|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3418-3431
Vascular Endothelial Genes That Are Responsive to Tumor Necrosis
Factor- In Vitro Are Expressed in Atherosclerotic Lesions,
Including Inhibitor of Apoptosis Protein-1, Stannin, and Two Novel
Genes
By
Anton J.G. Horrevoets,
Ruud D. Fontijn,
Anton Jan van Zonneveld,
Carlie J.M. de Vries,
Jan Wouter ten Cate, and
Hans Pannekoek
From the Departments of Biochemistry and Vascular Medicine of the
Academic Medical Center, University of Amsterdam, Amsterdam, The
Netherlands.
 |
ABSTRACT |
Activation and dysfunction of endothelial cells play a prominent
role in patho-physiological processes such as atherosclerosis. We
describe the identification by differential display of 106 cytokine-responsive gene fragments from endothelial cells, activated by
monocyte conditioned medium or tumor necrosis factor- . A minority of
the fragments (22/106) represent known genes involved in various processes, including leukocyte trafficking, vesicular transport, cell
cycle control, apoptosis, and cellular protection against oxidative
stress. Full-length cDNA clones were obtained for five novel
transcripts that were induced or repressed more than 10-fold in vitro.
These novel human cDNAs CA2_1, CG12_1, GG10_2, AG8_1, and GG2_1 encode
inhibitor of apoptosis protein-1 (hIAP-1), homologues of
apolipoprotein-L, mouse rabkinesin-6, rat stannin, and a novel 188 amino acid protein, respectively. Expression of 4 novel transcripts is
shown by in situ hybridization on healthy and atherosclerotic vascular
tissue, using monocyte chemotactic protein-1 as a marker for
inflammation. CA2_1 (hIAP-1) and AG8_1 are expressed by endothelial cells and macrophage foam cells of the inflamed vascular wall. CG12_1
(apolipoprotein-L like) was specifically expressed in endothelial cells
lining the normal and atherosclerotic iliac artery and aorta. These
results substantiate the complex change in the gene expression pattern
of vascular endothelial cells, which accompanies the inflammatory reaction of atherosclerotic lesions.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
ENDOTHELIAL CELLS play a central
communicative role between the blood in the lumen of the vessel and the
surrounding vascular tissue.1,2 These cells form the
nonthrombogenic lining of the vessel and play an active role in
maintaining the hemostatic balance, eg, via the expression of the
anticoagulant cofactor thrombomodulin, the secretion of the
profibrinolytic tissue-type plasminogen activator, and the production
of prostanoids that attenuate platelet activation. Invasion of the
vessel wall by circulating monocytes and other blood-borne cells is
actively promoted by the endothelial cells via the regulated expression of adhesion molecules. On the abluminal side there is constant communication between the endothelial cells and the underlying smooth
muscle cells, causing them to contract or relax in response to altering
blood pressure and flow via the production of signaling molecules such
as prostanoids, endothelins, and nitric oxide. As a result of these
functions, activation and dysfunction of endothelial cells play a
crucial role during the initiation and progression of
atherosclerosis.1-3 Furthermore, activation of endothelial
cells plays a definitive role in many other processes, such as
tumor-induced angiogenesis, allergic inflammation, adult respiratory
distress syndrome, and a variety of inflammatory and allergic disease
states.4-6 The altered properties of the endothelial cells
results from altered patterns of gene expression, eg, invasion of the
vessel wall by leukocytes can only occur after the induced expression
by the endothelial cells of adhesion molecules such as E-Selectin and
vascular cell adhesion molecule-1 (VCAM-1).1,7 Only a small
percentage of the total human gene repertoire will be subject to change
in a given pathological situation, because the majority of genes plays
a more basal housekeeping role within any given cell type. Many genes
and proteins have been implicated in atherosclerosis, based on studies
of single specific genes and proteins.2 However, the mere
fact that specific functions have been assigned to only 5% to 10% of
the estimated total of 50,000 to 100,000 human genes indicates that a
full understanding of the atherosclerotic process is far from
established, because it is conceivable that many of the presently
unknown genes might also play a prominent role in this
disease.8,9 The identification and characterization of
these novel atherogenesis-related genes is the goal of our studies to
define the difference between a resting (nonatherogenic) and an
activated (atherogenic) endothelial cell at the level of gene
expression. Next, the specific functional roles of the corresponding
gene-products in atherosclerosis will increase our knowledge about the
patho-physiology of the atherosclerotic vascular wall and may identify
targets for noninvasive early diagnosis and/or therapeutic agents.
The infiltration of the vessel wall by monocytes is a hallmark of the
initiation and progression of atherosclerosis. Activated monocytes and
their resulting macrophage foam cells will secrete a complex mixture of
cytokines,3,10,11 including tumor necrosis factor-
(TNF-), which greatly affects endothelial cell
function.12 Indeed, one of the cellular mediators of the
TNF- response, NF- B, has recently been identified in
atherosclerotic lesions.13 The secretion products of
monocytes, including TNF-, are known to have a profound influence on
the expression levels of certain endothelial cell genes, of which the
adhesion molecules E-selectin, VCAM-1, and intercellular adhesion
molecule-1 (ICAM-1) have been studied in great
detail.1,2 Little is known about other cytokine responsive
genes, although some important ones have been studied with respect to
the influence of endothelial cells on the hemostatic balance, such as
tissue factor, plasminogen activator inhibitor-1, tissue-type
plasminogen activator, and thrombomodulin.1 We have set out
to expand our knowledge of changes in endothelial cell gene expression
patterns in response to monocyte stimulation to describe the role of
endothelium in initiation and progression of atherosclerosis at the
molecular level. Unfortunately, the composition and architecture of
atherosclerotic lesions are extremely diverse: these lesions have
greatly varying cell-type composition and occur in many different
vessels, which are embedded in different tissues with cells derived
from different embryonic origin. Therefore, we have taken an unbiased
approach to identify candidate atherogenesis-involved known and novel
genes in pure cell cultures. Next, in vivo significance is tested by
studying expression of these genes in a variety of atherosclerotic
lesions. The differential display of gene expression technique by
random-primed reverse transcriptase-polymerase chain reaction
(DD/RT-PCR)14,15 was used to detect variations of mRNA
levels upon activation of cultured endothelial cells. This method
allowed us to identify genes that are either induced or repressed by a
given stimulus. Furthermore, the isolation of total RNA from resting
and cytokine-stimulated endothelial cells at various time points
enabled us to identify genes of different temporal kinetics, ie,
immediate early, delayed early, and late genes. In this report, we
describe the identification, in an unbiased way, of a large panel of
candidate genes that are potentially involved in atherosclerosis by
mimicking inflammatory conditions in vitro. The full-length cDNA for 5 novel genes is reported, and we started the localization of gene
expression for these novel genes in vascular endothelial cells by in
situ hybridization on human healthy and diseased vascular material.
| |
MATERIALS AND METHODS |
Cell culture and fluorescence-activated cell sorting
(FACS) analysis.
Endothelial cells were isolated from nontraumatized human umbilical
veins as described.16 These human umbilical vein
endothelial cells (HUVEC) were cultured in gelatin-coated
tissue-culture flasks (Nunc, Roskilde, Denmark) in medium composed of
equal parts of Medium-199 and RPMI-1640 (GIBCO-BRL, Paisley, Scotland),
supplemented with 20% (vol/vol) heat-inactivated, pooled human serum,
2 mmol/L glutamine, and a 1/100 dilution of antibiotic/antimyotic mix
(GIBCO-BRL; final concentrations: 100 U/mL penicillin, 100 U/mL streptomycin, and 2.5 mg/mL fungizone) at 37°C in a 5%
CO2 humidified air incubator. HUVEC were passaged once with
trypsin/EDTA (GIBCO-BRL) and only secondary cultures, which had been
confluent for 2 or 3 days, were used for all experiments described in
this report. Identity of the HUVEC was confirmed by their cobblestone
morphology and positive staining for von Willebrand factor. Primary
human monocytes were isolated from fresh buffy coats (Central
Laboratory of the Netherlands Blood Transfusion Service, Amsterdam, The
Netherlands) by Ficoll gradient centrifugation and attachment to
tissue-culture plastics. The monocytes (±106 cells per
flask) were washed twice with serum-free Iscove's modified Dulbecco's
modified Eagle's medium (DMEM; GIBCO-BRL) and were cultured overnight at 37°C in a humidified, 5% CO2/air
incubator in 8 mL of Iscove's supplemented with 2% human albumin,
insulin/transferin/selenite growth supplement (Sigma, St Louis,
MO), and antibiotic/antimyotic. The conditioned medium was collected
after 16 hours and stored in aliquots at  70°C. HUVEC
received fresh full-growth medium 16 hours before the beginning of the
activation experiment. Human recombinant TNF- was lot no. DOE 247/91
(Bayer AG, Wuppertal, Germany). Stimulation was performed either with
TNF- added to the full growth medium (20% human serum) or with
serial dilutions of the monocyte-conditioned medium in serum-free
medium. At different time points the cells were washed with
phosphate-buffered saline (PBS) and trypsinized for flow cytometric
analysis. Suspended cells were washed twice in cold PBS and
subsequently fixed in ice-cold PBS containing 1% bovine serum albumin
(BSA), 0.3 mmol/L EDTA, 0.01% (wt/vol) sodium azide, and 0.1%
(wt/vol) p-formaldehyde at a final concentration of 5 × 106 cells/mL. The primary antibodies used were directed
against tissue factor and ICAM-1, respectively. After the addition of
the primary monoclonal antibody to the suspension, the cells were
incubated for 30 minutes at 4°C and washed twice in cold PBS
containing 1% (wt/vol) BSA, 0.3 mmol/L EDTA, and 0.01% (wt/vol)
sodium azide. Subsequently, R-phycoerythrin (RPE)-conjugated
F(ab')2 fragments of rabbit-antimouse Igs (R 0439;
Dako A/S, Glostrup, Denmark) were added, and cells were incubated for
another 30 minutes at 4°C. After two washes, HUVEC were gated by
forward scatter and side scatter using a FACScan (Becton Dickinson,
Cowley, Oxford, UK) and 5,000 cells were counted. This established that
maximal stimulation, as evidenced by maximal fluorescence shift, was
obtained for both tissue factor and ICAM-1 at concentrations of 12 nmol/L TNF- and the 1/10 diluted monocyte-conditioned medium.
