethanolprecipitation. An in vitro transcription was performed to produce biotin-labeled cRNA from the cDNA. cRNA was synthesized using T7 RNApolymerase and biotin-labeled ribonucleotides and purified with affin-ity columns (Qiagen) followed by ethanol precipitation. No amplifica-tion procedure was performed to produce the final cRNA. The amount ofproduct was quantified by spectrophotometric analysis, and the qualityof the cRNA was assessed by gel electrophoresis and a test array (Test-2chip). Once prepared, cRNA was hybridized to Affymetrix U34A ratarrays (Affymetrix), which contains more than 7000 genes and 1000expressed sequence tags (ESTs are referred to as genes in the followingtext). The hybridization mixture, containing the cRNA sample, bovineserum albumin, and herring sperm DNA, was incubated at 99°C, trans-ferred to 45 °C, and then injected into the microarray chamber (Af-fymetrix). Hybridization was performed automatically in a chamber at45 °C for 16 h while being rotated at 60 rpm. The hybridized probe arraywas then automatically washed, dried, and scanned two times at anexcitation wavelength of 488 nm.

Microarray Data Analysis—For microarray analysis, we used totalRNA prepared from 2 animals for each group (6 groups in total) asfollows: 4 “hypoxia 3 h groups” with 0, 6, 18, or 24 h of re-oxygenationand 2 “controls groups” (6-day-old rat pups that correspond to thesham-control of the hypoxia 3 h with 0 h of re-oxygenation, and 7-day-old rat pups that correspond to the sham-control of the hypoxia 3 h with24 h of re-oxygenation) (Fig. 1). Two animals were used for each exper-imental group; thus 12 arrays were used in total. The Affymetrix ratU34A array contains more than 7000 genes and 1000 ESTs.

The Affymetrix Genechip software MAS 4.0 (Affymetrix) was usedfirst to collect and process the original expression data from the 12-ratAffymetrix arrays. Each gene on the array is assessed using 16 probepairs. Each probe pair consists of an oligomer (25 bases long) that isdesigned to be perfectly complementary to a particular message (calledthe perfect match or PM) and a companion oligomer that is identical tothe PM probe except for a single base difference in a central position(called the mismatch or MM probe). The mismatch probe serves as acontrol for hybridization specificity and helps subtract nonspecific hy-bridization. After hybridization intensity data are captured, the Af-fymetrix Genechip software automatically calculates intensity valuesfor each probe cell and uses these probe cell intensities to calculate anaverage intensity for each gene (called average difference), which di-rectly correlates with mRNA abundance. The software also gives eachgene a qualitative assessment of “present” or “absent” based on a“voting scheme,” with the number of instances in which the PM signalis significantly larger than the MM signal across the whole probe set.Prior to comparing any two measurements, a scaling procedure isperformed so that all signal intensities on an array are multiplied by afactor that makes the mean PM-MM value for each array equal to apreset value of 1500. The scaling corrects for any inter-array differencesor small differences in sample concentration, labeling efficiency, orfluorescence detection and makes inter-array more reproducible. In thecase of a pairwise comparison of two array results, the patterns ofchange of the whole probe set (with consistent voting) is used to makea qualitative call (called difference call) of “Increase,” “Decrease,” “Mar-ginally increase,” “Marginally decrease,” or “No change.” The foldchange is derived by the ratio of average differences from one experi-mental array compared with a control array.

In order to eliminate possible expression changes related to thedevelopment (difference of RNA expression between 6- and 7-day-oldrat pups), all groups were compared with both controls at 6 and 7 daysold. To obtain differentially expressed genes for each condition, Af-fymetrix Genechip software was used to compare 2 experimental arraysto the 4 control arrays (P6 and P7). For example, 6 h (array 1) ofre-oxygenation was compared with controls P6 (arrays 1 and 2) andcontrols P7 (arrays 1 and 2), respectively, and then 6 h (array 2) wascompared with controls P6 (arrays 1 and 2) and controls P7 (arrays 1and 2). As a result, there were 8 comparisons for each condition. Abso-

FIG. 1. Schematic representation of the hypoxic precondition-ing protocol that was used for the neonatal rat. Animals wereexposed to 3 h of hypoxia (8% oxygen) at 6 days of age. 0 and 24 h afterthis, HIF Western blots were performed. Microarray analyses wereperformed at 0, 6, 18, and 24 h after the hypoxia.

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lute calls (present, marginal, and absent) and the average difference(RNA abundance) for each gene on each chip were then imported intoGenespring software (Silicon Genetics, Redwood City, CA) for furtheranalysis.

By combining the fold change and the present calls derived from the8 comparisons, we obtained a list for each condition. Three criteria wereused for the lists as follows. 1) The fold change for each of the 8comparisons was at least 1.5-fold. 2) There were present calls in all thehypoxic groups for increasing gene expression and present calls in bothcontrols for decreasing gene expression. 3) Raw data are greater than100 in hypoxic samples for up-regulated genes and in control samplesfor down-regulated genes. To avoid developmental gene expressionchanges, genes that were increased or decreased more than 1.5-foldbetween 6- and 7-day-old controls were eliminated from the list ofhypoxic changes.

To obtain a general view of brain genomic response to hypoxia, genesregulated at different time points following hypoxia were selected bythe Significance Analysis of Microarrays (SAM) software (27). Amongthose genes, 285 genes with relatively high expression in most samples(raw data at of least 100 in 7 of 12 samples) were subjected to hierar-chical cluster analysis with Genespring software. A standard correla-tion coefficient of 0.95 was used as the measure for significant statisti-cal similarity. The branching behavior of the tree was controlled usinga separation ration setting of 0.5 and a minimum distance setting of0.001.

