A Comparison of Aorta and Vena Cava Medial MessageExpression by cDNA Array Analysis Identifies a Set of 68

Consistently Differentially Expressed Genes, All in Aortic MediaLawrence D. Adams, Randolph L. Geary, Bruce McManus, Stephen M. Schwartz

Abstract—We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles ofmacaque aorta and vena cava media using a cDNA array containing 4048 known human genes,'35% of currentlynamed human genes ('11 000). The data show extensive differences in RNA expression in artery versus vein media.Sixty-eight genes had consistent elevation in message expression by the aorta, but none were elevated in the vena cava.The most differentially expressed gene was regulator of G-protein signaling (RGS) 5, at an expression ratio of46.5612.6 (mean6SEM). The data set also contained 2 genes already known to be expressed in the aorta, elastin at5.061.4, and the aortic preferentially expressed gene 1 (APEG-1) at 2.360.6. We chose to analyze RGS5 expressionfurther because of its high level of differential expression in the aorta. Levels of RGS5 mRNA were confirmed byNorthern analysis and in situ hybridization. A human tissue RNA dot blot showed that RGS5 message is highest in aorta,followed by small intestine, stomach, and then heart. Northern analysis confirmed that RGS5 expression in human aortais higher than in any region of the heart. RGS5 is a G-protein signaling regulator of unknown specificity mosthomologous to RGS4, an inhibitory regulator of pressure-induced cardiac hypertrophy. The expression pattern of the 68differential genes as a whole is a start toward identifying the molecular phenotypes of arteries and veins on asystematic basis.(Circ Res. 2000;87:623-631.)

Key Words: cDNA arrayn aortan vena cavan expression profilen RGS5

We have begun a systematic analysis of expressiondifferences between vascular smooth muscle tissues.

We chose to begin with an analysis of the media of aorta andvena cava because of the possibility of isolating relativelyhomogenous cellular populations and because of the distinctfunctional requirements, embryological origins, and hemody-namic environments of these 2 tissues.

The present study, using a cDNA array containing 35% ofhuman named genes (4048 of the'10 900 functionally identi-fied human genes in UniGene Build No. 109 of the NationalCenter for Biotechnology Information human Unigene collec-tion), identified 68 genes that are consistently expressed athigher levels in the aorta. No genes were more consistentlyhighly expressed by the vena cava. One of these aortic markers,regulator of G-protein signaling (RGS) 5, showed extreme levelsof differential expression. Together, these genes offer a molec-ular definition of the phenotypes of these 2 types of smoothmuscle.

Materials and MethodsArray HybridizationResearch Genetics GF211 cDNA arrays (4048 human genes) wereused as specified. Equivalent amounts of RNA per vessel pair were

used to synthesize cDNA probes, and equivalent counts per minutewere added to hybridizations. Washed blots were exposed to phos-phor image screens and scanned on a Storm phosphor imager(Molecular Dynamics). Expression was quantified from scans withsimilar intensity and background, using Pathways software (Re-search Genetics). Ratios were calculated for each animal and thenaveraged.

Northern, Dot Blot, and In Situ HybridizationTotal RNA (15 mg) from macaque or rat was run, transferred, andhybridized as described1 with multiprime probes (Amersham) syn-thesized from sequence-verified clones of human and mouse RGS5(IMAGE clones 853809 and 1853118, respectively [American TypeCulture Collection]) and of human nonmuscle myosin heavy chain B(NMMHC-B) (IMAGE clone 823886), and then probed with a 28SrRNA primer as described.2 Washes were as follows: 3 cycles of 5minutes each at room temperature in 53SSC, 0.1% SDS, and 2cycles of 20 minutes each at 57°C (rat) or 55°C (macaque) in 0.33SSC, 0.1% SDS. Human Northern blot No. 7791-1 (ClontechLaboratories, Inc) containing 2mg poly(A) RNA from adult and fetalheart, aorta, and 5 regions of the heart was probed with humanRGS5, then G3PDH as specified. Human RNA master dot blot No.7770-1 (Clontech) containing 33 tissue, 14 brain region, and 7 fetalpoly(A) RNAs (89 to 514 ng each) was probed as specified withhuman clones for RGS5 and then human ubiquitin.

