© 2000 American Heart Association, Inc.
Molecular Medicine |
A Comparison of Aorta and Vena Cava Medial Message Expression by cDNA Array Analysis Identifies a Set of 68 Consistently Differentially Expressed Genes, All in Aortic Media
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 of British Columbia, Vancouver, British Columbia, Canada.
Correspondence to Lawrence D. Adams, Department of Pathology, University of Washington, Box 357335, 1959 NE Pacific St, Seattle, WA 98195-7335. E-mail ladams{at}u.washington.edu
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Abstract—We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles of macaque aorta and vena cava media using a cDNA array containing 4048 known human genes,
35% of currently
named 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 of 46.5±12.6 (mean±SEM). The data set also contained 2 genes
already known to be expressed in the aorta, elastin at 5.0±1.4, and
the aortic preferentially expressed gene 1 (APEG-1) at 2.3±0.6. We
chose to analyze RGS5 expression further because of its high
level of differential expression in the aorta. Levels of RGS5 mRNA were
confirmed by Northern 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 aorta is
higher than in any region of the heart. RGS5 is a G-protein signaling
regulator of unknown specificity most homologous to RGS4, an
inhibitory regulator of pressure-induced cardiac
hypertrophy. The expression pattern of the 68 differential
genes as a whole is a start toward identifying the molecular
phenotypes of arteries and veins on a systematic basis.
Key Words: cDNA array • aorta • vena cava • expression profile • RGS5
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Introduction |
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We have begun a systematic analysis of expression differences between vascular smooth muscle tissues. We chose to begin with an analysis of the media of aorta and vena cava because of the possibility of isolating relatively homogenous cellular populations and because of the distinct functional requirements, embryological origins, and hemodynamic environments of these 2 tissues.
The present study, using a cDNA array containing 35% of
human named genes (4048 of the 10 900 functionally identified human
genes in UniGene Build No. 109 of the National Center for
Biotechnology Information human Unigene collection), identified 68
genes that are consistently expressed at higher levels in the
aorta. No genes were more consistently highly expressed by the vena
cava. One of these aortic markers, regulator of G-protein signaling
(RGS) 5, showed extreme levels of differential expression. Together,
these genes offer a molecular definition of the phenotypes of
these 2 types of smooth muscle.
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Materials and Methods |
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Array Hybridization
Research Genetics GF211 cDNA arrays (4048 human genes) were used as specified. Equivalent amounts of RNA per vessel pair were used to synthesize cDNA probes, and equivalent counts per minute were added to hybridizations. Washed blots were exposed to phosphor image screens and scanned on a Storm phosphor imager (Molecular Dynamics). Expression was quantified from scans with similar intensity and background, using Pathways software (Research Genetics). Ratios were calculated for each animal and then averaged.
Northern, Dot Blot, and In Situ Hybridization
Total RNA (15 µg) from macaque or rat was run, transferred,
and hybridized as described1 with multiprime probes
(Amersham) synthesized from sequence-verified clones of human and mouse
RGS5 (IMAGE clones 853809 and 1853118, respectively [American Type
Culture Collection]) and of human nonmuscle myosin heavy chain B
(NMMHC-B) (IMAGE clone 823886), and then probed with a 28S rRNA
primer as described.2 Washes were as follows: 3 cycles of
5 minutes each at room temperature in 5x SSC, 0.1% SDS, and 2 cycles
of 20 minutes each at 57°C (rat) or 55°C (macaque) in 0.3x
SSC, 0.1% SDS. Human Northern blot No. 7791-1 (Clontech Laboratories,
Inc) containing 2 µg poly(A) RNA from adult and fetal heart, aorta,
and 5 regions of the heart was probed with human RGS5, then G3PDH as
specified. Human RNA master dot blot No. 7770-1 (Clontech) containing
33 tissue, 14 brain region, and 7 fetal poly(A) RNAs (89 to 514 ng
each) was probed as specified with human clones for RGS5 and then human
ubiquitin.