Real Time Q-RT-PCR—To validate the microarray data, TaqManquantitative one-step reverse transcriptase-polymerase chain reaction(Q-RT-PCR) was used to quantitate mRNA levels for selected genes.Two primers and one probe (TaqMan probe) (Applied Biosystems, Fos-ter City, CA) were designed for each gene using PerkinElmer LifeSciences PrimerExpress software (Applied Biosystems). Q-RT-PCR wasperformed for whole brain hemisphere RNA from at least 3 animals foreach of the groups (including the same RNA samples from 2 animals

employed for microarrays analysis) and from heart, kidney, liver, andlung RNA from 3 animals submitted or not to 3 h of hypoxia with orwithout re-oxygenation. Primer and probe sequences are listed forforward primers “F,” reverse primers “R,” and TaqMan probes “T” inTable I. TaqMan probes were labeled with VIC on the 5�-nucleotide andTAMRA (6-carboxytetramethylrhodamine) on the 3�-nucleotide. Assayswere run in triplicate on the PerkinElmer Life Sciences ABI 5700instrument under default conditions (RT, 48 °C for 30 min; Ampli-TaqGold activation, 95 °C for 10 min; PCR, 40 cycles of 95 °C, 15 s and60 °C, 1 min). The Q-RT-PCR protocol was done according to the man-ufacturer’s protocol using the TaqMan Gold RT-PCR kit (Applied Bio-systems) with 50 ng of RNA for each sample. The abundance of eachgene was determined relative to the glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) standard transcript using TaqMan GAPDH RNAcontrol reagents kits (Applied Biosystems). The abundance of each genewas also determined relative to the standard transcript 18 S usingTaqMan ribosomal control reagents kits (Applied Biosystems). The dataobtained with 18S are not shown because the results were identical tothose obtained with GAPDH (data not shown). To verify the presenceand the predicted size of amplified fragments, PCR products wereseparated by electrophoresis and visualized in 3% agarose gels withethidium bromide.

Preparation of Total and Nuclear Protein Extracts—Total and nu-clear protein extracts were prepared from whole brain hemispheretissue lysates after 3 h of hypoxia or 3 h of hypoxia followed by 24 h ofre-oxygenation and controls (Fig. 1) as described previously (16).

Western Blotting—Proteins (150 �g for total extracts and 75 �g fornuclear extracts) were heated at 100 °C for 3 min in the 2� loadingbuffer (EC-886, National Diagnostics, Atlanta, GA) and separated on8% SDS-PAGE gels for 1 h at 150 V. The electrophoresis running buffercontained 25 mM Tris base, 192 mM glycine, and 0.1% SDS. Proteinswere transferred onto a nitrocellulose membrane (Optitran BA-S 85,Schleicher & Schuell) in blotting buffer (25 mM Tris base, 192 mM

TABLE IList of primers used for real time RT-PCR

Gene name ACa Primers Position/size

bp

Adrenomedullin D15069 F, 5�-CAGGACAAGCAGAGCACGTC-3� 361 82T, 5�-CAAGCCAGCACTCAGAGCACAGCC-3� 391R, 5�-TCTGGCGGTAGCGTTTGAC-3� 442

CAR-1 U76714 F, 5�-ACCCGCTTCCATAAGGCTTTA-3� 52 142T, 5�-CAGCTTTGCTGTTCTTTGCCTTAGTTGTCC-3� 139R, 5�-CAGCCTTGTGCCGAAAGAC-3� 193

Casein kinase-1� U77582 F, 5�-CGGAAGATCGGATCTGGTTC-3� 61 99T, 5�-TCTAGCGATCAACATCACCAATGGCG-3� 96R, 5�-CCTGGCCTTCTGGGATTCTAG-3� 159

CELF M65149 F, 5�-CACGACCCCTGCCATGTATG-3� 142 118T, 5�-CTTCAGCGCCTACATTGATTCCATGGC-3� 181R, 5�-GAAGAGGTCGGCGAAGATCTC-3� 259

EPO D10763 F, 5�-GAATTGATGTCGCCTCCAGA-3� 449 91T, 5�-CCAAGCCGCTCCACTCCGAACA-3� 475R, 5�-TGGAGTAGACCCGGAAGAGCT-3� 539

EST 188825 AA799328 F, 5�-ATTGTCCCAGGTTGTGTCCAA-3� 481 102T, 5�-TCCTCCTAGCACTCTCCATTGTGAATCGTATC-3� 504R, 5�-GGACTTGAGTTGTGGAGAGCAA-3� 582

GLUT-1 S68135 F, 5�-GGTGTGCAGCAGCCTGTGTA-3� 1112 78T, 5�-CCATCGGCTCGGGTATCGTCAACAC-3� 1137R, 5�-GACGAACAGCGACACCACAGT-3� 1189

12-Lipoxygenase L06040 F, 5�-GATGGGTGTCTACCGCATCC-3� 19 98T, 5�-CTCCAAGTACGCGGGCTCCAACAAC-3� 55R, 5�-CCTCTCCATGCTGTCCAACC-3� 116

Metallothionein-1 M11794 F, 5�-CAAATGCACCTCCTGCAAGA-3� 142 120T, 5�-CTGCTCCAAATGTGCCCAGGGCT-3� 190R, 5�-TCACTTCAGGCACAGCACGT-3� 261