In situ hybridization was performed on cross sections of aorta andvena cava taken directly adjacent to vessel segments snap-frozen for

Received July 26, 2000; revision received August 22, 2000; accepted August 23, 2000.From the Department of Pathology (L.D.A., S.M.S.), University of Washington, Seattle, Wash; Department of Surgery (R.L.G.), Wake Forest

University School of Medicine, Winston-Salem, NC; and Department of Pathology and Laboratory Medicine (B.M.), St. Paul’s Hospital, University ofBritish Columbia, Vancouver, British Columbia, Canada.

Correspondence to Lawrence D. Adams, Department of Pathology, University of Washington, Box 357335, 1959 NE Pacific St, Seattle, WA98195-7335. E-mail ladams@u.washington.edu

© 2000 American Heart Association, Inc.

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array analyses. These tissues were immersion-fixed for 48 hours in10% formalin and embedded in paraffin. Sections were cut ontocoated slides and in situ hybridization was performed using35S-labeled riboprobes (sense and antisense) transcribed from humanRGS5 cDNA No. 853809 as described.3

Tissue Samples and RNA IsolationThree human aortas, 2 from males (age 32 and 33 years) and 1 froma female (age 47 years), were isolated from donor hearts obtainedfrom the University of British Columbia–St. Paul’s Hospital. Sam-ples were stripped of adventitia and endothelium and snap-frozen inliquid nitrogen.

Macaque tissues were obtained from 5 surgically menopausal(ovariectomized)female cynomolgus macaque monkeys (Macacafascicularis).4 These monkeys made up the control group from astudy of the effects of isoflavones on bypass graft intimal hyperplasiaand iliac artery angioplasty. Animals were not administered isofla-vones but underwent bypass graft placement between the distal aortaand right iliac artery and angioplasty of the left common iliac artery.The bypass graft originated at the most distal abdominal aorta, withthe proximal anastomosis adjacent to the aortic bifurcation and distalanastomosis to the external iliac artery. The vena cava and aorta usedfor arrays were removed well proximal to the site of surgery (distalthoracic aorta; vena cava from the diaphragm to just above the aorticanastomosis) 6 weeks after surgery. After sedation with ketamine (15mg/kg IM) and butorphanol (0.05 mg/kg IM), animals were antico-agulated with heparin (300 U/kg IV) and deeply anesthetized withsodium pentobarbital (100 mg/kg IV). The adventitia was strippedfrom both the aorta and the vena cava, and the endothelium wasremoved from the aorta but not the vena cava. Tissues weresnap-frozen in liquid nitrogen. All animal care and procedures wereperformed at the Comparative Medicine Clinical Research Center ofthe Wake Forest University School of Medicine in accordance withstate and federal law. Animal protocols were approved by the WakeForest University Animal Care and Use Committee and conformedto guidelines set forth in the Principles of Laboratory Animal Care(formulated by the National Society for Medical Research), and inthe Guide for the Care and Use of Laboratory Animals(NIHpublication No. 86-23, revised 1985).

Twenty male Sprague-Dawley rats (Rattus norvegicus) (Zivig-Miller) were housed and fed according to protocols approved by theAnimal Care Committee of the University of Washington. Rats wereanesthetized and euthanized, and tissues were processed as previ-ously described.2 All RNA was isolated as previously described.5

ResultsGeneral Comparison of Human and MacaqueArray HybridizationSamples of RNA from 5 pairs of macaque aortas and venacavas were used to probe a cDNA array containing 4048human genes. Genes were defined as expressed if hybridiza-tion was$1.5 times the average background (before normal-ization in Pathways software to overall hybridizationstrength). The informativeness of macaque probes on humanfilters in terms of the total number of positive respondingspots was a concern, because all arrayed targets consist of'1000 bp of the 39end of each gene and contain complete 39untranslated regions, areas of low interspecies homology. Toquantify general hybridization patterns, we hybridized cDNAprobes from human aorta (n53) and from macaque aorta andvena cava (both n55), then compared the average number ofgenes expressed by the above definition. The number ofgenes detected in human was'200 to 500 greater than inmacaque (human aorta, 38556115 [mean6SEM]; macaque

aorta, 33676115; and macaque vena cava, 36356181),indicating possible species specific differences in 39 noncod-ing domains or, possibly, species differences in expression.