In situ hybridization was performed on cross sections of aorta and vena cava taken directly adjacent to vessel segments snap-frozen for array analyses. These tissues were immersion-fixed for 48 hours in 10% formalin and embedded in paraffin. Sections were cut onto coated slides and in situ hybridization was performed using 35S-labeled riboprobes (sense and antisense) transcribed from human RGS5 cDNA No. 853809 as described.3
Tissue Samples and RNA Isolation
Three human aortas, 2 from males (age 32 and 33 years) and 1
from a female (age 47 years), were isolated from donor hearts obtained
from the University of British Columbia–St. Paul’s Hospital. Samples
were stripped of adventitia and endothelium and
snap-frozen in liquid nitrogen.
Macaque tissues were obtained from 5 surgically menopausal (ovariectomized) female cynomolgus macaque monkeys (Macaca fascicularis).4 These monkeys made up the control group from a study of the effects of isoflavones on bypass graft intimal hyperplasia and iliac artery angioplasty. Animals were not administered isoflavones but underwent bypass graft placement between the distal aorta and right iliac artery and angioplasty of the left common iliac artery. The bypass graft originated at the most distal abdominal aorta, with the proximal anastomosis adjacent to the aortic bifurcation and distal anastomosis to the external iliac artery. The vena cava and aorta used for arrays were removed well proximal to the site of surgery (distal thoracic aorta; vena cava from the diaphragm to just above the aortic anastomosis) 6 weeks after surgery. After sedation with ketamine (15 mg/kg IM) and butorphanol (0.05 mg/kg IM), animals were anticoagulated with heparin (300 U/kg IV) and deeply anesthetized with sodium pentobarbital (100 mg/kg IV). The adventitia was stripped from both the aorta and the vena cava, and the endothelium was removed from the aorta but not the vena cava. Tissues were snap-frozen in liquid nitrogen. All animal care and procedures were performed at the Comparative Medicine Clinical Research Center of the Wake Forest University School of Medicine in accordance with state and federal law. Animal protocols were approved by the Wake Forest University Animal Care and Use Committee and conformed to guidelines set forth in the Principles of Laboratory Animal Care (formulated by the National Society for Medical Research), and in the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985).
Twenty male Sprague-Dawley rats (Rattus norvegicus) (Zivig-Miller) were housed and fed according to protocols approved by the Animal Care Committee of the University of Washington. Rats were anesthetized and euthanized, and tissues were processed as previously described.2 All RNA was isolated as previously described.5
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Results |
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General Comparison of Human and Macaque Array Hybridization
Samples of RNA from 5 pairs of macaque aortas and vena cavas were used to probe a cDNA array containing 4048 human genes. Genes were defined as expressed if hybridization was
1.5 times the average
background (before normalization in Pathways software to overall
hybridization strength). The informativeness of macaque probes on human
filters in terms of the total number of positive responding spots was a
concern, because all arrayed targets consist of 1000 bp of the 3'
end of each gene and contain complete 3' untranslated regions, areas of
low interspecies homology. To quantify general hybridization patterns,
we hybridized cDNA probes from human aorta (n=3) and from macaque aorta
and vena cava (both n=5), then compared the average number of genes
expressed by the above definition. The number of genes detected in
human was 200 to 500 greater than in macaque (human aorta, 3855±115
[mean±SEM]; macaque aorta, 3367±115; and macaque vena cava,
3635±181), indicating possible species specific differences in 3'
noncoding domains or, possibly, species differences in expression.
Identification of Differentially Expressed Genes
To have objective criteria for identifying consistently
differentially expressed genes, we established categories of ratio and
array hybridization level for this study (Table 1
). After analyzing the data for the 5
independent macaque comparisons, there were 68 genes that fit these
criteria. All were higher in the aorta (Table 2). Of these, only RGS5 was expressed
>10-fold, at 46.5±12.6 SEM. RGS5 had the highest level of
differential expression in each macaque. Levels were undetectable, or
barely detectable, in vena cava, suggesting an "all-or-nothing"
expression. The array hybridization images for class IA genes are shown
in Figure 1A. Expression ratios of
the 68 differentially expressed genes are graphed in Figure 1B.
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Northern Analysis and In Situ Hybridization
We verified the array-based quantification by further examining
the expression of the 2 most highly expressed genes, RGS5 and NMMHC-B.