MKP-1 S81478 F, 5�-GCAGTGCTTACCATGCTTCC-3� 665 78T, 5�-ATATGCTCGACGCCTTGGGTATCACTGC-3� 692R, 5�-AATTGGCCGAGACGTTGATC-3� 742

Prolyl 4-hydroxylase � X78949 F, 5�-CTGTTCTGCCGCTACCATGA-3� 991 133T, 5�-CAAGCCTCGCATCATTCGTTTCCATG-3� 1068R, 5�-CGATCTCAATCTCGGCATCTG-3� 1123

t-PA M23697 F, 5�-CGTTGCCTGACCAGGGAATA-3� 81 120T, 5�-CTACAGAGCGACCTGCAGAGATGAACAGACTC-3� 130R, 5�-TGGGACGTAGCCATGACTGA-3� 200

VEGF M32167 F, 5�-GCAATGATGAAGCCCTGGAG-3� 261 78T, 5�-CACGTCGGAGAGCAACGTCACTATGC-3� 289R, 5�-GGTGAGGTTTGATCCGCATG-3� 338

a AC, GenBank™ accession number.

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glycine, and 20% methanol (v/v)) for 2 h at 300 mA. Membranes werestained with Ponceau S to verify equal protein loading and transfer.Membranes are blocked with 5% nonfat milk (Bio-Rad) in TBST (10 mM

Tris-HCl, pH 8.0, 200 mM NaCl, 0.05% Tween 20) for 2 h at roomtemperature and then incubated overnight at 4 °C with one of thefollowing antibodies diluted in the blocking solution: mouse monoclonalantibodies for HIF-1� (2 �g/ml, OSA-601; StressGen Biotechnologies,Victoria, British Columbia, Canada), HIF-1� (1 �g/ml, NB 100–124;Novas Biologicals, Littleton, CO), and rabbit polyclonal antibody forHIF-2� (2.2 �g/ml, NB 100–122; Novas Biologicals Inc.). After washingwith TBST (4 times, 15 min each), the blots were incubated 1 h at roomtemperature with peroxidase-coupled goat anti-rabbit IgG (1:15,000;Santa Cruz Biotechnologies, Santa Cruz, CA) or peroxidase-coupledgoat anti-mouse IgG (1:5,000; Santa Cruz Biotechnologies) diluted inblocking solution and then washed again in TBST. The blots weredeveloped using the chemiluminescence reagents (RPN 2106, Amer-sham Biosciences). The same blot was stripped (30 min at 50 °C in 62.5mM Tris base, 2% SDS, 100 mM �-mercaptoethanol) and re-stained witha monoclonal �-actin antibody (5441, Sigma) as an internal control.

RESULTS

Effect of Hypoxic Preconditioning on Brain HIF-1 and HIF-2Expression—HIF-1 is a heterodimer made of two protein sub-units, HIF-1� and HIF-1� (28). Whereas HIF-1� is constitu-tively expressed, HIF-1� expression is tightly regulated bycellular oxygen concentration (29). Thus HIF-1� determinesHIF-1 DNA binding activity and transcriptional activity duringhypoxia. As 3 h of hypoxia 8% O2 is known to induce expressionof HIF-1� and its target genes in neonatal brain (13, 14), weexamined the expression of this transcriptional factor in thecurrent study. As shown in Fig. 2, exposure of neonatal P6 ratsto hypoxia (8% O2, 3 h) induced an increase in HIF-1� ex-pression in total and nuclear extracts of the whole hemi-sphere (Fig. 2, 0 hr, �), when compared with control animals(Fig. 2, 0 hr, �). Twenty four hours following exposure tohypoxia, HIF-1� expression is reduced to control levels (Fig.2, 24 hr, �). Although no significant changes in HIF-1�expression were observed in total extracts, a slight increasewas observed in nuclear extracts after 3 h of hypoxia (Fig. 2,0 hr, �), suggesting that hypoxia did not change the totalcontent of HIF-1� but increased the translocation of HIF-1�into the nucleus (Fig. 2).

Recently, a novel hypoxia-inducible factor HIF-2� (alsoknown as EPAS1, MOP2, HRF, and HLF) was identified andalso binds as a heterodimer with HIF-1� to the hypoxia-re-sponse element. HIF-2� shares 48% identity with the HIF-1�protein (30). The protein levels of HIF-2� also increased follow-ing hypoxia in various cell lines (30). However, the influence ofhypoxia on HIF-2� expression in the brain is still completely

unknown. As shown in Fig. 2, one band (molecular massaround 120 kDa) appeared for HIF-2� in both normoxic andhypoxic brain total protein extracts. The results show thatHIF-2� is constitutively expressed in the brain and thatHIF-2� expression is higher than that of HIF-1�, but no sig-nificant increase in HIF-2� expression was observed after hy-poxia. These findings indicate that HIF-1� is more inducible byhypoxia than HIF-2� in the neonatal rat brain and suggest thatHIF-1� could play a role in mediating the preconditioningeffects of hypoxia.

Brain Genomic Response and Its Time Course FollowingHypoxic Preconditioning—High density oligonucleotide mi-croarrays (Affymetrix, U34A for rat) were used to study thetemporal profile of the brain genomic response in neonatal rat(6–7 days old) after 3 h of hypoxia (8% O2) followed by 0, 6, 18,or 24 h of re-oxygenation. For this purpose, we used 2 animals/time point.