Identification of Differentially Expressed GenesTo have objective criteria for identifying consistently differ-entially expressed genes, we established categories of ratioand array hybridization level for this study (Table 1). Afteranalyzing the data for the 5 independent macaque compari-sons, there were 68 genes that fit these criteria. All werehigher in the aorta (Table 2). Of these, only RGS5 wasexpressed.10-fold, at 46.5612.6 SEM. RGS5 had thehighest level of differential expression in each macaque.Levels were undetectable, or barely detectable, in vena cava,suggesting an “all-or-nothing” expression. The array hybrid-ization images for class IA genes are shown in Figure 1A.Expression ratios of the 68 differentially expressed genes aregraphed in Figure 1B.

Northern Analysis and In Situ HybridizationWe verified the array-based quantification by further exam-ining the expression of the 2 most highly expressed genes,RGS5 and NMMHC-B. First, we examined macaque aortaand vena cava by Northern analyses (Figure 2A). RGS5 wasmost highly expressed in macaque aorta, followed by carotidartery, heart, and vena cava, with little expression detected inskeletal muscle or liver. NMMHC-B was most highly ex-pressed in aorta, followed by vena cava and then the carotidartery. After normalization to 28S rRNA hybridization (Table3), the Northern blot aorta-to-vena cava ratio was quantifiedas 45-fold for RGS5 and 5-fold for NMMHC-B, very close tothe mean array values. Macaque message sizes were consis-tent with the published human sizes,6 with 2 major RGS5bands at'7.0 and'2.1 kb and 1 NMMHC-B band at'7.5 kb.7

We further examined RGS5 because of its large differentialexpression ratio. To compare human aorta RGS5 messagelevels to heart, the highest-expressing human tissue examinedin the literature,6 we probed a Clontech Northern blotcontaining RNA samples of aorta, fetal and adult heart, and 5distinct heart regions (Figure 2B). RGS5 message levels in

TABLE 1. Gene Expression CategoriesHybridization Classes for Genes, Defined by the Animal Sample With theLowest Fold Hybridization (Above the Average Background [AB] Before

Normalization) in the Higher-Expressing Tissue

Expression ClassLowest Sample

Expression Above AB

I $5

II 2 to 5

III 1.5 to 2

IV ,1.5

Ratio Classes Defined by the Lowest Animal Ratio

Ratio ClassLowest Animal Ratio of

Aorta/Vena Cava

A $1.5

B 1.3 to 1.5

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Figure 1. A, Array hybridization images of class IA genes foreach macaque (1–5). Ao indicates aorta; VC, vena cava. B,Graphs of expression levels of all 68 consistent aorta markergenes, sorted by ratio. Larger graph presents all genes exceptRGS5 sorted in descending ratio order, listed as 2 through 68(accession Nos., A490477, AA459308, AA486321, AA485748,R66310, H06516, H95960, AA487623, AA875933, H79047,AA489331, AA040703, R50953, AA490473, R22219, N53065,T96082, N64384, H25917, AA504461, AA873159, H09959,AA287323, T69450, R43778, H46554, AA777192, AA668681,T72076, AA845178, R75820, AA863149, R83876, H39192,AA127014, R76437, H59204, AA454160, R15111, AA630507,AA169645, AA455652, H23979, AA873152, AA706968,AA453750, R83837, AA644092, AA148548, AA877082,AA489201, AA448711, H17943, AA706987, AA188179, N73827,R92994, T73556, AA490945, AA421518, W51985, R48320,N32768, AA488406, AA599187, N79534, and R63811). Smaller,inset graph presents ratios for RGS5 (AA668470) and the next 9genes in descending ratio order, listed as 1 through 10. PAMindicates peptidylglycine a-amidating mono-oxygenase; SPARC,secreted protein acidic and rich in cysteine.

