Genomics of the Periinfarction Cortex After Focal Cerebral Ischemia

Genomics of the Periinfarction Cortex After FocalCerebral Ischemia

*Aigang Lu, *Yang Tang, *Ruiqiong Ran, *Joseph F. Clark, †Bruce J. Aronow, and*Frank R. Sharp

*Departments of Neurology, Pediatrics and the Neurosciences Program, University of Cincinnati, and †Division of MolecularDevelopmental Biology and Informatics, Children’s Hospital Research Foundation, Cincinnati, Ohio, U.S.A.

Summary: Understanding transcriptional changes in brain af-ter ischemia may provide therapeutic targets for treating strokeand promoting recovery. To study these changes on a genomicscale, oligonucleotide arrays were used to assess RNA samplesfrom periinfarction cortex of adult Sprague-Dawley rats 24 hafter permanent middle cerebral artery occlusions. Of the 328regulated transcripts in ischemia compared with sham-operatedanimals, 264 were upregulated, 64 were downregulated, and163 (49.7%) had not been reported in stroke. Of the functionalgroups modulated by ischemia: G-protein–related genes werethe least reported; and cytokines, chemokines, stress proteins,and cell adhesion and immune molecules were the most highlyexpressed. Quantitative reverse transcription polymerase chainreaction of 20 selected genes at 2, 4, and 24 h after ischemiashowed early upregulated genes (2 h) including Narp, Rad,

G33A, HYCP2, Pim-3, Cpg21, JAK2, CELF, Tenascin, andDAF. Late upregulated genes (24 h) included Cathepsin C,Cip-26, Cystatin B, PHAS-I, TBFII, Spr, PRG1, and LPS-binding protein. Glycerol 3-phosphate dehydrogenase, which isinvolved in mitochondrial reoxidation of glycolysis derivedNADH, was regulated more than 60-fold. Plasticity-relatedtranscripts were regulated, including Narp, agrin, and Cpg21.A newly reported lung pathway was also regulated in ischemicbrain: C/EBP induction of Egr-1 (NGFI-A) with downstreaminduction of PAI-1, VEGF, ICAM, IL1, and MIP1. Genes regu-lated acutely after stroke may modulate cell survival and death;also, late regulated genes may be related to tissue repair andfunctional recovery. Key Words: DNA microarrays—Functional genomics—Cerebral ischemia—Gene expression—Glycerol 3-phosphate dehydrogenase—egr-1—Cathepsins.

Recent clinical trials of neuroprotective drugs for theacute treatment of stroke have failed. These includedtrials of sodium and calcium channel antagonists, N-methyl-D-aspartate receptor antagonists, �-amino-butyricacid agonists, free radical scavengers, nitric oxide path-way modulators, blockers of adhesion molecules, andother drug classes (De Keyser et al., 1999; Barber et al.,2001; Albers et al., 2001; Fisher and Schaebitz, 2000). Inspite of these failures, there is still optimism that phar-macologic approaches can be developed to treat acutestroke or enhance recovery (White et al., 2000). One newapproach to search for targets is to perform genomic

studies at different times after stroke to try to identifytime-specific gene pathways or gene clusters related tospecific injury or recovery processes after stroke.

DNA microarrays can assay thousands of transcriptsin a single sample (Noordewier and Warren, 2001). Thefirst brain ischemia study used custom-designed 750gene arrays to examine RNA changes in cortex and stria-tum 3 h after focal ischemia (Soriano et al., 2000). Of the24 genes regulated more than twofold, most were imme-diate early genes such as c-fos, NGFI-A, NGFI-C, Krox-20, and Arc (Soriano et al., 2000). Subsequent studiesused oligodeoxynucleotide-based or complementaryDNA (cDNA) microarrays to study RNA expression inhippocampus of rats subjected to transient global ische-mia (Jin et al., 2001) and in cortex of rats at 6 h or 10days after focal ischemia (Kim et al., 2002; Keyvani etal., 2002). A recent study combined cDNA array analysisof 74 genes with brain metabolic status studied usingpositron emission tomography scanning in a baboon fo-cal cerebral ischemia model. A change in the pattern ofgene expression when the cerebral metabolic rate for

Received November 22, 2002; final version received January 16,2003; accepted January 20, 2003.

This study was supported by NIH grants NS28167, NS38743,NS42774, NS43252, AG19561 and a Bugher award from the AmericanHeart Association.

Address correspondence to Dr. Aigang Lu, Department of Neurol-ogy and the Neurosciences Program, University of Cincinnati, VontzCenter for Molecular Studies, Room 2327, 3125 Eden Avenue, Cin-cinnati, OH 45267-0536, U.S.A.; e-mail: [email protected]

Journal of Cerebral Blood Flow & Metabolism23:786–810 © 2003 The International Society for Cerebral Blood Flow and MetabolismPublished by Lippincott Williams & Wilkins, Inc., Baltimore

786 DOI: 10.1097/01.WCB.0000062340.80057.06

oxygen was reduced by 48% to 66% was suggested toserve as a molecular definition of the penumbra (Chu-quet et al., 2002).

The present study used rat Affymetrix U34A oligo-nucleotide arrays to assess 8,740 transcripts in the peri-infarction cerebral cortex at 24 h after permanent middlecerebral artery (MCA) occlusions in adult rats. Usingvery strict criteria, less than 4% of these transcripts wereregulated, and 49.7% of these had not been reportedpreviously. Real time reverse transcription polymerasechain reaction (RT-PCR) confirmed the expression of 20of these genes and showed two general classes: thoseinduced by 2 h, and hence might be targets for acutestroke therapy; and those induced at later times thatcould be targets for tissue repair and plasticity.

MATERIALS AND METHODS

Animal protocols were approved by the University of Cin-cinnati animal care committee and conform to the NationalInstitutes of Health Guide for Care and Use of LaboratoryAnimals. Male Sprague-Dawley rats weighed approximately300 g to 350 g, had unrestricted access to food and water, andwere housed two per cage with a 12-h light–dark cycle.

Stroke modelThe left MCA was occluded using the intraluminal filament

technique (Rajdev et al., 2000; Schwarz et al., 2002). AdultSprague-Dawley rats (n � 3) were anesthetized with isoflu-rane. During anesthesia, rectal temperature was monitored andbody temperature was maintained at 37 ± 0.2°C with a heatingblanket. The left common carotid artery, external carotid artery,and internal carotid artery were isolated via a ventral midlineincision. To occlude the MCA, a 3–0 monofilament nylon su-ture was inserted into the external carotid artery and advancedinto the internal carotid artery approximately 20 mm from thecarotid bifurcation until mild resistance was felt. The woundwas closed. Once animals recovered, they were returned totheir home cages with food and water available ad libitum. Oneday later (24 h), rats were reanesthetized and killed. Sham-operated animals (n � 3) were treated like ischemic animalsexcept that no suture was inserted into the carotid.

RNA preparationAt 24 h after cerebral ischemia, rats were reanesthetized with

ketamine (100 mg/kg) and xylazine (20 mg/kg) and killed. Theperiinfarction cortex was dissected according to a publishedmethod in rat filament model of unilateral proximal MCA oc-clusion (Ashwal et al., 1998; Schwarz et al., 2002; see thesearticles for diagram of dissected brain region). The brain wasquickly removed and cut coronally into three slices beginning3 mm from the anterior tip of the frontal lobe in a brain matrixin a cold room. A longitudinal cut approximately 2 mm fromthe midline through left hemisphere in the sections was made toavoid medial hemispheric structures, which are supplied pri-marily by the anterior cerebral artery. Then, a transverse di-agonal cut was made at approximately the “2 o’clock” posi-tion—avoiding obvious areas of infarction. The left parietal,periinfarction cortex was dissected. The parietal cerebral cor-texes in ischemic 2- or 4-h rats from the same location werealso dissected. The dissected brain tissues were homogenized ina Teflon–glass homogenizer with TRIzol Total RNA IsolationReagent (Life Technology, Rockville, MD, U.S.A.). TotalRNA was isolated according the manufacturer’s instructions.

Briefly, the brain homogenate was treated with chloroform;RNA was precipitated using isopropyl alcohol and cleaned us-ing a RNAeasy mini kit (Qiagen Inc., Valencia, CA, U.S.A.).

GeneChip expression analysis and database searchGeneChip expression analysis was performed according to

the Affymetrix expression analysis technical manual. Briefly,double-stranded cDNA was synthesized from total RNA with ahigh-performance liquid chromatography–purified oligo-dTprimer. Biotin-labeled complementary RNA (cRNA) was syn-thesized from cDNA using T7 RNA polymerase and biotin-labeled ribonucleotides. The quality of the cRNA was assessedusing gel electrophoresis. The cRNA was hybridized to Af-fymetrix U34A rat arrays (Affymetrix, Santa Clara, CA,U.S.A.). The U34A microarray was scanned with the GeneChipscanner.

The data were analyzed using Affymetrix GeneChip expres-sion analysis software according to the Affymetrix GeneChipAnalysis Suite (Tang et al., 2001). An absolute analysis re-ported the hybridization intensity data (average difference) andwhether transcripts were present, absent, or marginal in thetarget from each probe array. Then, a comparison analysis wasrun. The patterns of change of the whole probe set were used tomake a qualitative call of “Increase,” “Decrease,” “Marginalincrease,” “Marginal decrease,” or “No change.” Three chipswere used for each group (sham-operation and ischemia). Thecross-comparisons were made between sham-operation andischemia groups. Genes were included in the analysis only ifthey met all of the following criteria: they were present in allthree sham or all three ischemia samples; all three ischemiasamples for each gene showed either an “Increase or Decrease”when compared with all three sham samples for each gene; andthe fold change in each of the individual comparisons betweenischemia and sham had to be at least 1.7-fold (Jin et al., 2001).These are stringent criteria that probably eliminated manygenes that were actually regulated by ischemia.

Functional information for the regulated genes was obtainedusing LocusLink, OMIM, GeneCards, PubMed, and referenc-ing gene ontology (Ashburner et al., 2000). By searching Uni-Gene and doing Blast analyses of the GeneseqN database, wedetermined the similarity of the expressed sequence tags(ESTs) on the microarrays with known genes. For genes thatwere represented several times on an array, the alternate genenames are given, and the expression values for that gene wereaveraged. If the ESTs represented known or at least highlyhomologous genes, the known gene name is provided in thetables in the unigene/blast columns (Tables 1–14). Sometimesone EST may blast to short fragments of genes or blast toseveral genes, making these ESTs more difficult to interpret. Adraft rat genome covering more than 90% of the rat genomeis available. Most of the ESTs had known homologues(Butler, 2002).