RNA isolation and DD/RT-PCR.
Total RNA was isolated from resting and activated cells with TRIZOL
(GIBCO-BRL) with a typical yield of 80 µg/8 × 106
cells and stored at 70°C. The DD/RT-PCR reactions were
performed with 12 different anchored primers (T11XY; X = A, C, or G; Y = A, C, G, or T) and arbitrary decamers no. 1 through 12 as
described.15 In brief, 0.2 µg total RNA was reverse
transcribed with 20 U Superscript-RTII (GIBCO-BRL) in the presence of
2.5 µmol/L anchored primer and a dNTP concentration of 20 µmol/L in
a volume of 20 µL first-strand buffer. After the addition of 10 µL
water, 2 µL of first-strand cDNA was subjected to DD/RT-PCR in a
Perkin-Elmer 9600 thermocycler (Perkin Elmer, Norwalk, CT) using
GeneAmp tubes in a total volume of 20 µL under the following
conditions: 1× PCR-II buffer, 1.5 mmol/L MgCl2, 2 µmol/L dNTPs, 1 µmol/L anchored primer, 0.5 µmol/L decamer, 2 µCi (60 nmol/L) -33P-dATP (Redivue, Amersham, UK), and
1 U AmpliTaq (Perkin Elmer) for 40 cycles of 30 seconds at 94°C, 2 minutes at 40°C, and 30 seconds at 72°C, followed by 10 minutes
at 72°C. After completion of the reaction, 14 µL loading mix
(90% [vol/vol] formamide, 1 mmol/L EDTA, Xylene cyanol, and
BromoPhenol Blue) was added and a 2- to 5-µL sample was analyzed by
standard 6% sequencing polyacrylamide gel electrophoresis (PAGE; 8 mol/L urea) at 70 W. Subsequently, the gel was transferred to Whatman
paper (Whatman, Maidstone, UK) and vacuum-dried without
prior fixation. The gel was marked at the 4 corners with radioactive
ink and analyzed by autoradiography after exposure for 16 to 72 hours
at room temperature to BioMax film (Eastman Kodak, Rochester, NY).
Maximal comparability of the reactions that were to be analyzed in
concert was ensured by using core mixes for both RT-reactions and
DD/RT-PCR reactions and performing all reactions in concert in 96-well
format for a single anchored primer, all 12 decamers, and the 10 individual RNA preparations. These RNAs were isolated from unstimulated
HUVEC (0 and 20 hours) and HUVEC exposed to TNF- or
monocyte-conditioned medium for 3, 6, or 20 hours each.
Cloning and sequencing of DD/RT-PCR fragments.
DD/RT-PCR fragments of interest were recovered from sequencing gels and
reamplified by the DD/RT-PCR protocol at a dNTP concentration of 40 µmol/L. Reactions were analyzed on 1.5% agarose gel using the 100-bp
marker (GIBCO-BRL), and the bands of appropriate length were excised
and purified using QIAEX (Qiagen GmbH, Hilden, Germany). These inserts
were TA-cloned by ligation to either the pCR-II vector (Invitrogen,
Carlsbad, CA) or pGEM-T (Promega, Madison, WI) according to the
manufacturer's instructions. Sequencing of DD/RT-PCR fragment clones
was performed on purified plasmid DNA using the AutoRead Sequencing-kit
and Cy5-labeled T7 or SP6 oligonucleotides and analyzed on the
ALF-express automatic sequencer (materials and protocol: Pharmacia,
Uppsala, Sweden). Sequence files from the ALF-express were exported in
GCG-format and analyzed and stored using the GCG-program (Wisconsin
Package Version 9.1, Genetics Computer Group [GCG], Madison, WI) run
on a mainframe UNIX computer. A GCG database was constructed using
DATASET and all sequences were cross-analyzed by the FASTA algorithm.
Sequence identity was confirmed by BLAST searches on the combined
Genbank/EMBL nonredundant (nr) and expressed sequence tag libraries
(dbEST), accessed through the National Center for Biotechnology
Information homepage (http://www.ncbi.nlm.nih.gov./). Protein
homologies were traced using the Blitz algorithm run at the
EMBL/European Bioinformatics Institute (http://www.ebi.ac.uk), and
functional protein domains were traced using the BLOCKS WWW-server (http://www.blocks.fhcrc.org/). Multiple protein sequence alignments were performed using the ClustalW service at EBI.
Northern blotting analysis.
Formamide and heat-denatured 10 µg total RNA was electroforesed on a
standard formaldehyde 1% agarose gel and blotted to Hybond-N nylon
membranes (Amersham, Amersham, UK) according to the
manufacturer's protocol. Filters were prehybridized in 5× SSPE
(20× SSPE is 3.6 mol/L NaCl, 0.2 mol/L sodium phosphate, pH 7.7, 0.02 mol/L EDTA), 0.5% sodium dodecyl sulfate (SDS; wt/vol), 5×
Dennhardts' (100× Denhardt's solution is 2% [wt/vol] BSA,
2% [wt/vol] Ficoll, and 2% [wt/vol] polyvinylpyrrolidone), and
50% (vol/vol) deionized formamide at 42°C in a hybridization oven
for at least 4 hours. Hybridizations were performed at greater than 1 × 106 cpm/mL for 16 to 24 hours at 42°C in a
solution of 5× SSPE, 0.5% SDS (wt/vol), 10% dextrane-sulfate
(wt/vol), and 50% (vol/vol) deionized formamide. As probes, we used
agarose-purified restriction fragments corresponding to the original
DD/RT-PCR fragment or IMAGE-clones labeled to high specific
radioactivity using the random oligo labeling kit (GIBCO-BRL) and
-32P-dATP (Redivue; Amersham); unincorporated
nucleotides were removed by the Qiaquick nucleotide removal kit (Qiagen
GmbH). Filters were washed for 15 minutes in 2× SSPE at room
temperature, followed by 30 minutes in 1× SSPE at 65°C and
twice for 10 minutes in 0.2× SSPE at 65°C. Filters were
analyzed by autoradiography after exposure to Xomat-R films (Eastman
Kodak) for various periods at 70°C using intensifying
screens. Radioactivity was quantified using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
Construction and screening of an activated EC cDNA library.
Total RNA was isolated with Trizol from HUVEC that had been stimulated
for 6 hours with the conditioned medium from elutriated monocytes that
had been activated overnight with limitedly oxidized human low density
lipoprotein (LDL) (Sigma). First-strand cDNA synthesis was performed
with 10 µg of total RNA, using Superscript RT-II reverse
transcriptase and oligo-dT. The second-strand synthesis was performed
by the modified procedure of Gubler and Hoffmann.17 Subsequently, the cDNA was treated with T4 DNA polymerase and ligated
to an excess of nonpalindromic BstXI-linkers. Finally, the cDNA
was size fractionated on a low melting type agarose gel and cDNA
exceeding 600 bp was divided in two pools of sizes less than 1,500 bp
and greater than 1,500 bp. Both pools were separately ligated into the
BstXI sites of the vector prcCMV (Invitrogen). Ligation
mixtures were electroporated into Escherichia coli strain MC
1061/P3 and the cDNA library was plated as 1 × 105
and 6 × 104 independent colonies for sizes less than
1,500 bp and greater than 1,500 bp, respectively. The cDNA library was
screened by colony hybridization using radioactive probes, identical to
those used for Northern blotting analysis (see above). Hybridization of
the probe was performed at 65°C for 16 hours in the presence of
0.1% (wt/vol) SDS, 5× Denhardts' solution, 0.1 mg/mL herring sperm DNA, and 6× SSC (20× SSC is 3 mol/L NaCl, 0.3 mol/L
sodium citrate). The Hybond-N membranes were washed with increasing
stringency down to 0.2× SSC. IMAGE Consortium (LLNL)
cDNA-clones48 (http://www-bio.llnl.gov/image/) were
obtained from the American Type Culture Collection (ATCC; Rockville,
MD; http://www.atcc.org/) and their identity was confirmed by resequencing of purified plasmid from individual colonies.
Vascular tissue collection, morphological analysis, and
immuno-histochemistry.
Human vascular tissue specimens displaying various stages of
atherosclerosis were collected after obtaining informed consent during
organ transplantations from multiorgan donors who did not have a prior
history of vascular disease (approved by the AMC Medical Ethical
Committee #95/146). Tissues were fixed in saline, 3.8% (wt/vol)
p-formaldehyde, or formalin within 5 minutes after resection and
subsequently paraffin-embedded. Paraffin sections (5 µm) of vascular
tissue were mounted on 3-aminopropyl-triethoxysilane-coated slides.
Morphological analysis was performed by using the Masson Trichrome
staining. Cell-type specific staining was performed using Ulex
europaeus lectin for endothelial cells and antibodies 1A4 or HAM-56
(DAKO) directed against smooth muscle cell specific -actin or
monocytes/macrophages, respectively. Counterstaining was performed with
haematoxylin/eosin according to standard procedures.
In situ hybridization.