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TABLE 2. Class I to III Differentially Expressed Genes, All Aortic

Accession no. Mean R6SEM Mean Int Ao Mean Int VC

Class IA

RGS5 AA668470 46.5612.6 18 589 517

NMMHC-B AA490477 7.562.3 5147 898

Vimentin AA486321 4.961.4 18 440 4812

Peptidylglycine a-amidating mono-oxygenase R66310 4.261.8 3113 1080

SPARC/osteonectin H95960 3.860.9 12 178 3813

Class IB

a2-Macroglobulin, field 2* H06516 3.962.2 3980 1945

a2-Macroglobulin, field 1* H06516 3.561.7 3330 1555

FK506-binding protein precursor R75820 2.060.3 3134 1783

Thromboxane-A synthase R76437 2.060.3 2361 1152

Pregnancy specific b1 glycoprotein 5 W51985 1.760.1 1656 1022

Pregnancy-specific b1-glycoprotein 7 N32768 1.660.1 2779 1786

Nel-related protein N79534 1.560.1 2400 1535

Class IIA

Fibromodulin AA485748 4.660.6 1685 388

Cardiac gap junction protein AA487623 3.760.7 1545 487

Extracellular protein S1-5 AA875933 3.760.4 2324 666

Insulin-like growth factor binding protein 2 (36 kDa) H79047 3.360.5 961 328

Profilin 2 AA040703 2.860.3 1006 377

Proteasome subunit HsC7-I N53065 2.660.6 777 327

Integrin, aX N64384 2.560.6 1906 910

Transcription factor AREB6 H46554 2.260.3 1376 664

Proteasome subunit XAPC7 AA863149 2.060.2 1511 826

BMK1 a kinase H39192 2.060.2 1065 502

Mitochondrial NADH dehydrogenase-ubiquinone Fe-Sprotein 8, 23-kDa subunit precursor

AA127014 2.060.2 1657 903

Class IIB

Protein phosphatase 2A (b type) AA490473 2.761.2 1668 1017

Arp2/3 protein complex subunit p34-Arc H25917 2.560.4 2613 1162

Low-density lipoprotein receptor precursor AA504461 2.460.4 1423 576

DNA-repair protein complementing XP-C cells AA287323 2.360.8 1918 1201

APEG-1 T69450 2.360.6 1131 538

Plasma-cell membrane glycoprotein PC-1 R43778 2.360.4 818 386

DNA-directed RNA polymerase II (14.5 kDa) AA777192 2.260.3 1846 809

Cell division cycle 42 (GTP binding protein, 25 kDa) AA668681 2.160.3 1832 1054

Carboxypeptidase A1 AA845178 2.060.4 1033 609

ATP-binding cassette protein mRNA (06B09 clone) R83876 2.060.3 700 331

Superoxide dismutase 3, extracellular AA454160 1.960.3 2836 1628

RNA-binding protein CUG-BP/hNab50 R15111 1.960.2 1050 532

cAMP-dependent protein kinase (regulatory subunittype I)

AA630507 1.960.2 1753 1049

Arginyl-tRNA synthetase AA455652 1.960.2 817 462

OX-2 membrane glycoprotein precursor H23979 1.960.2 759 441

Complement C3 precursor AA873152 1.960.3 1273 791

M-phase phosphoprotein (mpp5) AA706968 1.860.2 1650 897

V-yes-1 oncogene homologue R83837 1.860.2 738 390

Nucleoside diphosphate kinase A AA644092 1.860.2 794 409

Zinc finger protein 10 (KOX 1) AA877082 1.860.3 1194 772

PHAPI2b protein AA489201 1.860.3 929 574

SA N73827 1.860.3 1292 832

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the aorta were at least 12-fold higher than any region of theheart (Table 4, top).

