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Content may be subject to copyright.
Coexisting proinflammatory and antioxidative
endothelial transcription profiles in a disturbed flow
region of the adult porcine aorta
Anthony G. Passerini*
†‡
, Denise C. Polacek*
‡
, Congzhu Shi*, Nadeene M. Francesco*, Elisabetta Manduchi
§
,
Gregory R. Grant
§
, William F. Pritchard
¶
, Steven Powell
储
, Gary Y. Chang*
†
, Christian J. Stoeckert, Jr.
§
**,
and Peter F. Davies*
†,††‡‡
*Institute for Medicine and Engineering, Departments of
†
Bioengineering,
††
Pathology and Laboratory Medicine, and **Genetics, and
§
Center for
Bioinformatics, University of Pennsylvania, Philadelphia, PA 19104;
¶
U.S. Food and Drug Administration, Rockville, MD 20852; and
储
AstraZeneca
Pharmaceuticals, Mereside Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved December 22, 2003 (received for review
September 15, 2003)
In the arterial circulation, regions of disturbed flow (DF), which are
characterized by flow separation and transient vortices, are suscep-
tible to atherogenesis, whereas regions of undisturbed laminar flow
(UF) appear protected. Coordinated regulation of gene expression by
endothelial cells (EC) may result in differing regional phenotypes that
either favor or inhibit atherogenesis. Linearly amplified RNA from
freshly isolated EC of DF (inner aortic arch) and UF (descending
thoracic aorta) regions of normal adult pigs was used to profile
differential gene expression reflecting the steady state in vivo.By
using human cDNA arrays, ⬇2,000 putatively differentially expressed
genes were identified through false-discovery-rate statistical meth-
ods. A sampling of these genes was validated by quantitative real-
time PCR and兾or immunostaining en face. Biological pathway analysis
revealed that in DF there was up-regulation of several broad-acting
inflammatory cytokines and receptors, in addition to elements of the
NF-
B system, which is consistent with a proinflammatory pheno-
type. However, the NF-
B complex was predominantly cytoplasmic
(inactive) in both regions, and no significant differences were ob-
served in the expression of key adhesion molecules for inflammatory
cells associated with early atherogenesis. Furthermore, there was no
histological evidence of inflammation. Protective profiles were ob-
served in DF regions, notably an enhanced antioxidative gene ex-
pression. This study provides a public database of regional EC gene
expression in a normal animal, implicates hemodynamics as a con-
tributory mechanism to athero-susceptibility, and reveals the coex-
istence of pro- and antiatherosclerotic transcript profiles in suscepti-
ble regions. The introduction of additional risk factors may shift this
balance to favor lesion development.
T
here is a strong correlation between flow characteristics and the
focal and regional nature of atherogenesis. Sites of disturbed
flow (DF) (e.g., the inner wall of curved vessels and the wall
opposite the flow divider at branches and bifurcations) are suscep-
tible to lesion development, whereas regions of undisturbed laminar
flow (UF) are relatively protected (1–3). Throughout the initiation
and development of atherosclerotic lesions, the endothelium is
retained as the interface between blood and arterial tissues (4, 5),
where its function both before and during lesion formation is critical
to the pathological outcome. It has been hypothesized that coor-
dinated regulation of endothelial gene expression in response to
local biomechanical forces results in differing regional phenotypes
that promote athero-protection or athero-susceptibility (6). This
hypothesis has been addressed in principle by gene expression
studies of cultured endothelium (7–20). However, it has only been
addressed in vivo for a limited number of candidate genes and
proteins (21–25).
Studies of candidate gene expression by endothelial cells (EC) in
culture (12, 18, 23) and in knock-out animals (22, 24, 25) suggest
that transcriptional activity is linked to flow disturbances. In EC
cultures, unidirectional laminar shear stress (LSS) was correlated
with protective profiles of gene expression (e.g., antioxidative,
antiinflammatory, and兾or antiproliferative) when compared to the
absence of flow (7–11, 13, 14, 17, 19) and selected examples of the
protective genes have been shown to be expressed in endothelium
in vivo (14, 19). A small number of microarray studies comparing
EC gene expression between DF and UF conditions in vitro have
been conducted (15, 16); however, transcriptional profiling in vivo
has been limited by the small sample size available within hemo-
dynamic regions of interest. The in vivo correlation between local
gene expression and athero-susceptibility has therefore been gen-
erated primarily by extrapolation of in vitro findings. Using RNA
amplification to overcome sampling limitations, we have profiled
regional EC gene expression in the normal adult pig aorta to
investigate athero-susceptibility as a function of differential hemo-
dynamics in vivo.