Real-time quantitative RT-PCRTwenty genes were selected for RT-PCR based on whether

they had important functions in cell death, were previouslyunreported, and had relatively high-fold changes on the micro-arrays. Real-time quantitative RT-PCR was performed on thesegenes (n � 3) using the ABI Prism 5700 Sequence Detectionsystem (Applied Biosystems, Foster City, CA, U.S.A.) (Tanget al., 2001). Primer and probe sequences were selected fromcoding regions of each of the genes with the aid of PrimerExpress 2.0 (Applied Biosystems). All primers and probeswere synthesized using PE Oligofactory (Applied Biosys-tems). Each probe was labeled at the 5�-end with the reporterdye VIC and at the 3�-end with quencher dye TAMRA

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(6-carboxytetramethyl-rhodamine) and was phosphate blockedat the 3�-end to prevent extension by AmpliTaq Gold DNApolymerase. One-step RT-PCR was performed according to theTaqman One-Step RT-PCR Master Mix Reagent kit protocol(Applied Biosystems). Fifty to 100-ng total RNA, 900-nmol/Lprimer and 250-nmol/L probe were added for the selectedgenes. Thermal cycling was carried out as follows. Reversetranscription: 48°C for 30 minutes; activation of hot startedAmpliTaq Gold DNA polymerase: 95°C for 10 minutes; ther-mal cycling: 95°C for 15 seconds, and 60°C for 1 minute for 40cycles. The amplified transcripts were quantified with the rela-tive standard curve method and using GAPDH as a loadingcontrol.

RESULTS

Total number of regulated genesA significant number of genes were regulated at 24 h

after permanent focal ischemia (Figs. 1 and 2; Tables1–15). Of the 328 transcripts (from 8,740 on the micro-arrays) that differed from the sham-operation group us-ing the criteria above (all present and fold change >1.7for all comparisons), 264 genes and ESTs were upregu-lated, and 64 genes and ESTs were downregulated. Fig-ure 1 shows a scatter plot of increased expression (brownand red) and decreased expression (green) of transcriptsin the 24-h ischemic samples compared with sham-operation controls. It is notable that many genes showedgreater than 10-fold increases of expression. Figure 2shows that the 328 regulated genes cluster into twogroups: those that increase in animals with stroke andthose that decrease in animals with stroke (Fig. 2). Thisunsupervised cluster also shows the relative consistencyof expression of upregulated (red, threefold increase) anddownregulated genes (blue, threefold decrease) in thethree stroke animals compared to the three sham-operated animals (Fig. 2).

By searching PubMed and performing Blast analyses,it was estimated that 165 (50%) of the 328 regulatedgenes had been reported in previous stroke studies. Ofnote, 147 known genes (45%) had not been reported tobe regulated after stroke or other type of ischemia, and16 (5%) were unknown ESTs. Thus, half (49.7%) of thetranscripts reported in this study have not been reportedin previous stroke or ischemia studies (Fig. 3).

Functional categories of differential expressed genesBy searching LocusLink, OMIM, GeneCards, and

PubMed and reference gene ontology, we divided theregulated genes into 14 different functional categories(Fig. 4, Tables 1–15). Although some of the categoriesare somewhat artificial because some genes fall into sev-eral categories, these categories help in assessing thelarge amount of data (Figs. 4A–C).

Transcription factors and metabolism- and signaltransduction–related categories had the most numbers ofregulated transcripts (Fig. 4A). The genes showing thehighest fold changes (more than fivefold) included thecytokines and chemokines, cell adhesion, motility and

immune response–related genes, stress proteins, andtranscriptional factors (Fig. 4B). Stress proteins, growthfactors, cytokines and chemokines, cell adhesion, motil-ity and immune response–related genes, and enzymesand enzyme inhibitors had been the most reported,whereas many of the G-protein–related genes and me-tabolism-related genes shown to be induced by stroke inthis study had not been previously reported (Fig. 4C).

Enzymes and inhibitors. Several enzymes and pro-teases well known in cerebral ischemia were detectedusing the microarrays including the ICE-like cysteineprotease, calpain and cathepsin L (cysteine protease),gelatinase B (collagenase), and TIM1 (metalloproteinaseinhibitor). Additional upregulated genes includedcathepsin K and C (cysteine protease) and PS-PLA1(phospholipase); enzyme inhibitors, such as cystatin B(cysteine protease inhibitor); contrapsin-like protease in-hibitor–related protein (serine protease inhibitor); and aribonuclease inhibitor (Table 1).

Metabolism-related genes. Several genes in Table 2known to be induced by stroke included GLUT1 (glucosetransporter), ornithine decarboxylase (rate-limiting en-zyme of polyamine biosynthesis), and PCNA/cyclin(DNA replication and repair). Additional regulated genesincluded glycerol 3-phosphate dehydrogenase (GPDH)that was induced more than 60-fold, PHAS-I (translationnegative regulation), PKBS (benzodiazepine receptor,flow of cholesterol into mitochondria), and TBFII (RNAbinding, splicing, and processing) (Table 2).

Stress response proteins. The transcripts of almost allheat shock proteins (Hsp) present on the microarrayswere induced. Hsp27, Hsp70, and heme oxygenase werehighly expressed as previously reported (Sharp et al.,2000). The DNA damage response gene GADD45 andglutathione peroxidase were also upregulated. The pre-viously unreported antioxidant protein 2 transcript wasdecreased (Table 3).

Neurotransmitter and hormone–related genes. Un-reported stroke-inducible genes, such as Ania-3 (metabo-tropic glutamate receptor signal pathway) and �-typecalcitonin gene–related peptide (vasodilator) werehighly expressed. Retinoic acid–related genes were sig-nificantly regulated, and the angiotensinogen transcriptdecreased. Reported stroke-inducible genes, such asNarp (extracellular aggregating for �-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [AMPA] receptor),�-type calcitonin gene-related peptide (vasodilator), cy-clooxygenase-2 (rate-limiting enzyme in the conversionof arachidonic acid to prostaglandins), and thyrotropin-releasing hormone were also highly expressed (Table 4).

Growth factor–related genes. Several growth factorsinduced by ischemia were detected including brain-derived neurotrophic factor, transforming growth factor-�-1, heparin binding epidermal growth factor-likegrowth factor and vascular endothelial growth factor

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FIG. 1. Scatter plot showing the av-erage hybridization signal intensity ofthe genes in the ischemic (n = 3)compared with the sham surgery (n =3) group. The diagonal lines indicatetwofold change in the ischemic groupcompared with the sham group (two-fold increase, upper line; twofold de-crease, lower line). Significantly up-regulated transcripts in the ischemiccompared with sham groups areshown in red–brown; significantlydownregulated transcripts in the ische-mic compared to the sham groups areshown in green.

FIG. 2. Hierarchical clustering ofregulated genes in the ischemicgroup compared with sham group.Red represents threefold increases ofexpression compared with themeans, and purple–blue indicatesthreefold decreases of expressioncompared with the means. The ex-pression of the three sham animals isshown in the top three rows, and theexpression of the three ischemic ani-mals is shown in the bottom threerows. The expression of individualgenes is shown in thin vertical col-umns. Note that a great many genesare induced in the three ischemic ani-mals compared with the shams (leftthree fourths of the cluster), and asmaller set of genes are decreased inthe three ischemic animals comparedwith the shams (right one fourth of thecluster).

FIG. 3. Pie chart showing the per-centages of genes identified as regu-lated by ischemia in the periinfarctioncerebral cortex in this study. The pro-portion of genes reported in previousischemic stroke studies (blue), not re-ported in previous ischemic strokestudies (lavender), and the percent-ages of expressed sequence tags(ESTs) (yellow) are shown.

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TABLE 1. Enzymes and inhibitors

Category Gene name Function summaryFold

changeGene

bank #

RNAchangein chip

RNAchangein paper Unigene/blast

Percent identity,aligned region

Oxidoreductase EST220459 Oxidoreductase, drug metabolismand synthesis of cholesterol,steroids, and other lipids. Eyemorphogenesis

7.8 AI176856 I I(CYP2E) CP1B ratcytochromeP450 1B1

100% 542 aa

Cytochrome P450(CYP1B1)

Oxidoreductase, drug metabolismand synthesis of cholesterol,steroids, and other lipids. Eyemorphogenesis

6.4 U09540 I I(CYP2E)

Cysteine proteasesand inhibitor

Calpain II 80 kDasubunit

Calcium-activated neutral proteases,nonlysosomal, intracellularcysteine proteases, catalyzinglimited proteolysis of substratesinvolved in cytoskeletalremodeling and signal tranduction.

1.9 L09120 I I

Cyclic Protein-2� cathepsinL proenzyme

Lysosomal cysteine (thiol) protease 1.8 S85184 I I

EST Lysosome cysteine-type peptidase 2.8 AA925246 I X Cathepsin Kprecursor

100% 328 aa

Cathepsin C Lysosomal cysteine (thiol) protease 3.2 D90404 I IICE-like cysteine

proteaseCysteine (thiol) protease, induction

of apoptosis3.8 U49930 I I

EST203339 Cysteine protease inhibitor, inhibitspapain (cathepsins L, h and b)

2.1 AI008888 I I Cystatin B 100% 97 aa

Major acute phasealpha-1

Inhibitors of thiol proteases,releasing bradykinin

3.8 K02814 I X

Serine proteaseand inhibitor

Tissue-typeplasminogenactivator (t-PA)

Serine protease, converts inactiveplasminogen to plasmin

2.6 M23697 I I

Plasminogenactivatorinhibitor-1(PAI-1)

Member of the serpin family ofserine protease inhibitors, “bait”for tissue plasminogen activator,urokinase, and protein c,regulation of fibrinolysis

17 M24067 I I

EST189815 Serine protease inhibitor, inhibitionof complement activation

2.1 AA800318 I X Complement C1inhibitorprecursor

81% 171 aa

Contrapsin-likeproteaseinhibitor relatedprotein (CPi-26)

Serine protease inhibitor, inhibitingneutrophil cathepsin g and mastcell chymase

∼71.6 D00753 I I

Metalloproteinaseand inhibitor

EST202002 Component of the neutrophilgelatinase complex, modulator ofinflammation, apoptosis

∼21 AA94650 I I NGAL ratneutrophilgelatinase-associatedlipocalinprecursor

100% 197 aa

Gelatinase B(GelB)

Collagenase, degrades type IV and Vcollagens

∼17.4 U24441 I I

EST215162 Inhibitors of the matrixmetalloproteinases, known to act onmmp-1, mmp-2, mmp-3, mmp-7,mmp-8, mmp-9, mmp-10, mmp-11,mmp-12, mmp-13 and mmp-16.Does not act on mmp-14.