In order to avoid gene expression changes related to devel-opment (difference of RNA expression between 6- and 7 day-oldrat pups), all groups were compared with both 6- and 7-day-oldcontrols, and genes that were increased or decreased more than1.5-fold between 6- and 7-day-old controls were eliminatedfrom the list of genes showing hypoxic changes. The develop-mentally regulated genes are presented in Table II. Amongthese genes, 14 are up-regulated and 31 are down-regulated.Its interesting that genes known to be implicated in angiogen-esis or neurogenesis (for example, VEGF-B, HES-5, andFGFR-2) (31–33), decreased with age in accordance with thefact that these processes may become less important with in-creased age.

For the analysis of up-regulated genes or down-regulatedgenes, there were 8 comparisons for each condition. Absolutecalls (present, marginal, and absent) and the average differ-ence (RNA abundance) for each gene on each chip obtainedfrom Affymetrix software were then imported into Genespringsoftware for further analysis. A general view of genes regulatedby hypoxia/re-oxygenation is presented in Figs. 3 and 4. Fig. 3shows genes grouped according to their temporal expressionpattern using a hierarchical clustering algorithm. Fig. 4 sum-marizes the number of genes up-regulated or down-regulatedusing the stringent criteria described under “ExperimentalProcedures.” Some of these genes have only been identified aspartially expressed sequence tags that encode for unknownproteins.

Up-regulated Genes—Hypoxia for 3 h increased the expres-sion of 18 genes, which then progressively decreased as a func-tion of re-oxygenation times (15 after 6 h, 4 after 18 h, or 5 after24 h of re-oxygenation) (Fig. 4). This strong up-regulation ofgenes during hypoxia rather than re-oxygenation is also shownwith the cluster analysis in Fig. 3. Interestingly, only 1 genewhich is an EST (GenBankTM accession number AA799328)was up-regulated at all the time points analyzed (0, 6, 18, and24 h). Because of the unique prolonged expression of this EST,real time Q-RT-PCR was performed for this gene in the brain ofadult rats submitted to the same degree of hypoxia (8% O2, 3 h).The results revealed a remarkable up-regulation of this EST inthe adult brain as well (35.0 � 4.6-fold, data not shown).

In addition to known hypoxic inducible genes such as MAPkinase phosphatase-1 (MKP-1) and metallothionein-1 (MT-1)(21, 23, 34) including those under HIF-1 control (VEGF, glucosetransporter-1 (GLUT-1), adrenomedullin, prolyl 4-hydroxylase�) (35–38), this study revealed new genes induced by hypoxiaand/or re-oxygenation in brain (Tables III and IV). The list ofgenes includes transcriptional factors, signal transduction mol-ecules, receptors, transporters, channels, proteases, genes im-plicated in apoptosis, detoxification, inflammation, vasodilata-

FIG. 2. Western blot analysis of HIF-1�, HIF-1�, HIF-2�, and�-actin in neonatal rat brain after 3 h of hypoxia (H) with nore-oxygenation (R, 0 hr) or with 24 h of re-oxygenation (R, 24 hr).

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tion, collagen synthesis, and membrane trafficking. Amongthese genes, it is worthwhile to point out that the EST (Gen-BankTM accession number AA799328), which is increased at allthe time points, also corresponds to the gene maximally in-creased by hypoxia/re-oxygenation (around 10-fold increase forall the time points) (Table III). To validate these microarraydata, real time Q-RT-PCR was performed on brain RNA pre-pared from 3 animals/time point for several candidates. Weconfirmed 10 up-regulated genes by Q-RT-PCR, and for nearlyall of the genes, the fold changes obtained with Q-RT-PCR werevery similar to those obtained by microarray analysis (Fig. 5).We focused our analysis on 4 HIF-1 target genes already known(VEGF, GLUT-1, adrenomedullin, and prolyl 4-hydroxylase �),the hypoxia-responsive genes MT-1 and MKP-1, and othernovel hypoxia-responsive genes CELF (C/EBP�), 12-lipoxygen-ase, tissue-type plasminogen activator (t-PA), and ESTAA799328. As shown in Fig. 5A, the time course of expressionafter hypoxia/re-oxygenation of the four HIF-1 target geneswas very similar; an increase of expression occurred duringhypoxia but then returned to control levels with re-oxygen-ation. A similar pattern of expression was also observed for

MKP-1, a phosphatase known to be induced by hypoxia (22, 23),but not known to be under HIF-1 control, and for 3 novelhypoxia-responsive genes (CELF, 12-lipoxygenase, and t-PA)(Fig. 5B). However, a different time course of induction wasobserved for MT-1 and EST AA799328 for which the increase ofexpression by hypoxia is maintained until 6 or 24 h of re-oxygenation, respectively (Fig. 5C). We were surprised not tofind an increase of EPO expression by hypoxia in the microar-ray data, because it has been widely described as an HIF-1target gene and is induced by hypoxia in many organs includ-ing the brain (26, 39–41). Indeed, the real time Q-RT-PCRresults showed an increase of EPO mRNA expression after 3 hof hypoxia (Fig. 5D). The raw data of EPO expression in themicroarray results were, at all the time points tested, equal tozero, suggesting that the probes designed for this gene were nothybridizing to the EPO cRNA.