Materials and Methods
Sample Collection and Characterization. Endothelial cells were
freshly harvested from eight adult pig aortas (Landrace X York-
shire males, 230–250 pounds; Hatfield Industries, Hatfield, PA).
The ascending aorta, aortic arch, and descending thoracic aorta
were dissected from the surrounding tissue (Fig. 1A), flushed with
cold sterile PBS, and incised lengthwise. EC were gently scraped
from a 1-cm
2
region located at the inner-curve and lateral walls of
the aortic arch (DF) and separately from the dorsal descending
thoracic aorta (UF) as illustrated in Fig. 1B, with each sample
representing ⬇10,000 cells. The cells were transferred directly to a
lysis buffer containing RNase inhibitors. Representative cell
scrapes were periodically transferred to glass microscope slides to
monitor cell purity by immunostaining with EC-specific anti-
platelet-endothelial cell adhesion molecule 1 and smooth muscle
cell (SMC)-specific anti-
␣
-actin antibodies. Additional tissue sam-
ples were fixed in 4% paraformaldehyde for en face immunostain-
ing, regional EC nuclear staining (Hoechst 33258), and cross-
sectional vessel histology.
Microarray Procedures. These procedures have been described (26)
and are provided in full in Supporting Materials and Methods, which
is published as supporting information on the PNAS web site. Total
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: DF, disturbed flow; UF, undisturbed flow; EC, endothelial cells; LSS, laminar
shear stress; SMC, smooth muscle cell; QRT-PCR, quantitative real-time PCR; VCAM-1,
vascular cell adhesion molecule 1; ROS, reactive oxygen species; GPX3, glutathione perox-
idase 3; TNF, tumor necrosis factor.
‡
A.G.P. and D.C.P. contributed equally to this work.
‡‡
To whom correspondence should be addressed at: Institute for Medicine and Engineer-
ing, University of Pennsylvania, 1010 Vagelos Laboratories, 3340 Smith Walk, Philadel-
phia, PA 19104. E-mail: pfd@pobox.upenn.edu.
© 2004 by The National Academy of Sciences of the USA
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no. 8 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0305938101
RNA (100 ng; ⬇1 ng of mRNA) was linearly amplified (27), and 2
g of amplified antisense RNA was used to synthesize
33
P-labeled
DNA probes. These probes were hybridized to nylon microarray
filters, custom designed, and printed by AstraZeneca Pharmaceu-
ticals (Alderley Park, UK), which contained 13,824 3⬘-biased,
sequence-verified human cDNA clones (1.5–2.0 kb) spotted in
duplicate. Matched DF and UF arrays for the same animal (n ⫽ 8)
were processed simultaneously.
Data Analysis and Bioinformatics. Image files were quantified with
ARRAYVISION 6.3 (Imaging Research, St. Catherine’s, ON, Canada)
as described (26). A putative set of differentially expressed genes
was identified by taking the union of predictions made through the
methods of
ARRAYSTAT 1.2 (Imaging Research) (28) in ‘‘unpaired’’
mode and
SAM 1.15 (Significance Analysis of Microarrays) (29) in
‘‘one class’’ mode. Both methods used an expected false-discovery
rate (30) of 5%. The putative set of differentially expressed genes
was imported into
GENESPRING (Silicon Genetics, Redwood City,
CA) for annotation by using information available in public data-
bases and hierarchical classification according to a simple gene
ontology construction. A detailed description of the data analysis is
provided in Supporting Materials and Methods. Results obtained by
other analytical approaches can be accessed at www.cbil.upenn.edu/
RAD/normal㛭pig㛭study. In accordance with proposed standards of
the Microarray Gene Expression Data Society (www.mged.org),
the complete annotated study is publicly available in a
MIAME
compliant framework through the RNA Abundance Database
(www.cbil.upenn.edu/RAD) (31).
Validation Studies. Immunostaining on fixed tissue sections was
performed according to standard procedures for selected proteins.
Quantitative real-time PCR (QRT-PCR) was performed on se-
lected genes as described (26). Expression was measured in paired
DF and UF samples from six animals, and a ratio was calculated for
each animal based on a minimum of three replicate observations.