∼21 AI169327 I I TIM1 rat metallo-proteinaseinhibitor 1precursor

100% 216 aa

Phospholipaseand inhibitor

PS-PLA1 Phospholipase A1,phosphatidylserine metabolism

∼13.1 D88666 I X

EST217956 Ca2+-dependentphospholipid-binding protein,inhibiting phospholipase A2 andantiinflammation

4.3 AI171962 I I Annexin I 100% 345 aa

Annexin II Calcium-dependentphospholipid-binding protein,regulation of cellular growth, andsignal transduction

5.1 L13039 I I

RNA helicase Nuclear RNAhelicase

ATP dependent RNA helicase,down-regulation of acute phasecytokine production (TNFalpha,IL-1, and IL-6)

2.7 AF063447 I X

Ribonucleaseinhibitor

Ribonucleaseinhibitor

Ribonuclease inhibitor 2.6 X62528 I X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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TABLE 2. Genes related to metabolism 1

Category Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

Carbohydratemetabolism

GLUT1�glucosetransporter 1,Rat brainglucose-transporter

Glucose transporter,transport ofdehydroascorbic acid

2.9 S68135, M13979

I I

EST189687 Glycogen catabolism −2.1 AA800190 D X Glycogen phosphorylase,brain form

99%, 137 aa

HK2 gene fortype IIhexokinase

Phosphorylating glucose toproduce glucose-6-phosphate, glycolysis

∼11 D26393 I X

6-phosphofructo-2-kinase/fructose-2,6bisphosphatase

Regulation of theconcentration offructose-2,6-bisphosphate,glycolysis

∼8.2 AB006710 I X

Glycerol3-phosphatedehydrogenase

Glycolysis, oxidoreductase ∼61.7 AB002558 I X

Amino acid,proteinmetabolism

EST226923 Serine synthesis −2.1 AI230228 D X Human phosphoserinetransaminase

54%, 108 aa

EST212157 Serine synthesis −2.5 AI102868 D X Phosphoserinetransaminase

54%, 108 aa

Cystathioninegamma-lyase

Cysteine metabolism ∼3.1 D17370 I X

L-cysteine oxygenoxidoreductase

Degradation of cysteine,Taurine and hypotaurinemetabolism, electrontransporter

2.9 E03229 I X

EST198184 Degradation of cysteine,Taurine and hypotaurinemetabolism, electrontransporter

2.5 AA942685 I X CYDX rat cysteinedioxygenase

100%, 199 aa

S-adenosyl-methioninesynthetase

Amino acid metabolism,biosynthesis ofpolyamines

2 J05571 I X

Ornithinedecarboxylase(ODC)

Rate-limiting enzyme of thepolyamine biosynthesis

2.8 J04791, J04792,X07944

I I

EST Mitochondrial ribosome 28Ssubunit protein

2.1 AI639387 I X Mitochondrial ribosomalprotein S6 (MRPS6)

Ribosomal proteinS27

Cytosolic small ribosomal(40S)-subunit

2.7 X59375 I I

PHAS-I Binding EIF4E, translationalnegative regulation

∼10.1 U05014 I X

EST195768 Fructosamine-3-kinase,deglycation

−2.8 AA891965 D X Fructosamine-3-kinase(FN3K gene)

EST197246 Nuclear and cytoplasmicprotein glycosylation

1.8 AA893443 I X UDP-N-acetylglu-cosamine-peptideN-acetylglucos-aminyltransferase 110Kda subunit (OGT1)

100%, 1035 aa

EST196352 N-glycosylation 2 AA892549 I X Mannosyl-oligosaccharidealpha-1,2-mannosidase

EST196578 Hydrolysis andtransglycosylation

2.9 AA892775 I I Rat lysozyme c, type 1precursor

100%, 146 aa

Lipidmetabolism

EST Glycolipids catabolism −2.5 AA859911 D X Cmp-n-acetyl-neuraminate-beta-galactosamide-alpha-2,3-sialyltransferase(alpha 2,3-st)(gal-nac6s) (st3gala.2)(siat4-b)

100%, 349 aa

Fatty acidtransportprotein

Membrane transporter forlong-chain fatty acids

−2.6 U89529 D X

Low molecularweight fattyacid bindingprotein

Lipid transporter, tumorsuppressor

−2.2 J02773 D X

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(VEGF) (Lipton, 1999). Other factors, not previouslydescribed in ischemic stroke, were also induced by is-chemia: PT N1 (negative regulation of insulin signaling),DOC-2 p59 isoform (CSF1 signal transduction pathway),and GRB2 (linking EGFR and PDGFRB to Ras and Racpathway). Of note, the Noggin transcript (binding andinactivating members of TGF-� superfamily signal pro-tein) decreased (Table 5).

Cytokine and chemokine–related gene. Ischemia isknown to induce many of these genes including tumornecrosis factor (TNF) receptor, interleukin (IL)-1�, IL-1

receptor antagonist, IL-6, CC chemokine ST38 precur-sor, MIP-1, and interferon-induced messenger RNA.Other inducible genes in this class included HCYP2(serine/threonine kinase, interacting with TNFR1and TRAF2), PRG1 (protection of cells from Fas orTNF�-induced apoptosis), gro, calgranulin, and IL-18(Table 6).

Signal transduction–related genes. GTP cyclohydro-lase I (NO-synthesis related), protein kinase C-�, oxida-tive stress-inducible protein tyrosine phosphatase,MKP-3 (inactivation of MAPK), and SOCS-3 (negative

TABLE 2—(Continued)

Category Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

Lipidmetabolism

C-FABP �cutaneous fattyacid-bindingprotein

Lipid binding,differentiation

2.4 S69874 I I

EST218294 Cholesterol biosynthesis 1.9 AI172293 I X RANP-1 100%, 292 aaEST Oxidoreductase,

cholesterol biosynthesis3.1 E12625 I X

Isopentenyldiphosphate-dimethylallyldiphosphateisomerase

Isoprenoid biosynthesis,steroids synthesis

3.5 AF003835 I X /C-4 methyl steroloxidase

Peripheral-typebenzodiazepinereceptor (PKBS)

Benzodiazepine receptor,involving in the flow ofcholesterol intomitochondria

11.3 J05122 I I

EST LDL receptor, proteolysisand peptidolysis

∼5.6 AI071531 I I JE0111 lectin-likeoxidized LDLreceptor-mouse

78%, 360 aa

Aminopeptidase-B Exopeptidase, eukotrieneA4 hydrolase

−1.8 D87515 D X

Nucleotidemetabolism

Adenosine kinase Conversion of adenosineto AMP, ribonucleosidemonophosphatebiosynthesis

−2 U57042 D X

Proliferating cellnuclear antigen(PCNA/cyclin)

Delta-DNA polymerasecofactor, DNAreplication and repair,cell cycle control

2.4 M24604 I I

EST Production of thepyrimidine nucleosidetriphosphates

∼11.8 AA859827 I X URK1 mouse uridinekinase

78%, 184 aa

Phosphodiesterase I Nucleotidepyrophosphatase, broadspecificity

−2.7 D28560 D X

Phosphodiesterase I Phosphodiesterase I.Nucleotidepyrophosphatase

∼6.2 D30649 I X

TBFII mRNA forpolypyrimidinetract bindingprotein

RNA binding, mRNAsplicing, mRNAprocessing

∼11.8 X74565 I X

EST189008 pre-mRNAs binding,pre-mRNA processing,and other aspects ofmRNA metabolism andtransport

2 AA799511 I X ROA2_humanheterogeneous nuclearribonucleoproteinsA2/B1

97%, 41 aa

EST SnoRNP assembly, pre-18srRNA processing

3 AF069782 I X /nucleolar protein 5(NOP5)

Gas-5 growth arresthomolognon-translatedmRNA sequence

Processing of ribosomalRNA

3 U77829 I X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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regulator in the JAK-STAT pathway) are induced byischemia as previously reported. Unreported genes in-cluded Ssecks 322 (PKA anchoring protein), Cpg21 (in-activating ERK1), JAK2 (JAK-STAT cascade), and sev-eral genes related to NO metabolism (Table 7).

G-protein–related genes. This was the most surpris-ing group of genes because ischemia was not generallyknown to regulate them. Highly expressed transcriptsincluded Rad (small monomeric GTPase) and G33A

(Rho GTPase binding and activator) (Table 8). G-protein–coupled receptors, GTPase binding molecules,and GTP signaling are remarkably regulated in the peri-infarction cortex after ischemia (Table 8).