RGS5 message is reported to be highly expressed in mouseheart and very low in liver,8 but has never been examined inrodent vessels. We compared RGS5 expression in rat aorta,carotid artery, and vena cava with that in heart and liver byNorthern analysis (Figure 2C). The RGS5 message level wasequally high in rat aorta and heart (Table 4, middle) and 2.3times higher than the vena cava, whereas the carotid arteryhad lower expression than the vena cava, differing frommacaque and human expression. The rat message consisted ofa single band the same size as mouse heart (data not shown).The rat and macaque data establish RGS5 as a markerdifferentiating aorta from carotid artery and aorta from venacava, but show that there are distinct species differences inexpression levels in vessels. Finally, we examined the expres-sion of RGS5 on a human dot blot (Clontech) containingRNAs from 47 adult and 7 fetal tissues (Figure 3). Twenty-nine tissues had higher expression than skeletal muscle, amoderate-expression tissue6; the top 15 are quantified inTable 4, bottom. The 6 highest-expressing tissues in order(beginning with the highest) were aorta, small intestine,

stomach, heart (4.6-fold lower than the aorta), adrenal gland,and fetal heart.

To localize the RGS5 transcript in vivo, we performed insitu hybridization on macaque aorta and vena cava (Figure 4).Hybridization in the aorta from the antisense probe was seenuniformly throughout the media, well above the sense probebackground level, whereas the vena cava antisense probehybridization was very similar to the background sensehybridization. No hybridization was seen in the endothelium.The presence of endothelium was confirmed by examinationof hematoxylin and eosin–stained sections (Figures 4a and4b) and by immunohistochemistry using Von Willebrandfactor antibody staining (data not shown). One possibleexplanation of differential expression of RGS5 could havebeen the presence of RNA from vasa vasorum, as they arepresent in arteries but not veins.9 This remains a formalpossibility for other aorta differentially expressed genes.

DiscussionOur analysis of aorta versus vena cava reveals 68 genes thatmark the aorta by overexpression, including 2 transcripts

TABLE 2. Continued

Accession no. Mean R6SEM Mean Int Ao Mean Int VC

Eph-like receptor tyrosine kinase R48320 1.760.1 3380 2171

hEphB1c

Phosphoglycerate kinase 1 AA599187 1.660.1 1013 697

Ribosomal protein S25 R63811 1.560.0 1219 804

Class IIIA

Elastin AA459308 5.061.4 1515 381

dsRNA adenosine deaminase DRADA2b AA489331 3.060.5 782 313

Low-Mr GTP-binding protein (RAB31) T96082 2.660.2 916 369

Choline kinase H09959 2.360.2 1840 777

Class IIIB

B-lymphocyte serine/threonine protein kinase AA873159 2.761.0 586 241

Phosphoethanolamine cytidylyltransferase T72076 2.760.9 705 308

Apolipoprotein CI H59204 2.460.4 929 474

Plasma retinol binding protein precursor AA169645 2.060.4 460 272

Cdc6-related protein AA148548 2.060.3 645 334

Splicing factor SRp55-2 H17943 1.960.2 456 249

Human D9 splice variant A mRNA T73556 1.860.3 611 336

Fatty acid binding protein AA453750 1.860.2 685 397

Zinc finger protein, clone:RES4-26 AA448711 1.860.2 665 361

AU RNA-binding protein/enoyl–coenzyme A hydratase AA706987 1.860.2 696 364

UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (T1) AA188179 1.860.2 752 440

Arp2/3 protein complex subunit p41-Arc AA490945 1.860.3 440 268

Secretory carrier-associated membrane protein (SCAMP) AA488406 1.760.2 805 442

Clathrin-associated protein R50953 1.760.2 654 430

Long-chain fatty acid acyl-coA ligase R22219 1.760.1 794 474

Pre-promegakaryocyte potentiating factor AA421518 1.660.1 487 300

Ratios are the average of individual macaque aorta (Ao) to vena cava (VC) comparisons (n55). Mean intensity (Int) values areincluded as a guide for expression levels; individual macaque intensities were used to calculate mean ratios (R). All 68 consistentlydifferentially expressed genes had SEMs less than two-thirds of their average ratios.