Down-regulated Genes—Hypoxia for 3 h decreased the ex-pression of 5 genes (Table V). However, the maximal number ofgenes down-regulated by hypoxia occurred at 6–24 h followingre-oxygenation (Table V and Figs. 3 and 4). Interestingly, only1 gene, which is an EST (GenBankTM accession number

TABLE IIGenes regulated by development in neonatal rat brain

ACa Gene name Change

fold

Up-regulatedAA875028 EST b

AI639301 EST 4.3X60651 Syndecan 3.1D17469 Thyrotropin-releasing hormone receptor, TRH receptor 2.9AI176021 EST 219597 2.9AB011666 Utrophin 2.6U32681 Ebnerin 2.3K00512 MBP, myelin basic protein 2.3AA925246 EST 2.3S68245 Carbonic anhydrase IV 2.2AI638946 EST 2.0M92433 NGFI-C, NGF-induced clone C, transcriptional regulator 1.8X57565 UDP-glucuronosyltransferase 2A1 precursor 1.7AA891614 EST 195417 1.6

Down-regulatedD29766 Crk-associated substrate � p130, complex with v-Crk, v-Src 12.3M93257 Cathechol O-methyltransferase 7.2AA892895 EST 196698 4.5AA891839 EST 195642 3.9AJ006295 AF-9, transcriptional activator 3.8AA799410 EST 188907 3.5AA925752 EST 3.4Z14030 TRAP-complex � subunit � SSR � subunit 3.3L10336 mss4 � guanine nucleotide-releasing protein 3.2AA900476 EST 3.1M77850 6-pyruvoyl-tetrahydropterin synthase 2.9AF051527 Class A calcium channel variant riA-II 2.5M27466 COX-VIc � liver cytochrome oxidase subunit VIc 2.1AI639441 EST 2.1AF100421 p80 � LYRIC, novel gene with elevated expression in tumors 2.0D12516 HES-5 � homologue of hairy enhancer of split 2.0X96394 Multidrug resistance protein � mrp gene 2.0AF041082 Transmembrane receptor Robo1, guidance receptor 2.0X70871 Cyclin G 1.9L20468 Cerebroglycan 1.9J05030 SCAD � short chain acyl-coenzyme A dehydrogenase 1.9U37138 Steroid sulfatase 1.9AA892294 EST 196097 1.9AA875615 EST 1.9D00512 Mitochondrial acetoacetyl-CoA thiolase precursor 1.8AF022952 Vascular endothelial growth factor B 1.7U18314 LAP-2 � lamina-associated polypeptide-2 1.7AA892942 EST 196745 1.7AA933181 EST PIM-2MF 1.7L19112 Heparin-binding fibroblast growth factor receptor 2, FGFR2 1.6AA893327 EST 197130 1.6

a AC, GenBank™ accession number.b Undefinable.

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AA799671), is down-regulated at all the time points analyzed(Table V). The list of down-regulated genes included genesimplicated in signal transduction, cell adhesion, DNA synthe-sis, cell cycle, structure, membrane trafficking, and metabo-lism. Among the down-regulated genes, we confirmed by realtime Q-RT-PCR that 3 h of hypoxia decreased the expression ofcell adhesion regulator-1 (CAR-1) mRNA, a gene encoding for acell adhesion regulator protein (Fig. 5E).

Expression of Hypoxia-regulated Genes in Neurons and As-trocytes—In order to elucidate the possible cellular source ofthe genes regulated by hypoxia, we studied the expression ofthese same genes in cultured neurons and astrocytes using realtime Q-RT-PCR (VEGF, GLUT-1, prolyl 4-hydroxylase �, ad-renomedullin, MKP-1, 12-lipoxygenase, t-PA, CELF, MT-1,EST AA799328, EPO, and CAR-1). RNA was obtained from ratprimary astrocytes or neuron cultures exposed to hypoxia orambient oxygen for 3 h. Fig. 5 shows that the basal expressionof the genes is not the same in astrocytes and neurons. Genessuch as GLUT-1, adrenomedullin, t-PA, CELF, MT-1, ESTAA799328, and CAR-1 were expressed more in astrocytes com-pared with neurons. In comparison, VEGF, EPO, MKP-1, and12-lipoxygenase were expressed more in neurons comparedwith astrocytes. Prolyl 4-hydroxylase � showed a similar levelof expression in both astrocytes and neurons. It was also nota-ble that some genes that are expressed more in neurons inbasal conditions were induced more in astrocytes followinghypoxia. This is the case for VEGF, EPO, and 12-lipoxygenase.Conversely, some genes that are expressed more in astrocytesin basal conditions are induced more in neurons in hypoxicconditions. This is the case for adrenomedullin. The ESTAA799328 showed a similar level of expression in both astro-cytes and neurons under hypoxic conditions. Some genes, forexample GLUT-1, MT-1, CELF, MKP-1, and t-PA, did not showany regulation by hypoxia in either astrocytes or neurons,suggesting that other cells present in the brain (endothelialcells, microglia, or oligodendrocytes) are responsible for theup-regulation of these genes in hypoxic brain. For CAR-1, theQ-RT-PCR results suggest that neurons are responsible for thedown-regulation of this gene observed in vivo in the hypoxicbrain. Of note, at least in vitro, hypoxia seems to induce CAR-1in astrocytes, the opposite of the neuronal response.

Comparison of the Genomic Response Following Hypoxic Pre-conditioning in Various Organs—To determine whether thehypoxia-induced gene expression changes observed in neonatalrat brain occur in other organs, we compared the brain expres-sion of the genes confirmed by Q-RT-PCR to other organs(heart, kidney, liver, and lung) after 3 h of hypoxia. The results,presented in Fig. 6, revealed 5 major groups: (a) genes up-regulated at a similar level in all organs (12-lipoxygenase, t-PA,MKP-1, and CELF); (b) genes more regulated in brain (ad-renomedullin, MT-1, VEGF, and CAR-1); (c) genes up-regu-lated at a similar level in brain and heart (EST GenBankTM

accession number AA799328); (d) genes up-regulated at a sim-ilar level in all organs except lung (prolyl 4-hydroxylase �); (e)genes much more up-regulated in liver or kidney (GLUT-1 andEPO).