Transcriptional regulatory proteins. Many tran-scription factors are known to be induced by ischemiaand were also regulated on the microarrays includingc-fos, Fra-1, c-jun, jun-B, c-myc, NGFI-A, krox20, andHIF1. Other highly regulated transcriptional factors de-

TABLE 3. Stress response genes

Category Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

Oxidativestress

EST211851 Heavy metal binding,detoxifing metals,antioxidation

8.4 AI102562 I I MT1 ratmetallothionein-1

100%, 60 aa

Metallothionein-2metallothionein-1

Heavy metal binding,detoxifing metals,antioxidation

8 M11794 I I

EST220041 Storage of trace-metals, cellstress

2.9 AI176456 I I Rat metallothionein-II 100%, 60 aa

GlutathioneS-transferaseYc1 subunit

Conjugation of reducedglutathione to a widenumber of hydrophobicelectrophiles

−2.2 S72505 D D

GlutathioneS-transferase Ycsubunit

Glutathione transferase −2.3 K01932 D D

EST195844 Peroxidase and antioxidantactivity

−2 AA892041 D X Antioxidant protein 2 100%, 223 aa

EST 190084 Selenium-dependentglutathione peroxidase,reduction of hydrogenperoxide, organichydroperoxide, and lipidperoxides

∼9.6 AA800587 I I A45207 glutathioneperoxidase

94%, 166 aa

Heat shockprotein

Heat shock protein70

Heat shock protein ∼103.7 L16764, Z27118 I I

EST, EST191323 Heat shock protein ∼91.6 AA818604,AA848563

I I Rat heat shock 70 Kdprotein 1/2

100%, 640 aa

EST Heat shock protein 2 AA875620 I I Heat shock 70 Kd protein3 (HSP70.3)

100%, 640 aa

Hsp70.2 Heat shock protein 52.6 Z75029 I IHeat shock protein

(Hsp27)Heat shock protein ∼358.7 M86389 I I

EST Heat shock protein 53 AA998683 I I Heat shock 27protein—rat

100%, 204 aa

EST220250 Heat shock protein 11.2 AI176658 I I Heat shock 27protein—rat

100%, 204 AA

Collagen-bindingprotein (gp46)

Heat shock protein 3.2 M69246 I I

EST216547 Heat shock protein 2 AI170613 I I 10 Kd heat shock protein,mitochondrial

99%, 101 aa

EST0020 Heat shock protein 2.8 AA108277 I I /Hsp105Alpha-crystallin B

chainMolecular chaperone 3.2 M55534, X60351 I I/−

EST223333 Heat shock protein 15.1 AI179610 I I A Chain A, Crystalstructure of rat hemeoxygenase-1 incomplex with heme

100%, 288 aa

Heme oxygenasegene

Heme oxygenase ∼88.5 J02722 I I

DNA damageresponse

Progressionelevated gene 3protein(Gadd34)

DNA damage response,initiation of proteintranslation, proapoptoticfunction

3.2 AF020618 I I

EST DNA damage response,inducing p38/JNKactivation and

7.8 AI070295 I I Growth arrest anddna-damage-inducibleprotein GADD45

100%, 164 aa

GADD45 DNA damage response,inducing p38/JNKactivation and

17.4 L32591 I I

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

GENOMICS OF PERIINFARCTION CORTEX AFTER ISCHEMIA 793

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TABLE 4. Neurotransmitter and hormone-related genes

Category Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

Glutamaterelated

Glutamatereceptor(GluR-B)

Subunit 2 of AMPA subtypeof ionotropic glutamatereceptor for synaptictransmission

−2 M38061 D D

Metabotropicglutamatereceptor 3

Metabotropic glutamatereceptor

−2.7 M92076 D D

Narp � neuronalactivityregulatedpentraxin(NPTX2)

Transportor, extracellularaggregating factor forAMPA receptors,synapses plasticity anduptake of extracellularmaterial

∼101.5 S82649 I I

AMPA receptorinteractingprotein GRIP

Adapter protein, lustering ofAMPA receptors

∼3.1 U88572 I X

VesI Modulation of metabotropicglutamate and IP3receptors

3 AB003726 I X

Activity andneurotrans-mitter-inducedearly gene 3(ania-3)

Metabotropic glutamatereceptor signalingpathway, neuronalimmediate early gene

∼6.0 AF030088 I X

GABA related EST225364 GABA transporter −2.1 AI228669 D X Sodium- and chloridedependent GABAtransporter 1

100%, 598 aa

GABA-A receptorgamma-2subunit

Benzodiazepine receptor,gamma-aminobutyricacid-inhibited chloridechannel

−2.2 L08497 D D/I

Angiotensinogen Angiotensinogen Angiotensinogen, hormone −5 M12112 D XEndothelin related Endothelin-

convertingenzyme

Endothelin-convertingenzyme,metalloendopeptidase

1.8 D29683 I X

EST Endothelin receptor, Gprotein-coupled receptor

3.3 AA818970 I I Rat endothelin Breceptor precursor

100%, 441 aa

Neuropeptide Neuropeptide Neuropeptide Y,neuropeptide hormone andinhibitory neuromodulator

2.3 M15880 I I

Alpha-typecalcitoningene-relatedpeptide

Ligands for receptors foundin nervous system- andperipheral tissues,inducing vasodilatation

∼9.5 M11597 I X

Beta-typecalcitoningene-relatedpeptide

Neuropeptide hormone,calcium ion homeostasis,vasodilatation

∼10.3 M11596 I I

Adrenomedullinprecurosr

A hypotensive peptide andvasodilatator agent

∼6.4 D15069 I I

Galanin (aneuropeptide)

Neuropeptide 5.5 J03624 I I

Prostaglandinrelated

Cyclooxygenaseisoform COX-2

Prostaglandin-endoperoxidesynthase, the firstrate-limiting step in theconversion of arachidonicacid to prostaglandins

7.6 S67722,L25925

I I

Thyrotropinreleasinghormone

Thyrotropinreleasinghormone (TRH)

A regulator of thebiosynthesis of TSHand as aneurotransmitter/neuromodulatorin the central andperipheral nervoussystems

∼11 M23643,M36317

I I

Retinoic acidrelated

Cellular retinoicacid-bindingprotein II(CRABP II)

Retinoic acid-binding protein 2.2 U23407 I X

Cytosolicretinol-bindingprotein (CRBP)

Retinoid binding andtransport

3.9 M19257 I I

Aldehydedehydrogenase(ALDH)

Aldehyde dehydrogenase −2.5 AF001898 D X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

A. LU ET AL.794

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tected on the microarrays included RL/IF-1 (I�B), reti-noic acid receptor �1, chromosomal protein HMG2,transcriptional repressor CREM, C/EBP-related tran-scription factor, and CELF (Tables 9 and 10).

Extracellular matrix. Osteopontin, a ligand of inte-grin and CD44, was highly expressed along with decorin(binding fibronectin and TGF-�), tenascin (ligand of in-tegrins), syndecan (receptor of extracellular matrix),and lysyl oxidase (protein modification of extracel-lular matrix) (Table 11). Other genes related to fibronec-tin and procollagen were also regulated as previouslyreported.

Cytoskeletal protein–related genes. Glial fibrillaryacidic protein � and � (intermediate filament) and tubu-

lin �-1 chain (microtubule protein) were regulated byischemia as expected. Other stroke-regulated genes in-cluded moesin (cross-linking actin to membranes), talin(linking cytoskeleton to extracellular matrix receptor),and spr (intermediate filament) (Table 12).

Genes-related ion, vesicular transport, and synap-tic transmission. Brain digoxin carrier protein (trans-porter of organic anions), potassium channel �-subunit,neuronal pentraxin I, and agrin expression decreased.Transferrin receptor and ceruloplasmin (ferroxidase,iron efflux) increased (Table 13).

Cell adhesion, motility, and immune response–related genes. CD14 (binding apoptotic cell, triggeringphagocytosis), CD44 (cell surface receptor, matrix adhe-

TABLE 5. Growth factor-related genes

Category Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

IGF-related Insulin-likegrowth factor II

Insulin-like growth factorreceptor ligand

−2.5 X17012 D I

EST Negative regulation ofinsulin signaling

2.2 AI113289 I X Protein-tyrosinephosphatase,non-receptor type 1(PTP-1b) (PTN1)

100%, 431 aa

EST203856 Insulin-like growth factorreceptor binding protein

∼13.4 AIl009405 I I IBP3 rat insulin-likegrowth factor bindingprotein 3

100%, 291 aa

Insulin-likegrowthfactor-bindingprotein(IGF-BP3)

Insulin-like growth factorreceptor binding protein

∼11.4 M31837 I I

BDNF EST Growth factor 5.1 AI030286 I I Brain-derivedneurotrophic factorprecursor

100% 248 aa

BDNF �brain-derivedneurotrophicfactor

Growth factor, neurogenesis,neuronal development

12, 7.3 S76758, D10938 I I

TGF related Transforminggrowth factorbeta 1 (TGFB1)

Ligand of TGF receptor, cellproliferation,differentiation, andapoptosis. Fibrosis.

∼29.1 X52498 I I

EST Binds and inactivatesmembers of TGF-betasuperfamily signalingproteins

−4.1 AA859752 D X Noggin 100%, 143 aa

Furin Processes hormoneprecursors: PTH, TGFb

2.2 X55660 I I

CSF related DOC-2 p59isoform

Csf-1 signal transductionpathway, tumor suppressor

2.7 U95178 I X

EGF related Heparin bindingEGF-likegrowth factor

Epidermal growth factorreceptor ligand, positivecontrol of cellproliferation

17.7 L05489 I I

EST216715 Linking EGFR andPDGFRB to Ras and Racsignaling pathways

1.9 AI170776 I X Growth factorreceptor-bound protein2 (GRB2)

100%, 202 aa

VEGF related EST193502 Vascular endothelial growthfactor I

2.3 AA850734 I I Mouse vascularendothelial growthfactor precursor

98%, 213 aa

Vascularendothelialgrowth factorform 3

Vascular endothelial growthfactor receptor ligand,induces endothelial cellproliferation and vascularpermeability

∼11.1 L20913 I I

FGF receptor Fibroblast growthfactor receptor1 beta-isoform

Fibroblast growth factorreceptor

4 S54008 I I

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

GENOMICS OF PERIINFARCTION CORTEX AFTER ISCHEMIA 795

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sion, and apoptosis), and immunoglobulin E binding pro-tein (galactose-specific lectin) increased as previouslyreported in other cerebral ischemia studies. Other regu-lated transcripts included lipopolysaccharide bindingprotein (carrier for lipopolysaccharide, interacting with

CD14), proteasome subunit RC1 (26S proteasome, classI-restricted antigen presentation), and DAF (inhibitingamplification of complement cascade) (Table 14).