Riboprobes for various gene transcripts, synthesized as described
below, contained the following sequences: human von Willebrand factor:
8239-8442 (192 bp; GB: X04385); monocyte chemotactic protein-1 (MCP-1):
86-741 (656 bp; GB: M24545); ferritin: 302-451 (149 bp; GB: M97164);
CA2_1: 2331-3317 (996 bp; this study; corresponding to 303-1289 of
MIHC/hIAP-1 GB: U37546.); GG2_1: 151-868 (718 bp; this study); GG10_2:
2375-2945 (571 bp; this study); AG8_1: 194-845 (652 bp; this study);
and CG12_1: 1687-2298 (611 bp; this study). Riboprobes were synthesized
by in vitro transcription of cDNA fragments cloned in various pGEM
vectors (Promega), containing T7 and SP6 RNA polymerase transcription initiation sites. In brief, the constructs were linearized and riboprobes were synthesized for 1 hour at 37°C in RNA polymerase buffer according to the manufacturer's instructions for SP6 RNA polymerase (Promega) or T7 RNA polymerase (Stratagene, La Jolla, CA)
and labeled with [35S]-UTP (Amersham). Paraffin sections
(5 µm) of vascular tissue were mounted on
3-aminopropyl-triethoxysilane-coated slides. In situ hybridization was
performed as described,18 with minor modifications. The
sections were pretreated with proteinase K (20 µg/mL) for 5 minutes,
refixed in 4% (wt/vol) p-formaldehyde, and treated for 10 minutes with
0.25% (wt/vol) acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0).
The riboprobes were added to and stored in hybridization mixture, which
consisted of 40% (vol/vol) formamide, 8% (wt/vol) dextrane sulfate,
0.8× Dennhardts', 0.5 mg/mL yeast tRNA, 4 mmol/L EDTA, 16 mmol/L
Tris-HCl (pH 8.0), and 0.24 mol/L NaCl. Hybridizations were performed
overnight at 50°C in 8 µL (0.5 µCi probe) per section under a
coverslip in a moist chamber. After hybridization, coverslips were
removed in 5× SSC, 10 mmol/L dithiothreitol (DTT) at
50°C (30 to 60 minutes), followed by a high stringency wash for 30 minutes at 65°C in 50% (vol/vol) formamide, 2× SSC, 10 mmol/L DTT. RNAse A digestion (20 µg/mL) was performed for 30 minutes
at 37°C in 10 mmol/L Tris-HCl (pH 8.0), 5 mmol/L EDTA, 500 mmol/L
NaCl. The high stringency wash was repeated, followed by washing for 15 minutes with 2× SSC. After dehydration, autoradiography emulsion
was applied as a 1:1 dilution of Ilford G5 emulsion (Ilford, Paramus,
NJ) with 2% (vol/vol) glycerol. After an exposure of 1 to 6 weeks,
slides were developed in Kodak D19 (Eastman Kodak), fixed in Kodak
UNIFIX (Eastman Kodak), and counterstained with haematoxylin and eosin.
| |
RESULTS |
Endothelial cell model system.
Cultured primary endothelial cells from the human umbilical vein
(HUVEC) were chosen as model cells to identify cytokine-responsive vascular endothelial cell genes. To mimic the situation in the fatty
streak, the earliest detectable onset of atheromas where monocytes have
entered the vessel wall and start to accumulate lipid, we have
incubated HUVEC with the conditioned medium from activated human
primary monocytes, containing a complex mixture of cytokines and
chemokines.3,10,11 As a second, independent activator, we
used purified, recombinant human TNF-, a cytokine released from
activated monocytes and macrophages, which is believed to play an
important role during inflammatory processes including atherogenesis
(reviewed in Sherry and Cerami19). Titration of these
stimuli was performed by FACScan analysis of the expression of an immediate early gene (tissue factor) and a late gene (ICAM-1) at
different dilutions of either TNF- or the conditioned medium from
activated monocytes. A uniform activation of the total population of
cells, necessary for unambiguous DD/RT-PCR interpretation, was obtained
at 12 nmol/L TNF- or the 1/10 dilution of the conditioned medium of
adherence-activated monocytes,10 as shown in
Fig 1.
View larger version (44K):
[in this window]
[in a new window]
| Fig 1.
Flow cytometric analysis of cytokine-activated HUVEC.
HUVEC were continuously exposed to 12 nmol/L TNF- (A and B) or the
1/10 diluted conditioned medium from activated human monocytes (C and
D), and at various timepoints aliquots of the cells were analyzed by
flow cytometry as described in Materials and Methods. In each case,
5,000 cells were counted (y-axis) and fluorescence intensity is
exponentially plotted starting from 100 (x-axis). Indicated
are the FACscan readouts for the immediate response gene tissue factor
(TF; A and C), and ICAM-1 (B and D), a late induced gene that is also
present at low amounts on nonstimulated cells. (C) and (D)
show nonactivated HUVEC (non stim.) or HUVEC activated by conditioned
medium from monocytes activated by adherence to plastic tissue culture
flasks with the addition of growth medium without additions (adherent)
or supplemented with 50 µg/mL of minimally modified LDL (mmLDL) or
oxidized LDL (oxLDL).
|
|
Differential display of gene expression.
An efficient and reproducible protocol for DD/RT-PCR was established,
based on published methods.14,15 This protocol enabled the
simultaneous identification of genes that are either induced or
repressed. Furthermore, by using RNA samples isolated at appropriate time points, immediate early, delayed early, and late genes were identified in a single experiment. Therefore, total RNA was isolated from resting cells (0 and 20 hours) and from cells activated by either
monocyte supernatant or purified human recombinant TNF- for various
time periods (1.5, 3, 6, and 20 hours). The quality and suitability of
each RNA preparation was assessed by Northern blotting using specific
probes for tissue factor, an early induced protein, and for
thrombomodulin, expression of which is repressed by TNF- (not
shown). Isolated total RNA was subjected to DD/RT-PCR and analyzed. The
specific set of 144 oligonucleotides we used was designed by Bauer et
al15 and is expected to display approximately 80% of the
different mRNA species that are expressed in a single cell based on
statistical grounds. Each primer combination yielded 50 to greater than
150 visible bands on gel for each RNA sample, and differentially
expressed bands were distributed randomly over the different primer
combinations, with an average of 16 (range, 8 to 23) for each anchored
primer with 12 arbitrary decamers.15 Differentially
displayed bands were identified by rigorous selection to prevent
inclusion of false positives, a problem frequently encountered during
DD/RT-PCR analyses (Fig 2).
View larger version (51K):
[in this window]
[in a new window]
| Fig 2.
DD/RT-PCR reproducibly identifies
cytokine-responsive genes in HUVEC. (A) and (B) show differential
display patterns for the primer sets T11GA with decamer 11 (A) and
decamer 7 (B). These represent the selection criteria we used to
exclude false-positives by only pursuing bands that show reproducible
differential expression on DD/RT-PCR gels. (A) and (B) show
enlargements for bands that are reproducibly induced at 3, 6, and 20 hours by both TNF- and monocyte-conditioned medium, whereas they are
not detectable in unstimulated cells. After cloning and sequencing,
these bands were shown to represent Mn-SOD and GM-CSF, respectively.
(A) also shows an unknown gene fragment that is specifically induced by
TNF- but not by monocyte-conditioned medium (unlabeled arrow) and
was excluded from our analysis. (C) shows confirmation of differential
expression of GM-CSF using the DD-fragment as probe and a Northern
blotting analysis of the same RNA sample as used for the DD/RT-PCR. The
insert shows the specific hybridization signal for GAPDH as internal
control for equal RNA loading.
|
|
Sequence analysis of DD/RT-PCR fragments.
The gene fragments that showed differential expression in a consistent
and reproducible way after stimulation of HUVEC by both TNF- (193 bands) and monocyte-conditioned medium (122 bands) were cloned and
sequenced in triplicate, totalling 119 fragments, 9 of which
represented repressed genes. The sequences obtained from DD/RT-PCR
fragments were cross-analyzed to trace redundancy: indeed, on 3 occasions a sequence appeared 3 times, whereas duplication was traced 6 times. In all cases, fragments of identical length and sequence had
been amplified by different anchored primers but identical arbitrary
decamer, with the exception of tissue factor, which was amplified with
the anchored primer only (Table 1). Only in
5 of 119 cases did we observe colonies of different sequence,
indicating contamination of the differentially displayed band by other
display products of the same length, whereas in only 3 of 119 cases the
sequence of the DD/RT-PCR fragment proved identical to mitochondrial
DNA. The resulting 106 unique sequences were compared with the
nonredundant Genbank/EMBL database at NCBI and EBI, using BLAST and
FASTA algorithms, showing that 22 of 106 gene fragments represented
mRNAs that encode known proteins (listed in Table 1). The remaining 84 sequences, representing novel genes, were compared with the Expressed
Sequence Tag-libraries at the National Center for Biotechnology
Information (dbEST) and The Institute for Genomic Research
(TIGR-HCI).16 We found that 33 of our novel sequences were
represented in these databases. The remainder of 51 DD-fragments
represent sequences not previously described.
Verification of differential expression by Northern blotting
analysis.