If the PCR ratios for at least four of six animals were ⬎1.1 or ⬍0.9,
then the PCR result was designated ‘‘up-regulation’’ or ‘‘down-
regulation,’’ respectively. If the PCR ratios for at least four of six
animals were between 0.9 and 1.1, then the designation was
‘‘unchanged.’’ In all other cases, no decision was made based on the
PCR results. Where the PCR ratios were ⬎1.1 in three animals and
⬍0.9 in the remaining animals, a note was made about this
interanimal discrepancy.
Results
Regional Characterization of EC. The presence of flow reversal in the
aortic arch of adult boars similar to that measured in humans (32)
was confirmed by ultrasound (data not shown). EC isolated from
DF and UF regions (Fig. 1 A and B) displayed differences in cell
shape and alignment that reflected the local hemodynamic condi-
tions (Fig. 4 A and B, which is published as supporting information
on the PNAS web site). In situ immunostaining with EC-specific
platelet-endothelial cell adhesion molecule 1 and SMC-specific
␣
-actin antibodies confirmed the presence of an intact endothelium
(Fig. 4 C and D). There was no histological evidence of inflamma-
tion at either site (Fig. 5, which is published as supporting infor-
mation on the PNAS web site). To monitor the specificity of the
isolation procedure for EC, cell scrapes were transferred to glass
microscope slides where cell-specific staining confirmed EC purity
of ⬎99% (Fig. 1 C and D). Thus near-pure isolates of EC showing
morphologies consistent with differential hemodynamic environ-
ments in vivo were rapidly obtained for transcript profiling.
Differential Expression. Putative sets of differentially expressed EC
genes (DF compared with UF) identified in the normal pig aorta
through the predictions of
ARRAYSTAT and兾or SAM are summa-
rized in Table 1, which also shows their annotation by biological
classifications of known relevance to the initiation and progression
of atherosclerosis.
SAM analysis identified a larger number of genes
(1,823) than did
ARRAYSTAT (1,048) at the same false-discovery
rate of 5%, while capturing ⬇75% of the
ARRAYSTAT set. An
expanded version of Table 1 with links to fully annotated gene lists
is available online (www.cbil.upenn.edu/RAD/normal㛭pig㛭study).
QRT-PCR. Twenty-seven genes identified as significantly differen-
tially expressed were selected for validation by QRT-PCR. Fig. 2
summarizes the concordance of differential expression determined
Fig. 1. (A and B) Illustration of the endothelial cell isolation procedure. About
10,000 cells were scraped from precisely defined regions (⬇1cm
2
) of the aortic
arch (DF) and the descending thoracic aorta (UF). (C and D) A representative field
of EC isolated by mechanical scraping and stained with anti-platelet-endothelial
cell adhesion molecule 1 (PECAM-1) and anti-
␣
-actin antibodies. EC purity (green)
was routinely ⬎99% with only occasional contamination by isolated SMC (red;
arrow).
Table 1. Genes identified as differentially expressed (DF vs. UF)
in the normal pig aorta by biological classification
Biological classification
No. represented
on array
No. differentially
expressed
All genes 13824 2091
Adhesion 298 44
Apoptosis 244 47
Coagulation 63 8
Complement 71 19
Extracellular matrix 270 47
Growth factors 238 50
Immune response 136 28
Inflammation 150 28
Lipid兾cholesterol metabolism 167 20
NF-
B5110
Oxidative mechanisms 166 27
Proliferation 391 73
Signal transduction 2430 412
TNF-
␣
69 8
Transcription factors 557 94
Passerini et al. PNAS
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by QRT-PCR with that predicted by array analysis. The array
prediction was validated for 60% (16兾27) of the genes sampled, and
there was disagreement with the array prediction for 18% (5兾27).
For 22% (6兾27) of the gene sample, high interanimal variability
prevented a definitive conclusion (reported as ‘‘undetermined’’ in
Fig. 2) despite highly reproducible measurements for each animal
(n ⱖ 3). The basis of the interanimal variability for these genes is
unclear, but it may be due to confounding factors other than the
influence of regional hemodynamics, and it may be genetic in origin.
The validation of about two of three genes after linear amplification
is consistent with our previous report, for which we used a model
system (26).