Others. Urinary plasminogen activator receptor 1,serine threonine kinase (pim-3), intracellular calcium–

TABLE 6. Genes related to cytokines and chemokines

Category Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

TNF related Tumor necrosisfactor receptor(TNF receptor)

Tumor necrosis factorreceptor, apoptosis andnf-kappa b signaling

4.2 M63122 I I

Homocysteinerespondentprotein HCYP2

Serine-threonine kinase,interacting with TNFR1and TRAF2 and cativtingcaspase

∼6.4 AF036537 I I

EST190110 Zinc finger protein,single-stranded RNAbiding, inhibiting,macrophage TNF-alphaproduction

5.2 AA800613 I I Rat tristetraproline 100%, 319 aa

PRG1(PACAP-responsive gene1)

Protection of cells from Fas-or tumor necrosis factoralpha-induced apoptosis

5.5 X96437 I X

Osteoprotegerin(OPG)

Decoy receptor for osteoclastdifferentiation factor

5 U94330 I X

IL related Interleukin 1-beta Interleukin-1 beta, apoptosis 3 M98820 I IInterleukin 1

receptorantagonist

Interleukin-1 receptorantogonist, inhibiting thebinding of IL-1-alpha andIL 1-beta

∼4.4 M63101 I I

Interleukin 6 (IL6) IL6R ligand growth factor ∼18 M26744,M26745

I I

Interleukin 18(IL-18)

Cytokine, immune response 2.9 AJ222813 I I

Chemokine CC chemokineST38 precursor

C-C chemokine, chemotaxis 22.3 AF053312 I I

EST201236 CXC chemokine receptor,mediating intracellularcalcium flux

∼3.2 AA945737 I X Chemokine receptorLCR1

100%, 331 aa

Macrophageinflammatoryprotein-1alpha

Chemokine cc,inflammatory, andchemokinetic properties

∼20.4 U22414 I I

Macrophageinflammatoryprotein-1 beta(MIP-1 beta)

Chemokine, inflammatory,and chemokineticproperties

∼6.5 U06434 I I

Immediate-earlyserum-responsiveJE gene

Chemotactic factor formonocyes, binding to ccr2and ccr4

61.5 X17053 I I

Gro Chemokine, inflammatoryprocesses

∼9.2 D11445 I I

Interferonrelated

Interferon inducedmRNA

Cell surface receptor linkedsignal transduction, thetransduction ofantiproliferative andhomotypic adhesionsignals

5.6 X61381 I I

EST207718 Regulating gene activity inthe proliferative and/ordifferentiative pathwaysinduced by ngf, may bean autocrine factor

5 AI014163 I X Interferon-related proteinPC4

100%, 448 aa

Others Platelet-activatingfactoracetylhydrolasealpha 1 subunit(PAF-AH alpha1)

Inactivates PAF 6.6 AF016047 I I

EST A member of the S100family of proteins,inhibition of casein kinaseand as a cytokine

∼3.4 AA957003 I X S108 RAT calgranulin A 100%, 88 aa

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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TABLE 7. Genes related to signal transduction 1

Category Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

Calciumrelated

Plasma membraneCA2+ ATPase-isoform 2

Calcium-transportingATPase, transport ofcalcium out of the cell

−2.3 J03754 D D

EST199283 Calcium-transportingATPase, transport ofcalcium out of the cell

−2.1 AA943784 D D Calcium-transportingatpase plasmamembrane, brainisoform 2

100%, 1197 aa

Calmodulin-sensitive plasmamembraneCa2+-transportingATPase (PMCA3)

Calcium-transportingATPase, transport ofcalcium out of the cell

−2.1 J05087 D D

EST Calcium-binding,actin-cytoskeletonassociated

−2.6 AI639532 D X Human troponin C, fastskeletal muscle

97%, 133 aa

Activity andneurotransmitter-induced earlygene 4 (ania-4)

Calcium/calmodulin-dependent proteinkinase-related

∼4 AF030089 I X

Kinase Arelated

cAMPphosphodiesterase

cAMP-specificphosphodiesterase

4.1 J04563 I I

cAMPphosphodiesterase(PDE4)

cAMP-specificphosphodiesterase

2.5 M25350 I I/D

Ssecks 322, PKCbinding protein,and substratemRNA

Protein kinase A anchoringprotein

∼6.6 U75404,U41453,U23146

I X

NO-kinaseG related

EST191798 Guanylate cyclase, NOmediated signaltransduction

−6.7 AA849036 D X Guanylate cyclasesoluble, alpha-1 chain

100%, 689 aa

Cyclic nucleotidephosphodiesterase(CaM-PDE)

CaM-dependentcyclic-nucleotidephosphodiesterase,hydrolase

−2.6 M94537 D X

EST NO mediated signaltransduction

2.1 AI058941 I X N(G),N(G)-dimethylargininedimethylaminohydrolase1

100%, 284 aa

GTP cyclo-hydrolase I

Synthesis oftetrahydrobiopterin,cofactor in aromaticamino acid hydroxylation,and nitric oxide synthesis

7.2 M58364 I I

Membranereceptortyrosinekinase

EST Transmembrane receptorprotein tyrosine kinase,neurogenesis

−1.7 M59814 D X Ephrin type-b receptor 1precursor (elk)

100%, 983 aa

Sky Transmembrane receptorsignaling protein tyrosinekinase, cell adhesion,particularly in CNS

−2.2 D37880 D X

EST Receptor tyrosine kinase,neural crest development

5.2 AI639318 I I Proto-oncogenetyrosine-protein kinasereeptor RET precursor

92%, 1115 aa

Inositolphosphatemetabolism/PKC

Phospholipase C-1 Inositol phosphatemetabolism, producingIP3 and DAG.Translocation of tubby

−2.7 M20636 D I/D/− /cornichon-like protein

PhospholipaseC-beta1b

Inositol phosphatemetabolism

−2.1 L14323 D I/D/−

Inositol1,4,5-triphosphate3-kinase

Inositol phosphatemetabolism, convertingIP3 to Ins(1, 3, 4, 5)P4

−2.2 X56917 D D/−

Protein kinase Cdelta subspecies

Diacylglycerol-activated/phosholipid dependentprotein kinase C

∼15.5 M18330 I I

MAPkinaserelated

Oxidativestress-inducibleprotein tyrosinephosphatase

Inactivation of MAP kinase(erk1/2), oxidative stressresponse

1.8 S81478 I I

Protein tyrosinephosphatase

Inactivation of MAPkinase(erk1/2), oxidativestress response

3.5 S74351U02553

I I

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binding protein, the major vault protein—which is in-volved in nucleo-cytoplasmic transport—and neuro-transmitter-induced early gene 6 were all regulated byischemia (Table 15).

Real-time RT-PCRReal-time RT-PCR was performed on 20 selected

genes at 2, 4, and 24 h after ischemia (Fig. 5). The earlytimes were chosen becausethese would be times when

TABLE 8. G protein-related genes

Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

EST G protein −2.9 AA925506 D D Guaninenucleotide-bindingprotein G(i)/G(s)/G(o)gamma-7 subunit

100%, 68 aa

N-chimaerin GTPase activator,cooperating with Rac1and Cdc42Hs

−1.5 X67250 D X

EST19812 GTPase activator −2.2 AA894317 D X N-CHIMAERIN/chimerin(chimaerin) 2 (CHN2)

100%, 333 aa

EST196299 GTPase activator −2.9 AA892496 D XSPA-1 like

proteinGTPase activator,

stimulating Rsr1GTPase

−1.8 AF026504 D X

Ras-related rab1Bprotein

Small GTP-bindingprotein

−1.7 X13905 D X

Ras-relatedprotein (rad)

Small monomericGTPase, member ofthe ras family of GTPbinding proteins

14.8 U12187 I I

G33A Rat mRNAfor gene 33polypeptide

RHO GTPase bindingand activator, stressresponse

∼37.2 X07266 I X

EST215655 RHO GTPase bindingand activator, stressresponse

3.8 AI169756 I X Gene 33 polypeptide-rat 97%, 136 aa

EST195743 RHO small monomericGTPase, ras-related,reorganization of theactin cytoskeleton

2.7 AA891940 I X RhoA:1 humanGTP-binding proteinrhoC 6

96%, 32 aa

Isoprenylated 67kDa protein

GTP-bindingprotein/GTPase,immune response

∼3.7 M80367 I X

Putative G-proteincoupledreceptor RA1c

G-protein coupledreceptor

−5 AF079864 D X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

TABLE 7—(Continued)

Category Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

MAP kinaserelated

Dual-specificity proteintyrosine phosphatase(rVH6)

Inactivation of ERK2 3.5 U42627 I I

Dual specificityphosphatase, MKP-3

Inactivation of MAPK 6.3 X94185 I I

MAP-kinase phosphatase(cpg21)

Inactivates ERK1 10.7 AF013144 I X

JAK-STATcascade

Protein-tyrosine kinase(JAK2)

Protein tyrosine kinase,JAK-STAT cascade

∼6.0 U13396,AJ000557

I X

Stat3 protein JAK-STAT cascade,transcription factor

3.2 X91810 I I

Suppressor of cytokinesignaling-3 (SOCS-3)

SH2-containing protein,negative regulator in theJAK-STAT pathway

∼25.7 AF075383 I I

Other EST Signaling role during T-cellactivation

−2.2 AA866291 D X /cornichon-like protein

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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acute stroke treatment might be initiated. The 24-h timewas chosen because this would be the time when infarctsize was nearly stable but tissue repair and plasticityresponses would likely have been initiated. The geneswere chosen based on their high fold induction or theirfunctions or functional classes.

The PCR results showed good agreement with the cor-responding microarray data in terms of increase and de-crease of expression. Early upregulated genes included

glutamate receptor–related gene (Narp), G-protein–related genes (Rad and G33A), kinases and phosphatases(HYCP2, Pim-3, Cpg21, JAK2), and the transcriptionalfactor CELF (Figs. 5A and 5B). Two other genes showedmodest regulation at early times and greater increases at24h including the extracellular matrix gene Tenascin andthe complement inhibitor, DAF (Fig. 5A). Early upregu-lated genes might be involved in acute neuronal injurycaused by stroke.