First, we examined macaque aorta and vena cava by Northern
analyses (Figure 2A). RGS5
was most highly expressed in macaque aorta, followed by carotid artery,
heart, and vena cava, with little expression detected in skeletal
muscle or liver. NMMHC-B was most highly expressed in aorta, followed
by vena cava and then the carotid artery. After normalization to 28S
rRNA hybridization (Table 3), the
Northern blot aorta-to-vena cava ratio was quantified as 45-fold for
RGS5 and 5-fold for NMMHC-B, very close to the mean array values.
Macaque message sizes were consistent with the published human
sizes,6 with 2 major RGS5 bands at 7.0 and 2.1
kb and 1 NMMHC-B band at 7.5 kb.7
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We further examined RGS5 because of its large differential expression
ratio. To compare human aorta RGS5 message levels to heart, the
highest-expressing human tissue examined in the
literature,6 we probed a Clontech Northern blot containing
RNA samples of aorta, fetal and adult heart, and 5 distinct heart
regions (Figure 2B
). RGS5 message levels in the aorta were at
least 12-fold higher than any region of the heart (Table 4, top).
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RGS5 message is reported to be highly expressed in mouse
heart and very low in liver,8 but has never been examined
in rodent vessels. We compared RGS5 expression in rat aorta, carotid
artery, and vena cava with that in heart and liver by Northern
analysis (Figure 2C). The RGS5 message level was equally
high in rat aorta and heart (Table 4, middle) and 2.3 times
higher than the vena cava, whereas the carotid artery had lower
expression than the vena cava, differing from macaque and human
expression. The rat message consisted of a single band the same size as
mouse heart (data not shown). The rat and macaque data establish RGS5
as a marker differentiating aorta from carotid artery and aorta from
vena cava, but show that there are distinct species differences in
expression levels in vessels. Finally, we examined the expression of
RGS5 on a human dot blot (Clontech) containing RNAs from 47 adult and 7
fetal tissues (Figure 3).
Twenty-nine tissues had higher expression than skeletal muscle, a
moderate-expression tissue6 ; the top 15 are quantified in
Table 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.
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To localize the RGS5 transcript in vivo, we performed in situ
hybridization on macaque aorta and vena cava (Figure 4). Hybridization in the aorta from the
antisense probe was seen uniformly throughout the media, well above the
sense probe background level, whereas the vena cava antisense probe
hybridization was very similar to the background sense hybridization.
No hybridization was seen in the endothelium. The
presence of endothelium was confirmed by examination of
hematoxylin and eosin–stained sections (Figures 4a and 4b) and
by immunohistochemistry using Von Willebrand factor antibody staining
(data not shown). One possible explanation of differential expression
of RGS5 could have been the presence of RNA from vasa vasorum, as they
are present in arteries but not veins.9 This remains a
formal possibility for other aorta differentially expressed genes.
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Discussion |
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Our analysis of aorta versus vena cava reveals 68 genes that mark the aorta by overexpression, including 2 transcripts previously identified with the aorta, aortic preferentially expressed gene 1 (APEG-1)10 and elastin.11 12
It is important to stress that we have examined 5 individual animals to produce this data set, eliminating false-positive results due to animal variation in elevations of gene expression or random measurement error. Genes were only declared differentially expressed if mean expression ratios were >1.5-fold. All 5 individual animal ratios were >1.3-fold; SEMs were less than two-thirds (given that a gene had to be differential in all animals by 1.3-fold, this fact in effect ensured that the SEM was a measurement of variation in differential ratio). All differential genes hybridized consistently above 1.5-fold the average background. Although an expression ratio of 1.3 is small, in an analysis of message expression using cDNA arrays, Geiss et al13 demonstrated that even ratios as low as 1.3 to 1.4 on the array can be statistically meaningful for genes with low SE. They showed that Northern blots produce higher ratios than do arrays. Thus, we expect that most of the 68 genes are truly differentially expressed. We have shown previously that this Research Genetics cDNA array produces values that agree with independent methods of expression measurement.14 Moreover, 44 of the 68 genes were expressed at ratios of 2-fold or greater.
The most striking aspect of the data set is the expression of one gene, RGS5. RGS5 is remarkably higher in average ratio than any other transcript compared between aorta and vena cava. Nevertheless, we suggest that it is the data set as a whole that begins the molecular description of the arterial and venous phenotypes. Golub et al15 have recently compared acute lymphoblastic leukemia with acute myeloid leukemia. Using more sophisticated analysis than currently available to us, these authors found a set of only 50 genes out of 6817 that could be used as a pattern to distinguish between these 2 generally related neoplasms. Similarly, the 68 genes reported here represent a consistently differentially expressed set that together constitute a phenotype for aortic and vena cava smooth muscle.