*Array housekeeping genes.

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previously identified with the aorta, aortic preferentiallyexpressed gene 1 (APEG-1)10 and elastin.11,12

It is important to stress that we have examined 5 individualanimals to produce this data set, eliminating false-positiveresults due to animal variation in elevations of gene expres-sion or random measurement error. Genes were only declareddifferentially expressed if mean expression ratios were.1.5-fold. All 5 individual animal ratios were.1.3-fold; SEMswere less than two-thirds (given that a gene had to bedifferential in all animals by 1.3-fold, this fact in effectensured that the SEM was a measurement of variation indifferential ratio). All differential genes hybridized consis-tently above 1.5-fold the average background. Although anexpression ratio of 1.3 is small, in an analysis of messageexpression using cDNA arrays, Geiss et al13 demonstratedthat even ratios as low as 1.3 to 1.4 on the array can bestatistically meaningful for genes with low SE. They showedthat Northern blots produce higher ratios than do arrays.Thus, we expect that most of the 68 genes are truly differen-tially expressed. We have shown previously that this Re-search Genetics cDNA array produces values that agree withindependent methods of expression measurement.14 More-over, 44 of the 68 genes were expressed at ratios of 2-fold orgreater.

The most striking aspect of the data set is the expression ofone gene, RGS5. RGS5 is remarkably higher in average ratiothan any other transcript compared between aorta and venacava. Nevertheless, we suggest that it is the data set as awhole that begins the molecular description of the arterial andvenous phenotypes. Golub et al15 have recently comparedacute lymphoblastic leukemia with acute myeloid leukemia.Using more sophisticated analysis than currently available tous, these authors found a set of only 50 genes out of 6817 that

Figure 2. Northern hybridizations of RGS5 and NMMHC-B.Blots were stripped and reprobed with 28S rRNA hybridizationsin panels A and C and with G3PDH in panel B for loading quan-tification. A, Macaque tissues. H indicates heart; Skm, skeletalmuscle; Ao, aorta; CA, carotid artery; VC, vena cava; and Lv,liver. B, Human aorta and heart tissue (Clontech). FH indicatesfetal heart; H, heart; Ao, aorta; AP, apex of heart; LA, left atrium;RA, right atrium; LV, left ventricle; and RV, right ventricle. C, Rattissues. Abbreviations as in panels A and B. Panels A and C,Locations of 28S and 18S rRNAs are shown on the left at 4.9and 1.7 kb, respectively.

TABLE 3. Normalized Expression Levels of RGS5 andNMMHC-B in Macaque Tissues

Lane Tissue

RatioTissue/Skeletal

Muscle

3 Aorta 184.3

4 Carotid 14.5

1 Heart 5.1

5 Vena cava 4.1

6 Liver 1.2

2 SkM 1.0

Lane TissueNMMHC-B

Normalized/Liver

3 Aorta 370.7

5 Vena cava 71.0

4 Carotid 55.6

1 Heart 1.5

6 Liver 1.3

2 SkM 1.0

RGS5 (top) and NMMHC-B (bottom) hybridizations were normalized, first to28S rRNA hybridization to adjust to loading differences, then to normalizedskeletal muscle (Skm) hybridization. All hybridizations were quantified fromphosphor imager scans.

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could be used as a pattern to distinguish between these 2generally related neoplasms. Similarly, the 68 genes reportedhere represent a consistently differentially expressed set that

together constitute a phenotype for aortic and vena cavasmooth muscle.