Mining of Biological Pathways. Table 2 shows selected differentially
expressed genes (DF vs. UF) in the biological classifications of
inflammation, cell adhesion, and oxidative mechanisms. Also
shown in Table 2 are the
ARRAYSTAT ratio (mean, n ⫽ 8) and the
putative positive or negative impact, deduced from the literature,
on mechanisms contributing to atherogenesis (references are pro-
vided as Supporting References to Table 2).
Proinflammatory gene expression in DF.
The differential regulation of
inflammatory and immune-related genes revealed an endothe-
lium primed for inflammation in DF. Consistent with a proin-
flammatory response, there was up-regulation of several broad-
acting cytokines, chemokines, and receptors (interleukin 1
␣
,
interleukin 1 receptor 1, interleukin 6, interleukin 8 receptor

,
advanced glycosylation end-product-specific receptor, monocyte
chemotactic protein 1) as well as down-regulation of the anti-
inflammatory interleukin 10 in DF when compared with UF.
Furthermore, up-regulation of elements of the NF-
B system
(nuclear factor of kappa light polypeptide gene enhancer in
B cells 1 and 2 and NF-
B inhibitor
␣
) and simultaneous
down-regulation of transcription for the I
B kinase-complex-
associated protein involved in assembling an active kinase
complex (33), and the cellular zinc finger anti-NF-
B protein
(Cezanne) which down-regulates NF-
B (34) were noted (Table
2). However, NF-
B was inactive in both DF and UF regions as
shown by cytoplasmic localization and nuclear exclusion of the
NF-
B complex (p65 antibody) (Fig. 3A and B). These obser-
vations are consistent with an NF-
B system primed but inactive
in DF as described by Hajra et al. (22). Differentially expressed
genes that may mitigate an inflammatory tendency included the
up-regulation of endothelial protein C receptor and the down-
regulation of platelet-activating factor receptor, chemokine re-
ceptor 4, complement component C3, several cathepsins, and the
MHC class II antigen HLA-DRB4.
Absence of differences in adhesion molecule expression.
Several adhe-
sion-related molecules were differentially regulated by flow type,
including von Willebrand factor, CD44, fibronectin 1, several
integrins, cadherins, and junction plakoglobin. The up-regulation of
von Willebrand factor (24) and CD44 (25) in particular may have
implications for atherogenesis. However, VCAM-1, intercellular
adhesion molecule 1, E-selectin, and P-selectin, all associated with
early NF-
B-mediated inflammatory responses and the onset of
atherogenesis, were not detected as differentially expressed despite
the increased transcriptional levels of proinflammatory cytokines
and NF-
B pathway components noted above. Because the statis-
tical methods aimed at detecting putative differential expression do
not allow us to make any definitive statement regarding unchanged
genes, we also measured differential expression of these genes by
QRT-PCR (data not shown). There was no clear pattern of
differential expression, with the results indicating slight up-
regulation in some animals, but slight down-regulation or no change
in others. Immunostaining for VCAM-1 was weak and indistin-
guishable between the two regions (data not shown). Collectively,
these observations in the context of normal histology suggest that,
although the endothelium in DF regions may be primed for an
inflammatory response, the process is held in check, possibly at the
level of NF-
B.
Protective antioxidative expression profile in DF.
Reactive oxygen spe-
cies (ROS) and prooxidative pathways are implicated in the initi-
ation and progression of atherosclerosis (35). Furthermore, ROS is
a potent activator of NF-
B (36, 37). Data mining revealed an
antioxidative profile in endothelium located in regions of DF that
may protect against NF-
B-mediated inflammation. Significant
suppression of a component of superoxide-generating NADH
oxidase was noted. An antioxidative state was supported by con-
comitant up-regulation of key antioxidative enzymes, including
GPX3, extracellular superoxide dismutase, NADH quinone oxi-
doreductase, heme oxygenase, and two forms of GST (GST-
1 and
microsomal MGST2). Several of these genes have been reported to
be up-regulated by laminar shear stress in vitro in a response
mediated by a shear-responsive antioxidant response element (38).
Down-regulation of components of the cytochrome c oxidase and
NADH dehydrogenase complexes (intramitochondrial sources of
ROS through their involvement in electron transport), and of
endothelial nitric oxide synthase 3 were noted. Also protective was
the down-regulation of thioredoxin-interacting protein [i.e., vitamin
D
3
-up-regulated protein 1 (VDUP-1)], an inhibitor of thioredoxin
that, in turn, is an important regulator of cell redox balance (39).