A few of the genes in this set of 68 have been examined previously in vascular smooth muscle contexts. Four genes showing aortic expression, APEG-1, elastin, NMMHC-B, and vimentin have previously been identified as markers of the intima. APEG-1 has been shown in rats to be differentially expressed between aorta and vena cava and downregulated after carotid balloon injury.16 NMMHC-B, also called SMemb, has been previously reported to be differentially expressed in vivo in vascular smooth muscle, in human normal intima and intima of coronary arteries after angioplasty,17 and in 2 rabbit models of intima formation.18 Vimentin has been shown to be expressed in balloon-injured rat carotid neointima, whereas another intermediate filament, desmin, disappears19 and is expressed in "synthetic" smooth muscle cells in culture.20 Elastin has also been shown to be a marker between rat smooth muscle cell types of different embryological origin and is upregulated after carotid artery balloon injury in vivo.21 Secreted protein acidic and rich in cysteine, seen here as an arterial marker, has also been shown to be differential between smooth muscle subtypes in vitro but is not modulated in expression after carotid artery balloon injury.22
As for the remainder of the data set, it is intriguing to see that only
a minority of these genes has been of traditional interest to vascular
biologists, suggesting that there are many novel aspects of arteries
and veins yet to functionally characterize. Among the aortic genes not
previously studied in vascular biology, the high level of differential
expression of RGS5 singles it out for our attention. RGS5 is currently
an "orphan" G-protein pathway regulator, and its exact functions
need to be characterized. The core function of RGS proteins is to
regulate G-protein signal strength and duration by binding to and
dephosphorylating G
subunits.8 22 23 24 25 26 Additionally,
there is evidence for both inhibitor and effector (signal
integration) G-protein signaling from 2 multidomain RGS proteins,
p115RhoGEF27 28 and PDZRhoGEF,29 suggesting
that the role RGS5 plays in arteries may be quite complex. RGS4, RGS5,
RGS8, and RGS16 are among the shortest RGS proteins and share homology
outside their RGS domains, suggesting they constitute a subfamily, with
RGS4 and RGS5 being the most similar.30 31 32 33 In transgenic
mouse overexpression studies, high levels of RGS4 inhibit cardiac
ventricular hypertrophy in response to cardiac
pressure overload after thoracic aortic constriction.34
These remodeling processes are required for adaptation and survival of
transgenics. Also, in transgenic mouse overexpression studies of Gq
(a binding partner of RGS4), high expression levels cause cardiac
hypertrophy.35 36
These data for RGS4 overexpression in heart, and our expression data for RGS5 in vessels, suggest that RGS5 could be a counterpart in arteries to the RGS4-regulated system of balance between activation and deactivation of G protein–induced hypertrophic signaling in the heart. Arteries, like hearts, adapt to increased tension by remodeling, producing a thick vessel wall able to generate a stronger contractile response.37 Considering the known role for RGS4 in heart accommodation to increased pressure and its structural similarities to RGS5, we speculate that RGS5 might also be involved in the adaptation of arteries to normal and pathological pressure changes. However, until a transgenic overexpression of RGS5 is performed and tested after aortic constriction, there will be no direct evidence to determine whether RGS5 is regulating pressure-induced hypertrophy in arteries.
In summary, our analysis of 4048 genes, combined with the
20 000 genes expected to be expressed in an average
tissue,38 suggests there will be roughly 340 genes marking
the aortic versus the venous phenotype, including perhaps 4
genes of the ratio magnitude of RGS5. It is intriguing that we found no
genes overexpressed in vein versus artery. One possible explanation is
that the arterial phenotype is built on a basic
veinlike phenotype. We speculate that the most highly
overexpressed aortic gene, RGS5, could play a role in control of
transcription of the entire aortic phenotype and, based on
homology to other RGS proteins, may play an aortic-specific role in
regulation of responses to G-protein–mediated vasoactive signals.