DISCUSSION

The results confirm that hypoxia induces HIF-1� and itstarget genes in newborn rat brain (13). Although HIF-2�, anHIF-1� homologue, is induced by cerebral ischemia (42) and byhypoxia in some cells (30), it was unknown whether hypoxiapreconditioning would induce HIF-2� in the brain. The datashow that HIF-2� is expressed in normoxia but is not inducedby 3 h of hypoxia in neonatal rat brain. This is consistent withprevious studies (30) showing higher expression of HIF-2� thanHIF-1� under normoxia and variable hypoxic induction ofHIF-2� in different cell types. Whereas HIF-1� is expressedand regulated by hypoxia in most cells, HIF-2� is expressedmainly in endothelial cells (30). HIF-2� appears to modulate

FIG. 3. Cluster analysis demonstrating genes grouped ac-cording to their temporal expression profiles in neonatal ratbrain after 3 h of hypoxia (hypoxia 3 h, 8% O2) followed bydifferent periods of re-oxygenation (R) (0, 6, 18, or 24 h) com-pared with 6-day-old controls (Control 6d) and 7-day-old con-trols (Control 7d).

FIG. 4. Number of genes up-regulated and down-regulated asdetermined by the microarray analyses in the neonatal ratbrain after 3 h of hypoxia (8% O2) followed by different periodsof re-oxygenation (R) (0, 6, 18, or 24 h).

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gene expression in endothelial cells during normoxia and lesssevere hypoxia (30). Therefore, HIF-1�- but not HIF-2�-medi-ated gene expression may be involved in brain hypoxia-inducedtolerance (13, 14, 43).

In addition to known HIF-1 target genes (VEGF, GLUT-1,prolyl 4-hydroxylase �, and adrenomedullin) (35–38) and otherknown hypoxic inducible genes (MT-1 and MKP-1) (21, 23, 34),this study revealed new genes regulated by hypoxia/re-oxygen-ation in brain. For example, hypoxic regulation of CELF, 12-lipoxygenase, t-PA, CAR-1, and many other transcripts shownin the tables has not been reported. Moreover, an EST (Gen-BankTM accession number AA799328) was induced to a greater

extent and for a longer time than any of the known genesexamined. Some genes (MKP-1, 12-lipoxygenase, CELF, t-PA)have the same profile of expression in response to hypoxia/re-oxygenation as those known to be under HIF-1 control. Thissuggests that these genes may be new HIF-1 target genes.Indeed, cobalt chloride and desferrioxamine, two agents acti-vating HIF-1 (17), mimic the hypoxic increase of MKP-1 gene inPC12 cells (23).

The possible contribution of these hypoxia-inducible genes inhypoxia-induced tolerance is supported by several previousstudies. VEGF (18) and EPO (11, 19, 20) protect the brainagainst ischemia/hypoxia. Astrocytes are an important sourceof endogenous VEGF and EPO during hypoxia (26, 39–41).GLUT-1, another HIF-1 target gene, may also be involved inhypoxia-induced ischemic tolerance (14). Although GLUT-1mRNA is up-regulated by hypoxia in neurons and astrocytes(44), our results suggest that other cells, like endothelial cells,may be involved. Indeed, GLUT-1 is highly expressed in endo-thelial cells at the blood-brain barrier (45), and hypoxia in-creases this expression (46). Prolyl 4-hydroxylase �, an active

TABLE IIIGenes up-regulated by 3 h of hypoxia (8% O2) followed by re-oxygenation (R) in neonatal rat brain

ACa Gene nameIncrease

0-h R 6-h R 18-h R 24-h R

-fold

Transcriptional factorM65149 CELF � C/EBP � transcriptional factor 3.9

Signal transduction system, receptorS81478 MKP-1 � MAP kinase phosphatase-1 2.7AF008650 SLC1 � somatostatin receptor-like protein 8.1L35921 G�8 � GTP-binding protein � subunit 8 2.0X52952 c-mos � protein kinase, proto-oncogene 5.2

ApoptosisAF065431 BOD � Bcl-2 related ovarian death agonist 2.5

DetoxificationM11794 MT-1, MT-2 � metallothionein-1 and -2 2.6 3.4

ProteaseM23697 t-PA 1.8

InflammationL06040 12-Lipoxygenase 2.0X71127 Clq � � complement protein 1q 1.7

Growth factorM32167 VEGF 2.8

Transporter, channelS68135 GLUT-1 � glucose transporter-1 2.5AF083341 SLON-1 � calcium-activated potassium channel 7.6X12589 Voltage-dependent potassium channel 2.6U80915 EAAT4 � Na�-dependent glutamate transporter 14.6

VasodilatationD15069 Adrenomedullin 2.1

Collagen synthesisX78949 Prolyl 4-hydroxylase � 1.8

Structural protein, membrane traffickingAF073839 Bithoraxoid-like protein � dynein light chain protein 2.0Z46614 Caveolin � caveolae-associated integral membrane protein b b

AA860030 �-Tubulin class I 2.7 2.3 2.8Others

X59993 Putative zinc finger protein 1.8X62951 pBUS19 with repetitive element 2.5D85035 DPD � dihydropyrimidine dehydrogenase 3.4

UnknownAA891949 EST 195752 1.7AI014135 EST 207690 3.0AI229924 EST 226619 2.7 2.0AA799467 EST 188964 1.6AI177054 EST 220661 1.8AA800750 EST 190247 2.3AA998683 EST 2.0AA946503 EST 202002 10.0AA891740 EST 195543 1.9AA799466 EST 188963 2.1AA799328 EST 188825 10.1 9.9 10.4 10.4

a AC, GenBank™ accession number.b Undefinable.