The fragments that were identified by differential display were
analyzed by Northern blotting analysis. We produced a series of 20 identical filters loaded with 10 µg of total RNA per lane of HUVEC
stimulated for 0, 0, 1.5, 3, 6, or 20 hours with 12 nmol/L human
recombinant TNF-. Filters were probed with highly radiolabelled purified DD/RT-PCR fragments, showing that all of the DD-fragments corresponding to known mRNAs and most of the novel DD-fragments corresponding to ESTs gave a signal on Northern blot. Only in 2 cases
did fragments turn out to be nondifferential, whereas others showed
induction or repression of threefold to greater than 10-fold. None of
the novel, non-EST transcripts could be detected in this way; these
await further analysis with more sensitive methods. This
is not unexpected, because most average abundant transcripts should at
present be represented in dbEST (NCBI). Furthermore, it should be
emphasized that DD/RT-PCR fragments do not constitute optimal probes
for Northern blotting analysis, because they are derived from the
3'-UTR of mRNAs, which are rich in repetitive sequences and
secondary structure. Figure 3 shows the
results of Northern blotting analysis for 5 novel transcripts that are
represented in dbEST (NCBI) and for which the full-length mRNA was
isolated as described below. Four probes were chosen that showed
greater than 10-fold induction by Northern blotting analysis, whereas
the fifth probe represented a gene that was totally repressed by 20 hours (Fig 3).
View larger version (75K):
[in this window]
[in a new window]
| Fig 3.
Northern blotting analysis for the expression of 5 novel
cytokine-responsive genes in resting and TNF--activated HUVEC.
Northern blotting analysis of 10 µg HUVEC total RNA with probes from
DD-fragments or corresponding EST-clones was performed as described in
Materials and Methods. Time periods of continuous stimulation by
TNF- are indicated, and GAPDH analysis is given as control for equal
loading. The approximate length of the transcripts was determined from
the position of 28S, 18S, and 5S ribosomal RNA at the following: CA2_1:
an abundant band of 5.2 kb and two minor bands of 6 and 4.4 kb,
respectively; GG10_2: 2.8 kb; GG2_1: 1.8 kb; CG12_1: 2.3 kb; and AG8_1:
3.2 kb. A detailed analysis of these 5 transcripts is given in the text
and in Fig 4. In the case of CA2_1, identical patterns and intensities
were found with radiolabelled probes from bases 639-1032 (DD-fragment)
and 2331-3317 (hIAP-1) from the full-length sequence. The other probes
that were used represent the following parts of the full-length
sequences: GG10_2: 2697-2883; GG2_1: 670-1882; CG12_1, 1690-2298;
AG8_1: 2880-3289; and GAPDH: 360-1070 (GB: M33197).
|
|
Cloning and analysis of 5 novel cytokine-responsive transcripts.
Specific details of each gene transcript can only be shown by
full-length cDNAs, despite the rapidly increasing information on
overlapping EST sequences at Unigene (NCBI). The IMAGE Consortium cDNA
Clones48 from which EST-sequences were derived can be
obtained through various distributors, but usually represent partial
cDNAs as a result of their average sizes ranging from 0.5 to 2 kb.
Therefore, a cDNA library was constructed from the mRNA isolated from
HUVEC that had been activated for 6 hours by monocyte-conditioned
medium. Next, differential display probes or corresponding IMAGE-clones were used to screen this cDNA library for the 5 mRNAs represented in
Fig 3, resulting in the isolation of several full-length and partial
clones. Full-length sequences for these five cDNAs can be obtained
through their Genbank accession numbers.2 Novel transcript
CA2_1 (5,212 bp, GB:AF070674) contained the identical DD/RT-PCR
fragments CA2_1_3, AC2_1_4, and GC2_1_5 (397 bp) at position 639-1032, which resulted from oligo-dT priming at a 27-bp polyA stretch in the
5'-UTR of the full-length message, rather than at the genuine
polyA-site at the 3'-end. A mRNA length of 5.4 kb has been
extensively documented for hIAP-1, but only partial cDNA sequences had
been deposited in Genbank. The sole open reading frame (ORF) encodes
the known protein human inhibitor of apoptosis protein-1 (hIAP-1; GB:
U45878), also known by the names MIHC (GB: U37546) and cIAP-2. This
protein blocks TNF- receptor-mediated cellular apoptosis by direct
inhibition of various caspases.22 The novel cDNA GG10_2
(2,872 bp, GB: AF070672) contains DD/RT-PCR fragment GG10_2 (188 bp) at
position 2697-2872 and represents a TNF--repressed mRNA. The
full-length sequence was constructed from the largest partial cDNA
obtained from our cDNA library (1.5 kb) and the overlapping IMAGE
Consortium (LLNL) cDNA Clone (clone ID: 613195).48 The
predicted 890 amino acid sequence is 86% identical (91% homologous;
Fig 4A) to the recently described murine protein Rabkinesin-6 (GB: Y09632); therefore, this cDNA
represents the human homolog.23 The novel cDNA AG8_1 (3,295 bp, GB: AF070673) contains DD/RT-PCR fragment AG8_1 (403 bp) at
position 2880-3289. We have isolated a partial cDNA of 2.0 kb from our
activated HUVEC library and completed the sequence with the
overlapping IMAGE Consortium cDNA Clone (clone ID:
0969636). The predicted ORF (Fig 4B) encodes the human homolog of
rat stannin25,26 (GB: M81639). The novel cDNA CG12_1 (2,298 bp, GB: AF070675) contains DD/RT-PCR fragment CG12_1 (180 bp) at the
extreme 3'-end at position 2127-2298. The full-length cDNA
sequence (2,298 bp) showed it to encode a 331 amino acid protein that
is 70% homologous (50% identical) to the recently described human
apolipoprotein-L, an HDL-associated lipoprotein produced by the
pancreas24 (Fig 4C). Finally, the novel cDNA GG2_1 (1,889 bp, GB: AF070671) contained DD/RT-PCR fragment GG2_1_2 (303 bp) at the
very 3'-end at position 1595-1889. The cDNA encodes an 188 amino
acid protein and shows no homology at either the nucleotide, amino
acid, or structural level with any protein or gene present in the
combined NCBI and EMBL databases. Therefore, we are at present unable
to assign a function to this gene.
View larger version (64K):
[in this window]
[in a new window]
| Fig 4.
Amino-acid sequence homology for 3 novel full-length
human cDNAs obtained from an activated HUVEC cDNA library. Amino acid
sequences from the coding sequence of 3 novel human cDNAs were compared
to known proteins using the ClustalW algorithm for (A) the novel human
cDNA GG10_2 and murine Rabkinesin-640; (B) the novel human
cDNA AG8_1 and rat stannin39; and (C) the novel human cDNA
CG12_1 and the human apolipoprotein-L.41
|
|
In situ hybridization on human vascular tissue.
The isolation of candidate genes as described so far gives detailed
information about the response of cultured HUVEC to cytokine stimulation. To determine whether this approach resulted in the isolation of novel genes that are actually expressed in arterial vascular endothelial cells, we performed in situ hybridization studies
on human vascular tissue. Endothelial cells are thought to play an
essential role, especially during the onset of lesion formation.
Therefore, vascular specimens displaying early stages of inflammatory
lesion formation were obtained from organ donors who did not have a
prior history of vascular disease. A series of vascular specimens was
analyzed by (immuno)histochemical staining and selected for displaying
either normal vascular wall or early and advanced lesions. Integrity of
the endothelial lining and its mRNA was substantiated by using probes
for human von Willebrand factor, being solely expressed in the
endothelial cells
(Fig 5). Cytokine activation of the endothelial lining was checked by a probe
for MCP-1, a cytokine-responsive gene that was identified in our
differential display approach and extensively described as a marker for
endothelial cell activation.3,11,27,28 MCP-1 is shown to be
an excellent marker both for endothelial cell activation and more
generally for inflammation of the vessel wall by virtue of its high
expression in macrophages (Fig 5). Expression by arterial endothelial
cells is exclusively observed when macrophages are present close to the
endothelium (Fig 5), whereas macrophages that have deeply infiltrated
into the vessel wall do not elicit such expression in endothelial cells
(not shown). Next, we tested expression of our 5 novel genes.
Expression of CA2_1 (hIAP-1) could be readily detected only in the
endothelial lining of atherosclerotic aorta and iliac arteries,
coinciding with MCP-1 expression, whereas we never detected hIAP-1 in
normal arteries or in arteries that did not display substantial
monocyte/macrophage infiltrates. Subsequently, we determined in vivo
expression of CG12_1, which shows low expression in unstimulated
cultured cells but is induced approximately 20-fold upon cytokine
activation in vitro (Fig 3). The mRNA for this apolipoprotein-L-like protein can be readily detected in the normal endothelial lining of
nonatherosclerotic iliac artery (Fig 5). In addition, expression is
seen in capillaries in the adventitia but not in smooth muscle cells.
Also, in atherosclerotic vascular material, expression is only seen in
endothelial cells and not in macrophages. This indicates that the
expression of this novel gene seems limited to endothelial cells in the
tissues examined so far. The novel human cDNA AG8_1 (stannin),
expressed at low levels in cultured cells, is detected in the
endothelial cells and in intimal macrophages of atherosclerotic
lesions, but was undetectable in the normal vessel wall (Fig 5).
Interestingly, GG2_1, which is expressed at high levels both in
unstimulated and cytokine-activated cultured cells (Fig 3), can be
detected only with great difficulty in the arterial vascular material
screened so far (Fig 5). The novel cDNA gene GG10_2 (Rabkinesin-6),
being constitutively expressed in HUVEC and repressed by TNF- in
vitro, could not be detected in the vessel wall so far, indicating low
steady-state mRNA levels in vivo.
View larger version (120K):
[in this window]
[in a new window]
| Fig 5.