Berk and colleagues have shown that shear stress influences
VDUP-1 gene and protein expression (B. Berk; personal commu-
nication). Down-regulation of VDUP-1 leads to increased thiore-
doxin activity and, ultimately, down-regulation of VCAM-1 expres-
sion through the mitogen-activated protein kinase兾Jun kinase
pathway. Few prooxidative genes were differentially expressed, and
the balance was decisively shifted in the direction of a protective
antioxidative state in DF regions in these healthy animals. The
expression of antioxidative enzymes GPX3 and heme oxygenase
were validated by QRT-PCR (Fig. 2). The prominent up-regulation
of GPX3 in DF was confirmed by immunostaining (Fig. 3 C and D).
The antioxidative expression profile observed here may be critical
to maintaining a delicate balance in gene expression in vulnerable
regions of normal animals.
Fig. 2. Comparison of QRT-PCR results with microarray predictions for selected
genes: red, up-regulated in DF; green, down-regulated in DF; gray, unchanged;
white, undetermined (high interanimal variability).
2484
兩
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0305938101 Passerini et al.
Gene expression in other biological classifications.
Although it is not
within the scope of this paper to exhaustively analyze the entire
database of differentially expressed genes, this study resulted in a
very rich dataset, which is now provided as a public resource.
Reference to the online database also revealed a balance of gene
expression related to coagulation mechanisms with a shift toward
protective anticoagulant and profibrinolytic mechanisms in the DF
region. Notably, all components of the plasminogen-activator sys-
tem identified by our analyses were regulated in a manner consis-
tent with antithrombotic mechanisms in DF. Lipid兾cholesterol
metabolism-related gene expression reflected a balance in DF
regions between the expression of genes, which might promote
cholesterol deposition and reverse cholesterol transport. Numerous
growth factors and receptors were shown to be differentially
regulated. The differential expression of numerous transcription
factors may provide new insight into the modulation of the endo-
thelial response to hemodynamics through the identification of
common regulatory binding elements.
Table 2. Selected genes identified as differentially expressed (DF vs. UF) in the context of a putative role in atherosclerosis
Up-regulated Down-regulated
Gene GenBank ID Ratio Putative effect* Gene GenBank ID Ratio Putative effect*
Inflammatory兾immune response
IL-1A X02851 2.01 ⫹ IL-10 M57627 0.67 ⫹
IL-1R1 M27492 1.46 ⫹ IKBKAP AF153419 0.77 ⫹
IL-6 X04430 1.68 ⫹ CEZANNE AJ293573 0.66 ⫹
MCP-1 M37719 1.57 ⫹ IL-8 M28130 0.62 ⫺
RAGE AB036432 4.02 ⫹ CXCR4 AF005058 0.29 ⫺
IL-8RB M99412 1.65 ⫹ FOS BC004490 0.43 ⫺
NFKB1 M58603 1.34 ⫹ PAFR D10202 0.66 ⫺
NFKB2 S76638 1.35 ⫹ C3 J04763 0.27 ⫺
NFKBIA BC004983 2.76 ⫹ HLADRB4 M19556 0.55 ⫺
EPCR L35545 1.90 ⫺ TRAF6 H12612 0.62 ⫺
PTGS2 D28235 1.55 ⫺ IGFBP1 R81994 0.66 ⫺
IL-14 L15344 0.67 ?
Oxidative mechanisms
GPX3 D00632 5.64 ⫺ TXNIP S73591 0.44 ⫺
SOD3 U10116 1.39 ⫺ COX6B BC001015 0.61 ⫺
NQO1 BC000474 1.50 ⫺ COX7A2 AY007643 0.74 ⫺
POR AF258341 1.36 ⫺ NDUFC1 AK023115 0.69 ⫺
HMOX1 X06985 1.66 ⫺ NDUFS4 AF020351 0.64 ⫺
GSTT1 AB057594 1.55 ⫺ NDUFS6 AF044959 0.56 ⫺
MGST2 U77604 2.83 ⫺ NDUFS3 AL135819 0.52 ⫺
NDUFA7 Y16007 1.40 ⫹ NDUFA3 AF044955 0.42 ⫺
NDUFB7 AF112200 1.35 ⫹ NDUFV2 M22538 0.75 ⫺
NDUFB10 AF088991 1.35 ⫹ NDUFA6 BC002772 0.76 ⫺
NDUFS8 U65579 0.70 ⫺
CYBB X04011 0.46 ⫺
NOS3 M93718 0.69 ⫹
SOD1 K00065 0.77 ⫹
Cell adhesion
†
VWF X04385 5.04 ⫹ CTNND1 AF062343 0.59 ?