TABLE IVGenes known to be up-regulated by hypoxia via HIF-1 revealed by

microarray analyses

VEGFGLUT-1, glucose transporter-1AdrenomedullinProlyl 4-hydroxylase �

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subunit that catalyzes oxygen-dependent hydroxylation of pro-line residues in procollagen, is also an HIF-1 target gene (38).The loss of basal lamina, including type IV collagen, compro-mises microvascular integrity in cerebral ischemia that likelycontributes to edema and hemorrhage (47). Hypoxia precondi-tioning may protect in part by increasing prolyl 4-hydroxylase� expression and therefore decreasing loss of vascular integrityinduced by ischemia. Adrenomedullin, a vasodilatory peptide,is also transcriptionally regulated by HIF-1 (37) and reduces

ischemic brain injury in rats (48). Adrenomedullin appears tobe induced by hypoxia in neurons. MKP-1, a MAP kinase phos-phatase and another putative HIF-1 target (23), improves sur-vival of PC12 and other cells by antagonizing c-Jun N-terminalkinase (JNK) activation (49, 50). JNK levels increase afterischemia. This can be abrogated by ischemia preconditioning,which also protected against ischemic injury (51). Thus, hy-poxic or ischemic preconditioning may reduce ischemic JNKactivation and protect the brain through a common MKP-1

FIG. 5. The left panels show real time Q-RT-PCR analysis (black bars) of RNA isolated from neonatal rat brain after 3 h of hypoxia(8% O2) followed by different periods of re-oxygenation (R) (0, 6, 18, or 24 h) compared with microarray analyses (gray bars). Theright panels show real time Q-RT-PCR analysis of RNA isolated from primary cultures of astrocytes (Astro) or primary cultures of neurons (Neuron)that were exposed to control ambient air (white bars, or C) or were exposed to 3 h of hypoxia (H, black bars). White bars are from normoxic controlsin both panels. Data represent fold change versus controls (normoxic brain or normoxic cells) or fold change in neurons versus astrocytes at thebasal expression for cerebral cells. The RT-PCR values were normalized to GAPDH (mean � S.E., n � 3 for each conditions). A, known HIF-1 targetgenes. B, genes having a similar pattern of expression after hypoxia as the HIF-1 target genes. C, genes up-regulated by both hypoxia andre-oxygenation. D, EPO, a gene not detected by the microarray studies but shown to be up-regulated by hypoxia by Q-RT-PCR. E, CAR-1, adown-regulated gene. C, control; H, hypoxia; R, re-oxygenation.

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pathway. Hypoxic induction of MKP-1 expression does not ap-pear to be localized to neurons or astrocytes. This is the firstdemonstration that hypoxia induces 12-lipoxygenase in brainand that this occurs in astrocytes. There is no report on the roleof 12-lipoxygenase in cerebral ischemia. However, ischemicpreconditioning-induced cardio-protection is impaired in 12-lipoxygenase-deficient mice and by 12-lipoxygenase inhibitors(52, 53). These data support the possibility that 12-lipoxygen-ase could also be involved in hypoxia-induced ischemic toler-ance. The transcription factor CELF (also named C/EBP�,CRP3, and NF-IL6�) is also induced by hypoxia, likely in cellsother than neurons or astrocytes. CELF participates in thetranscriptional regulation of many molecules that have beenshown to be protective and/or detrimental in ischemia such ascyclooxygenase-2 (54), nerve growth factor (55), complementcomponent C3 (56), and peroxisome proliferator-activated re-ceptor �2 (57). Cyclooxygenase-2 inhibitors decrease injury(58), whereas cyclooxygenase-2 mediates the protective effectsof cardiac ischemic preconditioning (59). Thus, depending on

the cell type expressing CELF and CELF target genes, CELFcould either help or harm. A similar hypothesis could be pro-posed for t-PA because hypoxia increased its expression inwhole brain, and t-PA can protect or be detrimental in cerebralischemia (60). Hypoxia-preconditioning has been shown to in-duce both MT-1 and MT-2 (61), and both molecules protectagainst cerebral ischemia (62, 63).

Hypoxia not only increased but also decreased gene expres-sion. For example, the cell adhesion regulator CAR-1 decreasedfollowing hypoxia. In addition, hypoxia decreased its expres-sion in neurons but increased it in astrocytes. The differenceobserved in CAR-1 expression from different cell types suggeststhat the cellular mechanisms by which CAR-1 expression isregulated by hypoxia are different among various cell types inbrain. This may be true for other genes as well.

The current results along with those in literature stronglysupport an important role of VEGF, EPO, GLUT-1, prolyl 4-hy-droxylase �, adrenomedullin, MKP-1, 12-lipoxygenase, t-PA,CELF, MT-1, and CAR-1 in brain ischemic tolerance induced

TABLE VGenes down-regulated by 3 h of hypoxia (8% O2) followed by re-oxygenation (R) in neonatal rat brain

ACa Gene nameDecrease

0-h R 6-h R 18-h R 24-h R

-foldSignal transduction system, receptor

AB001576 NTAK�2-1p � activator for ErbB kinases 1.8M83681 RAB16 � ras-like GTP-binding protein 1.9AF091563 Isolate QIL-LD1 olfactory receptor 2.5L19699 Ral B � GTP-binding protein 2.1AF085693 GIT-1 � GTPase-activating protein 6.6M96159 Adenylyl cyclase type V 67U40999 RRG4 � retinal protein, photoreceptor 3.6

