CHAPTER 7

The endothelial genomic response to chronic hypoxia

Robert E. Verloop1 Anton J. G. Horrevoets2 Marten A. Engelse1 Oscar L. Volger3 Sophie Nadaud4 Pieter Koolwijk1 Victor W.M. van Hinsbergh1

1Laboratory for Physiology and 2Department of Molecular Cell Biology and Immunolo- gy, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, the Netherland 3Department of Biochemistry, Academic Medical Center, Amsterdam, the Netherlands 4UPMC University Paris 06 and INSERM, UMR_S 525, F-75005, Pa- ris, France 196 Chapter 7 lysis pathway was well represented in the that displayed a gradual increase a gradual lysis pathwaywaswellrepresentedinthegenesthatdisplayed glyco the while oxygen, 5% not but 1% at regulated specifically genes dependent hypoxia/HIF- showed analysis Pathway conditions. after theseculture into 8panels grouped genes, defined 435 of regulation significant a revealed expressions parably orbetterthanat 21% oxygenfor up to 2 weeks.Microarrayanalysisof and healthy com at 1%and5%oxygenatmosphere properties their proliferation maintained remained cells Endothelial tensions. oxygen defined under renewal and medium culture, inspection continuous that allowed under cultureconditions vely. cells Therefore, westudied humanumbilicalveinandmicrovascularendothelial extensi because onlyshort-termhypoxia(upto24hours)hasbeeninvestigated chronic hypoxiaonthesecells as duringmanyconditions, experienced pathological of theeffectsLittle isknown tension. in oxygen tothechange adjust tabolically of Exposure of endothelial cells to hypoxia shifts their geneexpressionpattern to me Abstract: identify a novel interaction between KLF2 and HIF activity and their regulated genes. interactionbetweenKLF2andHIFactivitytheirregulated identify anovel circuit, and for chronichypoxia,despitethecomplexadjustments initsregulatory enhanced. These resultsidentifyHIF-1 as the most important markedly were KLF2 by suppressed normally genes responsive TGF-β and matory proinflam contrast, In eNOS. and thrombomodulin like targets direct mRNAits and KLF2 of decrease a observed we as KLF2, as identified was regulation additional for such One ofthecandidates of HIF-1regulation. complexity additional suggesting HIF-3 inhibitory thepotentially whereas induced, remained levels but theircellular were suppressedafter2weeksofhypoxia, of HIF-1 Still,theexpression 24 hoursand2weeksexposure. deed, only 8 out 55 genes, including PDH3, showed a significant difference between chronic hypoxia, but tended to increase to higher levelsafterhypoxia.In prolonged or 2 weeks to 1% oxygen.Most genes showedsimilarregulationbybothacuteand (RT)-PCRreal-time 24 hours for either exposed that were cultures insixadditional of 55genesselectedfromourstudieswasstudiedindetailby results andpublished in identityofthegenes.Next,apanel substantial overlap cells showed endothelial previously publishedmicroarraystudiesonshort-term (24 hours)hypoxia-exposed five with comparison Surprisingly, concentration. oxygen 1%) and (5% lowering at a /HIF-3 a 4 also markedly increased, markedly 4 also a andHIF-2 a mRNA ------Endothelial During Chronic Hypoxia 197

Introduction Oxygen is required for proper functioning of tissues. It is delivered by the blood and replenished via the circulatory system that is lined by endothelial cells. When hypox- ia occurs, tissue cells switch to a change in metabolic activity and an altered gene expression pattern, which is largely determined by hypoxia inducible transcription factors (HIF) as well as by additional mechanisms1, 2. This results in reduced energy expenditure, enhanced glycolysis, and the production of factors, such as VEGF and erythropoietin, that help to restore vascularization and oxygen transfer. Being part of the circulation, the vascular endothelium is normally well oxygenated, and can respond to angiogenic factors produced by hypoxic tissues3, 4. However, after occlu- sion of a proximal vessel, by high local oxygen consumption e.g. in inflamed tissues or tumors, and in transplanted tissue, endothelial cells become part of a hypoxic environment. In such cases the endothelium has to maintain its survival and to alter its properties towards a phenotype that restores the circulation and blood supply, i.e. inducing vasodilation and participating in angiogenesis5, 6. This requires a change in gene expression, which may be dependent on the duration and severity of hypoxia.

7 Hypoxia is defined as insufficient delivery of O2 to meet the demands of the tissue . While normal tissues, such as resting skeletal muscles and heart, are exposed to a

4 - 6.5% O2 tension, the oxygen concentration of hypoxic regions is usually reduced 8-14 to 0.5-2% O2 , but can incidentally decrease to anoxic values . The lack of oxygen causes the immediate availability of HIF and induces several HIF-dependent and independent pathways that contribute to hypoxia tolerance 2, 15, 16. HIF are heterodi- meric transcription factors composed of a stable, constitutively expressed, HIF-1β (ARNT) subunit and a HIF-α protein, either HIF-1α or HIF-2α, which are negatively regulated by oxygen17, 18. HIF-1α is ubiquitously expressed, while HIF-2α, also called EPAS1, is encountered only in a limited number of cell types, including endothelial cells19-22. The HIF-α subunits are continuously synthesized. However, when oxygen is present, they are degraded after hydroxylation of specific proline residues by oxy- gen-sensitive prolyl hydroxylases (PHD), after which they bind to the von Hippel- Lindau protein and become degraded in the proteasome15, 23. An additional aspara- gine hydroxylation by factor-inhibiting hypoxia (FIH-1) prevents the formation of an active HIF-containing transcription complex. Conversely, in the absence of sufficient oxygen the HIF-a-subunits escape from hydroxylation and degradation and become able to bind as active HIF complexes to many hypoxia-responsive elements (HRE), thereby inducing a large number of different genes. 198 Chapter 7 al. hypoxia. Chaplin by prolonged and chronicinfection,areusuallyaccompanied like cancer,conditions, disease since This israthercontradictory heart ischemia geneexpression. tohypoxiaonhypoxia-regulated influexposure ence ofprolonged the ignored largely have studies date to limitations, technical to due Mostly hypoxic oranoxicconditions,usuallystudiedwithina24-hourperiodoftime. in downregulated or up- were that genes individual identified studies of number a a differentanother.with one overlapped that onlypartially setofgenes Inaddition response a hypoxia-like that induced agents of HIF-2 overexpression genes byevaluating 1 lo groups havestudiedthe genomic responsesof EC to hypoxic exposure Because of the centralroleofcells(EC)inangiogenesis,anumber endothelial survival pathwayduringchronic hypoxia. this essential of initiating cells arecapable genes todeterminewhether endothelial Weculture condition. dard of angiogenesis-regulating the expression on focused of hypoxia(1%O nomic analysisof endothelialcellssubjectedto short periods and prolonged ge wemadeadetailed Because ofthelackdataonchronichypoxicexposure chronic hypoxiathaninchronicallyhypoxiclegs after acute-on- legs ischemic in tissuesectionsofhuman SDF-1 werepresent and of HIF-1 concentrations that muchhigher the recentobservation underlie system for a rapidresponseto an acutemoreseverehypoxicstimulus cell deathandthusto adapt themtochronichypoxia.In addition italsopreparesthe of the PHD. These feedbackmechanisms vitro HIF-1 induced and demonstratedthatchronichypoxia hypoxia prolonged effects adverse of papershavedemonstrated HIF systemduring active ofafully genes mediate crucial beneficial short-term biological adaptations, a limited number of hypoxia. durations for various of humantumorsexposed HIF-regulated Although areas specific of radio-sensitivity the to relation with hypoxia (prolonged) chronic a 28 et al. overexpression. On theotherhand, overexpression. Takeda , was among the first who described the concepts of acute (short-term) and acute(short-term) of the concepts described who first the among was , and 5 selected genes that were both induced/reduced by hypoxia as well by HIF- as well by hypoxia both induced/reduced that were selectedgenes in vivo . This was accomplished by increasing the abundance and activity the abundance by increasing This wasaccomplished 2 ), (5%O normoxia 29, 30 . Ginouvès et al. 2 ), and hyperoxia(21% O 31 made a detailed analysis oftheHIF madeadetailedsystem 31, 32 a 24, 25,27 aimed to protect cells againstnecrotic inEC.Other studies utilizedchemical et al. 34 . . Each of the studies came with . Eachofthestudies

26 selectedhypoxia-responsive α and-2 2 ), which isthestan α desensitization 5, 24, 25 33 . This may . This a , VEGF . Mana et in - - - - Endothelial Gene Expression During Chronic Hypoxia 199

Materials and Methods Materials Medium 199 (M199), penicillin/streptomycin, Hank’s balanced salt solution (HBSS) and trypsin were purchased from Lonza (Basel, Switzerland). Tissue culture plastics were from Corning Life Sciences (Lowell, MA, USA) and Greiner Bio-One (Krem- smünster, Austria). Heparin was from Leo Pharmaceutical Products (Weesp, The Netherlands). A crude extract of endothelial cell growth factor (ECGF) was isolated from bovine brain as described by Maciag et al.35. Newborn calf serum (NBCS) was obtained from Invitrogen (Carlsbad, CA, USA). For HUVEC culture, human serum was purchased from the local blood bank (Sanquin Blood Supply Foundation, Am- sterdam, The Netherlands) and was prepared from at least 20 healthy donors (after informed consent). hMVEC were cultured in human serum from PAA Laboratories (Pasching, Austria); both sera were heat-inactivated and stored at 4ºC. L-glutamine and ethylenediaminetetraacetic acid (EDTA) were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human bFGF was purchased from PeproTech (Rocky Hill, NJ, USA), dispase II from Roche Diagnostics (Penzberg, Germany) and collagenase type II from Invitrogen. For immunohistochemistry and FACS analysis, we used anti- CD31, PE-conjugated, from BD Biosciences (San Diego, CA, USA). The RNeasy mini kit and RNeasy RNA cleanup kit were purchased from Qiagen (Venlo, Nether- lands). The cloned AMV cDNA synthesis kit was from Invitrogen. For RT-PCR we purchased MESA SYBR green qPCR mastermix with low ROX and qPCR master- mix plus low ROX for probe assays from Eurogentec (Liege, Belgium), and TaqMan human β-2-microglobulin endogenous control from Applied Biosystems (Foster City, CA, USA).

Hypoxic Cell culture Human umbilical vein endothelial cells (HUVEC) and foreskin microvascular endo- thelial cells (hMVEC) were isolated, cultured and characterized as previously des- cribed36. hMVEC were isolated and purified by fluorescence-activated cell sorting (FACS) using anti-CD31-PE (1:10) (purity 97.8 ± 1.7%). For the prolonged hypoxic culture of endothelial cells, completely independent iso- lates of HUVEC and hMVEC were cultured for 3 passages (HUVEC ~2 weeks, hM- VEC ~4 weeks) inside a custom-designed hypoxia workstation (T.C.P.S., Rotselaar,

Belgium), consisting of two CO2 and O2 controlled incubators (Sanyo, Etten-Leur, Netherlands) placed inside a T4 glovebox (Jacomex, Dagneux, France) equipped with an O2X1 oxygen transmitter (GE Panametrics, Billerica, MA, USA). The oxygen concentration (vol%) inside the incubators was continuously monitored with an inter- nal zirconia sensor and periodically checked with oxygen test tubes (Drager Safety, 200 Chapter 7 proceeding toRT-PCR. kit (Invitrogen). All cDNAwater before 1:5 inDEPC-treated diluted were samples synthesis cDNA first-strand AMV cloned the for instructions manufacturer’s lowing fol cDNA to converted was RNA USA). MA, Waltham, Scientific, Fisher (Thermo 1000 ontheNanoDrop and purity(260/280nmratio)weredetermined concentration (Qiagen, RNA kit Netherlands). Cleanup Venlo, MinElute RNeasy the using purified Venlo,(Qiagen, cells animal If necessary,Netherlands). RNAfurther were samples DNAfor protocol to manufacturer’s DNase set,bothaccording bytheRNase-free RNA was isolated using the RNeasy Minikit and eliminatedof residual genomic RNA IsolationandPurificationcDNA synthesis oxygen (vol%O percentage volume as defined are chapter this in concentrations oxygen given All oftheculturemedium. concentration Germany) determinedtheoxygen gensburg, Zoetermeer, Netherlands). The Sensordish ReaderwithOxoPlates(PreSens,Re composed of a poolofRNA from different HUVECisolates. QualityofRNA (28S:18S RNAreference a common using performed were microarray experiments dual-color from purchased were sequences Agilent Technologies(Santa Clara,CA,USA). All 60-mer gene-specific ~41,000 containing chips microarray genome human Whole Microarray transcriptome analysis = Ct was calculatedbasedonthethresholdcycle microglobulin (Ct) as 2 were done in duplo. Fold change in expression of each target mRNA relative to β-2- electrophoresis. to gel the amplicons subjecting and analysis reactions All PCR curve melting checking by determined was reaction PCR the of Specificity edu/). (http://frodo.wi.mit. software Primer3 using designed were primers Gene-specific β-2- of expression and GAPDH(seealsoLeJan the with interfere β-actin 18S, genes housekeeping used regularly the to compared not as microglobulin, did concentrations oxygen Varying for 15secondsand60°C conditions: in housekeeping gene β-2-microglobulin. The real-time PCR reactions were performed tec) for target genes andqPCRMastermixfor probe assays(Eurogentec)for the RT-PCR wasperformedusingMESA SYBRGreenqPCRMastermixPlus(Eurogen Real time(RT)-PCR

an the following Biosystems) under (Applied Detector Sequence ABI PRISM7500 target – Ct

50°C for 2 minutes, 95°C for 10 minutes, followed by 50°C for2minutes,95°C10followed β-2-microglobulin 2 ). and Δ(ΔCt) = ΔCt

1 minute. et al 27 . 1% - ΔCt 21% .

40 cyclesat95°C -Δ(ΔCt) , where ΔCt - - - Endothelial Gene Expression During Chronic Hypoxia 201 ratio) was determined using the Agilent 2100 bioanalyzer. Up to 500 ng of total RNA from both sample and common reference were amplified for one round using the MessageAmp II aRNA Amplification Kit (Applied Biosystems/Ambion, Foster City, CA, USA). Aminoallyl-modified amplified RNA (aRNA) was generated using In Vitro Transcription in combination with the Amino Allyl cDNA Labeling kit (Applied Bio- systems/Ambion). The purified aminoallyl-aRNA samples were then labeled with mono-reactive cyanine dyes (samples Cy5, common reference Cy3; GE Healthcare, Uppsala, Sweden). RNA concentration as well as dye incorporation was measured using the Nanodrop 1000. Equal amounts of labeled aRNA (500 ng) were hybridized to the Agilent 44K whole microarray chips according to manufactu- rer’s protocol.