CD44 AJ251595 1.47 ⫹ CDH1 Z13009 0.50 ?
CDH3 X63629 1.43 ⫺ CDH13 L34058 0.65 ?
FN1 AJ276395 2.51 ? ITGA6 X53586 0.43 ?
ITGB4 X51841 1.43 ? ITGA5 BC008786 0.69 ?
MO1A J03270 1.34 ? JUP Z68228 0.19 ?
IL-1A, IL-1
␣
; IL-1R1, IL-1 receptor 1; IL-6, IL-6 (interferon

2); MCP-1, monocyte chemotactic protein 1; RAGE, advanced glycosylation end product-specific
receptor; IL-8RB (CXCR2), IL-8 receptor

; NFKB1, nuclear factor of
light polypeptide gene enhancer in B cells 1 (p105); NFKB2, nuclear factor of
light
polypeptide gene enhancer in B cells 2 (p49兾p100); NFKBIA, NF-
B inhibitor
␣
; EPCR, endothelial protein C receptor; PTGS2, prostaglandin endoperoxide
synthase-2 (COX-2); IKBKAP, inhibitor of
light polypeptide gene enhancer in B-cells, kinase complex-associated protein; CEZANNE, cellular zinc finger
anti-NF-
B; CXCR4, chemokine (C-X-C motif) receptor 4; FOS, v-fos FBJ murine osteosarcoma viral oncogene; PAFR, platelet-activating factor receptor; C3,
complement component C3; HLADRB4, MHC class II HLA-DRw53-beta (DR4,w4); TRAF6, tumor necrosis factor receptor-associated factor 6; IGFBP1, insulin-like
growth factor binding protein 1; GPX3, glutathione peroxidase 3; SOD3, superoxide dismutase 3; NQO1, NADH quinone oxidoreductase; POR, cytochrome P450
oxidoreductase; HMOX1, heme oxygenase (decycling) 1; GSTT1, GST
1; MGST2, microsomal GST 2; NDUFA7, NADH ubiquinone oxidoreductase; NDUFB7, NADH
dehydrogenase (ubiquinone) component B7; NDUFB10, NADH dehydrogenase (ubiquinone) component B10; TXNIP, thioredoxin interacting protein (VDUP1);
COX6B, cytochrome c oxidase subunit VIb; COX7A2, subunit VIIa; NDUFC1, NADH dehydrogenase (ubiquinone) component C1; NDUFS4, component S4; NDUFS6,
component S6; NDUFS3, component S3; NDUFA3, component A3; NDUFV2, component V2; NDUFA6, component A6; NDUFS8, component S8; CYBB, cytochrome
b-245 beta (NADPH oxidase component p91 phox); NOS3, endothelial nitric oxide synthase; SOD1, superoxide dismutase 1; VWF, von Willebrand factor; CD44,
cell adhesion molecule (CD44); CDH3, cadherin 3 (P-cadherin); FN1, fibronectin 1; ITGB4, integrin

4; MO1A, leukocyte adhesion glycoprotein Mo1
␣
; CTNND1,
catenin
␦
1; CDH1, cadherin 1 (E-cadherin); CDH13, cadherin 13 (H-cadherin); ITGA6, integrin
␣
6; ITGA5, integrin
␣
5(fibronectin receptor); JUP, junction
plakoglobin.
⫹, pro-atherosclerotic; ⫺, anti-atherosclerotic; ?, unknown.
*References to the putative effect provided as Supporting References to Table 2, which is published as supporting information on the PNAS web site.
†
Vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1, P-selectin, E-selectin were not significantly different.