Pattern of in situ hybridization of mRNA
expression in human vascular tissue. In situ hybridization and
immuno-histochemical staining was performed on serial sections (5 µm)
of formaline-fixed and paraffin-embedded vascular tissue as described
in Materials and Methods. Tissues were derived from the normal iliac
artery of a 12-year-old male (A through D), 0.5 cm past the aortic
bifurcation and the abdominal aorta of a 49-year-old woman (E through
H), or a 39-year-old woman (I through T), both 1 cm before the
bifurcation. (A) Masson Trichrome staining showing an overview through
the iliac artery with the lumen on top, endothelial cells lining the
vessel wall, the internal elastic lamina, the smooth muscle
cell-containing media, and the spongy-appearing adventitia. (B)
Specific staining of smooth muscle cells, using an antibody directed
against SMC-specific -actin (1A4). (C) Autoradiographic image of the
specific hybridization of an antisense probe for the endothelial cell
specific protein von Willebrand factor, showing the integrity of
endothelial cell mRNA and specificity of hybridization conditions. (D)
Expression of novel cDNA CG12_1 is restricted to endothelial cells
lining the vessel and the capillaries of the adventitia, identical to
von Willebrand factor (C). (E) Masson Trichrome staining showing an
overview through the aorta with the lumen on top, endothelial cells
lining the vessel wall, a fibrous neo-intima (blue), the internal
elastic lamina, the smooth muscle cell-containing media (purple), and
the spongy-appearing adventitia. The boxed area is represented in (F)
through (H). (F) Immunohistochemical staining using a
monocyte/macrophage-specific antibody (HAM-56), showing the presence of
extensive infiltration of the neointimal layer of the vessel wall,
indicative of an inflammatory lesion. (G) Specific expression of MCP-1
by both macrophages and endothelial cells in this inflamed vessel wall.
(H) Specific expression of ferritin by monocytes/macrophages and
endothelial cells. (I) Masson Trichrome staining showing an overview
through the aorta with the lumen on top, endothelial cells lining the
vessel wall, a fibrous neo-intima (gray), the internal elastic lamina,
the smooth muscle cell-containing media (blue/purple), and the
spongy-appearing adventitia. The boxed area is represented in (J). (J)
Immunostaining for macrophages (HAM-56) in this aorta section showing
inflammation of the intima; the boxed area is represented in the in
situ hybridizations shown in (K) through (N). (K) Expression pattern of
MCP-1 in endothelial cells and macrophage/foam cells. (L) Specific
expression of CA2_1 (hIAP-1) by the endothelial cells. (M) Expression
of novel cDNA GG2_1 by endothelial cells. (M) and (N) are a darkfield
representation of the autoradiographic images for greater clarity of
hybridization of radiolabeled probes for sense mRNA for novel cDNA
GG2_1 (M) and, as control for specificity, for antisense GG2_1 (N). (O)
Masson Trichrome staining showing an overview through the aorta with
the lumen on top, endothelial cells lining the vessel wall, a fibrous
neo-intima (gray), the internal elastic lamina, the smooth muscle
cell-containing media (blue/purple), and the spongy-appearing
adventitia. (P) The integrity of the endothelial cell lining is shown
with the specific lectin from Ulex europaeus, and specific
expression of novel cDNA AG8_1 (stannin) is detected in endothelial
cells (Q, darkfield representation). A more heavily inflamed area of
the same aorta is shown by Masson Trichrome (R) and in the boxed area
by immunostaining for macrophages (S) and expression of AG8_1 (T) in
both endothelial cells and macrophages.
|
|
| |
DISCUSSION |
We have identified 106 nonredundant HUVEC gene fragments that are
differentially expressed upon cytokine stimulation of these cells. In a
substantial number of cases, differential expression was observed with
TNF- (193 fragments) but not with monocyte supernatant (119 fragments), as exemplified in Fig 2. These results strongly suggest the
presence of counteracting cytokines in the more complex mixture, eg,
interleukin-4 (IL-4), IL-10, and IL-13 are well known to attenuate gene
induction by TNF-, whereas interferon- (IFN-) can act
synergistically.29 Even for a single function as
prostacyclin production by endothelial cells, different combinations of
cytokines have differential effects on cells of different vascular origin.30 The minority of our panel of cytokine-responsive
genes (22/106) represents mRNAs for known proteins, most of which have been shown previously to be induced by cytokines. Many of the corresponding proteins have been implicated in
atherogenesis,2 showing the specificity of our approach. It
seems remarkable that the transcripts for adhesion molecules VCAM-1 and
ICAM-1, purported for their role in atherogenesis, were not among the
106 genes identified (Table 1), although FACScan and Northern blotting analysis showed them to be induced by both monocyte CM and TNF- (Fig
1 and data not shown). However, it should be noted that, by definition,
DD/RT-PCR uses a statistically defined set of arbitrary primers, and
the identification of specific transcripts is merely a chance
process.15 This is in fact the main asset of this approach, because it ensures an unbiased sampling of induced and repressed known
and novel genes of both low and high abundance in any given situation.
Therefore, we limited our primer set to 144 combinations to ensure an
unbiased sampling of activation-sensitive genes especially to find
novel genes, although statistics predict that a portion of transcripts
will then be missed. Nevertheless, many expected transcripts for known
genes were identified, as shown in Table 1. The corresponding gene
products are involved in a variety of intercellular and intracellular
processes (Table 1). As expected, we find induced expression of a
number of proinflammatory (proatherogenesis) genes, such as PTGS2
(Cox2) and several genes involved in leukocyte trafficking, such as
IL-8, RANTES, granulocyte-monocyte colony-stimulating factor
(GM-CSF), and the recently described galectin PCTA-1. In addition, the endothelial cell shows induced expression of a number of
protective (antiatherogenesis) genes. Protection against oxidative stress, which accompanies many inflammatory processes as a result of
monocyte/macrophage activation,28 is accomplished by the increased expression of ferritin and manganese superoxide dismutase, an
enzyme essential in withstanding the intracellular oxidative burst that
accompanies TNF- activation.31,32 Identification of
ferritin as a TNF- responsive gene shows the sensitivity of our
DD/RT-PCR approach, which did not identify only strongly induced genes.
Like PTGS-2, ferritin expression is easily detected in unstimulated
cells and is increased only threefold after stimulation. The
significance of identifying moderately regulated genes is underscored
by the fact that increased ferritin expression has been documented for
both human and rabbit aortic atherosclerotic lesions,33 as
confirmed in Fig 5. TNF- is known for its ability to induce
apoptosis in many cell types under certain conditions through
activation of its cognate receptor subtype I.34,35 Apparently, the endothelial cell protects itself very efficiently against TNF--induced apoptosis35,36 by expressing
hIAP-1, a protein that is able to block cellular
apoptosis.22,35,37 In addition, the antiapoptotic A20
protein, which was previously identified from a similar screen of
differential endothelial cell gene expression in response to TNF-,
is expressed.38 The latter protein has recently been shown
to attenuate TNF--induced endothelial cell activation by blocking
NF-B-dependent mechanisms.39 In our study, yet another
gene (AG8_1) was identified, encoding an 88 amino acid residue protein
that is the human homolog of rat stannin. Stannin is a protein that is
essential for a sensitive apoptotic response by neuronal cells to
organo-tin compounds. The exact functional role of this protein in this
cytotoxic process has not yet been established.25,26
Although apoptosis seems to be counteracted efficiently, proliferation
appears to be shut down. This is apparently accomplished by repressing
two genes involved in cell cycle initiation (GSPT1, RGS5) and the
upregulation of an antiproliferative protein (BTG1). Endothelial cells
function as an important selective barrier to separate the blood in the lumen of the vessel from the surrounding tissue. Transport of nutrients
and other plasma constituents is actively regulated by the endothelium.
Changes in cell shape, one of the affected functions described in Table
1, could greatly compromise this barrier function, similar to
observations in lung tissue, where lung fluid balance is greatly
disturbed by actin rearrangements in the endothelial
cells.40 Indeed, the most striking visual effect of TNF-
activation of HUVEC is the gross change of cell morphology from
cobblestone to spindle shape, a well-documented phenomenon41 that we also observed upon incubation with
monocyte-conditioned medium. Furthermore, several proteins involved in
vesicular transport show cytokine responsive regulation, including the
novel human cDNA GG10_2. As shown in Fig 4, GG10_2 represents the human
homologue for the recently described murine protein Rabkinesin-6
(Y09632), a kinesin-like protein involved in intra-Golgi vesicle
transport via micro-tubuli, that is regulated by the GTPase
Rab-6.23
The remaining 84 of the DD/RT-PCR fragments represent genes of
presently unknown function, but in 33 cases (including our 5 novel
transcripts) their identity as genuine human transcripts is verified by
their presence in Expressed Sequence Tag (EST) libraries.9
These EST libraries contain at present more than 1,000,000 partial
sequences of human cDNA clones, mostly from the IMAGE
consortium48 or TIGR,9 together with
information about tissue- or disease-specific expression data and
possible functional data based on sequence homologies with known genes (Unigene; NCBI). In addition, a chromosomal location has been determined to aid genetic analysis, complementary to the human genome
project (SCIENCE Map of the Human Genome; NCBI). Most of the ESTs that
we have identified using DD/RT-PCR have been described in activated or
fetal tissues: DD/RT-PCR fragment CA2_1 was identified by ESTs from
adult parathyroid tumor (GB: W32947), adenoma-transformed lung
cell-line (GB: U54711), and ulcerative Colitis Mucosa (GB: AA190195).
The full-length cDNA CA2_1 (5.2 kb) is shown to encode hIAP1, a protein
that blocks TNF- receptor-mediated cellular
apoptosis,35,37 a function that is indeed expected to be
found preferentially in tumor or inflamed tissues. Similarly, expression of GG2_1 is found in EST libraries from adult multiple sclerosis lesions and HeLa carcinoma, CG12_1 in fetal tissues and
ovarian cancer, and AG8_1 in neuroepithelium, fetal heart, and multiple
sclerosis lesions. Similar expression profiles were found for the
remaining DD-fragments corresponding to ESTs; only rarely has
expression been observed in resting tissue-specific libraries such as
pancreas, brain, or retina. This is remarkable given the fact that the
large majority of ESTs has been obtained from resting tissue, although
this is rapidly changing. This bias towards activated tissue expression
in vivo, in combination with our in vitro Northern blots, strongly
suggests an in vivo role in EC activation of the ESTs that remain to be
studied in more detail.