Statistical Analyses Whole Genome Array gene expression profiling Microarray spot intensities were extracted using Agilent feature extraction software version 8.5 (Agilent Technologies). Intra-array Cy3 and Cy5 intensities were norma- lized using LOWESS, and Cy3/Cy5-ratios were next normalized using inter-array Quantile normalization, both using R/Bioconductor (www.bioconductor.org). Diffe- rential expression in response to the oxygen concentration was assessed by a Bay- esian statistics t-test (Cyber-T)37 and adjusted for multiple testing by using Benjamini and Hochberg’s method38. Genes were considered significant if the q-values were <0.05, thereby controlling the False Discovery Rate (FDR) to be <5%. Identification of specific panels of gene expression in response to different levels of oxygen was done using Spotfire DecisionSite version 9.1 (TIBCO Software Inc, Palo Alto, CA, USA). Differential modulation of specific pathways and transcription factor networks was assessed using Gene Set Enrichment Analysis (GSEA)39, 40 using MSigDB ver- sion 2.5 (http://www.broad.mit.edu/gsea/), and by the MetaCore Systems Biology Suite (GeneGo Inc., St Joseph, MI, USA).

Results and Discussion Hypoxic and normoxic endothelial cell cultures Human endothelial cells (EC) were cultured for prolonged periods (2-4 weeks) in am- bient air atmosphere (21% O2), 5% O2 (physiological normoxia) or 1% O2 (hypoxia). Oxygen concentrations were monitored during the incubation period and remained constant during culture and medium renewal. During the whole incubation period, visual inspection of both HUVEC and hMVEC did not demonstrate apparent diffe- rences under reduced oxygen atmosphere (Fig. 1A) and propagated well (Fig. 1B). 202 Chapter 7 gen. oxy 5% and 21% between expression regulated significantly with probes gene 293 and oxygen, 1% and 5% between probes gene regulated significantly 583 q<0.05), (FDR oxygen 1% and 21% between altered significantly were which probes, gene and Hochberg’smultiple testingbyusingBenjamini method statistics trations wasassessedbyaBayesian concen oxygen different three the between expression gene differential Significant taining ~41,000gene-specific60-mersequences. concentrations (1%, 5%, and 21%O from different atthreedifferentcontinuously for 2-4weeks cultured donors) oxygen cultures (each on fourHUVECandthreehMVECprimaryendothelial performed was analysis expression EC, gene in human tension in oxygen changes longed In order to determine the complete repertoireof genes that are regulatedby pro Microarray analysis obtained withhMVEC fromanotherdonorandHUVEC. time points. During the culture periodthe cells weresubculturedafter days 5, 9 and 19. Similar datawere at various counting by cell determined was of hMVEC 5% CO2). (B)Proliferation with (all atmosphere oxygen 1%, 5%or21% for twoweeksunder cultured ofHUVEC contrast photomicrographs (A) Phase Fig 1.Endothelialcellsduringprolonged exposuretohypoxia Next, these probeswerematchedto unique UniGeneentries and sortedintodistinct tent whenprobedbyindependentRT-PCR analysis(datanotshown). for aselection samples Validationof microarray A 1% oxygen 1% 21% oxygen 21%

of 30genes. ex to asimilar regulated tested were All genes

expression datawasperformedbyRT-PCR oftheoriginal 2 ) using oligonucleotide-based B cells/cm2 10000 15000 20000 25000 30000 35000 40000 5000 0 =0 t= =1 t= t =2 t= -test (Cyber-T) =5 t= =6 t= =7 t= =8 t= =9 t= days 14 E =12 t= 38 =13 t= . We identified 346 identified We . 37 =14 t= andadjustedfor

microarrays con =15 t= =16 t= =19 t= =20 t= =21 t= 14 21% E 14 1% E - - - - - Endothelial Gene Expression During Chronic Hypoxia 203

1.6 A B C D 84 genes 56 genes 28 genes 101 genes

1.4 1.0 1.4

1.4

1.2 0.8 1.2 1.2

1.0 0.6 1.0 1.0

1% 5% 21% 1% 5% 21% 1% 5% 21% 1% 5% 21%

1.1 1.1 E F G H 32 genes 45 genes 31 genes 59 genes

1.4 1.0 1.0 1.0

0.9 0.9

1.2 0.8 0.9 0.8

0.7

0.7 1.0

0.6 0.8 1% 5% 21% 1% 5% 21% 1% 5% 21% 1% 5% 21%

Fig 2. Eight gene expression panels Different patterns were obtained when significant differences were compared between gene expressions of endothelial cells (4 HUVEC, 3 hMVEC of different donors) cultured for 2-4 weeks under 1%, 5% and 21% oxygen atmosphere.

groups based on their regulation at the three oxygen concentrations. This hierarchi- cal clustering resulted in 8 different expression patterns (Fig. 2). Pattern A and E depict 84 and 31 genes that are specifically up- or down-regulated at 1% oxygen respectively. Pattern B and F show all the genes that are differentially regulated at

5% O2 compared to 1% and 21% (56 up, 45 down). The genes which are affected at

21% O2 are depicted in panel C and G (28 up, 31 down). Finally, panel D and H show the number of genes with a gradual O2 concentration dependent expression profile, 101 genes were upregulated at 1% and downregulated at 21% (D), while a total of 59 genes displayed the opposite pattern (H). These data are summarized in Table 1.

Pathway analysis of microarray transcriptomes during chronic hypoxia We utilized the online tools Gene Set Enrichhment Analysis (GSEA) and MetaCore Systems Biology Suite to assign the 435 genes with a single UniGene entry match to particular molecular pathways. With unsupervised pathway analysis, the largest categories of significantly regulated genes in canonical pathways were involved in cytokine/cytokine interactions, glycolysis/glyconeogenesis, MAPK signal- 204 Chapter 7 genase 1β(ADH1B), (PFKP), (LDHA), platelet phosphofructokinase enolase2(ENO2), alcohol dehydro (GPI), mutase1(PGAM1),lactatedehydrogenase phosphoglycerate A (TPI1), aldolase isomerase-1 Aphosphate (ADLOA)and C(ALDOC),glucose triosephophate hypoxia: prolonged by induced significantly being as genes related negative feedback loops of metabolites, microarray analysis identified 11 glycolysis- and positive through at theenzymelevel regulated glycolysis ismainly Although both hypoxiasignalingaswellglycolysis/glyconeogenesis. from 21to1%O upregulation D (gradual those inpanel whereas O displayed that hypoxia-induced genes in panel A (specific upregulation from 5 to 1% panels gene eight individual the of analysis Pathway expression. gene specific type cell and endothelial signaling in hypoxia were involved lated inchemicalpathways of theactincytoskeleton. and regulation ing regu of genes The largestcategories regulated endothelial cell transcripts with our list of 334 genes that were significantly of cells. Comparisonthisjointliterature listof on humanendothelial 836 hypoxia- al. (Ning studies five from generated was genes HIF-2α-regulated and HIF-1α and/or hypoxia of list A refreshing schemes). (CO conditions standard culture maintaining tion, while concentra oxygen to alow exposed of time(uptofourweeks) period a prolonged or HIF-2 of oxygen tensions.Previousstudiesevaluatedhypoxia oroverexpressionHIF-1 published fromallthepreviously The mostimportantfactorthatmakesthisworkdiscriminative Comparison withpreviousmicroarraystudiesonhypoxicendothelialcells reduced found PFKM as the only one of glycolysis-stimulating that was significantly O to 21% as compared kinase (PFKM),by53±23%at1%oxygen,and41±9%5% was reduced ingly,phosphofructo enzymes, muscle glycolysis-stimulating of all onlyoneenzyme in hypoxicEC also induced cells, butapparently in neuronal pressed hypoxia 1A2 (ALDH1A2). on short-term studies with previous agreement close This isin 2 26 ) in clustered predominantlyhypoxia-andHIF1-signaling pathways(seebelow), ), that evaluated short-term (24 hours)effects of hypoxia or HIF overexpression 24, 25, 41 41 a . Itisuncertainwhetherthisrelatedtoanadditionalroleofenzyme. for a period up to 24 hours. Our cells were grown and evaluated during up to24hours.Ourcellsweregrownandevaluated foraperiod in vitro . Furthermore, ENO2and ALDOC areisoformsthat are normallyex studies on hypoxia is the prolonged exposure timetolowered studiesonhypoxiaistheprolonged et al. 2 exposure. Indeed, a recent study on short-term hypoxia also on short-termhypoxia study a recent exposure.Indeed, 24 acylphosphatase 1 (ACYP1), and aldehyde dehydrogenase , Manalo et al. 5 , Chi et al. 25 , Le Jan 2 percentage, humidity and humidity percentage, et al. 2 ) were involved in involved ) were 27 and Takeda 42, 43 . Interest et a ------

Endothelial Gene Expression During Chronic Hypoxia 205

regulated between 1% and 21% O2 displayed an overlap of only 66 genes (19% of our gene set; 8% of literature gene set). The majority of genes of this overlapping population may be designated as ‘common hypoxia genes’, since their expression has been described in a number of cell types exposed to hypoxia. These common hypoxia genes include genes encoding with central roles in metabolism (VLDLR, ENO2, GPI, SLC2A1, PFKP, LDHA, TPI1), angiogenesis (ADM, ANGPTL4, PDGFB, VEGFC, RNASE4, SERPINE1, PLAT), inflammation (IL8, CXCL1, SELE), cell proliferation and apoptosis (DUSP6, CDKN1C, INHBA, BNIP3, CASP1, PA2G4 ), survival (NOTCH4, DLL1), extracellular matrix synthesis (P4HA2, LOX, LOXL2, COL4A1, COL4A2, EFEMP1, BGN), transcription factors (BHLHB2, MXI1, MAFF) and hypoxia signaling (EGLN1, EPAS1).

Transcriptome changes during prolonged versus acute hypoxia It is plausible to assume that the duration of hypoxic exposure is responsible for the apparent differences in gene expression profiles between short-term and prolonged hypoxia. Alternatively, the differences may be explained by overcorrection during statistical analysis, by which only the most significant genes would match, but only a part of the moderately significant genes are recognized at random in various stud- ies. To test these possibilities, new endothelial cell cultures (6 individual HUVEC and 4 hMVEC donors) were exposed to a short (24 hours) and prolonged (2 weeks for

HUVEC 4 weeks for hMVEC) periods of exposure to hypoxia (1% O2) and ambient oxygen concentration (21% O2). Next, 55 genes were analysed by real-time PCR, as summarized in Table 2.

First, we compared fifteen genes which were significantly regulated in our chronic hy- poxic cells with 9 genes overlapping with one or several of the acute hypoxia studies (Fig. 3, upper part). The RT-PCR data of all 15 genes were fully comparable with the microarray data that we obtained and with the literature data, although due to varia- tion between cultures of different donors the data of uPAR and HIF-1α (2 weeks) did not reach significance (Fig. 3; see Table 2 for RT-PCR data). Second, twenty genes were evaluated that were recognized as significantly regulated by hypoxia by one or several of the other published reports on acute hypoxia, but not in our microarray analysis of chronic hypoxia (Fig. 3, lower part). Surprisingly, we found similar re- sponses for 12 of these genes in our chronic hypoxic cultures when compared to the acute hypoxic transcriptomes, including all 9 genes identified by Manaloet al.5 and 4 out of 5 and 3 out of 4 of the genes identified by Takedaet al.26 (all except FLT1) and Le Jan et al.27 (all except PLAT), respectively. Furthermore, 6 genes responding to acute hypoxia as identified by Ning24, and Chi et al.25 (GYS1, CXCL3, CCND, , 206 Chapter 7 are independent experiments. cultured under1% and21%oxygenaregivenin Table 2.ND:too lowfordetection;arrayandRT-PCR (1% oxygen). to 24hoursor2weeksofhypoxia that wereexposed of theRT-PCRThe values dataofcells were comparedwith RT-PCRindependent data obtainedfrom 6 HUVEC cultures(from different donors) et al.25),4(LeJanal.27)and5 (Takedaet al.5),3(Chi 1 (Ningetal.24),2(Manalo et al.26). These data tained on short-term (24 hours)hypoxia(takentogetheras a joint list, and presentedasstudies individual array dataob with previously published hypoxia data (arrayHUVECandhMVEC) onprolonged The effect of hypoxiaongeneexpression35geneswascomparedbetweenthepresentmicroarray Fig 3. N vs Y N Y vsN Yvs E2F6 E2F3 CXCL3 PCNA CCND1 PLAT IGFBP3 DPP3 HK2 GYS1 EDN1 CXCR4 MMP2 COL5A1 MMP10 FLT1 PGF VEGFA EGLN3 HIF3A TFRC HIF1A HMOX1 EPAS1 PLAUR ICAM1 CXCL1 SELE EGLN1 SERPINE1 VEGFC ANGPLT4 LDHA ADM SLC2A1 CyclinD1 tPA Glyc synt ET-1 PlGF PHD-3 Transfer R HO-1 HIF2A PDH-2 PAI-1 Glut1 prolonged array 1% orange: bordelineintherightdirection yellow: notaltered green: reduced red: induced HUVEC RT-PCR variabel HUVEC 24h RT-PCR

1,2,3,4,5 literature joint list 5 excl 2,3,4 1,2,5 1,3,5 2,3,5 3,4 1,4 1,4 3,5 3,4 2,3 3,5 1 5 3 1 1 1 1 1 2 2 1 3 5 5 3 3 3 blue disagreemntwithourdata red agreementwithourdata M: agreementwithMVEC M 1 individual datalit 2 3 M 4 5 - Endothelial Gene Expression During Chronic Hypoxia 207

E2F6 en DPP3) also showed regulation by chronic hypoxia. Third, we extended the RT-PCR analysis with 20 additional genes that were selected as potentially of inter- est in angiogenesis or hypoxia, but which were neither recognized as statistically significant hypoxia-regulated genes in our microarray analysis nor in the published reports on acute hypoxic responses. This analysis identified: 5 genes that were significantly altered both after 24 hours and 2 weeks of hypoxia (HK1, KLF2, BMP4, CXCR4, NQO1), 2 genes only responded to acute hypoxia (PDH1, VEGFR3), and 6 genes only responded to chronic hypoxia (HIF3α4, VEGFR2, CXCL12, CCL2, THBD, RGS5, NOS3). Finally, 6 genes did not respond at all in HUVEC (FIH1, DLL4, NOTCH1, PLAU, MMP14, IGFBP2)

The detailed RT-PCR analysis clearly resolves several issues related to the different transcriptomes reported, as our data point to a considerable underestimation of the number of hypoxia-induced genes identified by the individual microarray analyses. Although different conditions, such as used by Chi et al.25, and Ning et al.24, may contribute to the observed differences, the overall trend indicates a large overlap be- tween our data and those published by Manalo et al.5, as well with those of Takeda et al.26 and le Jan et al.27. More importantly, when we compared the effects of acute ver- sus chronic hypoxia, it became clear that the differences were limited and reflected, if any, a quantitative rather than a qualitative effect. Indeed, only eight genes (PHD3, VEGFC, CCL2, ICAM1, HMOX1, PLAT, NOS3, E2F3) responded specifically to the duration of hypoxia, based on statistical analysis (Table 2).