TABLE 9. Transcriptional Regulation 1

Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

C-fos Transcription factor 15.9 X06769 I IFBR-murine

osteosarcomaprovirus genome

Transcription factor 14.3 X03347 I I /c-fos

Fos-related antigen(Fra-1)

Transcription factor,similar to Fos

∼21 M19651 I I

EST Transcription factor 3.5 AA875032 I I /fos-related antigen-2(fra-2 gene)

c-jun oncogene Transcription factor ∼17.6 X17163 I IEST201366 Transcription factor 2.3 AA945867 I I /C-junEST219534 Transcription factor 2.8 AI175959 I I /C-junEST194844 Transcription factor,

growth factor response6.4 AA891041 I I Rat transcription factor

jun-B100%, 343 aa

pJunB Transcription factor,growth factor response

7.1 X54686 I I

c-myc Transcription factor,activating and repressingexpression of targetgenes

∼10.4 Y00396 I I

EST191797 Transcriptional regulatoryprotein, cell growth,and/or maintenance

−2.1 AA849035 D X MYCB 100%, 177 aa

Nerve growthfactor-induced(NGFI-A) gene(egr1)

Transcription factor,mitogenesis, anddifferentiation

2.1 M18416 I I

Immediate early genetranscription factorNGFI-B

Transcriptional factor,orphan nuclear receptor,steroid receptor family

2.3 U17254 I I

NGFI-C3E exon#1-2 Transcription factor,positive control of cellproliferation

2.5 M92433 I I

krox20 Transcription factor,nervous systemdevelopment

7.2 U78102 I I

EST Transcription factor,repression oftranscription, negativecontrol of cellproliferation

3.6 AI071299 I X Mouse transforminggrowth factor-beta-inducible early growthresponse protein 1(MGIF)

93%, 479 aa

Hypoxia-induciblefactor 1

Transcription factor, stressresponse

2.7 Y09507 I I

RL/IF-1 Transcription factorbinding and cytoplasmicsequestering

∼16.4 X63594 I I

NGF-inducibleanti-proliferativeputative secretedprotein (PC3)

Transcription factor, DNArepair, negative controlof cell proliferation

4.1 M60921 I I

EST199655 Transcription factor, DNArepair, negative controlof cell proliferation

2.6 AA944156 I I BTG2 rat btg2 proteinprecursor

100%, 157 aa

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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Late upregulated transcripts included enzymes and inhibi-tors (Cathepsin C, Cip-26, Systatin B), metabolism-relatedgenes (PHAS-I, TBFII), cytoskeletal proteins (Spr), andthe lipopolysaccharide binding protein (Figs. 5C and 5D).These genes may be related to tissue immune and repair re-sponses or possibly to some aspect of plasticity after thestroke. Several transcripts were also downregulated on themicroarrays (Tables 1–15). The expression of two of these

transcripts, brain digoxin carrier protein and a potassiumchannel, was confirmed by RT-PCR (Fig. 5E).

DISCUSSION

Various members of virtually every class of genesthroughout the entire genome appear to be regulated in

TABLE 10. Trancriptional Regulation 2

Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

FSH-regulatedprotein

Transcription factor,negative control of cellproliferation

∼5.5 L26292 I X /Kruppel-like factor 4(KLF4)

RYB-a Cell proliferation and DNArepair, repression oftranscription

3 D28557 I X

Sry-relatedHMG-box proteinSox11

Transcription activatingfactor, neurogenesis

∼5.0 AJ004858 I X

EST198080 Transcription factor, neuraldevelopment

−3.1 AA894277 D X /Soggy precursor andTEAD-2

BHF-1 Differentiation factor duringneurogenesis

∼−7.7 D82074 D X

EST Transcription factor,differentiation factorduring neurogenesis

∼−5.8 AI639109 D X /Neurogenic differentiation(NEUROD1, BHF-1,NIDDM)

LIM homeodomainprotein (LH-2)

Transcription factor, celldifferentiation

−2 L06804 D X

Retinoic acidreceptor alpha1

Nuclear hormone receptor,differentiation

∼9.8 AJ002940 I X

Inhibitor ofDNA-binding,splice variantId1.25

Transcription factor,inhibiting dna binding,negative regulation of celldifferentiation

1.7 L23148 I X

High MobilityGroup Protein I(Y)

AT DNA binding,transcription regulation

2.3 X62875 I X

Chromosomalprotein HMG2

DNA-binding protein,affecting transcription andcell differentiation, DNArepairing

5.2 D84418 I X

Leucine zipperprotein

Transcription factor,transcription co-repressor,cAMP responsive elementbinding

2.6 M63282 I X

Transcriptionalrepressor CREM

Regulator of thetranscription ofcAMP-inducible genes

∼17.1 S66024 I I

Interferon regulatoryfactor 1 (IRF-1)

Transcription activatingfactor, regulatingapoptosis andtumor-suppression

2.6 M34253 I I

EST, EST234097 Transcription factor 2.2 U53184, AI237535 I X Human LPS-inducedTNF-alpha factor

80%, 134 aa

rNFIL-6�C/EBP-relatedtranscriptionfactor

Transcription factor, immuneand inflammatoryresponses, antiapoptosis

∼14.2 S77528 I X

Sfb mRNA forsilencer factor B

Transcription factor 6.2 X60769 I I

CELF Transcription factors,immune and inflammatoryresponses

14 M65149 I I

EST Transcription activatingfactor, immune response

∼43.8 AI045030 I X Rat CCAAT/enhancerbinding protein delta

100%, 267 aa

Zinc finger protein Transcriptional activator,b-cell growth anddevelopment, fibrogenesis

∼15.4 AF001417 I X

EST Transcription factor,activator

6.5 AA900476 I X MRG1 mouse MSG-relatedprotein 1

98%, 268 aa

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

A. LU ET AL.800

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response to cerebral ischemia. The challenge will be todevelop strategies for identifying the best targets to treatacute stroke or to promote recovery and repair. Our pre-vious studies identified groups of molecules that wereregulated in common by several types of injuries thatmight provide such targets (Tang et al., 2002). The pre-sent study focuses on genes with high expression levelsor on groups of genes that appear to signify activation ofspecific pathways. The multiplicity of genes induced af-ter focal ischemia would suggest that pharmacologic ap-proaches that targeted multiple pathways simultaneouslymight be more successful than single gene or even singlegene pathway approaches. High-throughput drug screen-ing might be designed to detect single compounds thatmodulated several different but critical injury or recov-ery pathways.

RNAs for calpain, ICE-like cysteine protease, cathep-sin L, cathepsin K, cathepsin C, cystatin B, and contrap-sin-like protease inhibitor–related protein (Cpi-26) wereall induced after stroke. The ICE-like cysteine protease ishomologous to caspase-3 (Juan et al., 1996). Caspase-3,a central executioner in mitochondrial apoptotic path-ways (Hengartner, 2000), is induced along withcaspase-9 in the penumbra of some (Benchoua et al.,2001; Namura et al., 1998) but not all focal ischemiastudies of adult brain (Gill et al., 2002; Loetscher et al.,2001). The present results support the induction of sev-eral caspase-like RNAs by focal ischemia (Dirnagl et al.,1999). However, it is likely that regulation of mostcaspases occurs by cleavage-induced activation of theirproteolytic domains rather than being transcriptionallyregulated (Phan et al., 2002; Dirnagl et al., 1999).

TABLE 11. Extracellular matrix-related genes

Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

Fibronectin gene 3 end Extracellular matrix, celladhesion and migration,signal transduction

1.7 X05834 I I

Brevican core protein Chondroitin sulfateproteoglycan, extracellularmatrix, differentiation,gliotic response

−2.3 U37142 D X

Dermatan sulfateproteoglycan-II(decorin)

Small proteoglycan, bindingcollagen, fibronectin, andtgf-beta. Suppressing thegrowth of various tumorcell lines

∼10.6 Z12298 I X

EST206481 Calcium-binding of theextracellular matrix;inhibitor of calcification

5 AI012030 I I MGP rat matrix gla-proteinprecursor

100%, 102 aa

Osteopontin Ligand for integrinalpha-v/beta-3 and CD44

60.9 M14656 I I

Tenascin Extracellular matrixglycoprotein, a ligand forintegrins

∼7.7 U09401, U15550 I X

Syndecan Cell surface proteoglycanand receptor for theextracellular matrix, celladhesion, action of growthfactors

5.2 X60651, S61865 I I

Ryudocan�heparansulfate proteoglycancore protein

Cell surface proteoglycan,neutrophil migration,receptors or coreceptors

2.2 S61868 I I

Neuroglycan C precursor Chondroitin sulfateproteoglycan,CNS-specific functions

−2.3 U33553 D X

UDP-glucosedehydrogenase

Excellular matrixbiosynthesis

1.7 AB013732 I X

Protein disulphideisomerase (PDI)

Procollagen-prolinehydroxylation,extracellular matrixmaintenance

2.3 X02918 I I

Iodothyronine5-monodeiodinase(5-MD)

Procollagen-prolinehydroxylation,extracellular matrixmaintenance

3.5 M21476 I I

EST230748 Protein modification ofextracellular matrix, tumorsuppressor

1.8 AI234060 I X Protein-lysine 6-oxidaseprecursor

100%, 410 aa

Lysyl oxidase Protein modification ofextracellular matrix, tumorsuppression

∼12.6 S77494, S66184 I X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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Calpain is a calcium-activated nonlysosomal, intracel-lular cysteine protease that is induced and activated dur-ing cerebral ischemia (Neumar et al., 2001; Wang,2000). Calpains can compromise lysosomal membraneintegrity and lead to cathepsin B and L activation. Duringcerebral ischemia and neuronal apoptosis, cathepsin Band L were activated (Islekel et al., 1999; Yoshida et al.,2002). Cathepsin B activates caspase-3 via caspase-11during liver cell apoptosis, and cathepsin L activates a

caspase-3–like protease in liver cells. Calpain can alsodirectly activate caspase-12 and cause endoplasmic re-ticulum stress. Calpain and caspase-3 cleave severalcommon cytoskeletal, cytosolic, and nuclear protein sub-strates (Huang and Wang, 2001; Yamashima, 2000;Wang, 2000). It is not known whether caspase-11 or 12are activated after cerebral ischemia, but it is likely thatcalpain and some caspase-related proteases are effectorsof injury and tissue repair.

TABLE 12. Genes related to cytoskeletal proteins

Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

Lamina associatedpolypeptide 2 (LAP2)

Binding lamin B1 andchromosomes, regulationof nuclear architecture

1.7 U18314 I X

EST Lamin B receptor,lamin/chromatin binding

AI145490 I X Rat NBP60 100%, 619 aa

EST189270 Crosslinking actin tomembrane glycoproteins,transducing ligand-receptor binding to actinreorganization, neuronalmigration

∼13.9 AA799773 I X ABP2_human endothelialactin-binding protein

60%, 124 aa

EST Cross-linkers betweenplasma membranes andactin-based cytoskeletons,cell-cell recognition andsignaling and cellmovement

∼15.3 AF004811 I I Mouse moesin 99%, 576 aa

EST190459 Cytoskeletal protein, linkingcytoskeleton toextracellular matrixreceptors

∼7.1 AA800962 I X TALI_human talin 96%, 113 aa

Growth factor (Arc) Actin binding, endodermdevelopment

2.9 U19866 I I

Myosin regulatory lightchain (RLC)

Cytoskeleton organizationand biogenesis

2.1 X05566 I X

RLC-A gene for myosinregulatory light chain,exon 4

Cytoskeleton organizationand biogenesis

2.5 X54617 I X

SM22 Actin-binding proteins,muscle development

∼21 M83107 I X

Tropmyosin (TM-4) Binds actin filament 3 J02780 I XEST Tropomyosin, cytoskeletal

type, binds actin filament5 AA859305 I X Tropomyosin isoform 6-rat 100%, 247 aa

CLP36 (clp36) Cytoskeletal protein, adapter,actin binding

2.6 U23769 I X

Striated musclealpha-tropomyosin

Actin-binding andtroponin-binding proteins

1.9 X02412 I X

EST199921 Actin-binding proteins,inhibiting actomyosinMg(2+)-ATPase

2.4 AA944422 I I Calponin, acidic isoform 100%, 329 aa

Nestin Intermediate filament,neurogenesis

1.9 M34384 I I

Vimentin Intermediate filament,cytoskeletal protein

3.2 X62952 I I

Glial fibrillary acidic proteinalpha (GFAP) and glialfibrillary acidic proteindelta (GFAP)

Intermediate filament,cell-specific marker

∼22.6 AF028784

Small proline-rich protein(spr)

Intermediate filament, cellshape and cell size control

5.6 L46593 I I

EST195714 Member of the cornifiedenvelope precursor proteinfamily, structural protein,epidermal differentiation

13.5 AA891911 I X CORA rat cornifin alpha 100%, 151 aa

EST196136 Microtubule, cytoskeletalstructural protein

16.8 AA892333 I D Tubulin alpha-1 chain 100%, 450 aa

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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Cystatin B is an inhibitor of cathepsins B, H, and L(Cimerman et al., 2001). Cystatin B–deficient mice haveincreased expression of apoptosis/glial activation genes(Lieuallen et al., 2001), suggesting that Cystatin B mayprotect. Cathepsin C, a lysosomal cysteine protease, pro-cesses granzyme A and B. Granzymes are expressed inthe granules of activated cytotoxic lymphocytes, thegranules entering target cells via perforin after cytotoxiclymphocytes synapse with the target cells. Granzyme Bcan act upstream and downstream of caspases. GranzymeA causes apoptosis via a caspase-independent pathway(Podack, 1999). Since lymphocytes infiltrate ischemicbrain (Jander et al., 1995), these cells may mediate ap-optosis of target ischemic brain cells via these genes. It isnot known whether cystatins, cathepsins, and granzymesplay a role in neurons or glia after ischemia, althoughkeratinocytes can express both perforin and granzymes(Berthou et al., 1997).

Contrapsin-like protease inhibitor related protein (Cpi-26), a member of the ‘serpins‘ subfamily, is homologousto antichymotrypsin (Ohkubo et al., 1991). Antichymo-trypsin can inhibit caspase activity and is antiapoptotic(Ikari et al., 2001). If Cpi-26 functions like antichymo-trypsin, it may protect against focal cerebral ischemia.

Glycerol 3-phosphate dehydrogenase expression in-creased more than 60-fold at 1 d after focal ischemia.Glycerol 3-phosphate dehydrogenase is localized to mi-tochondria and is involved in mitochondrial hydrogenshuttles necessary for the reoxidation of glycolysis-derived NADH. Activated GPDH increases intracellularlevels of NADH and increases cellular resistance againstH2O2 injury (Hwang et al., 1999). It is possible thatGPDH and PARP interact to modulate NADH/NADPHlevels and cell survival and death.

Translation-related transcripts, including PHAS-I andpolypyrimidine track-binding protein (TBFII), were also

TABLE 13. Ion, vesicular transport, and synaptic transmission

Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

Brain digoxin carrier protein Na(+)-independenttransporter of organicanions

∼−14.7 U88036 D I

EST Glycine-inhibited chloridechannel

−2 AI145044 D D Glycine receptor alpha-2chain variant A precursor

100%, 451 aa

Potassium channel, alphasubunit

Potassium channel regulator ∼−11 Y17606 D X

Serum and glucocorticoid-regulated kinase (sgk)

Protein serine/threoninekinase, sodium transport

2.2 L01624 I I

Sodium myo-inositoltransporter (SMIT)

Small molecule transport,regulation of membranepotential and tissueosmolyte

2.9 AJ001290 I I

Na-K-2Cl cotransporter(Nkcc1)

Sodium:chloride/potassium:chloridesymporter

5 AF086758 I X

Transferrin receptor Iron transport 2.9 M58040 I XCeruloplasmin Ferroxidase, iron efflux 5 L33869 I IEST Ferroxidase, iron efflux ∼5.1 AA817854 I X CERU rat ceruloplasmin

precursor100%, 1058 aa

Complexin II Docking protein, exocytosis,neurotransmitter release

−1.8 U35099 D X

Alpha-soluble NSFattachment protein

Membrane fusion,exocytosis, andintra-Golgi transport

−1.8 X89968 D X

B/K protein Calcium-dependentphospholipid binding

−3.6 U30831 D X

EST Membrane fusion, bindsphospholipid vesicles

−3.4 AI639118 D X Calcium-dependentactin-binding protein

80%, 41 aa

Synaptotagmin IV homolog Ca2+-dependent vesiculartrafficking and exocytosis

1.9 U14398 I I

S-100 related protein Calcium binding, annexin IIligand, exocytosis andendocytosis

5 J03627 I I

Clathrin assembly proteinshort form (CALM)

Retrieving synaptic vesicles 2.3 AF041373 I X

EST Synaptic transmission,synaptic uptake ofextracellular material

−2 AI072943 D D Neuronal pentraxin Iprecursor (np-I) (np1)(47 kd taipoxin-bindingprotein)

100%, 431 aa

Secretogranin II Packaging or sorting ofpeptide hormones andneuropeptides intosecretory vesicles

2.2 M93669 I I

Agrin Synaptogenesis, extracellularmatrix

−2 M64780 D X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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induced after focal ischemia. PHAS-I binds eIF4E andnegatively regulates translation initiation. It dissociatesfrom eIF4E when phosphorylated. Insulin treatment ofadipose cells increased the phosphorylation of 4EBP1and reduced the interaction of 4EBP1 with eIF4E (Pauseet al., 1994). PHAS-I undergoes caspase-dependentcleavage in cells undergoing apoptosis. The N-terminaltruncated polypeptide binds to eIF4E and fails to become

sufficiently phosphorylated on insulin stimulation tobring about its release from eIF4E (Tee and Proud,2002). Binding initiation factor eIF4E can rapidly induceapoptosis (Herbert et al., 2000). During global cerebralischemia, eIF4E and 4E-BP1 were significantly dephos-phorylated (Martin de la Vega et al., 2001). PHAS-Ilevels and phosphorylation state could modulate isch-emic brain damage. Overexpression of polypyrimidine

TABLE 14. Genes related to cell adhesion, motility, and immune responses

Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

CD14 Lps receptor, bindinglipopolysaccharide binding protein,lbp/lps complex, and apoptoticcells, triggering phagocytosis

10.3 AF087943, AF087944 I I

Lipopolysaccharidebinding protein

A carrier for LPS and controlLPS-dependent monocyteresponses, the lbp/lps complexseems to interact with the cd14receptor

∼15.3 L32132 I X

EST188970 Cell adhesion −2.1 AA799473 D X Lactadherin precursor(mfg-e8)

79%, 101 aa

Intercellular adhesionmolecule-1

Binding integrin alpha-I/beta-2 andleukocyte adhesion IFA-1 protein,cell adhesion

3 D00913 I I

P-Meta-1 mRNA forCD44 surfaceprotein

Cell surface receptor for hyaluronate,matrix adhesion, lymphocyteactivation and apoptosis

∼4 A30543 I I

Glycoprotein CD44(CD44)

Cell surface receptor for hyaluronate,matrix adhesion, lymphocyteactivation and apoptosis

∼35.9 M61875 I I

P41-Arc Assembly of the actin cytoskeleton,cell motility and cell elongation

6.2 AF083269 I I

Protein p9Kahomologous tocalcium-bindingprotein

Calcium binding, motility, invasion,and tubulin polymerization

3.8 X06916 I I

EST197807 Barbed-end actin capping,macrophage motile

2 AA894004 I X Mouse macrophage-capping protein

92%, 226 aa

MHC class IRT1.C-type protein

Human leukocyte antigen,presentation of foreign antigens

2.4 L40362 I I

Proteasome subunitRC1

26S proteasome, non-lysosomalproteolytic pathway, class I-restricted antigen presentation

∼6.9 D10729 I I

MHC class I antigen(RT1.EC2)

Human leukocyte antigen,presentation of foreign antigens

∼7.0 AF074608, M24324,M64795

I I

MHC class II antigenRT1.B beta chain

Human leukocyte antigen,presentation of foreign antigens

∼2.3 U65217 I I

MHC class II-like betachain (RT1.DMb)

Human leukocyte antigen,presentation of foreign antigens

5.4 U31599 I I

MHC-associatedinvariant chaingamma

Gamma chain antigen associatedwith MHC class II antigen

∼8.1 X13044 I I

Tumor-associatedglycoprotein E4(Tage4)

A member of the immunoglobulinsupergene family

∼7.9 L12025 I X

Fc-gamma receptor Fc fragment of IgG, low affinityIIIb, receptor

3.7 M32062 I X

Fc gamma receptor Receptor for the fc region ofimmunoglobulins gamma, lowaffinity receptor, downmodulationof cell activation state

∼11.2 X73371 I I

IgE binding protein Galactose-specific lectin, bindingIgE, cell growth regulation,chemoattractant activity

21.7 J02962 I I

Complement proteinC1q beta chain

Complement component 2.9 X71127 I I

Decay acceleratingfactor GPI-formprecursor (DAF)

Preventing the formation of c4b2aand c3bbb; inhibiting theamplification of the complementcascade

∼23.9 AF039583 I X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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track-binding protein stimulates Apaf-1 internal ribo-some entry site function (Mitchell et al., 2001). Polypy-rimidine track-binding protein also modulates alternativesplicing of caspase-2 (Cote et al., 2001), although therole for this gene in ischemic brain injury is unclear.

Narp expression, increased by 2- to 5-fold at 2 to 4 hof ischemia, increased more than 25-fold at 24 h afterischemia. Narp is a secreted immediate-early gene in-duced by a wide variety of stimuli. Since it is an extra-cellular aggregating factor for AMPA receptors (O’Brien

et al., 1999), it might exacerbate early ischemic excito-toxicity. Agrin, another aggregating factor, was de-creased after cerebral ischemia. Agrin is an extracellularheparan sulfate proteoglycan that signals AChR cluster-ing, particularly during the formation of the neuromus-cular junction. Agrin is also widely expressed in the cen-tral nervous system (Fong and Craig, 1999), althoughany effects of decreased agrin expression on central ace-tylcholine neurotransmission after ischemia are uncer-tain. The chronic changes of Narp and agrin and effects

TABLE 15. Others

Gene name Function summaryFold

changeGene

bank #

RNAchangein chips

RNAchangein paper Unigene/blast

Percent identity,aligned region

MG87 Iron-sulfur protein −1.8 AF095741 D X Probable iron-sulfur proteinb0947

32%, 279 aa

EST196401 Nucleotide binding 2.4 AA892598 I X /Putative nucleotide bindingprotein, estradiol-induced

EST221135 Compaction of DNA 2.1 AI177503 I X Human histone H3.3 100%, 135 aaEST Probably binding Ca2+ and an

unknown ligand2.6 AI639058 I X Human protein c18ORF1 73%, 141 aa

CD9 CD9 antigen, platelet activationand aggregation

2.1 X76489 I X

ad1-antigen Antigens, mast cell marker,platelet activation marker

2.9 X61654 I X

Urinary plasminogenactivator receptor 1(uPAR-1)

Receptor for urokinaseplasminogen activator,localizing and promotingplasmin formation,proteolysis-independentsignal transduction

∼16.3 X71898 I X

EST189300 Egf-like module 2.1 AA799803 I X Structure of the Egf-likemodule of human C1r, Nmr,19 structures

76%, 134 aa

Major vault protein Nucleocytoplasmic transport 3.1 U09870 I XSerine threonine kinase

(pim-3)Protein serine-threonine kinase,

developmental processes5.3 AF086624 I X

EST105188 Similar to protein kinases ∼4.1 H31287 I X GS3955 61%, 85aaIntracellular calcium-

binding protein(MRP14)

Inhibition of casein kinase ∼16.2 L18948 I X

EST229907 The domain of insulin growthfactor-binding proteinhomologues

∼5.2 AI233219 I X MEGF6 protein-rat 37%, 78 aa

Epithelial celltransmembraneprotein antigenprecursor (RTI40)

An apical integral plasmamembrane protein expressedin type I but not type IIalveolar epithelial cells

10.6 U92081 I I

EST197083 Globule surface membranematerial, indication ofadipocyte differentiation

12.3 AA893280 I X Mouse adipose differentiation-related protein

86%, 180 aa

Epithelial membraneprotein-1

Membrane glycoproteins, celldifferentiation

∼8.9 Z54212 I X

ASM15 Encoding an untranslated RNA,lies at the end of a cluster ofimprinted genes; tumorsuppressor activity

∼40.3 X59864 I I

Ri1 A LIM domain protein,LIM/double zinc fingerdomains

∼31.2 X76454 I X

Activity andneurotransmitter-induced early gene 6(ania-6)

Unclear; low similarity tocyclins

∼6.8 AF030091 I X

EST196323 Unclear 3.5 AA892520 I X /Membrane protein ofcholinergic synaptic vesicles(VATI), human BRCA1,Rho7 and vatI genes,complete cds, and ipf35gene, partial cds

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.

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FIG. 4. (A) Percentages of the 328 genesregulated by ischemia in this study thatwere in each functional category. (B) Per-centages of the genes in each functionalcategory that were regulated more thanfivefold. (C) Percentages of genes in eachfunctional category that had not been re-ported in previous stroke studies. EST, ex-pressed sequence tag.

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on glutamate and acetylcholine neurotransmission mightalso play important roles in synaptic plasticity and re-covery after stroke.

Brain-derived neurotrophic factor RNA upregulationon the microarrays confirms many previous cerebralischemia studies. Brain-derived neurotrophic factor mayprotect against cerebral ischemia in part by phosphory-lation of ERK1/2 (Han and Holtzman, 2000; Encinas etal., 1999). Ischemia also induced the MAP-kinase phos-

phatase (cpg21) that inactivates ERK1 (Ishibashi et al.,1994). This might decrease the protective effects ofbrain-derived neurotrophic factor. However, ischemia-induced glutamate accumulation activates the P44/42MAP-kinase pathway. Since inhibition of the MAP-kinasepathways protected neurons against glutamate excitotox-icity (Grant et al., 2001), induction of the cpg21 MAP-kinase phosphatase may also protect against brain isch-emia. It is notable that cpg21 is induced more than 20-

FIG. 5. Real-time quantitative reverse transcription polymer-ase chain reaction showing the expression of selected genes(y-axis) at 2, 4, and 24 h after cerebral ischemia in the peri-infarction cerebral cortex. (A and B) Early upregulated genesthat show a twofold or greater increase of expression at 2 or4 h after cerebral ischemia compared with the sham-controls.(C and D) Late upregulated genes that showed a twofold orgreater increase of expression at 24 h after cerebral ischemiacompared with the sham-controls. (E) Downregulated genes.LPS, lipopolysaccharide; TBF, track-binding protein; DAF, de-cay accelerating factor.

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fold after 2 h of ischemia and more than 120-fold by 24h of ischemia, suggesting an important but unstudied rolefor this gene.

Interleukin-6, Jak2, SOCS-3, and Pim-3 RNAs werealso regulated by cerebral ischemia. Interleukin-6 in-duces gp130-homodimerization and activates Jak1, Jak2,Tyk2, STAT1, and STAT3. STAT1 and STAT3 translocateto the nucleus to activate transcription (Heinrich et al.,1998). Interleukin-6 protects the brain against focal andglobal ischemia (Loddick et al., 1998; Matsuda et al.,1996). SOCS-3 binds all four JAK kinases and inhibitstheir activity (Chen et al., 2000), possibly suggesting thatSOCS-3 might counter the protective effects of IL-6. Co-expression of Pim kinases with Socs-1 results in phos-phorylation and stabilization of Socs-1 protein (Chen etal., 2002). Lymphocytes from SOCS-1−/− mice undergoaccelerated apoptosis associated with increased Bax, andmurine embryonic fibroblasts lacking SOCS-1 are moresensitive to TNF-induced cell death (Chen et al., 2000).Some Pim kinases prolong cell survival and inhibit ap-optosis activity (Lilly et al., 1999). Thus, although therole of SOCS-3 is uncertain, IL-6, Pim kinases, andSOCS-1 may protect against cerebral ischemia.

rNFIL-6 (C/EBP �) and CELF (C/EBP �) were highlyinduced. C/EBP plays a role in regulating other genes,since Egr-1 (NGFI-A) and MKP-1 mRNA were signifi-cantly reduced in liver of C/EBP knockout mice (Green-baum et al., 1998). Our data show that, after stroke, bothEgr-1 and oxidative stress-inducible protein tyrosinephosphatase (MKP-1) RNAs increased. The target genesof Egr-1, including PAI-1, VEGF, ICAM-1, IL1-�, andimmediate-early serum-responsive JE gene (MIP-1)were also induced. Recent lung studies show that Egr-1regulates many downstream genes and could have a cen-tral role in the pathogenesis of ischemic lung tissue dam-age (Yan et al., 2000). The present data support the pos-sibility that C/EBP regulation of Egr-1 (NGFI-A) and itsdownstream genes including PAI-1, VEGF, ICAM-1, IL-1�, and MIP-1 could be an important signaling pathwayin cerebral ischemia as well.

Transcripts for CD14, IgE binding protein (lectin),lectin-like oxidized LDL receptor (LOX-1), complementprotein C1q, PS-PLA1 (serine-phospholipid-selectivephospholipase A), and lipopolysaccharide binding pro-tein were all increased after cerebral ischemia. CD14,lectin, and LOX-1 are phagocyte transmembrane mol-ecules. CD14 and LOX-1 can bind phosphatidylserine(PtdSer) and other surface molecules to initiate apopto-sis. C1q is an adhesive “bridging” molecule betweenapoptotic cells and phagocytes. These molecules cantether apoptotic cells and shuttle them to the phagocyticmachinery (Savill and Fadok, 2000; Oka et al., 1998).PS-PLA1 hydrolyzes a fatty acyl residue at the sn-1 po-sition of lysophosphatidylserine and phosphatidylserine(Sato et al., 1997). Lipopolysaccharide binding protein

can bind CD14 (Gutsmann et al., 2001). The increase ofthe latter molecules can decrease phagocytic recognitionof apoptotic cells, which can enhance cell survival whencells are subjected to weak proapoptotic signals (Hoepp-ner et al., 2001). During cerebral ischemia, microglia–macrophages are markedly activated (Stevens et al.,2002). Although inhibition of microglial activation is re-ported to protect against global brain ischemia (Yrjan-heikki et al., 1998), macrophage and microglial engulf-ment of apoptotic cells can suppress the secretion ofproinflammatory mediators such as tumor necrosis fac-tor-� and suppress the inflammatory response to isch-emia (Savill and Fadok, 2000). There seems to be abalance of the detrimental and neuroprotective effects ofactivated microglia and macrophages that may vary overtime and with the severity of injury (Stoll et al., 1998).Exactly how these pathways influence acute injury orrecovery after stroke is still unclear.

The focus of this study was to examine the periinfarc-tion cortex at 24 h after a permanent MCA occlusion, ata time well beyond that when any treatment for acutestroke would likely be useful. The changes in many ofthe 328 transcripts may be related to the onset of tissuerepair and the beginning of recovery. As many as two tothree times as many molecules may be identified oncethe entire rat and human genomes are studied as a func-tion of time and area of brain injured after stroke. Futureapproaches at defining those pathways crucial for recov-ery might include looking for genes shared with toler-ance models, models where recovery is improved or ispoor, and other approaches that will help refine thesearch for clinically important pathways. We have pre-viously reported genes that are induced in common byseveral types of injury in brain and in blood (Tang et al.,2001, 2002). These common genes may relate to com-mon mechanisms of injury and could represent commontargets for a common therapy.

Acknowledgments: The authors thank Melinda Reilly forexcellent technical assistance, and regret the inability to refer-ence all of the publications related to the regulated genes in thisstudy.

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