Excessively long untranslated regions were found in the 4 TNF--induced novel transcripts. This is in remarkable contrast to
the relatively short UTRs of the constitutively expressed GG10_2 (Rabkinesin-6). Long untranslated regions are frequently found in the
3'-UTR of induced transcripts, as exemplified by our novel human
cDNA AG8_1, which, like the rat mRNA for stannin, contains almost 3 kb
of 3'-UTR. Interestingly, the 4-kb message for PCTA1 (GB:
L78132), identified as a differentially expressed mRNA in prostate
carcinoma (and Table 1), has the same coding sequence as galactin-8
(GB: X91790), an mRNA from normal tissue of only 1.1 kb.
Remarkably, the untranslated region of the CA2_1 (hIAP-1) transcript
was located in the 5'-UTR rather than in the 3'-UTR. The
functional significance of this unusually long 5'-UTR (2,750 bp)
remains to be established. The DD/RT-PCR initially identified these as
novel transcripts, whereas the coding sequence has already been
described. At present, we cannot exclude the possibility that some of
our novel sequences will turn out to represent novel transcripts for
known genes, because many alternate mRNAs are already appearing in
dbEST and Unigene, showing that the sequences deposited in Genbank for
known mRNAs usually represent but one of several (possibly cell-type
specific) transcripts.
The in vivo significance of any one of the candidate genes we
identified in vitro obviously relies on in situ expression in the
endothelial lining of the vascular bed. Using aorta and iliac arteries
obtained during organ transplantations, our in situ hybridization approach confirms that expression of MCP-1 is a reliable marker for
inflamed vascular lesions (Fig 5), as has been
described.3,11,27,28 Furthermore, to our knowledge, we are
the first to show in vivo expression of the antiapoptotic hIAP-1
(CA2_1), which we detected in endothelial cells overlying lesions
heavily infiltrated by monocytes and foam cells (Fig 5). So, despite
the fact that hIAP-1 (CA2_1) is an immediate early gene in culture, it
can be detected during a chronic disease such as atherosclerosis,
stressing the fact that its molecular processes are of an episodal
nature. Expression of AG8_1 (stannin), expressed at low levels in
cultured cells, could be confirmed in vivo, but does not seem to be
absolutely specific for the endothelial cells, because occasionally
monocytes/macrophages of the atherosclerotic lesions express this gene
(Fig 5). Expression in vivo of GG10_2 (Rabkinesin-6), being one of the
few constitutively expressed and cytokine-repressed genes in vitro has
not yet been established, probably due to a low steady-state mRNA
level, and awaits confirmation by an immuno-histochemical approach.
Two interesting novel genes are not yet assigned to a gene panel in
Table 1. The novel cDNA fragment GG2_1 encodes an 188 amino acid
protein that shows no sequence or structural homology with any protein
in the EMBL databases. No structural or functional protein
motifs/domains could be determined using PROSITE and Blocks, indicating
that it seems to represent a totally novel protein. Although this
greatly complicates its functional study, exactly these kind of
proteins will be the most significant outcome of unbiased approaches
such as DD/RT-PCR, because they will lead the way to more fundamental
knowledge on basic processes. Interestingly, expression of GG2_1, which
is expressed at relatively high levels both in unstimulated and
cytokine-activated cultured cells, could be detected in the arterial
vascular material screened so far only with great difficulty, although
it seems restricted to arterial lesion endothelial cells (Fig 5). This
might indicate that its expression level in vivo is quite low and is
markedly induced by culturing the cells in vitro, something that has
been documented for other genes as well.42 We cannot
speculate about the significance of this at present, because its
protein sequence does not yield clues about a possible function of this
novel protein. Equally significant is the novel cDNA CG12_1, which
represents an endothelial cell-specific, 331 amino acid protein. We
determined in vivo expression of CG12_1, for which we detected low
expression in unstimulated cultured cells (Fig 3), that was upregulated
approximately 20-fold. The mRNA for this apolipoprotein-L-like protein
can be detected readily in the normal endothelial lining of
nonatherosclerotic iliac artery and aorta (Fig 5). In addition,
expression is seen in capillary endothelial cells in the adventitia,
but not in smooth muscle cells. Also, in atherosclerotic vascular
material, expression was restricted to endothelial cells, indicating
that the expression of this novel gene seems limited to endothelial
cells in all tissues examined so far. Interestingly, the deduced amino
acid sequence for CG12_1 is 70% homologous (50% identical) to the
recently described human apolipoprotein-L, an HDL-associated
lipoprotein produced by the pancreas.24 The fact that
CG12_1 is highly homologous to an HDL-associated protein and seems to
be expressed in the vessel wall in an endothelial cell-specific way
might indicate a role for the vessel wall in lipoprotein homeostasis.
The next stage of our studies will embody the analysis of the
expression of our panel of 106 EC genes in a quantitative way in
healthy and diseased vascular tissue to confirm which gene or gene
panels play a dominant in vivo role during atherosclerosis. A thorough
mapping of gene expression patterns in various (patho-)physiological situations will give indispensable information concerning alterations in functional properties related to altered gene expression patterns. Prescreening of the total human gene repertoire for potential candidate
genes will greatly accelerate gene expression mapping of cellular
anomalies that are characteristic for a specific
disease.8,9 The results from the present study on
cytokine-responsive genes may then be combined with data from other
differential screening procedures on vascular endothelial cells in a
variety of processes, such as hemodynamic forces,43
hyperhomocysteinemia,44 endothelial differentiation,42 endothelial cell
proliferation,45 and IL-146 or angiotensin
II47 stimulation of endothelium. This will yield detailed
information on the exact nature of what distinguishes an activated or
dysfunctional endothelial cell at the gene expression level from a
healthy or resting cell, information needed to describe the
patho-physiology of the atherosclerotic vessel wall at the molecular
level. The 5 novel cDNAs that we described in this report illustrate
that many genes potentially related to these molecular processes remain
to be identified.
| |
FOOTNOTES |
Submitted September 15, 1998; accepted January 11, 1999.
Supported by the Molecular Cardiology Program of the Netherlands Heart
Foundation (The Hague) (Grant No. M93.007).
The sequences of all novel cDNAs were deposited into Genbank and can be
accessed freely through their respective Genbank numbers: GG2_1:
AF070671; GG10_2: AF070672; AG8_1: AF070673; CA2_1: AF070674; and
CG12_1: AF070675.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Anton J.G. Horrevoets, PhD, Department
of Biochemistry, Academic Medical Center Room K1-160,
Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands;
e-mail: a.j.horrevoets{at}amc.uva.nl.
| |
REFERENCES |
1.
Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, Pober JS, Wick TM, Konkle BA, Schwartz BS, Barnathan ES, McCrae KR, Hug BA, Schmidt A-M, Stern DM:
Endothelial cells in physiology and in the pathophysiology of vascular disorders.
Blood
91:3527, 1998[Free Full Text]
2.
Ross R:
Atherosclerosis: A perspective for the 1990's.
Nature
362:801, 1993[Medline]
[Order article via Infotrieve]
3.
Wilcox JN, Nelken NA, Coughlin SR, Gordon D, Schall TJ:
Local expression of inflammatory cytokines in human atherosclerotic plaques.
J Atheroscler Thromb
1:S10, 1994 (suppl 1)
4.
Mazzuchelli L, Hauser C, Zgraggen K, Wagner HF, Hess MW, Laissue JA, Muller C:
Differential in situ expression of the genes encoding the chemokines MCP-1 and RANTES in human inflammatory bowel disease.
J Pathol
178:201, 1996[Medline]
[Order article via Infotrieve]
5.
Miller A, Lanir N, Shapiro S, Revel M, Honigman S, Kinarty A, Lahat N:
Immunoregulatory effects of interferon-beta and interacting cytokines on human vascular endothelial cells. Implications for multiple sclerosis autoimmune diseases.
J Neuroimmunol
64:151, 1996[Medline]
[Order article via Infotrieve]
6.
Weinberger MS, Davidson TM, Broide DH:
Differential expression of vascular cell adhesion molecule mRNA and protein in nasal mucosa in response to IL-1 or tumor necrosis factor.
J Allergy Clin Immunol
97:662, 1996[Medline]
[Order article via Infotrieve]
7.
Konstantopoulos K, McIntire LV:
Effects of fluid dynamic forces on vascular cell adhesion.
J Clin Invest
100:S19, 1997 (suppl 1997)
8.
Nowak R:
Entering the postgenome era.
Science
270:368, 1995[Abstract/Free Full Text]
9.
Adams MD, Kerlavage AR, Fleischmann RD, Fuldner RA, Bult CJ, Lee NH, Kirkness EF, Weinstock KG, Gocayne JD, White O, Venter C:
Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence.
Nature
377:3, 1995[Medline]
[Order article via Infotrieve] (suppl)
10.
Sporn SA, Eierman DF, Johnson CE, Morris J, Martin G, Ladner M, Haskill S:
Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair.
J Immunol
144:4434, 1990[Abstract]
11.
Mazzucchelli L, Hauser C, Zgraggen K, Wagner HE, Hess MW, Laissue JA, Mueller C:
Differential expression of the genes encoding the chemokines MCP-1 and RANTES in human inflammatory bowel disease.
J Pathol
178:201, 1996
12.
Lukacs NW, Strieter RM, Elner V, Evanoff HL, Burdick MD, Kunkel SL:
Production of chemokines, interleukin-8 and monocyte chemoattractant protein-1, during monocyte:endothelial cell interactions.