Comparison of hMVEC with HUVEC responses In parallel, hMVEC (4 primary isolates from different donors) were cultured under the same conditions, i.e. ambient air (21% O2) and 1% O2 for 24 hours and 2 weeks. mRNA levels of the same set of genes were determined by RT-PCR (Table 3). In general, the data on hMVEC support the data on HUVEC with a few exceptions. Interestingly, CCND1 (cyclin D1) identified by Chi et al.25 and Ning et al.24 (compare Table 3 and Fig. 3) was downregulated in hMVEC but not HUVEC, which may point to a cell-type specific regulation of this gene. When comparing the 24 hours hypoxia with 2 weeks of hypoxia, again only, 7 genes responded differently (PHD3, DLL4, RGS5, EDN1, CXCL1, CXCL3, IGFBP3), of which only PHD3 overlapped with the HUVEC set. Of particular interest are the acute responses of delta-like ligand 4 (DLL4), endothelin 1 (EDN1) and CXCL3, which peaked around 24 hours after hy- poxia and returned almost to baseline after chronic hypoxia. DLL4 may be involved in modulating angiogenesis44-46, but requires further study for a functional interpre- tation. Endothelin-1 is a well known target of HIF and hypoxia47. An increased endo- 208 Chapter 7 ANKRD37 ANGPTL4 ANG AKAP12 ADSSL1 ADM GENE SYMBOL Table 1-A VLDLR VEGFC TUSC3 TUBB2B TPM1 TPI1 TNR TMCC1 TM2D1 TLR7 SYTL2 SSX5 SPRY1 SPAG4 SOD2 SLC6A7 SLC39A13 SLC2A1 SLC16A3 SGCE SERPINE1 SELE RHBDF2 RGNEF PYCARD PTPRF PTPRB PTENP1 PSMAL PPARG PLOD1 PIM1 PHF10 PFKP PDIA5 PDGFB PDE1C PAWR P4HA2 P4HA1 P2RX4 NOX4 NAALADL1 N4BP3 MYH9 MIF MGLL LOC158014 LOC154761 LEPROTL1 JUB INHBA HYI HSD17B2 GLT8D1 GALNTL2 FOLH1 F13B ERO1L EGLN1 DNAJC18 DLC1 DDIT4 CYR61 CXCL14 CTSZ CTBS COL4A2 COL4A1 CCDC74B CCDC74A C1orf86 C10orf9 BNIP3 BLCAP BCAR1 BACH1 ARRDC3 very lowdensitylipoproteinreceptor vascular endothelialgrowthfactorC tumor suppressorcandidate3 tubulin, beta2B tropomyosin 1(alpha) triosephosphate isomerase1 tenascin R(restrictin,janusin) transmembrane andcoiled-coildomainfamily1 TM2 domaincontaining1 toll-like receptor7 synaptotagmin-like 2 synovial sarcoma,Xbreakpoint5 sprouty homolog1,antagonistofFGFsignaling(Drosophila) sperm associatedantigen4 superoxide dismutase2,mitochondrial member 7 solute carrierfamily6(neurotransmittertransporter, L-proline), solute carrierfamily39(zinctransporter),member13 solute carrierfamily2(facilitatedglucosetransporter),member1 transporter 4) solute carrierfamily16,member3(monocarboxylicacid sarcoglycan, epsilon inhibitor type1),member1 serpin peptidaseinhibitor, cladeE(nexin,plasminogenactivator selectin E(endothelialadhesionmolecule1) rhomboid 5homolog2(Drosophila) Rho- nucleotideexchangefactor PYD andCARDdomaincontaining protein tyrosinephosphatase,receptortype,F protein tyrosinephosphatase,receptortype,B cancers 1),pseudogene1 phosphatase andtensinhomolog(mutatedinmultipleadvanced growth-inhibiting protein26 peroxisome proliferativeactivatedreceptor, gamma procollagen-lysine 1,2-oxoglutarate5-dioxygenase1 pim-1 oncogene PHD fingerprotein10 phosphofructokinase, platelet protein disulfideisomerasefamily A,member5 viral (v-sis)oncogenehomolog) platelet-derived growthfactorbetapolypeptide(simiansarcoma phosphodiesterase 1C,calmodulin-dependent70kDa PRKC, apoptosis,WT1,regulator 4-hydroxylase), alphapolypeptideII procollagen-proline, 2-oxoglutarate4-dioxygenase(proline 4-hydroxylase), alphapolypeptideI procollagen-proline, 2-oxoglutarate4-dioxygenase(proline purinergic receptorP2X,ligand-gatedionchannel,4 NADPH oxidase4 N-acetylated alpha-linkedacidicdipeptidase-like1 Nedd4 bindingprotein3 myosin, heavychain9,non-muscle factor) macrophage migrationinhibitoryfactor(glycosylation-inhibiting monoglyceride lipase hypothetical LOC158014 hypothetical LOC154761 leptin receptoroverlappingtranscript-like1 jub, ajubahomolog(Xenopuslaevis) inhibin, beta A (activin A, activin AB alphapolypeptide) hydroxypyruvate isomerasehomolog(E.coli) hydroxysteroid (17-beta)dehydrogenase2 glycosyltransferase 8domaincontaining1 saminyltransferase-like 2 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalacto folate (prostate-specificmembraneantigen)1 coagulation factorXIII,Bpolypeptide ERO1-like (S.cerevisiae) egl ninehomolog1(C.elegans) DnaJ (Hsp40)homolog,subfamilyC,member18 deleted inlivercancer1 DNA-damage-inducible transcript4 cysteine-rich, angiogenicinducer, 61 chemokine (C-X-Cmotif)ligand14 cathepsin Z chitobiase, di-N-acetyl- collagen, typeIV, alpha2 collagen, typeIV, alpha1 coiled-coil domaincontaining74B coiled-coil domaincontaining74A 1openreadingframe86 chromosome 10openreadingframe9 BCL2/adenovirus E1B19kDainteractingprotein3 bladder cancerassociatedprotein breast canceranti-estrogenresistance1 factor 1 BTB andCNChomology1,basicleucinezippertranscription arrestin domaincontaining3 ankyrin repeatdomain37 angiopoietin-like 4 angiogenin, ribonuclease,RNase A family, 5 A kinase(PRKA)anchorprotein(gravin)12 adenylosuccinate synthaselike1 adrenomedullin OFFICIAL NAME(EntrezGene) FOLD 1.068 1.309 1.553 1.138 1.094 1.078 1.101 1.153 1.137 1.077 1.086 1.101 1.193 1.167 1.182 1.173 1.170 1.131 1.100 1.174 1.181 1.170 1.126 1.146 1.222 1.143 1.241 1.085 1.384 1.219 1.094 1.121 1.091 1.066 1.131 1.076 1.101 1.098 1.054 1.120 1.136 1.269 1.243 1.108 1.107 1.139 1.217 1.143 1.298 1.306 1.381 1.048 1.200 1.177 1.380 1.187 1.181 1.164 1.098 1.129 1.121 1.073 1.056 1.172 1.077 1.186 1.131 1.195 1.165 1.077 1.465 1.192 1.096 1.205 1.248 1.122 1.085 1.082 1.093 1.167 1.116 1.110 1.111 1.111 21 vs.1 BAYES (p) < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.007 0.018 0.001 0.035 0.039 0.007 0.038 0.006 0.010 0.000 0.002 0.010 0.005 0.005 0.043 0.001 0.008 0.000 0.039 0.005 0.002 0.001 0.000 0.010 0.008 0.012 0.001 0.010 0.030 0.035 0.021 0.023 0.003 0.000 0.024 0.003 0.034 0.022 0.002 0.010 0.046 0.014 0.019 0.002 0.019 0.019 0.003 0.004 0.024 0.002 0.000 0.036 0.038 0.001 0.004 0.005 0.002 0.003 0.000 0.008 0.003 0.001 0.026 0.003 0.007 0.002 0.004 0.001 0.021 0.017 0.000 0.012 0.021 0.001 0.005 0.011 0.011 0.011 FOLD -1.021 -1.004 -1.061 -1.016 -1.002 -1.052 -1.012 -1.063 -1.014 -1.043 -1.032 -1.026 -1.021 -1.004 -1.009 -1.029 -1.004 -1.010 -1.022 -1.013 -1.034 -1.035 -1.074 -1.019 -1.050 -1.040 -1.026 -1.021 -1.022 -1.043 -1.020 -1.035 -1.012 -1.023 -1.011 1.009 1.009 1.004 1.026 1.053 1.045 1.036 1.026 1.048 1.064 1.014 1.031 1.028 1.036 1.055 1.052 1.066 1.025 1.015 1.030 1.020 1.006 1.021 1.031 1.025 1.016 1.010 1.015 1.002 1.150 1.012 1.010 1.027 1.008 1.032 1.025 1.067 1.032 1.025 1.038 1.034 1.000 1.004 1.010 1.004 1.011 1.011 1.011 1.011 21 vs.5 BAYES (p) 0.539 0.899 0.856 0.178 0.796 0.680 0.945 0.920 0.595 0.288 0.422 0.266 0.264 0.408 0.768 0.402 0.220 0.207 0.809 0.777 0.590 0.602 0.452 0.183 0.254 0.209 0.441 0.625 0.576 0.322 0.697 0.446 0.579 0.883 0.654 0.880 0.860 0.474 0.449 0.794 0.904 0.554 0.618 0.626 0.897 0.593 0.841 0.789 0.693 0.372 0.354 0.948 0.352 0.318 0.667 0.715 0.804 0.758 0.471 0.308 0.808 0.371 0.509 0.482 0.728 0.672 0.202 0.483 0.696 0.492 0.591 0.334 0.529 0.772 0.824 0.796 0.479 0.909 0.997 0.955 0.613 0.740 0.698 0.892 FOLD 1.070 1.122 1.141 1.091 1.056 1.107 1.205 1.190 1.166 1.068 1.124 1.161 1.215 1.220 1.288 1.339 1.063 1.168 1.136 1.308 1.128 1.108 1.159 1.201 1.126 1.102 1.095 1.088 1.096 1.060 1.150 1.070 1.197 1.107 1.229 1.153 1.081 1.421 1.164 1.079 1.217 1.275 1.136 1.075 1.066 1.130 1.208 1.066 1.406 1.351 1.159 1.081 1.068 1.089 1.123 1.195 1.069 1.130 1.129 1.156 1.192 1.154 1.100 1.134 1.156 1.073 1.131 1.232 1.131 1.139 1.133 1.246 1.183 1.256 1.084 1.378 1.247 1.083 1.109 1.114 1.116 1.116 1.110 1.112 5 vs.1 BAYES (p) < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.004 0.026 0.002 0.041 0.010 0.020 0.022 0.039 0.001 0.010 0.001 0.038 0.018 0.017 0.029 0.001 0.000 0.000 0.008 0.014 0.006 0.015 0.025 0.033 0.007 0.007 0.010 0.005 0.048 0.048 0.022 0.045 0.042 0.007 0.001 0.009 0.007 0.025 0.001 0.049 0.015 0.005 0.039 0.016 0.029 0.002 0.032 0.003 0.014 0.000 0.043 0.006 0.004 0.001 0.048 0.012 0.001 0.003 0.002 0.018 0.038 0.002 0.006 0.035 0.047 0.008 0.012 0.003 0.022 0.013 0.001 0.027 0.012 0.007 0.040 0.003 0.004 0.011 0.011 0.011 Endothelial Gene Expression During Chronic Hypoxia 209