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Discussion
Although the localization of atherosclerotic lesions to predict-
able regions in mammalian arteries has been recognized for over
a century, compelling evidence implicating the local hemody-
namics is more recent (6). We have exploited RNA amplification
to demonstrate the heterogeneity of endothelial phenotypes
from hemodynamically distinct regions in vivo, demonstrating
that a delicate balance of pro- and antiatherosclerotic mecha-
nisms may exist simultaneously in endothelium of lesion-prone
sites of the adult porcine aorta to create a setting of vulnerability
to atherogenesis. Atherosclerosis has been described as a chronic
inflammatory disease involving many cell types and complex
cross-talk between them (40). Many interrelated physiological
mechanisms are recruited very early in the process, including cell
adhesion, oxidative metabolism, lipid metabolism, apoptosis,
innate and adaptive immunity, and coagulation (recently re-
viewed in refs. 40–46). The predisposition for expression of key
molecules in multiple pathways is therefore likely to be impor-
tant in determining the susceptibility of a site to lesion initiation
when additional risk factors are prevalent, thereby influencing
the threshold or timing for atherogenesis.
NF-
B is a transcription factor proposed to play a central role as
a mediator兾integrator of atherogenesis (36). Its association with
inhibitory I
Bs masks the nuclear localization sequence, retaining
the complex in the cytoplasm. Activation of I
B kinase results in
phosphorylation of I
Bs and their targeting for degradation by the
proteosome, releasing NF-
B dimers for translocation to the nu-
cleus, where they activate transcription in target genes. These genes
include cytokines, chemokines, adhesion molecules, and genes
involved in cell proliferation and cell survival. NF-
B is controlled
by the redox state of the cell, which may be a common signaling
pathway leading to NF-
B activation through ROS (37). Activation
of NF-
B and NF-
B-regulated genes has been observed in ath-
erosclerotic plaques (47), and NF-
B-activated by shear stress has
been observed in cultured cells (48, 49) and regions of DF (22).
Collins et al. (36) have suggested that the survival兾protective genes
induced by NF-
B limit the inflammatory response at low levels,
although a strong challenge results in the expression of adhesion
molecules兾cytokines.
In one of the few in vivo regional arterial studies, Hajra et al. (22)
evaluated NF-
B regulation in regions of high and low probability
for atherosclerosis in mouse aorta by using en face immunostaining
and confocal microscopy. They demonstrated preferential activa-
tion of NF-
B (translocation to the nucleus) and induction of
NF-
B-responsive gene expression in high probability regions of
the aortas of lipopolysaccharide-treated or hypercholester-
olemic LDLR
⫺/⫺
mice. In normal mice, however, although p65 and
I
Bs were elevated in such regions, NF-
B activation remained
low, suggesting that the pathway was primed but not activated.
Our findings are consistent with and greatly extend such an
interpretation.
Several recent reports have used microarrays to assess gene
expression in EC exposed to unidirectional steady flow in vitro (9,
13, 20) and compared expression profiles to those of control cells
in no-flow conditions. The results, however, are not easily com-
pared with our study, which captures the profile of two distinct
regions long-adapted to differential hemodynamic environments in
vivo. More relevant are two in vitro microarray studies that com-
pared flow conditions analogous to DF and UF.
In the first, Garcia-Cardena et al. (16) compared differential
expression in cultured human umbilical vein endothelial cells
between turbulent shear stress (TSS) and LSS, analogous to DF vs.
UF regions in vivo in this study. One hundred genes were identified
as differentially expressed (TSS vs. LSS) after 24 h (68 up-regulated
and 32 down-regulated). The authors identified a set of genes that
was down-regulated in LSS but up-regulated in TSS as potentially
the most pathologically relevant. They considered highly regulated
genes of known or putative function in signaling, response to injury,
or atherogenesis and focused on linking the protective effects of
LSS to matrix biology and cell cycle. Brooks et al. (15) compared
gene expression in cultured human aortic EC under pulsatile DF
conditions with LSS by using microarrays (588 genes) and subtrac-
tion cloning. They identified ⬎100 genes as differentially expressed
at 24 h, many of which were up-regulated genes associated
with mechanisms known to be proatherosclerotic, particularly
inflammatory molecules, adhesion factors, and oxidation-related
molecules.
Few genes identified as differentially expressed by these in vitro
studies were common to those identified by our in vivo study.
Furthermore, there was little agreement between the in vitro
studies. The reasons may be the different nature of the DF in each
study, different cell origins, sensitivities of microarray analysis, or a
myriad of technical considerations. Despite significantly different
outcomes, however, both of these in vitro studies demonstrated that
conditions of DF resulted in significant differential endothelial gene
expression compared with conditions of unidirectional steady flow.