Blood
86:2767, 1995[Abstract/Free Full Text]
13.
Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle D, Neumeier D:
Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion.
J Clin Invest
97:1715, 1996[Medline]
[Order article via Infotrieve]
14.
Liang P, Pardee AB:
Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.
Science
257:967, 1992[Abstract/Free Full Text]
15.
Bauer D, Muller H, Reich J, Riedel H, Ahrenkiel V, Warthoe P, Strauss M:
Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR).
Nucleic Acids Res
21:4272, 1993[Abstract/Free Full Text]
16.
Jaffe EA, Nachman RL, Becker CG, Minick CR:
Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.
J Clin Invest
52:2745, 1973
17.
Gubler U, Hoffman BJ:
A simple and very efficient method for generating cDNA libraries.
Gene
25:263, 1983[Medline]
[Order article via Infotrieve]
18.
Wilkinson D, Green J:
In situ hybridization and the three-dimensional reconstruction of serial sections, in
Copp AJ,
Cockroft DL
(eds):
Postimplantation Mammalian Embryos: A Practical Approach. Oxford, UK, Oxford, 1990, p 155.
19.
Sherry B, Cerami A:
Cachectin/tumor necrosis factor exerts endocrine, paracrine, and autocrine control of inflammatory responses.
J Cell Biol
107:1269, 1988[Free Full Text]
20.
Rothe M, Pan M-G, Henzel WJ, Ayres TM, Goeddel DV:
The TNF--TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins.
Cell
83:1243, 1995[Medline]
[Order article via Infotrieve]
21.
Uren AG, Pakusch M, Hawkins CJ, Puls KL, Vaux DL:
Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors.
Proc Natl Acad Sci USA
93:4974, 1996[Abstract/Free Full Text]
22.
Quinn L, Devereaux RN, Stennicke HR, Van Arsdale T, Zhou Q, Srinivasula SM, Alnemri ES, Salvesen GS, Reed JC:
IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.
EMBO J
17:2215, 1998[Medline]
[Order article via Infotrieve]
23.
Echard A, Jollivet F, Martinez O, Lacapere JJ, Rousselet A, Janoueix-Lerosey I, Goud B:
Interaction of a Golgi-associated kinesin-like protein with Rab6.
Science
279:580, 1998[Abstract/Free Full Text]
24.
Duchateau PN, Pullinger CR, Orellana RE, Kunitake ST, NayaVigne J, O'Connor PM, Malloy MJ, Kane JP:
Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas.
J Biol Chem
272:25576, 1997[Abstract/Free Full Text]
25.
Toggas SM, Krady JK, Billingsley ML:
Molecular neurotoxicity of trimethyltin: Identification of stannin, a novel protein expressed in trimethyltin-sensitive cells.
Mol Pharmacol
42:44, 1992[Abstract]
26.
Thompson TA, Lewis JM, Dejneka DS, Severs WB, Polavarapu R, Billingsley ML:
Induction of apoptosis by organotin compounds in vitro: Neuronal protection with antisense oligonucleotides directed against stannin.
J Pharmacol Exp Ther
276:1201, 1996[Abstract/Free Full Text]
27.
Yu X, Dluz S, Graves DT, Zhang L, Antoniades HN, Hollander W, Prusty S, Valente AJ, Schwartz CJ, Sonensheim GE:
Elevated expression of monocyte chemoattractant protein 1 by vascular smooth muscle cells in hypercholesterolemic primates.
Proc Natl Acad Sci USA
89:6953, 1992[Abstract/Free Full Text]
28.
Kilgore KS, Imlay MM, Szaflarski JP, Silverstein FS, Malani AN, Evans VM, Warren JS:
Neutrophils and reactive oxygen intermediates mediate glucan-induced pulmonary granuloma formation through the local induction of monocyte chemoattractant protein-1.
Lab Invest
76:191, 1997[Medline]
[Order article via Infotrieve]
29.
Marfaing-Koka A, Devergne O, Gorgone O, Portier A, Shall TJ, Galanaud P, Emilie D:
Regulation of the RANTES chemokine by endothelial cells. Synergistic induction by IFN- plus TNF- and inhibition by IL-4 and IL-13.
J Immunol
154:1870, 1995[Abstract]
30.
Ristimaki A, Viinikka L:
Modulation of prostacyclin production by cytokines in vascular endothelial cells.
Prostaglandins Leukot Essent Fatty Acids
47:93, 1992[Medline]
[Order article via Infotrieve]
31.
Balla G, Jacobs HS, Balla J, Rosenberg M, Nath K, Apple F, Eaton JW, Vercellotti GM:
Ferritin: A cytoprotective antioxidant strategem of endothelium.
J Biol Chem
267:18148, 1992[Abstract/Free Full Text]
32.
Wong GHW, Elwell JH, Oberley LW, Goeddel DV:
Mangenous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor.
Cell
58:923, 1989[Medline]
[Order article via Infotrieve]
33.
Pang JH, Jiang MJ, Chen YL, Wang FW, Wang DL, Chu SH, Chau LY:
Increased ferritin gene expression in atherosclerotic lesions.
J Clin Invest
97:2204, 1996[Medline]
[Order article via Infotrieve]
34.
Fraser A, Evan G:
A licence to kill.
Cell
85:781, 1996[Medline]
[Order article via Infotrieve]
35.
Clem RJ, Duckett CS:
The iap genes: Unique arbitrators of cell death.
Trends Cell Biol
7:337, 1997[Medline]
[Order article via Infotrieve]
36.
Lindner H, Holler E, Ertl B, Multhoff G, Schreglman M, Klauke I, Schultz-Hector S, Eissner G:
Peripheral blood mononuclear cells induce programmed cell death in human endothelial cells and may prevent repair: Role of cytokines.
Blood
89:1931, 1997[Abstract/Free Full Text]
37.
Chu ZL, McKinsey TA, Liu L, Gentry JJ, Malim MH, Ballard DW:
Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-B control.
Proc Natl Acad Sci USA
94:10057, 1997[Abstract/Free Full Text]
38.
Dixit VM, Green S, Sarma V, Holzman LB, Wolf FW, O'Rourke K, Ward PA, Prochownik EV, Marks RM:
Tumor necrosis factor- induction of novel gene products in human endothelial cells including a macrophage-specific chemotaxin.
J Biol Chem
265:2973, 1990[Abstract/Free Full Text]
39.
Cooper JT, Stroka DM, Brostjan C, Palmetshofer A, Bach FH, Ferran C:
A20 blocks endothelial cell activation through a NF-kB-dependent mechanism.
J Biol Chem
271:18068, 1996[Abstract/Free Full Text]
40.
Ermert L, Rossig R, Hansen T, Schutte H, Aktories K, Seeger W:
Differential role of actin in lung endothelial and epithelial barrier properties in perfused rabbit lungs.
Eur Resp J
9:93, 1996[Abstract]
41.
Nelimarkka L, Kainulanen V, Schonherr E, Moisander S, Jortikka M, Lammi M, Elenius K, Jalkanen M, Jarvelainen H:
Expression of small extracellular chondroitin/dermatan sulfate proteoglycans is differentially regulated in human endothelial cells.
J Biol Chem
272:12730, 1997[Abstract/Free Full Text]
42.
Shima DT, Saunders KB, Gougos A, D'Amore PA:
Alterations in gene expression associated with changes in the state of endothelial differentiation.
Differentiation
58:217, 1995[Medline]
[Order article via Infotrieve]
43.
Topper JN, Cai J, Falb D, Gimbrone MA:
Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: COX-2, Mn-SOD, and e-NOS are selectively up-regulated by steady laminar shear stress.
Proc Natl Acad Sci USA
93:10417, 1996[Abstract/Free Full Text]
44.
Kokame K, Kato H, Miyata T:
Homocysteine-responsive genes in vascular endothelial cells identified by differential display analysis.
J Biol Chem
271:29659, 1996[Abstract/Free Full Text]
45.
Kozian DH, Augustin HG:
Rapid identification of differentially expressed endothelial cell genes by RNA display.
Biochem Biophys Res Commun
209:1068, 1995[Medline]
[Order article via Infotrieve]
46.
Introna M, Breviario F, D'Aniello EM, Golay J, Dejana E, Mantovani A:
IL-1 inducible genes in human umbilical vein endothelial cells.
Eur Heart J
14:78, 1993 (suppl K)
47.
Feener EP, Northrup JM, Aiello LP, King GL:
Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.
J Clin Invest
95:353, 1995
48.
Lennon G, Auffray C, Polymeropoulos M, Soares MB:
The I.M.A.G.E. Consortium: An integrated molecular analysis of genomes and their expression.
Genomics
33:151, 1996[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
J. Rohlena, O. L. Volger, J. D. van Buul, L. H.P. Hekking, J. M. van Gils, P. I. Bonta, R. D. Fontijn, J. A. Post, P. L. Hordijk, and A. J.G. Horrevoets
Endothelial CD81 is a marker of early human atherosclerotic plaques and facilitates monocyte adhesion
Cardiovasc Res,
January 1, 2009;
81(1):
187 - 196.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Pluvinet, R. Olivar, J. Krupinski, I. Herrero-Fresneda, A. Luque, J. Torras, J. M. Cruzado, J. M. Grinyo, L. Sumoy, and J. M. Aran
CD40: an upstream master switch for endothelial cell activation uncovered by RNAi-coupled transcriptional profiling
Blood,
November 1, 2008;
112(9):
3624 - 3637.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. D. Fontijn, O. L. Volger, J. O. Fledderus, A. Reijerkerk, H. E. de Vries, and A. J. G. Horrevoets
SOX-18 controls endothelial-specific claudin-5 gene expression and barrier function
Am J Physiol Heart Circ Physiol,
February 1, 2008;
294(2):
H891 - H900.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Christian, R. Winkler, I. Helfrich, A. M. Boos, E. Besemfelder, D. Schadendorf, and H. G. Augustin
Endosialin (Tem1) Is a Marker of Tumor-Associated Myofibroblasts and Tumor Vessel-Associated Mural Cells
Am. J. Pathol.,
February 1, 2008;
172(2):
486 - 494.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Chauvin, S. Salhi, and O. Jean-Jean
Human Eukaryotic Release Factor 3a Depletion Causes Cell Cycle Arrest at G1 Phase through Inhibition of the mTOR Pathway
Mol. Cell. Biol.,
August 15, 2007;
27(16):
5619 - 5629.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. L. Volger, J. O. Fledderus, N. Kisters, R. D. Fontijn, P. D. Moerland, J. Kuiper, T. J. van Berkel, A.-P. J.J. Bijnens, M. J.A.P. Daemen, H. Pannekoek, et al.