A few of the genes in this set of 68 have been examinedpreviously in vascular smooth muscle contexts. Four genesshowing aortic expression, APEG-1, elastin, NMMHC-B,and vimentin have previously been identified as markers ofthe intima. APEG-1 has been shown in rats to be differen-tially expressed between aorta and vena cava and downregu-lated after carotid balloon injury.16 NMMHC-B, also calledSMemb, has been previously reported to be differentiallyexpressed in vivo in vascular smooth muscle, in humannormal intima and intima of coronary arteries after angio-plasty,17 and in 2 rabbit models of intima formation.18

Vimentin has been shown to be expressed in balloon-injuredrat carotid neointima, whereas another intermediate filament,desmin, disappears19 and is expressed in “synthetic” smoothmuscle cells in culture.20 Elastin has also been shown to be amarker between rat smooth muscle cell types of differentembryological origin and is upregulated after carotid arteryballoon injury in vivo.21 Secreted protein acidic and rich incysteine, seen here as an arterial marker, has also been shownto be differential between smooth muscle subtypes in vitrobut is not modulated in expression after carotid artery ballooninjury.22

TABLE 4. Quantification of RGS5 Expression in the HumanHeart and Human Tissues

Lane Tissue

RatioRGS5/Tissue/Fetal Heart*

3 Aorta 31.5

5 Left atrium 2.6

2 Heart 2.5

8 Right ventricle 2.4

7 Left ventricle 2.1

6 Right atrium 1.6

4 Heart apex 1.4

1 Fetal heart 1.0

Lane Tissue

RatioTissue/Skeletal

Muscle†

1 Heart 3.1

3 Aorta 3.0

5 Vena cava 1.3

2 Skeletal muscle 1.0

4 Carotid artery 0.2

6 Liver 0.2

BlotLocation Tissue

RatioTissue/Skeletal

Muscle‡

C2 Aorta 23.0

E3 Small intestine 6.7

C8 Stomach 6.6

C1 Heart 5.0

D5 Adrenal gland 3.8

G1 Fetal heart 3.7

D6 Thyroid gland 3.6

F1 Appendix 3.0

C5 Bladder 2.5

C7 Prostate 2.5

G7 Fetal lung 2.1

B6 Subthalamic nucleus 2.0

E1 Kidney 1.9

E7 Lymph node 1.8

A8 Medulla oblongata 1.8

*Quantified Northern analysis of RGS5 in human aorta and heart. RGS5hybridizations were normalized first to GAPDH hybridizations for loadingadjustment and then to the fetal heart hybridization level.

†Quantified expression of RGS5 in rat tissues. RGS5 levels were normalizedfirst to 28S rRNA levels to adjust to loading differences and then to normalizedskeletal muscle RGS5 hybridization. Hybridizations were quantified fromphosphor imager scans.

‡The 15 tissues with highest RGS5 message levels. RGS5 hybridizationswere normalized first to ubiquitin hybridization to adjust to loading differencesand then to normalized skeletal muscle RGS5 hybridization. Hybridizations werequantified from phosphor imager scans.

Figure 3. Dot blot of human tissue poly(A) RNAs (89 to 514 ngeach) (Clontech). A1 through A8, respectively, Whole brain, amyg-dala, caudate nucleus, cerebellum, cerebral cortex, frontal lobe,hippocampus, and medulla oblongata. B1 through B8, Occipitallobe, putamen, substantia nigra, temporal lobe, thalamus, subtha-lamic nucleus, spinal chord, and blank. C1 through C8, Heart,aorta, skeletal muscle, colon, bladder, uterus, prostate, and stom-ach. D1 through D8, Testis, ovary, pancreas, pituitary gland, adre-nal gland, thyroid gland, salivary gland, and mammary gland. E1through E8, Kidney, liver, small intestine, spleen, thymus, peripher-al leukocyte, lymph node, and bone marrow. F1 through F4,Appendix, lung, trachea, and placenta. F5 through F8, Blank. G1through G8 (fetal tissue), brain, heart, kidney, liver, spleen, thymus,lung, and blank. H1 through H8 (controls), 100 ng yeast total RNA,100 ng yeast tRNA, 100 ng Escherichia coli rRNA, 100 ng E. coliDNA, 100 ng poly r(A), 100 ng human Cot-1 DNA, 100 ng humanDNA, and 500 ng human DNA.