Table 1-B 21 vs. 1 21 vs. 5 5 vs. 1 GENE SYMBOL OFFICIAL NAME (EntrezGene) FOLD BAYES (p) FOLD BAYES (p) FOLD BAYES (p) ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 -1.023 0.735 1.295 0.027 -1.325 0.021 ADAMTS9 ADAM metallopeptidase with thrombospondin type 1 motif, 9 1.025 0.447 1.095 0.015 -1.069 0.022 ADK adenosine kinase 1.038 0.332 1.147 0.002 -1.105 0.004 AFG3L2 AFG3 ATPase family gene 3-like 2 (yeast) 1.007 0.843 1.082 0.039 -1.075 0.019 ALDH1A2 aldehyde dehydrogenase 1 family, member A2 -1.034 0.348 1.162 0.030 -1.201 0.009 ARNTL2 aryl hydrocarbon receptor nuclear translocator-like 2 1.015 0.641 1.088 0.012 -1.071 0.039 ARRDC4 arrestin domain containing 4 -1.033 0.625 1.208 0.043 -1.248 0.015 BCL2A1 BCL2-related protein A1 1.098 0.135 1.436 0.000 -1.309 0.002 CASC5 cancer susceptibility candidate 5 1.005 0.919 1.126 0.045 -1.119 0.032 CEP170 centrosomal protein 170kDa 1.048 0.495 1.163 0.023 -1.109 0.025 CEP192 centrosomal protein 192kDa -1.011 0.778 1.096 0.024 -1.107 0.007 CIT citron (rho-interacting, serine/threonine kinase 21) 1.001 0.991 1.132 0.011 -1.132 0.002 COL17A1 collagen, type XVII, alpha 1 -1.005 0.887 1.109 0.006 -1.114 0.001 CTPS CTP synthase -1.013 0.655 1.083 0.031 -1.097 0.011 DMXL2 Dmx-like 2 -1.005 0.860 1.071 0.027 -1.077 0.016 FEZ2 fasciculation and elongation protein zeta 2 (zygin II) -1.001 0.962 1.068 0.043 -1.069 0.016 GLUD1 glutamate dehydrogenase 1 1.012 0.750 1.093 0.024 -1.081 0.030 GLUD2 glutamate dehydrogenase 2 1.001 0.982 1.080 0.033 -1.079 0.012 GRB14 growth factor receptor-bound protein 14 1.024 0.586 1.277 0.009 -1.247 0.016 GSR glutathione reductase -1.008 0.835 1.097 0.027 -1.106 0.008 HERC4 hect domain and RLD 4 -1.005 0.876 1.074 0.046 -1.080 0.019 HMGA2 high mobility group AT-hook 2 1.037 0.372 1.116 0.012 -1.076 0.043 HMGB2 high-mobility group box 2 1.011 0.845 1.164 0.028 -1.152 0.022 HSPA4L heat shock 70kDa protein 4-like -1.041 0.381 1.135 0.019 -1.182 0.002 IARS isoleucine-tRNA synthetase -1.004 0.913 1.073 0.044 -1.077 0.027 IGF2BP3 insulin-like growth factor 2 mRNA binding protein 3 1.023 0.720 1.169 0.032 -1.143 0.028 KNTC1 kinetochore associated 1 -1.003 0.926 1.122 0.011 -1.125 0.007 LTB4DH leukotriene B4 12-hydroxydehydrogenase 1.047 0.351 1.180 0.007 -1.126 0.034 MCFD2 multiple coagulation factor deficiency 2 1.005 0.882 1.083 0.030 -1.077 0.016 MGST1 microsomal glutathione S- 1 1.043 0.549 1.248 0.021 -1.197 0.038 NAV3 neuron navigator 3 1.030 0.496 1.177 0.016 -1.144 0.040 NQO1 NAD(P)H dehydrogenase, quinone 1 -1.028 0.547 1.134 0.018 -1.166 0.003 PA2G4 proliferation-associated 2G4, 38kDa -1.001 0.986 1.102 0.031 -1.103 0.007 PAQR3 progestin and adipoQ receptor family member III -1.042 0.523 1.140 0.022 -1.188 0.005 PLAT plasminogen activator, tissue -1.088 0.253 1.186 0.047 -1.291 0.001 phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange PREX1 factor 1 -1.005 0.905 1.118 0.037 -1.124 0.019 PRKAR2B protein kinase, cAMP-dependent, regulatory, type II, beta -1.008 0.811 1.135 0.010 -1.144 0.003 PSD3 pleckstrin and Sec7 domain containing 3 1.002 0.938 1.082 0.019 -1.079 0.026 PTGER4 prostaglandin E receptor 4 (subtype EP4) -1.001 0.977 1.095 0.034 -1.096 0.030 PTPN22 protein tyrosine phosphatase, non-receptor type 22 (lymphoid) 1.009 0.732 1.077 0.024 -1.067 0.039 phosphorylase, glycogen; liver (Hers disease, glycogen storage PYGL disease type VI) 1.050 0.249 1.137 0.004 -1.082 0.026 RRM2B ribonucleotide reductase M2 B (TP53 inducible) 1.004 0.913 1.147 0.030 -1.142 0.032 SLC20A1 solute carrier family 20 (phosphate transporter), member 1 1.003 0.918 1.105 0.010 -1.102 0.006 SLC27A5 solute carrier family 27 (fatty acid transporter), member 5 -1.009 0.708 1.068 0.036 -1.078 0.018 SLCO4A1 solute carrier organic anion transporter family, member 4A1 -1.032 0.420 1.085 0.029 -1.120 0.002 SRPX sushi-repeat-containing protein, X-linked 1.056 0.073 1.131 0.002 -1.071 0.043 SRXN1 sulfiredoxin 1 homolog (S. cerevisiae) 1.001 0.967 1.078 0.032 -1.077 0.032 TARBP1 Tar (HIV-1) RNA binding protein 1 1.016 0.595 1.086 0.025 -1.068 0.050 TncRNA non-protein coding RNA 84 -1.045 0.199 1.120 0.031 -1.169 0.004 TRAP1 TNF receptor-associated protein 1 -1.000 0.992 1.101 0.036 -1.102 0.010 TRIM14 tripartite motif-containing 14 1.008 0.844 1.123 0.005 -1.114 0.002 TUBGCP5 tubulin, gamma complex associated protein 5 1.011 0.695 1.079 0.014 -1.068 0.015 TXNIP thioredoxin interacting protein -1.067 0.594 1.298 0.003 -1.385 0.003 USP13 ubiquitin specific peptidase 13 (isopeptidase T-3) 1.004 0.888 1.076 0.050 -1.072 0.043 YES1 v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 -1.018 0.520 1.123 0.022 -1.143 0.009 ZNF185 protein 185 (LIM domain) 1.045 0.542 1.206 0.020 -1.155 0.023 210 Chapter 7 ZNF167 TMTC1 TMOD1 SRP14 SLC26A4 PSCD3 PRKCZ PIK3R3 NPAS4 MGP MGC7036 LOC641467 LAPTM4A IFIT2 HIST1H4F GJA4 GDF3 FZD2 FLRT2 FGF8 FAM118B DAB2 CYSLTR2 CLU ATP1B3 ARL4A ADI1 ABLIM1 GENE SYMBOL Table 1-C H1F0 GPNMB GPI GNA14 GAPDHS FAIM ERGIC1 ENO2 ENO1B DUSP6 DULLARD CTHRC1 CSNK1E CSF2 COPZ2 CD200R1 CCDC88 CAV2 CASP1 CARS C5orf23 C3orf22 BHLHB2 BANP BAG1 BACE2 ASGR1 ANKZF1 ALLC ALDOC ALDOAP2 ALDOA AKT1 AK3L1 ADH1B ADAM23 ACIN1 SYMBOL GENE Table 1-D H1 histonefamily, member0 glycoprotein (transmembrane) nmb glucose phosphateisomerase guanine nucleotidebindingprotein(Gprotein),alpha14 glyceraldehyde-3-phosphate dehydrogenase,spermatogenic Fas apoptoticinhibitorymolecule endoplasmic reticulum-golgiintermediatecompartment (ERGIC)1 enolase 2(gamma,neuronal) enolase alpha,lung-specific dual specificityphosphatase6 dullard homolog(Xenopuslaevis) collagen triplehelixrepeatcontaining1 casein kinase1,epsilon colony stimulatingfactor2(granulocyte-macrophage) coatomer proteincomplex,subunitzeta2 CD200 receptor1 coiled-coil domaincontaining88 caveolin 2 caspase 1,apoptosis-relatedcysteinepeptidase(interleukinbeta,convertase) cysteinyl-tRNA synthetase chromosome 5openreadingframe23 chromosome 3openreadingframe22 basic helix-loop-helixdomaincontaining,classB,2 BTG3 associatednuclearprotein BCL2-associated athanogene beta-site APP-cleaving enzyme2 asialoglycoprotein receptor1 ankyrin repeatandzincfingerdomaincontaining1 allantoicase aldolase C,fructose-bisphosphate aldolase A, fructose-bisphosphatepseudogene2 fructose-bisphosphate aldolase A, v-akt murinethymomaviraloncogenehomolog1 adenylate kinase3-like1 alcohol dehydrogenaseIB(classI),betapolypeptide ADAM metallopeptidasedomain23 apoptotic chromatincondensationinducer1 OFFICIAL NAME(EntrezGene) zinc fingerprotein167 transmembrane andtetratricopeptiderepeatcontaining1 tropomodulin 1 signal recognitionparticle14kDa(homologous Alu RNA bindingprotein) solute carrierfamily26,member4 cytohesin 3 protein kinaseC,zeta phosphoinositide-3-kinase, regulatorysubunit3(gamma) neuronal PAS domainprotein4 matrix Glaprotein Rab interactinglysosomalprotein-like2 hypothetical LOC641467 lysosomal proteintransmembrane4alpha interferon-induced proteinwithtetratricopeptiderepeats2 histone cluster1,H4f gap junctionprotein,alpha4,37kDa growth differentiation factor3 frizzled homolog2(Drosophila) fibronectin leucinerichtransmembraneprotein2 fibroblast growthfactor8(androgen-induced) family withsequencesimilarity118, memberB disabled homolog2,mitogen-responsivephosphoprotein(Drosophila) cysteinyl leukotrienereceptor2 clusterin ATPase, Na+/K+transporting,beta3polypeptide ADP-ribosylation factor-like4A acireductone dioxygenase1 actin bindingLIMprotein1 OFFICIAL NAME(EntrezGene) FOLD -1.098 -1.129 -1.202 -1.091 -1.087 -1.099 -1.099 -1.062 -1.419 -1.087 -1.095 -1.069 -1.086 -1.238 -1.448 -1.100 -1.147 -1.069 -1.064 -1.100 -1.134 -1.327 -1.061 -1.128 -1.096 -1.198 -1.113 -1.114 FOLD 21 vs.1 1.140 1.076 1.061 1.104 1.106 1.075 1.055 1.124 1.160 1.080 1.399 1.055 1.158 1.086 1.084 1.097 1.075 1.068 1.078 1.079 1.053 1.125 1.062 1.060 1.127 1.089 1.108 1.084 1.075 1.228 1.074 1.126 1.349 1.076 1.087 1.076 1.119 21 vs.1 BAYES (p) 0.000 0.039 0.002 0.025 0.008 0.013 0.001 0.004 0.022 0.009 0.048 0.028 0.031 0.003 0.034 0.035 0.021 0.002 0.002 0.017 0.021 0.001 0.001 0.018 0.012 0.048 0.004 0.036 BAYES (p) 0.012 0.029 0.036 0.015 0.008 0.021 0.034 0.004 0.016 0.024 0.047 0.000 0.038 0.040 0.026 0.035 0.017 0.024 0.026 0.020 0.037 0.037 0.019 0.035 0.016 0.028 0.038 0.035 0.038 0.020 0.000 0.032 0.042 0.000 0.038 0.048 0.021 FOLD -1.455 -1.096 -1.069 -1.077 -1.091 -1.237 -1.323 -1.164 -1.164 -1.085 -1.073 -1.138 -1.466 -1.061 -1.180 -1.073 -1.224 -1.106 -1.153 -1.200 -1.078 -1.099 -1.135 -1.101 -1.064 -1.090 -1.110 -1.110 FOLD 21 vs.5 1.075 1.028 1.034 1.060 1.056 1.019 1.035 1.057 1.095 1.044 1.021 1.178 1.010 1.145 1.037 1.008 1.047 1.046 1.032 1.030 1.028 1.015 1.052 1.037 1.020 1.023 1.042 1.024 1.013 1.028 1.122 1.034 1.080 1.080 1.013 1.029 1.028 21 vs.5 BAYES (p) BAYES (p) 0.000 0.032 0.036 0.026 0.033 0.003 0.002 0.027 0.001 0.007 0.016 0.022 0.020 0.004 0.009 0.046 0.004 0.017 0.001 0.019 0.003 0.014 0.017 0.003 0.043 0.039 0.014 0.011 0.121 0.339 0.265 0.125 0.171 0.624 0.141 0.126 0.061 0.468 0.569 0.130 0.730 0.090 0.351 0.841 0.215 0.170 0.364 0.398 0.378 0.519 0.303 0.257 0.430 0.687 0.317 0.645 0.695 0.235 0.050 0.371 0.184 0.038 0.727 0.570 0.432 FOLD -1.024 -1.021 -1.001 -1.095 -1.021 -1.002 -1.013 -1.046 1.026 1.009 1.007 1.022 1.059 1.015 1.015 1.008 1.009 1.004 1.105 1.000 1.046 1.022 1.007 1.022 1.010 1.033 1.002 1.027 FOLD 1.061 1.046 1.026 1.042 1.048 1.055 1.020 1.058 1.026 1.058 1.188 1.045 1.012 1.048 1.076 1.048 1.027 1.035 1.047 1.049 1.038 1.069 1.024 1.039 1.102 1.045 1.082 1.070 1.045 1.094 1.039 1.043 1.249 1.062 1.056 1.047 1.111 5 vs.1 5 vs.1 BAYES (p) BAYES 0.588 0.815 0.825 0.386 0.446 0.971 0.150 0.094 0.760 0.658 0.743 0.792 0.922 0.147 0.999 0.276 0.480 0.624 0.817 0.586 0.970 0.667 0.794 0.463 0.957 0.233 0.375 0.511 (p) 0.234 0.192 0.331 0.321 0.200 0.145 0.378 0.144 0.327 0.135 0.051 0.105 0.897 0.216 0.074 0.292 0.242 0.230 0.226 0.184 0.383 0.078 0.074 0.304 0.068 0.121 0.072 0.191 0.421 0.000 0.067 0.237 0.158 0.112 0.118 0.113 0.114 Endothelial Gene Expression During Chronic Hypoxia 211