Cell adhesionU76714 CAR-1 � cell adhesion regulator-1 2.4L21711 Galectin-5 � �-galactoside binding lectin 2.2

DNA synthesis, cell cycleM13100 LINE 3 � L1Rn, long interspersed repetitive DNA sequence 2.0X05472 ORF2 � 2.4-kb repeat DNA right terminal region 2.6X64589 Cyclin B 23.3U15149 Putative DNA-binding protein —b —b

Structural protein, membrane traffickingAA851887 EST194656 � Dynamin 2 � GTPase, role in endocytosis 3.4S61868 Ryudocan � heparan sulfate proteoglycan core protein 2.3 2.7AI105348 EST214637 � Cofilin-1 � actin-binding protein 1.9 2.0 2.8

MetabolismAF044574 Putative peroxisomal 2,4-dienoyl-CoA reductase 2.0D17310 Steroid 3-�-dehydrogenase 2.0 2.0

OthersAA875033 Fibulin5 � EVEC � vascular EGF-repeat-containing protein 2.2D49955 BST-1 � bone marrow stromal cell antigen-1 2.2X95850 Novel gene expressed in circadian manner 2.0AB014722 rSALT-1(806) no. human SART-1, tumor rejection antigen 2.0U17133 ZnT-1 � zinc transporter 2.7

UnknownAA892284 EST 196087 2.5AA874897 EST 1.7AA874791 EST 2.2AI639187 EST 1.9AA875042 EST 1.8AA875577 EST 2.2AA891664 EST 195467 4.2AA799438 EST 188935 —b

AA800318 EST 189815 2.1AA894030 EST 197833 6.7AI639490 EST 2.3AA893869 EST 197672 4.6AA799396 EST 188893 5.3AI179027 EST 222709 7.0 5.1AI104012 EST 213301 3.6AI171462 EST 217424 2.0AA799721 EST 189218 3.2AA799475 EST 188972 2.8AA799671 EST 189168 7.2 4.4 3.9 3.2

a AC, GenBank™ accession number.b Undefinable.

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by hypoxia in neonatal rats. Moreover, the data showed thathypoxia increased expression of glutamate transporter excita-tory amino acid transporter 4 (EAAT4) in addition to that ofexcitatory amino acid carrier 1 (EAAC1), glutamate transport-er-1 (GLT-1), EAAT2, and EAAT3 as published previously (64,65). Thus, hypoxic preconditioning may counteract the de-crease of glutamate transporters induced by hypoxia-ischemia(66) and protect cells from high extracellular concentrations ofglutamate by increasing its uptake. Furthermore, the presentstudy shows that, in addition to ATP-dependent potassiumchannel known to participate in ischemic tolerance (67), calci-um- and voltage-dependent potassium channels may be impor-tant. In addition, many of the genes not discussed here andmany more genes not surveyed by the current microarrays arealso likely to be involved in conferring hypoxia-induced ische-mic tolerance.

Most of the genes that appear to be regulated by hypoxia in

brain may be of importance in many other organs and mayrepresent common mechanisms of hypoxia-induced tolerance inall organs. However, some genes may play a specific role in thehypoxic brain. For example, adrenomedullin was induced byhypoxia in brain but not in heart, kidney, liver, and lung inneonatal rats, pointing to a unique but unknown hypoxia-induced role for brain adrenomedullin in the neonate.

The limitations of the present study should be mentioned.First, it is the protein expression in cells that would mediatehypoxia-induced tolerance, and increased transcription doesnot always imply increased translation. Technical issues arelimiting, because the entire rat genome was not surveyed here.In addition, as the EPO data show, some of the microarray datamay be incomplete or wrong. The experimental paradigm usedhas limitations. For example, Egr-1 (early growth-responsegene) is induced by hypoxia and therefore represents anotherimportant oxygen-regulated transcription factor independentof HIF-1 (68). However, our results only showed a marginalregulation of this gene by hypoxia. Because induction of Egr-1mRNA reaches a maximum at 30 min of hypoxia and thendecreases (69), and that the shortest time of hypoxia used inthe present study was 3 h, Egr-1 could mediate the braingenomic response to hypoxia. This could also be true for anumber of other genes like c-fos and other immediate earlygenes. This could mean that many of the hypoxia-induciblegenes observed here are immediate early gene targets requir-ing the co-activation by HIF-1, Egr-1, and/or other immediateearly transcription factors.

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FIG. 6. Real time Q-RT-PCR analysis of RNA isolated fromneonatal rat brain, heart, kidney, liver, and lung after 3 h ofhypoxia (8% O2). Black bars represent hypoxic samples, and whitebars represent control (normoxic) samples. Data represent fold changeversus controls, and values were normalized to GAPDH (mean � S.E.,n � 3 for each condition). C, control; H, hypoxia. A, genes up-regulatedto a similar level in all organs; B, genes that were highly regulatedin brain; C, genes up-regulated at a similar level in brain and heart;D, genes up-regulated to a similar level in all organs except the lung;E, genes highly regulated in the liver or kidney.

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Brain Genomic Response to Hypoxia39738

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Myriam Bernaudin, Yang Tang, Melinda Reilly, Edwige Petit and Frank R. SharpHYPOXIA-INDUCED ISCHEMIC TOLERANCE

Rat: IDENTIFICATION OF GENES THAT MIGHT CONTRIBUTE TO Brain Genomic Response following Hypoxia and Re-oxygenation in the Neonatal

doi: 10.1074/jbc.M204619200 originally published online July 26, 20022002, 277:39728-39738.J. Biol. Chem. 

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