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As for the remainder of the data set, it is intriguing to seethat only a minority of these genes has been of traditionalinterest to vascular biologists, suggesting that there are manynovel aspects of arteries and veins yet to functionally char-acterize. Among the aortic genes not previously studied invascular biology, the high level of differential expression ofRGS5 singles it out for our attention. RGS5 is currently an“orphan” G-protein pathway regulator, and its exact functionsneed to be characterized. The core function of RGS proteinsis to regulate G-protein signal strength and duration bybinding to and dephosphorylating Ga subunits.8,22–26 Addi-tionally, there is evidence for both inhibitor and effector(signal integration) G-protein signaling from 2 multidomainRGS proteins, p115RhoGEF27,28 and PDZRhoGEF,29 sug-gesting that the role RGS5 plays in arteries may be quitecomplex. RGS4, RGS5, RGS8, and RGS16 are among theshortest RGS proteins and share homology outside their RGSdomains, suggesting they constitute a subfamily, with RGS4and RGS5 being the most similar.30–33 In transgenic mouseoverexpression studies, high levels of RGS4 inhibit cardiacventricular hypertrophy in response to cardiac pressure over-load after thoracic aortic constriction.34 These remodelingprocesses are required for adaptation and survival of trans-genics. Also, in transgenic mouse overexpression studies ofGaq (a binding partner of RGS4), high expression levelscause cardiac hypertrophy.35,36

These data for RGS4 overexpression in heart, and ourexpression data for RGS5 in vessels, suggest that RGS5 could

be a counterpart in arteries to the RGS4-regulated system ofbalance between activation and deactivation of G protein–induced hypertrophic signaling in the heart. Arteries, likehearts, adapt to increased tension by remodeling, producing athick vessel wall able to generate a stronger contractileresponse.37 Considering the known role for RGS4 in heartaccommodation to increased pressure and its structural sim-ilarities to RGS5, we speculate that RGS5 might also beinvolved in the adaptation of arteries to normal and patho-logical pressure changes. However, until a transgenic over-expression of RGS5 is performed and tested after aorticconstriction, there will be no direct evidence to determinewhether RGS5 is regulating pressure-induced hypertrophy inarteries.

In summary, our analysis of 4048 genes, combined withthe '20 000 genes expected to be expressed in an averagetissue,38 suggests there will be roughly 340 genes marking theaortic versus the venous phenotype, including perhaps 4genes of the ratio magnitude of RGS5. It is intriguing that wefound no genes overexpressed in vein versus artery. Onepossible explanation is that the arterial phenotype is built ona basic veinlike phenotype. We speculate that the most highlyoverexpressed aortic gene, RGS5, could play a role in controlof transcription of the entire aortic phenotype and, based onhomology to other RGS proteins, may play an aortic-specificrole in regulation of responses to G-protein–mediated vaso-active signals.

AcknowledgmentsThis work was supported by NIH Grants RO1HL 57557,RO1HL58083, and PO1HL03174. We thank Isa Werny, JonathanMcBride, and Colette Norby-Slycord for their excellent technicalassistance. We thank Dr Linda Harris for critical reading of themanuscript and many helpful discussions on cDNA array issues, aswell as Dr Roger Bumgarner and Dr Eileen Mulvihill for usefuldiscussions on cDNA array analysis methodologies.

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Adams et al Expression Profiles of Aorta and Vena Cava 631

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Lawrence D. Adams, Randolph L. Geary, Bruce McManus and Stephen M. SchwartzMedia

Analysis Identifies a Set of 68 Consistently Differentially Expressed Genes, All in Aortic A Comparison of Aorta and Vena Cava Medial Message Expression by cDNA Array

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