Table 1-D HAO2 hydroxyacid oxidase 2 (long chain) 1.116 0.035 1.042 0.268 1.072 0.183 HOXA4 A4 1.065 0.047 1.047 0.230 1.017 0.658 HOXB3 homeobox B3 1.074 0.013 1.028 0.333 1.044 0.128 HTATIP2 HIV-1 Tat interactive protein 2, 30kDa 1.140 0.001 1.065 0.074 1.071 0.070 ICAM1 intercellular adhesion molecule 1 (CD54), human rhinovirus receptor 1.243 0.002 1.122 0.353 1.107 0.359 IL8 interleukin 8 1.292 0.041 1.289 0.087 1.002 0.988 IQCA IQ motif containing with AAA domain 1.173 0.014 1.068 0.232 1.098 0.153 KLF7 Kruppel-like factor 7 (ubiquitous) 1.077 0.035 1.029 0.474 1.046 0.207 LOC158402 hypothetical protein LOC158402 1.065 0.038 1.014 0.654 1.050 0.132 LOC283663 hypothetical LOC283663 1.121 0.027 1.091 0.125 1.027 0.656 LOC286016 hypothetical protein LOC286016, triosephosphate isomerase 1 pseudogene 1.123 0.013 1.051 0.248 1.069 0.139 LOC338579 hypothetical protein LOC338579 1.076 0.046 1.059 0.137 1.016 0.686 LOC375295 hypothetical protein LOC375295 1.181 0.013 1.095 0.202 1.079 0.239 LOC401152 chromosome 4 open reading frame 3 1.055 0.046 1.003 0.896 1.051 0.076 LOX lysyl oxidase 1.186 0.002 1.068 0.221 1.111 0.054 LOXL2 lysyl oxidase-like 2 1.130 0.002 1.076 0.091 1.050 0.266 MAFF v- musculoaponeurotic fibrosarcoma oncogene homolog F (avian) 1.168 0.005 1.065 0.320 1.097 0.115 MAMDC2 MAM domain containing 2 1.180 0.045 1.098 0.188 1.074 0.370 MAML3 mastermind-like 3 (Drosophila) 1.070 0.040 1.008 0.802 1.062 0.064 MAPK13 mitogen-activated protein kinase 13 1.067 0.017 1.030 0.341 1.036 0.193 MDK midkine (neurite growth-promoting factor 2) 1.126 0.009 1.056 0.250 1.067 0.115 MTHFD1L methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like 1.113 0.005 1.068 0.108 1.042 0.205 MXI1 MAX interactor 1 1.078 0.021 1.016 0.605 1.062 0.092 MYL9 myosin, light chain 9, regulatory 1.176 0.005 1.040 0.574 1.131 0.063 NEDD9 neural precursor cell expressed, developmentally down-regulated 9 1.158 0.026 1.061 0.347 1.091 0.118 NFE2L3 nuclear factor (erythroid-derived 2)-like 3 1.146 0.005 1.079 0.099 1.062 0.169 NOB1 NIN1/RPN12 binding protein 1 homolog (S. cerevisiae) 1.074 0.028 1.039 0.260 1.034 0.229 NOTCH4 Notch homolog 4 (Drosophila) 1.107 0.011 1.061 0.115 1.043 0.325 OK/SW-CL.92 - 1.106 0.049 1.060 0.154 1.043 0.439 OSBPL10 oxysterol binding protein-like 10 1.092 0.016 1.041 0.169 1.049 0.193 PBX2 pre-B-cell leukemia transcription factor 2 1.057 0.047 1.015 0.632 1.042 0.202 PCK2 phosphoenolpyruvate carboxykinase 2 (mitochondrial) 1.106 0.049 1.077 0.189 1.027 0.569 PCSK1 proprotein convertase subtilisin/kexin type 1 1.140 0.005 1.044 0.201 1.092 0.065 PDK1 pyruvate dehydrogenase kinase, isozyme 1 1.079 0.019 1.054 0.109 1.024 0.395 PGAM1 phosphoglycerate mutase 1 (brain) 1.094 0.006 1.035 0.228 1.056 0.064 PHGDH phosphoglycerate dehydrogenase 1.184 0.001 1.094 0.167 1.082 0.206 PLAC8 placenta-specific 8 1.239 0.041 1.064 0.442 1.164 0.141 PLD3 phospholipase D family, member 3 1.091 0.045 1.045 0.386 1.044 0.395 PPP1R13L protein phosphatase 1, regulatory (inhibitor) subunit 13 like 1.100 0.025 1.002 0.973 1.098 0.056 PPP4R1 protein phosphatase 4, regulatory subunit 1 1.082 0.022 1.038 0.274 1.042 0.142 PROM1 prominin 1 1.112 0.044 1.007 0.846 1.105 0.056 RASSF8 Ras association (RalGDS/AF-6) domain family 8 1.108 0.033 1.071 0.142 1.034 0.435 RIPK3 receptor-interacting serine-threonine kinase 3 1.139 0.008 1.082 0.159 1.053 0.347 RNASE4 ribonuclease, RNase A family, 4 1.101 0.011 1.022 0.538 1.076 0.058 RPL29P2 ribosomal protein L29 pseudogene 2 1.052 0.043 1.036 0.173 1.015 0.576 SEC24B SEC24 related gene family, member B (S. cerevisiae) 1.072 0.047 1.056 0.107 1.016 0.538 SHB Src homology 2 domain containing adaptor protein B 1.061 0.039 1.011 0.682 1.049 0.110 SHMT2 serine hydroxymethyltransferase 2 (mitochondrial) 1.099 0.008 1.063 0.078 1.034 0.313 SLC5A3 solute carrier family 5 (inositol transporters), member 3 1.201 0.049 1.046 0.255 1.148 0.138 SPAG16 sperm associated antigen 16 1.068 0.020 1.040 0.177 1.027 0.239 STC2 stanniocalcin 2 1.294 0.003 1.101 0.415 1.175 0.122 TACSTD1 tumor-associated calcium signal transducer 1 1.072 0.043 1.050 0.163 1.021 0.446 TBX3 T-box 3 (ulnar mammary syndrome) 1.071 0.047 1.059 0.219 1.012 0.795 TCTEX1D1 Tctex1 domain containing 1 1.093 0.025 1.071 0.122 1.021 0.654 TGFA transforming growth factor, alpha 1.240 0.022 1.064 0.470 1.165 0.131 TGFB1I1 transforming growth factor beta 1 induced transcript 1 1.098 0.049 1.050 0.319 1.046 0.371 TSNAX translin-associated factor X 1.079 0.027 1.061 0.091 1.018 0.634 TUSC1 tumor suppressor candidate 1 1.068 0.035 1.021 0.459 1.046 0.110 TXNRD2 thioredoxin reductase 2 1.074 0.027 1.010 0.735 1.063 0.061 UGCG UDP-glucose ceramide glucosyltransferase 1.166 0.026 1.061 0.260 1.099 0.072 ULBP1 UL16 binding protein 1 1.109 0.009 1.044 0.222 1.062 0.069 VWCE von Willebrand factor C and EGF domains 1.076 0.030 1.075 0.121 1.001 0.982 WDR54 WD repeat domain 54 1.087 0.012 1.067 0.085 1.019 0.608 WHDC1L1 WAS protein homology region 2 domain containing 1-like 1 1.184 0.009 1.063 0.337 1.114 0.132 212 Chapter 7 PPAP2B POP1 NANOS1 MYO3A MGAT4A METTL7A MAP7 IL6R HSPA4 HSPA14 HS3ST1 HMOX1 HMGCS1 HIF1A GAGE3 FRY EPAS1 EBP DHCR24 CXCL11 CTSL2 CRTAC1 CEP72 CAB39L BRCA2 GENE SYMBOL Table 1-E ZNF549 YWHAQ TSPAN5 TIMELESS TEX10 TEKT3 STAT1 zinc fingerprotein549 tyrosine 3-monooxygenase/tryptophan5-monooxygenaseactivationprotein,thetapolypeptide tetraspanin 5 timeless homolog(Drosophila) testis expressedsequence10 tektin 3 signal transducerandactivatoroftranscription1,91kDa phosphatidic acidphosphatasetype2B processing ofprecursor1,ribonucleaseP/MRP subunit(S.cerevisiae) nanos homolog1(Drosophila) myosin IIIA mannosyl (alpha-1,3-)-glycoproteinbeta-1,4-N-acetylglucosaminyltransferase,isozyme A methyltransferase like7A microtubule-associated protein7 interleukin 6receptor heat shock70kDaprotein4 heat shock70kDaprotein14 heparan sulfate(glucosamine)3-O-sulfotransferase1 heme oxygenase(decycling)1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase1(soluble) hypoxia-inducible factor1,alphasubunit(basichelix-loop-helixtranscriptionfactor) G antigen3 furry homolog(Drosophila) endothelial PAS domainprotein1 emopamil bindingprotein(sterolisomerase) 24-dehydrocholesterol reductase chemokine (C-X-Cmotif)ligand11 cathepsin L2 cartilage acidicprotein1 centrosomal protein72kDa calcium bindingprotein39-like breast cancer2,earlyonset OFFICIAL NAME(EntrezGene) FOLD -1.077 -1.084 -1.092 -1.207 -1.326 -1.104 -1.142 -1.128 -1.109 -1.172 -1.061 -1.128 -1.165 -1.190 -1.252 -1.047 -1.159 -1.102 -1.206 -1.076 -1.145 -1.068 -1.079 -1.094 -1.101 -1.076 -1.080 -1.082 -1.165 -1.066 -1.193 -1.081 21 vs.1 BAYES (p) 0.047 0.010 0.017 0.030 0.004 0.016 0.009 0.006 0.007 0.001 0.046 0.002 0.018 0.001 0.001 0.040 0.003 0.029 0.032 0.025 0.001 0.050 0.023 0.020 0.037 0.024 0.018 0.036 0.022 0.023 0.003 0.032 FOLD -1.019 -1.001 -1.041 -1.029 -1.029 -1.080 -1.030 -1.027 -1.047 -1.048 -1.077 -1.012 -1.018 -1.005 -1.009 -1.024 -1.005 -1.010 -1.003 -1.028 -1.007 1.079 1.016 1.043 1.059 1.036 1.022 1.063 1.028 1.034 1.043 1.111 21 vs.5 BAYES (p) 0.146 0.507 0.670 0.991 0.669 0.410 0.588 0.455 0.286 0.092 0.281 0.090 0.679 0.665 0.481 0.433 0.263 0.248 0.385 0.722 0.631 0.414 0.873 0.774 0.604 0.892 0.742 0.920 0.535 0.795 0.596 0.424 FOLD -1.162 -1.063 -1.206 -1.273 -1.151 -1.096 -1.174 -1.085 -1.099 -1.253 -1.131 -1.159 -1.195 -1.070 -1.106 -1.172 -1.120 -1.063 -1.125 -1.098 -1.073 -1.084 -1.075 -1.071 -1.069 -1.079 -1.134 -1.058 -1.234 -1.127 -1.110 -1.110 5 vs.1 BAYES (p) 0.004 0.033 0.006 0.019 0.015 0.009 0.039 0.020 0.004 0.034 0.006 0.002 0.033 0.012 0.003 0.019 0.047 0.006 0.012 0.032 0.006 0.005 0.013 0.033 0.037 0.047 0.043 0.039 0.026 0.048 0.002 0.019 Endothelial Gene Expression During Chronic Hypoxia 213

Table 1-F 21 vs. 1 21 vs. 5 5 vs. 1 GENE SYMBOL OFFICIAL NAME (EntrezGene) FOLD BAYES (p) FOLD BAYES (p) FOLD BAYES (p) ADARB1 adenosine deaminase, RNA-specific, B1 (RED1 homolog rat) 1.007 0.770 -1.060 0.050 1.068 0.039 BGN biglycan 1.066 0.370 -1.169 0.012 1.247 0.001 C12orf50 chromosome 12 open reading frame 50 -1.004 0.887 -1.094 0.011 1.089 0.030 C2orf27 open reading frame 27 1.051 0.115 -1.165 0.000 1.225 0.000 C6orf145 chromosome 6 open reading frame 145 1.030 0.250 -1.077 0.033 1.109 0.004 C9orf61 chromosome 9 open reading frame 61 1.025 0.633 -1.113 0.030 1.141 0.021 CCL2 chemokine (C-C motif) ligand 2 1.094 0.424 -1.227 0.035 1.342 0.010 CD24 CD24 molecule -1.041 0.359 -1.162 0.001 1.116 0.024 CDKN1C cyclin-dependent kinase inhibitor 1C (p57, Kip2) -1.003 0.962 -1.262 0.002 1.258 0.000 DNAJB2 DnaJ (Hsp40) homolog, subfamily B, member 2 -1.009 0.783 -1.078 0.028 1.068 0.032 EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 -1.058 0.141 -1.178 0.000 1.114 0.021 EFNA1 ephrin-A1 1.047 0.449 -1.162 0.010 1.217 0.001 FNDC3B fibronectin type III domain containing 3B 1.026 0.450 -1.101 0.012 1.130 0.001 GABARAPL1 GABA(A) receptor-associated protein like 1 1.015 0.596 -1.077 0.026 1.093 0.009 GP1BB glycoprotein Ib (platelet), beta polypeptide -1.030 0.368 -1.113 0.008 1.081 0.032 HOXD1 homeobox D1 1.063 0.146 -1.097 0.013 1.165 0.001 ITM2C integral membrane protein 2C -1.008 0.860 -1.096 0.018 1.088 0.026 KLHL26 kelch-like 26 (Drosophila) -1.005 0.846 -1.086 0.013 1.080 0.018 LAMP1 lysosomal-associated membrane protein 1 1.007 0.889 -1.105 0.039 1.113 0.021 LOC440895 LIM and senescent cell antigen-like domains 3-like 1.059 0.209 -1.123 0.019 1.189 0.006 MAGED2 melanoma antigen family D, 2 -1.012 0.819 -1.119 0.037 1.106 0.004 MGC4677 non-protein coding RNA 152 -1.031 0.297 -1.098 0.010 1.064 0.024 MMP21 matrix metallopeptidase 21 1.027 0.564 -1.096 0.006 1.125 0.025 MSL3L1 male-specific lethal 3-like 1 (Drosophila) 1.007 0.779 -1.078 0.014 1.085 0.004 NNMT nicotinamide N-methyltransferase -1.008 0.836 -1.110 0.036 1.101 0.047 PLEKHO1 pleckstrin homology domain containing, family O member 1 1.071 0.093 -1.100 0.034 1.178 0.001 PLXDC2 plexin domain containing 2 -1.073 0.365 -1.297 0.001 1.209 0.012 PODXL2 podocalyxin-like 2 -1.060 0.268 -1.154 0.021 1.089 0.047 RAB3C RAB3C, member RAS oncogene family -1.083 0.353 -1.240 0.014 1.145 0.025 RGS3 regulator of G-protein signalling 3 -1.012 0.858 -1.242 0.002 1.227 0.000 RHOB ras homolog gene family, member B 1.054 0.256 -1.118 0.038 1.178 0.000 RNF130 ring finger protein 130 1.011 0.684 -1.073 0.043 1.085 0.019 SLC25A29 solute carrier family 25, member 29 -1.044 0.312 -1.130 0.006 1.082 0.039 SNCAIP synuclein, alpha interacting protein (synphilin) 1.014 0.746 -1.122 0.012 1.138 0.002 SOX11 SRY (sex determining region Y)-box 11 1.051 0.181 -1.055 0.046 1.109 0.011 SPSB1 splA/ryanodine receptor domain and SOCS box containing 1 -1.030 0.441 -1.153 0.002 1.119 0.005 TLE2 transducin-like enhancer of split 2 (E(sp1) homolog, Drosophila) 1.031 0.506 -1.119 0.016 1.154 0.000 TMEM132A transmembrane protein 132A -1.025 0.650 -1.149 0.028 1.121 0.027 TMEPAI transmembrane, prostate androgen induced RNA -1.024 0.480 -1.151 0.004 1.124 0.010 TNFRSF21 tumor necrosis factor receptor superfamily, member 21 -1.069 0.180 -1.168 0.007 1.093 0.034 TRIM47 tripartite motif-containing 47 -1.003 0.907 -1.089 0.021 1.086 0.017 TSPAN6 tetraspanin 6 -1.016 0.594 -1.101 0.006 1.084 0.007 VGLL3 vestigial like 3 (Drosophila) 1.004 0.933 -1.090 0.041 1.094 0.034 WIPI1 WD repeat domain, phosphoinositide interacting 1 -1.039 0.236 -1.122 0.002 1.080 0.015 ZNF553 zinc finger protein 553 -1.042 0.329 -1.113 0.010 1.068 0.047 214 Chapter 7 WDR51B TBX4 SPOCD1 RBM3 RASSF4 PRTFDC1 PLAUR PHF11 OVOL1 NFKBIZ NAV1 MYSM1 MTHFD2 MATN3 LOC54103 LOC285216 LDHA JUN IMPDH2 GALNT6 ESD EMILIN2 DARS CXCL6 CXCL2 CXCL1 C14orf94 C10orf59 AP4B1 AEBP1 ACYP1 GENE SYMBOL Table 1-G WD repeatdomain51B T-box 4 SPOC domaincontaining1 RNA bindingmotif(RNP1,RRM)protein3 Ras association(RalGDS/AF-6)domainfamily4 phosphoribosyl transferasedomaincontaining1 plasminogen activator, urokinasereceptor PHD fingerprotein 11 ovo-like 1(Drosophila) nuclear factorofkappalightpolypeptidegeneenhancerinB-cellsinhibitor, zeta neuron navigator1 -like, SWIRMandMPNdomains1 methylenetetrahydrofolate dehydrogenase(NADP+dependent)2,methenyltetrahydrofolatecyclohydrolase 3 matrilin hypothetical proteinLOC54103 hypothetical proteinLOC285216 dehydrogenase A lactate jun oncogene IMP (inosinemonophosphate)dehydrogenase2 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase6(GalNAc-T6) esterase D/formylglutathionehydrolase elastin microfibrilinterfacer2 aspartyl-tRNA synthetase chemokine (C-X-Cmotif)ligand6(granulocytechemotacticprotein2) chemokine (C-X-Cmotif)ligand2 chemokine (C-X-Cmotif)ligand1(melanomagrowthstimulatingactivity, alpha) chromosome 14openreadingframe94 chromosome 10openreadingframe59 adaptor-related proteincomplex4,beta1subunit AE bindingprotein1 acylphosphatase 1,erythrocyte(common)type OFFICIAL NAME(EntrezGene) FOLD 1.245 1.099 1.078 1.147 1.103 1.221 1.190 1.071 1.200 1.083 1.085 1.200 1.257 1.490 1.084 1.134 1.066 1.140 1.082 1.092 1.100 1.108 1.089 1.221 1.131 1.120 1.102 1.078 1.163 1.093 1.115 21 vs.1 BAYES (p) 0.026 0.047 0.008 0.024 0.031 0.000 0.000 0.047 0.017 0.046 0.022 0.005 0.027 0.002 0.004 0.028 0.006 0.038 0.038 0.028 0.047 0.014 0.010 0.038 0.001 0.001 0.010 0.009 0.021 0.001 0.009 FOLD 1.279 1.070 1.145 1.125 1.141 1.137 1.087 1.254 1.130 1.096 1.180 1.154 1.319 1.105 1.125 1.100 1.157 1.100 1.092 1.164 1.155 1.151 1.171 1.135 1.078 1.122 1.070 1.116 1.117 1.112 1.113 21 vs.5 BAYES (p) 0.018 0.025 0.020 0.012 0.025 0.006 0.021 0.024 0.003 0.016 0.013 0.004 0.009 0.030 0.042 0.006 0.007 0.021 0.018 0.021 0.031 0.015 0.001 0.043 0.000 0.008 0.003 0.018 0.003 0.034 0.011 FOLD -1.027 -1.015 -1.020 -1.015 -1.045 -1.031 -1.013 -1.010 -1.019 -1.032 -1.015 -1.017 -1.000 -1.058 -1.003 -1.060 -1.035 -1.014 -1.010 -1.001 1.007 1.002 1.070 1.047 1.017 1.089 1.129 1.008 1.061 1.036 1.021 5 vs.1 BAYES (p) 0.719 0.753 0.778 0.979 0.684 0.082 0.256 0.589 0.492 0.377 0.751 0.670 0.847 0.284 0.381 0.488 0.843 0.410 0.799 0.634 0.993 0.354 0.929 0.060 0.380 0.368 0.759 0.790 0.987 0.446 0.488 Endothelial Gene Expression During Chronic Hypoxia 215

Table 1-H 21 vs. 1 21 vs. 5 5 vs. 1 GENE SYMBOL OFFICIAL NAME (EntrezGene) FOLD BAYES (p) FOLD BAYES (p) FOLD BAYES (p) ABCG2 ATP-binding cassette, sub-family G (WHITE), member 2 -1.119 0.009 -1.082 0.105 -1.034 0.387 ALG10 asparagine-linked glycosylation 10 homolog (yeast, alpha-1,2-glucosyltransferase) -1.126 0.006 -1.064 0.170 -1.058 0.263 ANXA2 annexin A2 -1.078 0.016 -1.018 0.581 -1.059 0.082 AR (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) -1.160 0.022 -1.133 0.086 -1.024 0.704 C20orf152 chromosome 20 open reading frame 152 -1.059 0.039 -1.036 0.188 -1.022 0.486 C20orf160 chromosome 20 open reading frame 160 -1.159 0.043 -1.015 0.825 -1.142 0.081 CAMK2N1 calcium/calmodulin-dependent protein kinase II inhibitor 1 -1.162 0.031 -1.112 0.118 -1.045 0.460 CCRL1 chemokine (C-C motif) receptor-like 1 -1.149 0.044 -1.078 0.325 -1.065 0.317 CLTC clathrin, heavy chain (Hc) -1.073 0.041 -1.043 0.213 -1.029 0.267 CNOT7 CCR4-NOT transcription complex, subunit 7 -1.062 0.037 -1.037 0.218 -1.024 0.397 DDX1 DEAD (Asp-Glu-Ala-Asp) box polypeptide 1 -1.059 0.043 -1.054 0.066 -1.005 0.864 DLL1 delta-like 1 (Drosophila) -1.102 0.038 -1.034 0.513 -1.066 0.109 FLJ21075 hypothetical protein FLJ21075 -1.123 0.028 -1.037 0.362 -1.083 0.081 FLJ34515 hypothetical gene supported by AK091834 -1.092 0.038 -1.044 0.197 -1.046 0.272 FRMD4B FERM domain containing 4B -1.127 0.012 -1.084 0.092 -1.041 0.327 GDF11 growth differentiation factor 11 -1.079 0.039 -1.037 0.310 -1.040 0.255 GMPR reductase -1.079 0.008 -1.039 0.182 -1.038 0.173 HOXB1 homeobox B1 -1.072 0.032 -1.054 0.091 -1.017 0.588 HSPA8 heat shock 70kDa protein 8 -1.065 0.050 -1.023 0.508 -1.041 0.198 IFIT3 interferon-induced protein with tetratricopeptide repeats 3 -1.102 0.010 -1.035 0.393 -1.065 0.067 IHPK1 inositol hexaphosphate kinase 1 -1.066 0.030 -1.050 0.084 -1.016 0.593 KIAA0984 TBC1 domain family, member 30 -1.061 0.042 -1.001 0.980 -1.061 0.050 KIAA1045 KIAA1045 -1.103 0.020 -1.043 0.225 -1.057 0.155 KPNA2 karyopherin alpha 2 (RAG cohort 1, importin alpha 1) -1.120 0.015 -1.023 0.639 -1.095 0.050 KRT18 keratin 18 -1.081 0.042 -1.073 0.069 -1.008 0.830 KRTAP4-4 keratin associated protein 4-4 -1.069 0.043 -1.060 0.097 -1.009 0.791 LAPTM4B lysosomal associated protein transmembrane 4 beta -1.172 0.029 -1.118 0.150 -1.048 0.463 LCN6 lipocalin 6 -1.102 0.010 -1.052 0.224 -1.047 0.229 LHFPL2 lipoma HMGIC fusion partner-like 2 -1.116 0.022 -1.067 0.226 -1.045 0.315 LOC643650 hypothetical protein LOC643650 -1.107 0.018 -1.036 0.402 -1.069 0.092 MEIS2 Meis1, myeloid ecotropic viral integration site 1 homolog 2 (mouse) -1.087 0.029 -1.059 0.165 -1.026 0.539 MYO1H myosin IH -1.053 0.042 -1.020 0.462 -1.033 0.257 NDRG4 NDRG family member 4 -1.108 0.019 -1.072 0.130 -1.034 0.487 NDUFA4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa -1.062 0.045 -1.041 0.206 -1.020 0.532 NFYA nuclear transcription factor Y, alpha -1.072 0.043 -1.010 0.785 -1.061 0.052 NUDT4 nudix (nucleoside diphosphate linked moiety X)-type motif 4 -1.156 0.003 -1.105 0.068 -1.046 0.270 PDCD4 programmed cell death 4 (neoplastic transformation inhibitor) -1.105 0.007 -1.074 0.073 -1.029 0.312 PDZD8 PDZ domain containing 8 -1.072 0.032 -1.004 0.889 -1.067 0.061 PIK3C2B phosphoinositide-3-kinase, class 2, beta polypeptide -1.176 0.002 -1.084 0.232 -1.085 0.179 PLVAP plasmalemma vesicle associated protein -1.125 0.021 -1.104 0.083 -1.020 0.703 PPEF1 protein phosphatase, EF-hand calcium binding domain 1 -1.130 0.038 -1.030 0.346 -1.097 0.098 PPFIBP2 PTPRF interacting protein, binding protein 2 (liprin beta 2) -1.171 0.011 -1.054 0.453 -1.111 0.101 PPIA peptidylprolyl isomerase A (cyclophilin A) -1.057 0.042 -1.007 0.818 -1.050 0.059 PPM1D protein phosphatase 1D magnesium-dependent, delta isoform -1.056 0.030 -1.008 0.803 -1.048 0.131 PRDX1 peroxiredoxin 1 -1.074 0.022 -1.026 0.466 -1.048 0.196 PROCR protein C receptor, endothelial (EPCR) -1.100 0.030 -1.014 0.758 -1.084 0.071 PTPRG protein tyrosine phosphatase, receptor type, G -1.070 0.022 -1.046 0.123 -1.023 0.447 SCAMP5 secretory carrier membrane protein 5 -1.114 0.046 -1.082 0.118 -1.029 0.580 SERPINB9 serpin peptidase inhibitor, clade B (ovalbumin), member 9 -1.074 0.024 -1.021 0.473 -1.053 0.062 SLC40A1 solute carrier family 40 (iron-regulated transporter), member 1 -1.229 0.002 -1.126 0.155 -1.092 0.247 SNX2 sorting nexin 2 -1.074 0.010 -1.029 0.325 -1.044 0.154 SRPRB signal recognition particle receptor, B subunit -1.079 0.010 -1.024 0.466 -1.054 0.112 TFRC transferrin receptor (p90, CD71) -1.122 0.014 -1.101 0.054 -1.019 0.717 THSD1 thrombospondin, type I, domain containing 1 -1.085 0.044 -1.041 0.381 -1.042 0.315 TIMP1 TIMP metallopeptidase inhibitor 1 -1.106 0.019 -1.051 0.162 -1.053 0.201 TRPA1 transient receptor potential cation channel, subfamily A, member 1 -1.070 0.028 -1.027 0.280 -1.041 0.199 ULBP2 UL16 binding protein 2 -1.064 0.039 -1.041 0.251 -1.022 0.547 ZMPSTE24 zinc metallopeptidase (STE24 homolog, yeast) -1.057 0.039 -1.022 0.477 -1.034 0.271 ZNF17 zinc finger protein 17 -1.069 0.026 -1.007 0.825 -1.061 0.084 216 Chapter 7 gulate theactivityofHIF-1circuitduringchronichypoxia. core factors other that imply apparently data These 4). (Fig. decreased also finally but hours, 24 than more for lasted HIF-2α of activation transient the while hours, 12 level, HIF-1α was transiently induced by hypoxia, but it decreased rapidly again after lar accumulation and activation of HIF-1α and HIF-2α proteins (Fig. 4). At the protein cellu the rapid, causes indeed oxygen to 1% (Fig. 4).Exposure analysis tern blotting which arecontrolledbyoxygentension by itssteadystateproteinlevels, activity ofHIFismainlycontrolled transcriptional the as Hence, Table3. from noted be can HIF-2α and HIF-1α for levels mRNA of by HIF-1 hypoxia.Still, also duringprolongeda rapid as well aspersistentdecrease The RT-PCRgene expression of endothelial regulation analysishintsataprominent HIF-1 andHIF-2proteinanalysis general hypertension and suchaspulmonary conditions, a roleinpathological plays production thelin-1 hypoxia Functional implications of altered transcriptomes during chronic versus acute poxia, althoughthisawaits futurefunctionalvalidation. interaction betweenKLF2- and HIF-inducedgene regulationalsoduringchronichy function inendothelialcells duringacutehypoxia.Our data thus suggestamutual Recently, Kawanami MCP1/CCL2, PAI1/SERPINE1,including E-selectin/SELEandendothelin/EDN1. by KLF2 are repressed that genes profibrotic and proinflammatory the of many Indeed, PAI-1. and VEGF in they areindicated Tables3 asKLF2/TGF 2 and endothelium in signaling TGF-β block to known is KLF2 KLF2. As to indirectly or evaluate theexpressionsof KLF2 and a number of genes that respond eitherdirectly eNOS and targets, thrombomodulin that bothKLF2anditsdirectdownstream show indeed factor KLF2 transcription the endothelial lap betweenthechronichypoxiatranscriptomewithpublishedof shown). Furtheranalysisofgeneexpression,however, identified asignificantover (datanot arose module transcriptional specific no clear, but identified, tentatively GSEAusing elements andgenomeVISTA of thedifferentthe promoters response conserved (Fig. 2)forevolutionary panels HIF-1, In searchofpotentialothertranscriptionfactorscoregulatingweanalysed Candidate transcriptionfactorscoregulatingHIFactivity:KLF2 51 , were downregulated after hypoxia(Tableprolonged 2). This prompted usto 48 et al. , butitsroleinmicrovascularendotheliumremainsobscure. 49, 52

show an increased expression after prolonged hypoxia, after prolonged expression an increased show 53 reported that KLF2 inhibited HIF-1α expression and expression HIF-1α inhibited KLF2 that reported 3, 15 50 . The RT-PCT dataofHUVECandhMVEC 49 , we evaluatedtheirproteinsusingWes . Afactors was of transcription multitude b -regulated genes and include and genes -regulated 52 - - - - - , Endothelial Gene Expression During Chronic Hypoxia 217

The results of our transcriptome analysis by combined microarray analysis, biblio- graphical analysis, RT-PCR validation and Western blot analysis can be discussed by means of its functional implications on pathways relevant to endothelial cells in pathological conditions and angiogenesis, exemplified by the subheadings of Tables 2 and 3.