Distinctive Expression of Chemokines and Transforming Growth Factor-{beta} Signaling in Human Arterial Endothelium during Atherosclerosis
Am. J. Pathol.,
July 1, 2007;
171(1):
326 - 337.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. O. Fledderus, J. V. van Thienen, R. A. Boon, R. J. Dekker, J. Rohlena, O. L. Volger, A.-P. J. J. Bijnens, M. J. A. P. Daemen, J. Kuiper, T. J. C. van Berkel, et al.
Prolonged shear stress and KLF2 suppress constitutive proinflammatory transcription through inhibition of ATF2
Blood,
May 15, 2007;
109(10):
4249 - 4257.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. P. Blanc-Brude, E. Teissier, Y. Castier, G. Leseche, A.-P. Bijnens, M. Daemen, B. Staels, Z. Mallat, and A. Tedgui
IAP Survivin Regulates Atherosclerotic Macrophage Survival
Arterioscler. Thromb. Vasc. Biol.,
April 1, 2007;
27(4):
901 - 907.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. V. van Thienen, J. O. Fledderus, R. J. Dekker, J. Rohlena, G. A. van IJzendoorn, N. A. Kootstra, H. Pannekoek, and A. J.G. Horrevoets
Shear stress sustains atheroprotective endothelial KLF2 expression more potently than statins through mRNA stabilization
Cardiovasc Res,
November 1, 2006;
72(2):
231 - 240.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. J. Dekker, R. A. Boon, M. G. Rondaij, A. Kragt, O. L. Volger, Y. W. Elderkamp, J. C. M. Meijers, J. Voorberg, H. Pannekoek, and A. J. G. Horrevoets
KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium
Blood,
June 1, 2006;
107(11):
4354 - 4363.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. G. Rondaij, R. Bierings, A. Kragt, J. A. van Mourik, and J. Voorberg
Dynamics and Plasticity of Weibel-Palade Bodies in Endothelial Cells
Arterioscler. Thromb. Vasc. Biol.,
May 1, 2006;
26(5):
1002 - 1007.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. S. E. Albert, P. N. Duchateau, S. S. Deeb, C. R. Pullinger, M. H. Cho, D. C. Heilbron, M. J. Malloy, J. P. Kane, and B. G. Brown
Apolipoprotein L-I is positively associated with hyperglycemia and plasma triglycerides in CAD patients with low HDL
J. Lipid Res.,
March 1, 2005;
46(3):
469 - 474.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Berger, G. Bergers, B. Arnold, G. J. Hammerling, and R. Ganss
Regulator of G-protein signaling-5 induction in pericytes coincides with active vessel remodeling during neovascularization
Blood,
February 1, 2005;
105(3):
1094 - 1101.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Liu, H. Lu, Z. Jiang, A. Pastuszyn, and C.-a. A. Hu
Apolipoprotein L6, a Novel Proapoptotic Bcl-2 Homology 3-Only Protein, Induces Mitochondria-Mediated Apoptosis in Cancer Cells
Mol. Cancer Res.,
January 1, 2005;
3(1):
21 - 31.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
R. Kleemann, H. M.G. Princen, J. J. Emeis, J. W. Jukema, R. D. Fontijn, A. J.G. Horrevoets, T. Kooistra, and L. M. Havekes
Rosuvastatin Reduces Atherosclerosis Development Beyond and Independent of Its Plasma Cholesterol-Lowering Effect in APOE*3-Leiden Transgenic Mice: Evidence for Antiinflammatory Effects of Rosuvastatin
Circulation,
September 16, 2003;
108(11):
1368 - 1374.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. R. Lareu, M. D. Lacher, C. K. Bradley, R. Sridaran, R. R. Friis, and A. M. Dharmarajan
Regulated Expression of Inhibitor of Apoptosis Protein 3 in the Rat Corpus Luteum
Biol Reprod,
June 1, 2003;
68(6):
2232 - 2240.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Ho, E. Yang, G. Matcuk, D. Deng, N. Sampas, A. Tsalenko, R. Tabibiazar, Y. Zhang, M. Chen, S. Talbi, et al.
Identification of endothelial cell genes by combined database mining and microarray analysis
Physiol Genomics,
May 13, 2003;
13(3):
249 - 262.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Han and J.-T. Zhang
Regulation of Gene Expression by Internal Ribosome Entry Sites or Cryptic Promoters: the eIF4G Story
Mol. Cell. Biol.,
November 1, 2002;
22(21):
7372 - 7384.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. J. Dekker, S. van Soest, R. D. Fontijn, S. Salamanca, P. G. de Groot, E. VanBavel, H. Pannekoek, and A. J. G. Horrevoets
Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Kruppel-like factor (KLF2)
Blood,
August 13, 2002;
100(5):
1689 - 1698.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. J. Gordon, K. Appasani, J. P. Parcells, N. K. Mukhopadhyay, M. T. Jaklitsch, W. G. Richards, D. J. Sugarbaker, and R. Bueno
Inhibitor of apoptosis protein-1 promotes tumor cell survival in mesothelioma
Carcinogenesis,
June 1, 2002;
23(6):
1017 - 1024.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. L. Tyson, P. L. Weissberg, and C. M. Shanahan
Heterogeneity of gene expression in human atheroma unmasked using cDNA representational difference analysis
Physiol Genomics,
May 10, 2002;
9(2):
121 - 130.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Mimmack, M. Ryan, H. Baba, J. Navarro-Ruiz, S. Iritani, R. L. M. Faull, P. J. McKenna, P. B. Jones, H. Arai, M. Starkey, et al.
Gene expression analysis in schizophrenia: Reproducible up-regulation of several members of the apolipoprotein L family located in a high-susceptibility locus for schizophrenia on chromosome 22
PNAS,
April 2, 2002;
99(7):
4680 - 4685.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. T.B. Crawley, D. A. Goulding, V. Ferreira, N. J. Severs, and F. Lupu
Expression and Localization of Tissue Factor Pathway Inhibitor-2 in Normal and Atherosclerotic Human Vessels
Arterioscler. Thromb. Vasc. Biol.,
February 1, 2002;
22(2):
218 - 224.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Cooper, S. Potter, B. Mueck, S. Yousefi, and G. Jarai
Identification of genes induced by inflammatory cytokines in airway epithelium
Am J Physiol Lung Cell Mol Physiol,
May 1, 2001;
280(5):
L841 - L852.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. D. Fontijn, B. Goud, A. Echard, F. Jollivet, J. van Marle, H. Pannekoek, and A. J. G. Horrevoets
The Human Kinesin-Like Protein RB6K Is under Tight Cell Cycle Control and Is Essential for Cytokinesis
Mol. Cell. Biol.,
April 15, 2001;
21(8):
2944 - 2955.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
P. N. Duchateau, C. R. Pullinger, M. H. Cho, C. Eng, and J. P. Kane
Apolipoprotein L gene family: tissue-specific expression, splicing, promoter regions; discovery of a new gene
J. Lipid Res.,
April 1, 2001;
42(4):
620 - 630.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. E. Rectenwald, L. L. Moldawer, T. S. Huber, J. M. Seeger, and C. K. Ozaki
Direct Evidence for Cytokine Involvement in Neointimal Hyperplasia
Circulation,
October 3, 2000;
102(14):
1697 - 1702.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Walsh and J. M. Isner
Apoptosis in inflammatory-fibroproliferative disorders of the vessel wall
Cardiovasc Res,
February 1, 2000;
45(3):
756 - 765.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Kumar, T. L. Whiteside, and U. Kasid
Identification of a Novel Tumor Necrosis Factor-alpha -inducible Gene, SCC-S2, Containing the Consensus Sequence of a Death Effector Domain of Fas-associated Death Domain-like Interleukin- 1beta -converting Enzyme-inhibitory Protein
J. Biol. Chem.,
January 28, 2000;
275(4):
2973 - 2978.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. You, H. Ouyang, D. Lopatin, P. J. Polver, and C.-Y. Wang
Nuclear Factor-kappa B-inducible Death Effector Domain-containing Protein Suppresses Tumor Necrosis Factor-mediated Apoptosis by Inhibiting Caspase-8 Activity
J. Biol. Chem.,
July 6, 2001;
276(28):
26398 - 26404.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. J. M. de Vries, T. A. E. van Achterberg, A. J. G. Horrevoets, J. W. ten Cate, and H. Pannekoek
Differential Display Identification of 40 Genes with Altered Expression in Activated Human Smooth Muscle Cells. LOCAL EXPRESSION IN ATHEROSCLEROTIC LESIONS OF smags, SMOOTH MUSCLE ACTIVATION-SPECIFIC GENES
J. Biol. Chem.,
July 28, 2000;
275(31):
23939 - 23947.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION