Genomics of the Periinfarction Cortex After Focal Cerebral Ischemia

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Genomics of the Periinfarction Cortex After Focal Cerebral Ischemia
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  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 Molecular  Developmental 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,  and  DAF.  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 genomicstudies 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 of gene 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:  Journal of Cerebral Blood Flow & Metabolism 23: 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 model The left MCA was occluded using the intraluminal filamenttechnique (Rajdev et al., 2000; Schwarz et al., 2002). Adult Sprague-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 preparation At 24 h after cerebral ischemia, rats were reanesthetized withketamine (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 these articles 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 search GeneChip expression analysis was performed according tothe 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 if they 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-PCR Twenty genes were selected for RT-PCR based on whetherthey 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 GENOMICS OF PERIINFARCTION CORTEX AFTER ISCHEMIA 787   J Cereb Blood Flow Metab, Vol. 23, No. 7, 2003  (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. RESULTSTotal number of regulated genes A significant number of genes were regulated at 24 hafter 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. Of note, 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 genes By searching LocusLink, OMIM, GeneCards, andPubMed 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 of regulated transcripts (Fig. 4A). The genes showing thehighest fold changes (more than fivefold) included thecytokines and chemokines, cell adhesion, motility andimmune 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 cysteine protease, calpain  and  cathepsin L  (cysteine protease), gelatinase B  (collagenase), and  TIM1  (metalloproteinaseinhibitor). Additional upregulated genes included cathepsin 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  and glutathione 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  A. LU ET AL.788  J Cereb Blood Flow Metab, Vol. 23, No. 7, 2003  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-miccomparedtotheshamgroupsareshown 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. GENOMICS OF PERIINFARCTION CORTEX AFTER ISCHEMIA 789  J Cereb Blood Flow Metab, Vol. 23, No. 7, 2003  TABLE 1.  Enzymes and inhibitors Category Gene name Function summaryFoldchangeGenebank #RNAchangein chipRNAchangein paper Unigene/blastPercent identity,aligned regionOxidoreductase EST220459 Oxidoreductase, drug metabolismand synthesis of cholesterol,steroids, and other lipids. Eyemorphogenesis7.8 AI176856 I I(CYP2E) CP1B ratcytochromeP450 1B1100% 542 aaCytochrome P450(CYP1B1)Oxidoreductase, drug metabolismand synthesis of cholesterol,steroids, and other lipids. Eyemorphogenesis6.4 U09540 I I(CYP2E)Cysteine proteasesand inhibitorCalpain II 80 kDasubunitCalcium-activated neutral proteases,nonlysosomal, intracellularcysteine proteases, catalyzinglimited proteolysis of substratesinvolved in cytoskeletalremodeling and signal tranduction.1.9 L09120 I ICyclic Protein-2   cathepsinL proenzymeLysosomal cysteine (thiol) protease 1.8 S85184 I IEST Lysosome cysteine-type peptidase 2.8 AA925246 I X Cathepsin Kprecursor100% 328 aaCathepsin C Lysosomal cysteine (thiol) protease 3.2 D90404 I IICE-like cysteineproteaseCysteine (thiol) protease, inductionof apoptosis3.8 U49930 I IEST203339 Cysteine protease inhibitor, inhibitspapain (cathepsins L, h and b)2.1 AI008888 I I Cystatin B 100% 97 aaMajor acute phasealpha-1Inhibitors of thiol proteases,releasing bradykinin3.8 K02814 I XSerine proteaseand inhibitorTissue-typeplasminogenactivator (t-PA)Serine protease, converts inactiveplasminogen to plasmin2.6 M23697 I IPlasminogenactivatorinhibitor-1(PAI-1)Member of the serpin family of serine protease inhibitors, “bait”for tissue plasminogen activator,urokinase, and protein c,regulation of fibrinolysis17 M24067 I IEST189815 Serine protease inhibitor, inhibitionof complement activation2.1 AA800318 I X Complement C1inhibitorprecursor81% 171 aaContrapsin-likeproteaseinhibitor relatedprotein (CPi-26)Serine protease inhibitor, inhibitingneutrophil cathepsin g and mastcell chymase ∼ 71.6 D00753 I IMetalloproteinaseand inhibitorEST202002 Component of the neutrophilgelatinase complex, modulator of inflammation, apoptosis ∼ 21 AA94650 I I NGAL ratneutrophilgelatinase-associatedlipocalinprecursor100% 197 aaGelatinase B(GelB)Collagenase, degrades type IV and Vcollagens ∼ 17.4 U24441 I IEST215162 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 1precursor100% 216 aaPhospholipaseand inhibitorPS-PLA1 Phospholipase A1,phosphatidylserine metabolism ∼ 13.1 D88666 I XEST217956 Ca2+-dependentphospholipid-binding protein,inhibiting phospholipase A2 andantiinflammation4.3 AI171962 I I Annexin I 100% 345 aaAnnexin II Calcium-dependentphospholipid-binding protein,regulation of cellular growth, andsignal transduction5.1 L13039 I IRNA helicase Nuclear RNAhelicaseATP dependent RNA helicase,down-regulation of acute phasecytokine production (TNFalpha,IL-1, and IL-6)2.7 AF063447 I XRibonucleaseinhibitorRibonucleaseinhibitorRibonuclease inhibitor 2.6 X62528 I XRNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; −, Unchange; X, not reported.  A. LU ET AL.790  J Cereb Blood Flow Metab, Vol. 23, No. 7, 2003
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