Hypoxia Inducible Factors Because of their prominent role in translating low oxygen concentrations to gene transcription during states of hypoxia3, 15, we evaluated the expression of the indi- vidual HIF and PHD family members. While exposure to 1% oxygen causes the rapid, but transient cellular accumulation and activation of HIF-1α and HIF-2α pro- teins (Fig. 4), hypoxia decreased HIF-1α and HIF-2α transcript levels by around 20% already after 24 hours of hypoxia (Table 2), in agreement with previous studies. This decrease in mRNA remained after 2 weeks of hypoxia. At the protein level, HIF-1α was transiently induced by hypoxia, but it decreased rapidly again after 12 hours, while the transient activation of HIF-2α lasted for more than 24 hours, but finally also decreased (Fig. 4). Interestingly, the levels of HIF-3α and HIF-3α4, an alternatively spliced variant of HIF-3α with a similar HRE-inhibiting domain structure as the murine inhibitory PAS protein (IPAS)54, 55, were increased after 1 day (6- and 4-fold, respectively) in HUVEC, with a trend towards further increase after prolonged

HIF1α

actin

HIF2α

actin 24 48h 0 3 6 24 48h 0 3 6 24h

21% O2 1%O2 after 7 d 21%O2 1%O2 after 7d 1%O2

Fig 4. Western blots of HIF-1α and HIF-2α proteins after exposure of HUVEC during various time periods to 1% oxygen. Similar data were obtained in hMVEC, which express relatively more HIF-2α than HUVEC. 218 Chapter 7 The increase of HIF-3α/HIF-3α4 is of interest as HIF activity is inhibited by iPAS, by inhibited is activity HIF as interest of is HIF-3α/HIF-3α4 of increase The cell types proteins, remainedunalteredinbothendothelialduringhypoxia(Table 2). of HIF domain of thetransactivation for hydroxylation FIH1, responsible hydroxylase ter 24 hoursandincreasedto over 500-fold inductionafter 4 weeks. The asparagine af induction an138-fold reached expression PHD3 in hMVEC,which pronounced data with previous which wasinagreement tern ofexpression, in HUVEC. after 14daysofhypoxia to 28-fold after 24hours,and 16-fold This pat expressed to a limited extent at ambient (21%)oxygenconcentration,it increased hypoxia. Furthermore,whilePHD3was HUVEC, bothinshort-termandprolonged However,exposure. hypoxic upon in 3-fold induced was PHD2 expressed thehighly unaltered remained and expressed little was PHD1 of HIFproteins, degradation With respect to the PHD family, for responsible prolylhydroxylationandsubsequent by hypoxia(datanotshown). hypoxia (15- and 14-fold, respectively). HIF-1β (ARNT) expression was not affected been unequivocal been by otherauthors sis, isinconcertwithobservations crease, which was significant in the RT-PCR analysis but not in the microarray analy in mRNA of thepivotalreceptorVEGFR2,bothinHUVEC andhMVEC. expression This de decrease significant a caused hypoxia to cells endothelial of exposure is stimulatedbyhypoxia,theprolonged days. WhileVEGF-inducedangiogenesis a wentupafter role inlymphangiogenesis, 1daytransiently andnormalizedafter 14 while VEGFR3,whichplays conditions, under ourexperimental unaltered remained 4.1-fold inductionafter two weeks. Among the VEGF receptors, VEGFR1 expression after24hoursand with a2.7-fold induction regulation a time-dependent displayed extent by short-term and prolongedhypoxia(Table 2). On the otherhand,VEGFC of VEGFBandPlGF,together withtheinductions to asimilar were allinduced which sion is very low. By RT-PCR analysisa clear inductionwasobservedinHUVEC, Although VEGFA expression isimportantforcells,thelevelof endothelial expres VEGF andtheirreceptors may contributetoenhancedsproutformation PDH2 of expression enhanced that and hypoxia, towards responsiveness specific in chronichypoxia.However, cellsmaydisplaya it shouldbenotedthatendothelial mice anddesensitizedtheHIFresponse. contributed tocellsurvival This reduction of HIF-1 theaccumulation reduced hypoxia Ginouvès from HIF-3 is derived which et al. 31 showed that PHD overexpression and -activation during chronic during and-activation that PHDoverexpression showed 59 . In interaction with VEGF signaling, Delta-like 4 (Dll4)-mediated Delta-like with VEGF signaling, . Ininteraction a , and by the splice variant HIF-3 , andbythesplice a /-2 56 . a proteins in various tumor cells and tumor cells in various proteins 57, 58 , although data havenot , although a 4 31 54, 55 , wasevenmore . Furthermore, - - - - - Endothelial Gene Expression During Chronic Hypoxia 219

Notch signaling is another key pathway in angiogenesis regulation. Endothelial Dll4 expression activates Notch1 resulting in reduced VEGF signaling46 and restriction of new sprout development60, 61. In HUVEC neither Dll4 nor Notch1 were affected by hypoxia, in hMVEC Dll4 was transiently induced by 24 hours of hypoxia (2-fold), but has returned to baseline after 4 weeks. Notch1 expression remained unaltered.

Proliferation genes CCND1 and PCNA The expression of the proliferation regulator cyclin D1, which remained unaltered in HUVEC, was markedly reduced in hMVEC (by 27% after 24 hours, 44% after 28 days). This observation contrasts to a previous observation that VHL depletion caused HIF-dependent upregulation of cyclin D1 in renal carcinoma cells62. Further- more, while proliferating cell nuclear antigen (PCNA) was not identified by microarray analysis as being significantly regulated, RT-PCR demonstrated significant reduction of their gene expression by hypoxia, similar as found by Chi et al. 25. Apparently, the reduction of cyclin D1 and PCNA in hMVEC had no adverse effect on the prolifera- tion of hMVEC in hypoxia, as shown in figure 1. However, the enhanced proliferation at 1% O2 as compared to 21% O2 may partly be due to a slightly improved survival of the cells due to less oxidation damage at the lower oxygen tension.

Matrix remodeling proteases While there is general consensus about the importance of proteases in the regula- tion of angiogenic sprout formation, the data on the regulation of proteases by hy- poxia are unequivocal. Despite its high initial expression, PAI1 (SERPINE1) expres- sion was enhanced more than 2-fold by hypoxia. A PAI1 induction was consistently found in other studies5, 24, 27, 63, 64. Wojta et al.65 also found an increase in plasminogen activator activity (both TPA and UPA). However, neither the present data, nor those of Kroon et al.66 could confirm an increase in UPA. Furthermore, TPA was markedly reduced after prolonged hypoxia (45% reduction) in our present analysis, but not in earlier studies65, 66. Although our microarray data confirmed the increase in UPAR, previously reported by Kroon et al.66, another study indicated no difference at the mRNA level67. However, it should be noted that a considerable variation in UPAR expression was observed between different cultures. Prolonged hypoxia also in- creased the expression of MMP2 and MMP10, while that of MT1-MMP and MMP9 remained unaltered. As pointed out by Ben-Yosef et al. 68, the regulation of MMP2 expression and activity by hypoxia is complex and mainly involves posttranscrip- tional regulation. 220 Chapter 7 oxygen inHUVEC. Dataexpressedasmean+/-std. of genesafter exposure(28days)to Regulation hypoxia-responsive short-term(24h)andprolonged 1% Table 2 IGFBP-2 DPP-3 receptor Transferrin 1 Heme oxygenase E2F6 E2F3 CXCL3 CXCL1 Angiopoietin like-4 ICAM-1 Miscellaneous PCNA Cyclin D1 Proliferation Hexokinase 2 Hexokinase 1 dehydrogenase A Lactate synthase 1 Glycogen transporter 1 Glucose Glycolysis eNOS Endothelin-1 E-selectin VEGF-A PAI-1 RGS-5 NADH quinone1 CXCR4 Collagen Vα1 BMP-4 Adrenomedullin Thrombomodulin MCP-1 KLF-2 regulated genes KLF-2/TGFβ MT1-MMP MMP-10 MMP-9 MMP-2 PAI-1 tPA uPAR uPA Proteases Notch1 DLL-4 VEGFR-3 VEGFR-2 VEGFR-1 PlGF VEGF-D VEGF-C VEGF-B VEGF-A VEGF familly FIH-1 PHD-3 PHD-2 PHD-1 HIF-3α4 HIF-3α HIF-2α HIF-1α HIF regulation Table 2HUVEC SDF-1 CXCR4 IGFBP-3 SERPINE1 SERPINE1 ANGPTL4 NOTCH1 COL5A1 CXCL12 SLC2A1 Symbol IGFBP3 IGFBP2 HIF1AN HMOX1 MMP14 MMP10 CCND1 VEGFC CXCR4 VEGFB CXCR4 PLAUR VEGFA VEGFA EGLN3 EGLN1 EGLN2 CXCL3 CXCL1 EPAS1 ICAM1 MMP9 MMP2 NQO1 HIF3A HIF1A PCNA NOS3 RGS5 BMP4 THBD GYS1 EDN1 TFRC DPP3 LDHA PLAU SELE CCL2 Gene PLAT DLL4 E2F6 E2F3 KLF2 FIGF FLT4 FLT1 ADM KDR PGF HK2 HK1 31.16 ±1.90 24.48 ±2.24 25.43 ±1.31 20.94 ±0.80 24.30 ±0.53 20.08 ±0.74 19.45 ±2.72 24.17 ±1.01 22.33 ±1.00 29.30 ±0.85 23.44 ±0.97 20.78 ±0.57 30.56 ±1.74 20.16 ±1.06 23.27 ±0.74 27.05 ±1.33 21.20 ±0.46 21.56 ±1.57 20.44 ±0.47 23.90 ±0.63 24.47 ±1.34 32.58 ±0.78 29.91 ±1.35 34.80 ±0.91 16.42 ±1.38 19.15 ±1.23 18.34 ±1.38 24.48 ±2.24 19.40 ±0.63 20.19 ±0.76 23.90 ±1.56 25.76 ±0.56 19.87 ±1.02 22.89 ±0.64 20.01 ±0.63 24.04 ±0.98 17.19 ±1.22 16.42 ±1.38 21.65 ±0.97 25.97 ±1.56 25.27 ±1.28 24.26 ±1.67 25.01 ±0.84 32.12 ±0.70 22.12 ±0.82 22.84 ±0.91 22.28 ±0.84 30.90 ±0.83 25.89 ±1.19 26.82 ±1.46 34.80 ±0.91 23.00 ±1.76 30.46 ±0.93 22.62 ±0.55 29.09 ±1.27 31.73 ±1.67 34.37 ±1.81 20.55 ±0.76 21.90 ±1.11 Ambient oxygen(21%) Avg. Ct ± 14.03 ±0.58 14.49 ±0.32 12.49 ±0.89 12.35 ±0.81 14.49 ±0.32 10.42 ±0.72 12.83 ±1.95 14.95 ±2.17 11.12 ±1.59 -0.28 ±0.32 -3.29 ±1.48 -1.57 ±1.07 -1.57 ±1.20 -0.24 ±1.19 -0.25 ±0.58 -0.78 ±0.88 -2.73 ±0.99 -3.29 ±1.48 -0.30 ±2.39 2.55 ±0.73 4.62 ±1.39 2.45 ±0.64 5.37 ±1.05 7.77 ±0.50 1.16 ±0.58 2.28 ±2.67 4.82 ±0.47 9.19 ±1.22 4.44 ±1.87 4.76 ±0.92 6.34 ±1.19 1.46 ±0.43 4.62 ±1.50 1.89 ±1.41 6.83 ±2.48 3.54 ±1.53 4.29 ±0.61 2.21 ±0.60 2.52 ±0.36 2.86 ±1.47 6.25 ±1.46 6.78 ±1.69 2.96 ±1.40 2.59 ±0.35 9.33 ±1.99 1.86 ±0.76 0.51 ±0.42 4.44 ±1.87 4.71 ±1.42 0.50 ±0.66 4.68 ±1.40 0.04 ±0.43 3.45 ±0.78 1.89 ±1.04 9.74 ±1.24 4.16 ±1.36 1.63 ±1.28 9.84 ±1.81 0.24 ±0.81 Avg. ΔCt * ± Fold** of21% 13.43 ±9.27 13.43 ±9.27 15.56 ±6.32 8.29 ±2.91 0.71 ±0.15 1.10 ±0.29 1.65 ±0.86 2.39 ±0.81 1.24 ±0.15 3.12 ±1.12 1.05 ±0.16 0.92 ±0.23 3.48 ±2.10 2.17 ±0.69 1.10 ±0.65 0.64 ±0.14 1.98 ±0.45 1.86 ±0.34 2.09 ±0.57 4.28 ±0.94 0.96 ±0.37 1.30 ±0.32 0.94 ±0.24 2.08 ±0.65 1.73 ±0.32 2.17 ±0.69 0.94 ±0.28 1.24 ±0.56 1.29 ±0.86 1.70 ±1.08 1.64 ±0.36 0.85 ±0.22 1.29 ±0.59 2.25 ±0.46 1.24 ±0.51 2.70 ±0.63 1.90 ±0.21 1.16 ±0.18 2.89 ±0.59 1.20 ±0.10 4.60 ±3.83 6.35 ±3.30 0.87 ±0.09 0.80 ±0.10 2.80 ±2.04 1.98 ±0.45 3.48 ±2.42 1.10 ±0.28 0.91 ±0.26 0.65 ±0.04 0.54 ±0.15 1.25 ±0.37 1.00 ±0.14 2.31 ±0.68 1.02 ±0.43 4.38 ±1.81 4.41 ±3.08 0.81 ±0.18 2.47 ±1.11 24h, 1%oxygen NA < 0.001 < 0.001 < 0.001 0.008 0.001 0.001 0.022 0.002 0.001 0.005 0.014 0.004 0.003 0.001 0.005 0.006 0.042 0.049 0.009 0.009 0.006 0.002 0.023 0.034 0.022 0.009 0.001 0.003 0.002 0.006 0.005 0.010 0.003 0.009 0.011 Sign n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. NA Fold** of21% 13.69 ±10.86 16.16 ±11.32 13.35 ±5.36 28.28 ±3.34 13.35 ±5.36 1.36 ±0.64 1.25 ±0.42 1.19 ±0.52 0.65 ±0.30 1.10 ±0.39 1.78 ±0.76 1.24 ±0.47 4.09 ±1.31 1.79 ±0.51 1.10 ±0.22 2.70 ±0.71 1.08 ±0.57 0.80 ±0.29 0.84 ±0.29 4.97 ±3.19 1.98 ±0.68 0.96 ±0.34 1.00 ±0.23 0.62 ±0.23 0.25 ±0.09 1.26 ±0.35 1.33 ±0.36 1.63 ±0.63 0.73 ±0.39 4.03 ±1.61 8.36 ±3.30 0.69 ±0.16 1.22 ±0.48 2.88 ±0.92 1.21 ±0.32 2.93 ±1.83 0.97 ±0.22 8.19 ±3.67 0.62 ±0.12 1.77 ±0.70 7.88 ±5.50 2.29 ±1.06 0.76 ±0.29 0.76 ±0.31 1.98 ±0.68 1.90 ±0.45 2.04 ±0.45 4.29 ±1.93 0.79 ±0.22 2.52 ±1.06 0.65 ±0.24 0.92 ±0.21 1.38 ±0.76 1.67 ±0.42 2.29 ±1.06 0.55 ±0.24 0.90 ±0.34 1.32 ±0.69 3.22 ±1.11 28 days,1%oxygen NA < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.002 0.005 0.002 0.001 0.037 0.001 0.026 0.008 0.006 0.031 0.044 0.004 0.003 0.001 0.001 0.017 0.022 0.007 0.001 Sign n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. NA Differential regulation < 0.001 0.026 0.021 0.049 0.026 0.002 0.015 0.007 Sign ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns Endothelial Gene Expression During Chronic Hypoxia 221

Table 3 MVEC Gene Ambient oxygen (21%) 24h, 1% oxygen 28 days, 1% oxygen Differential regulation

Fold** of 21% Fold** of 21% HIF regulation Symbol Avg. Ct Avg. ΔCt * Sign Sign Sign HIF-1α HIF1A 20.26 ± 0.57 0.75 ± 0.40 0.54 ± 0.17 0.012 0.70 ± 0.28 0.021 ns HIF-2α EPAS1 20.54 ± 0.38 1.04 ± 0.41 0.77 ± 0.11 0.028 0.59 ± 0.24 0.002 ns HIF-3α HIF3A 28.65 ± 0.87 9.24 ± 0.82 3.31 ± 1.05 0.022 2.31 ± 1.07 0.010 ns HIF-3α4 28.59 ± 1.28 9.18 ± 1.17 2.14 ± 0.17 0.016 2.80 ± 2.41 n.s. ns PHD-1 EGLN2 28.65 ± 0.64 9.14 ± 0.61 1.00 ± 0.16 n.s. 1.34 ± 0.84 n.s. ns PHD-2 EGLN1 21.68 ± 0.34 2.17 ± 0.38 1.89 ± 0.72 n.s. 1.50 ± 0.53 0.033 ns PHD-3 EGLN3 32.10 ± 1.27 12.60 ± 1.10 138.31 ± 28.69 0.002 516.68 ± 235.06 < 0.001 0.011 FIH-1 HIF1AN 22.96 ± 0.30 3.45 ± 0.39 0.94 ± 0.09 n.s. 0.91 ± 0.31 0.042 ns

VEGF familly VEGF-A VEGFA 37.21 ± 1.94 17.71 ± 1.71 18.72 ± 14.15 n.s. 9.84 ± 5.76 0.003 ns VEGF-B VEGFB 27.90 ± 1.30 8.40 ± 1.30 1.71 ± 0.65 n.s. 1.23 ± 0.47 n.s. ns VEGF-C VEGFC 25.74 ± 0.72 6.24 ± 0.74 1.32 ± 0.33 n.s. 1.35 ± 0.66 n.s. ns VEGF-D FIGF 31.10 ± 0.65 11.84 ± 0.72 0.83 ± 0.16 n.s. 0.78 ± 0.21 0.021 ns PlGF PGF 21.55 ± 0.92 2.28 ± 0.78 1.22 ± 0.30 n.s. 1.44 ± 1.33 n.s. ns VEGFR-1 FLT1 26.49 ± 0.90 6.99 ± 0.68 0.95 ± 0.46 n.s. 1.03 ± 0.74 n.s. ns VEGFR-2 KDR 22.36 ± 0.69 2.85 ± 0.47 0.65 ± 0.07 0.002 0.71 ± 0.27 0.021 ns VEGFR-3 FLT4 30.22 ± 0.67 10.71 ± 0.56 1.06 ± 0.25 n.s. 0.87 ± 0.57 n.s. ns DLL-4 DLL4 26.77 ± 0.42 7.36 ± 0.31 1.93 ± 0.86 n.s. 0.95 ± 0.33 n.s. 0.015 Notch1 NOTCH1 23.47 ± 0.49 4.06 ± 0.45 1.07 ± 0.29 n.s. 0.94 ± 0.34 n.s. ns

Proteases uPA PLAU 22.19 ± 1.05 2.68 ± 1.01 0.89 ± 0.19 n.s. 0.89 ± 0.29 n.s. ns uPAR PLAUR 24.99 ± 0.58 8.29 ± 0.59 0.66 ± 0.14 0.019 1.45 ± 1.47 n.s. ns tPA PLAT 22.15 ± 0.99 2.65 ± 1.19 0.85 ± 0.10 n.s. 0.68 ± 0.24 0.007 ns PAI-1 SERPINE1 17.21 ± 0.88 -2.30 ± 0.93 1.32 ± 0.23 n.s. 1.10 ± 0.61 n.s. ns MMP-2 MMP2 19.34 ± 1.03 0.08 ± 0.90 1.11 ± 0.09 n.s. 1.21 ± 0.52 n.s. ns MMP-9 MMP9 > 40 NA*** NA NA NA NA NA MMP-10 MMP10 > 40 NA NA NA NA NA NA MT1-MMP MMP14 20.19 ± 0.59 0.93 ± 0.51 0.81 ± 0.13 n.s. 0.74 ± 0.22 0.011 ns

KLF-2/TGFβ regulated genes KLF-2 KLF2 19.68 ± 0.89 1.44 ± 1.01 0.71 ± 0.20 n.s. 0.66 ± 0.20 0.002 ns MCP-1 CCL2 23.45 ± 1.84 4.04 ± 1.96 2.47 ± 1.92 n.s. 1.14 ± 0.70 n.s. ns Thrombomodulin THBD 22.84 ± 1.10 3.57 ± 1.13 0.87 ± 0.35 n.s. 0.71 ± 0.19 0.003 ns Adrenomedullin ADM 21.19 ± 1.11 4.11 ± 1.20 4.64 ± 1.51 0.017 5.23 ± 4.20 0.025 ns BMP-4 BMP4 21.26 ± 2.88 1.85 ± 2.95 2.59 ± 0.81 0.029 2.68 ± 1.40 0.011 ns Collagen Vα1 COL5A1 22.40 ± 0.54 2.90 ± 0.54 1.24 ± 0.07 0.008 1.28 ± 0.33 0.046 ns CXCR4 CXCR4 25.38 ± 0.77 5.87 ± 0.67 1.77 ± 0.69 n.s. 1.88 ± 0.62 0.005 ns NADH quinone 1 NQO1 17.59 ± 1.10 -1.92 ± 1.05 0.50 ± 0.03 < 0.001 0.51 ± 0.08 < 0.001 ns RGS-5 RGS5 24.01 ± 1.35 4.60 ± 1.46 0.96 ± 0.11 n.s. 0.41 ± 0.24 0.000 0.002 PAI-1 SERPINE1 17.21 ± 0.88 -2.30 ± 0.93 1.32 ± 0.23 n.s. 1.10 ± 0.61 n.s. ns VEGF-A VEGFA 37.21 ± 1.94 17.71 ± 1.71 18.72 ± 14.15 n.s. 9.84 ± 5.76 0.003 ns E-selectin SELE 31.98 ± 4.26 12.57 ± 4.38 27.71 ± 34.38 n.s. 8.22 ± 10.58 n.s. ns Endothelin-1 EDN1 34.36 ± 1.36 15.10 ± 1.51 10.91 ± 9.45 n.s. 2.88 ± 1.32 0.005 0.033 eNOS NOS3 21.74 ± 0.38 2.33 ± 0.42 0.69 ± 0.19 0.043 0.74 ± 0.24 0.020 ns

Glycolysis Glucose transporter 1 SLC2A1 23.76 ± 0.76 5.61 ± 0.71 10.16 ± 2.06 0.003 9.87 ± 5.48 0.003 ns Glycogen synthase 1 GYS1 19.48 ± 0.37 1.32 ± 0.24 1.10 ± 0.15 n.s. 0.87 ± 0.25 n.s. ns A LDHA 22.35 ± 2.80 5.26 ± 2.71 2.20 ± 1.76 n.s. 2.42 ± 1.76 n.s. ns Hexokinase 1 HK1 21.40 ± 0.41 2.13 ± 0.36 1.01 ± 0.05 n.s. 0.99 ± 0.35 n.s. ns Hexokinase 2 HK2 26.84 ± 0.47 9.83 ± 0.52 1.32 ± 0.42 n.s. 1.82 ± 1.02 n.s. ns

Proliferation Cyclin D1 CCND1 16.14 ± 0.42 -2.02 ± 0.34 0.73 ± 0.13 0.024 0.56 ± 0.15 < 0.001 ns PCNA PCNA 19.89 ± 0.49 0.38 ± 0.50 0.71 ± 0.11 0.012 0.76 ± 0.17 0.004 ns

Miscellaneous ICAM-1 ICAM1 27.91 ± 0.74 8.50 ± 0.74 3.05 ± 2.71 n.s. 1.50 ± 0.42 0.012 ns Angiopoietin like-4 ANGPTL4 18.85 ± 0.95 1.77 ± 0.94 2.72 ± 1.59 n.s. 3.40 ± 2.32 0.023 ns CXCL1 CXCL1 20.15 ± 0.24 1.99 ± 0.35 0.88 ± 0.08 n.s. 0.49 ± 0.07 < 0.001 p < 0.001 CXCL3 CXCL3 27.41 ± 1.25 9.26 ± 1.20 1.54 ± 0.17 0.008 1.19 ± 0.20 0.030 0.012 E2F3 E2F3 20.87 ± 0.30 2.71 ± 0.31 0.91 ± 0.15 n.s. 0.87 ± 0.19 n.s. ns E2F6 E2F6 22.22 ± 0.44 4.07 ± 0.40 0.97 ± 0.23 n.s. 0.92 ± 0.22 n.s. ns Heme oxygenase 1 HMOX1 19.84 ± 0.95 0.34 ± 1.08 0.23 ± 0.11 0.001 0.25 ± 0.15 < 0.001 ns Transferrin receptor TFRC 22.25 ± 0.43 2.74 ± 0.55 0.25 ± 0.13 0.001 0.38 ± 0.22 < 0.001 ns DPP-3 DPP3 23.55 ± 0.26 5.39 ± 0.28 0.83 ± 0.12 n.s. 0.72 ± 0.11 < 0.001 ns IGFBP-2 IGFBP2 26.05 ± 3.40 7.89 ± 3.48 1.04 ± 0.34 n.s. 0.62 ± 0.27 0.006 0.043 IGFBP-3 IGFBP3 28.36 ± 1.90 10.20 ± 1.97 2.38 ± 1.34 n.s. 5.64 ± 3.57 0.008 ns CXCR4 CXCR4 25.38 ± 0.77 5.87 ± 0.67 1.77 ± 0.69 n.s. 1.88 ± 0.62 0.005 ns SDF-1 CXCL12 26.77 ± 1.30 7.26 ± 1.21 0.82 ± 0.16 n.s. 0.83 ± 0.51 n.s. ns

Table 3 Regulation of hypoxia-responsive genes after short-term (24h) and prolonged exposure (28 days) to 1% oxygen in MVEC 222 Chapter 7 fects of KLF2-repressed TGF-β effectsand anti-angiogenic anti-migratory ofKLF2 anti-permeability,are thusfullyconsistentwiththeknown implications functional The with angiogenesis. associated normally transcriptome profibrotic and matory proinflam the establishes This expression. KLF2 reduced a of effect expected the effectorDownstream consistent with or reduced genesofKLF2areeitherinduced HUVEC andhMVEC, which wouldresultinlossof its inhibitory actionsonHIF-1. exposure tohypoxia(24hoursand2weeks)inboth ally arereducedafterprolonged KLF2- andHIF-inducedgene regulations.We nowobservedthat KLF2 levelsactu baseline levelswithinseveralhours. This suggests a mutual interactionbetween to return shown from VHL,butKLF2wasalso independent cells in endothelial function and expression protein HIF-1α inhibited and hypoxia, of hour 1 by induced ogy biol endothelial of modulator transcriptional central a as identified been has KLF2 KLF2-regulated genes and KLF2transcriptomeeffects during chronichypoxia. of HIF-1 and anintegration and KLF2regulation hypoxia between gest aninteraction totheirknownresponseKLF2-knockdown. that wasidentical These resultssug RT-PCRgenes pattern ofKLF2-dependent byanexpression accompanied analysis of HIF1α and HIF2α proteins. Finally, we observed a reduced expression of KLF2 by reduction of HIF-1α and HIF-2α mRNA expression resulting in a limited accumulation induction of HIF3 and HIF3α4, the induction of PDH2 and particularly PDH3, and the during prolonged hypoxia in both HUVEC and hMVEC, exemplified by the increased 1α and HIF-2α protein levels. Apparently, a rewiring of the HIF-1 circuitry takes place of HIF- levels variable highly despite hypoxia, prolonged tome aftershort-termand transcrip genes werestudiedinmoredetailshowingalargeoverlap inendothelial by thestringentanalysis underestimated genes isconsiderably of hypoxia-induced the number array analyses gene published all that inthisand specific additional conclude on we based analysis but RT-PCR altered, significantly were genes of 435 that theexpressions EC showed human data on of microarray siderably, with a pivotal responseof genes involvedinglycolysis.Stringent analysis EC reprogramtheirtranscriptionofgenescon the changeinoxygenconcentration to Related toproliferate. their ability maintaining riods in1%and5%oxygen,while time pe be culturedforprolonged can cells that endothelial we show In thisreport Conclusion between transcriptionalprogramsofHIF11 andKLF2duringprolongedhypoxia. 50, 69,70 . In a recent report, Kawanami report, Kawanami In arecent 52, 72 . As such,ourdatasuggestsanintricateinterplay et al. 53 reported that KLF2istransiently reported 50, 71 , and the proangiogenic ef , andtheproangiogenic 5, 24,27 . Collectively, 55 ------Endothelial Gene Expression During Chronic Hypoxia 223

Acknowledgements The study was supported by the European Vascular Genomics Network (EU grant LSHM-CT-2003-503254), and the Dutch Program on Tissue Engineering (DTPE, grant VGT67.47).

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