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Acknowledgments |
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This work was supported by NIH Grants RO1HL 57557, RO1HL58083, and PO1HL03174. We thank Isa Werny, Jonathan McBride, and Colette Norby-Slycord for their excellent technical assistance. We thank Dr Linda Harris for critical reading of the manuscript and many helpful discussions on cDNA array issues, as well as Dr Roger Bumgarner and Dr Eileen Mulvihill for useful discussions on cDNA array analysis methodologies.
Received July 26, 2000; revision received August 22, 2000; accepted August 23, 2000.
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 E. Lutgens, B. Faber, K. Schapira, C. T.A. Evelo, R. van Haaften, S. Heeneman, K. B.J.M. Cleutjens, A. P. Bijnens, L. Beckers, J. G. Porter, et al. Gene Profiling in Atherosclerosis Reveals a Key Role for Small Inducible Cytokines: Validation Using a Novel Monocyte Chemoattractant Protein Monoclonal Antibody Circulation, June 28, 2005; 111(25): 3443 - 3452. [Abstract] [Full Text] [PDF]
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N. Kaplan-Albuquerque, Y. E. Bogaert, V. Van Putten, M. C. Weiser-Evans, and R. A. Nemenoff Patterns of Gene Expression Differentially Regulated by Platelet-derived Growth Factor and Hypertrophic Stimuli in Vascular Smooth Muscle Cells: MARKERS FOR PHENOTYPIC MODULATION AND RESPONSE TO INJURY J. Biol. Chem., May 20, 2005; 280(20): 19966 - 19976. [Abstract] [Full Text] [PDF]
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N. Grillet, A. Pattyn, C. Contet, B. L. Kieffer, C. Goridis, and J.-F. Brunet Generation and Characterization of Rgs4 Mutant Mice Mol. Cell. Biol., May 15, 2005; 25(10): 4221 - 4228. [Abstract] [Full Text] [PDF]
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T. Yoshida and G. K. Owens Molecular Determinants of Vascular Smooth Muscle Cell Diversity Circ. Res., February 18, 2005; 96(3): 280 - 291. [Abstract] [Full Text] [PDF]
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 A. P. J. J. Bijnens, A. Gils, B. Jutten, B. C. G. Faber, S. Heeneman, P. J. E. H. M. Kitslaar, J. H. M. Tordoir, C. J. M. de Vries, A. A. Kroon, M. J. A. P. Daemen, et al. Vasculin, a novel vascular protein differentially expressed in human atherogenesis Blood, October 15, 2003; 102(8): 2803 - 2810. [Abstract] [Full Text] [PDF]
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S. E. Levy and J. A.S. Muldowney 3rd Microarray Analysis of Neointima: Flowing Toward a Clear Future Arterioscler. Thromb. Vasc. Biol., December 1, 2002; 22(12): 1946 - 1947. [Full Text] [PDF]
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R. L. Geary, J. M. Wong, A. Rossini, S. M. Schwartz, and L. D. Adams Expression Profiling Identifies 147 Genes Contributing to a Unique Primate Neointimal Smooth Muscle Cell Phenotype Arterioscler. Thromb. Vasc. Biol., December 1, 2002; 22(12): 2010 - 2016. [Abstract] [Full Text] [PDF]
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Q. J. Zhang, M. Goddard, C. Shanahan, L. Shapiro, and M. Bennett Differential Gene Expression in Vascular Smooth Muscle Cells in Primary Atherosclerosis and In Stent Stenosis in Humans Arterioscler. Thromb. Vasc. Biol., December 1, 2002; 22(12): 2030 - 2036. [Abstract] [Full Text] [PDF]
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Q. Wang, M. Liu, B. Mullah, D. P. Siderovski, and R. R. Neubig Receptor-selective Effects of Endogenous RGS3 and RGS5 to Regulate Mitogen-activated Protein Kinase Activation in Rat Vascular Smooth Muscle Cells J. Biol. Chem., July 5, 2002; 277(28): 24949 - 24958. [Abstract] [Full Text] [PDF]
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 S. M. McCormick, S. G. Eskin, L. V. McIntire, C. L. Teng, C.-M. Lu, C. G. Russell, and K. K. Chittur DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells PNAS, July 31, 2001; 98(16): 8955 - 8960. [Abstract] [Full Text] |