The methods used here have refined endothelial transcriptional
profiling to regions small enough to represent exposure to discrete
flow characteristics in vivo, but these methods also provide suffi-
cient cells to amplify RNA and profile gene expression with
reasonable confidence. We have previously evaluated the linear
amplification protocol by using similar amounts of human matched
probe兾target and found it to identify differential expression with
fidelity and enhanced sensitivity (26). In agreement with this
previous study, the QRT-PCR results reported in the current study
corroborate approximately two of three genes tested. When con-
sidering differential expression with cross-species hybridization,
differences in sequence homology are expected to lead to fewer
genes hybridizing than for the equivalent same-species hybridiza-
tion, thus contributing to false negatives. However, both samples
should hybridize in the same way so that false positives will not be
Fig. 3. En face immunostaining in DF and UF regions. (A and B) Subcellular
localization of NF-
B complex (p65 antibody) was visualized by 2D projection of
confocal imaging through the EC layer. The staining was predominantly cyto-
plasmic with nuclear exclusion in both aortic regions. Also clearly illustrated are
the EC morphologies characteristic of DF (A) and UF (B). (C and D) Epifluorescence
microscopy for the prominent differentially expressed antioxidative enzyme
GPX3 revealed strong expression in DF (C) in contrast to low expression in UF (D),
consistent with microarray predictions.
2486
兩
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0305938101 Passerini et al.
affected. The linear amplification protocol minimizes bias possibly
introduced by other amplification methods, but it is clear that
certain transcripts may be reproducibly misrepresented. Although
we verified EC purity at ⬎99%, we cannot rule out the possibility
that a small number of contaminating SMC could lead to some false
positives with the use of amplification. Given the increased sensi-
tivity of amplification, it is possible that strongly differentially
expressed genes in a small number of contaminating cells could be
detected.
Inflammatory responses leading to atherogenesis in DF regions
may share common signaling pathways as responses mediated by
cytokines, such as tumor necrosis factor (TNF)-
␣
. In particular, the
two may share common mechanisms mediated by NF-
B. We have
previously profiled EC gene expression in response to TNF-
␣
in
vitro (26), and we find the overlap with the present in vivo study to
be small. Up-regulated genes common to both studies included
monocyte chemotactic protein 1, interleukin 1
␣
, NF-
B1, NF-
B2,
NF-
B inhibitor
␣
, TNF-
␣
-induced protein 6, and cycloxygenase-2,
whereas common down-regulated genes included TNF receptor-
associated factor 6 and the chemokine receptor CXCR4. An
important distinction between these studies is that differential
expression of VCAM-1, intercellular adhesion molecule 1, and
E-selectin was induced by TNF-
␣
, but not by differential hemody-
namics in the normal animal.
Because this is the first in vivo study profiling regional endothelial
gene expression on a large scale, we have attempted to bridge the
findings to published candidate gene studies in vivo (21–24) and
experiments of flow disturbance in vitro (8, 15, 16, 19, 23). Fur-
thermore, we have presented specific observations relating to some
pathways that are central to atherogenesis. Further mining of these
data with emphasis on different biological pathways (such as those
highlighted in Table 1) will provide additional insights into the
regional susceptibility to atherosclerosis.
In summary, a baseline is established for gene expression pro-
filing by using endothelium from DF and UF regions in the normal
adult pig aorta, which defines in vivo the existing relationships
between many classes of molecules responsible for vascular ho-
meostasis and implicated in early atherogenesis. This study revealed
distinctly different patterns of gene expression than reported by in
vitro studies to date. The hemodynamic characteristics of DF may
prime the endothelium toward inflammation (and by inference,
atherogenesis) but protective profiles in gene expression (e.g., a net
antioxidative profile) prevent disease initiation in the absence of
additional risk factors.
We thank Drs. Garret FitzGerald, Aron Fisher, and Paul Janmey of the
University of Pennsylvania for critical reading of the manuscript, Re-
becca Riley for expert technical assistance, and Dr. Richard Magid for
help with NF-
B imaging. This work was supported by National Insti-
tutes of Health Grants HL62250, HL70128, K25-HG-02296, and K25-
HG-00052; National Space Biomedical Research Institute (National
Aeronautics and Space Administration) Grant NSBRI-01-102; and a
Sponsored Research Award from AstraZeneca Pharmaceuticals.
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MEDICAL SCIENCES
