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Prepublished online as a Blood First Edition Paper on May 13, 2002; DOI 10.1182/blood-2002-01-0046.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Biochemistry and Medical
Physics, Academic Medical Center, University of Amsterdam, The
Netherlands; and the Department of Hematology, University Medical
Center, Utrecht, The Netherlands.
The endothelium expresses a large repertoire of genes under
apparent transcriptional control of biomechanical forces, many of which
are neither cell-type nor flow specific. We set out to identify genes
that are uniquely flow responsive in human vascular endothelial cells.
Transcriptional profiling using commercial DNA microarrays identified
12 of 18 000 genes that were modulated at least 5-fold after 24 hours
of steady laminar flow (25 dyne/cm2). After a 7-day
exposure to unidirectional pulsatile flow (19 ± 12 dyne/cm2), only 3 of 12 remained elevated at least 5-fold.
A custom microarray of ~300 vascular cell-related gene fragments was
constructed, and expression analysis revealed that many flow-induced
genes are also induced by at least one of the following agents: tumor necrosis factor- Atherosclerosis is a chronic multifactorial disease
of the arteries that initiates and develops from young age until it
manifests itself clinically later in human life. Although the risk
factors for atherosclerosis are of a systemic nature, the localization of lesions is confined to specific and reproducible positions in the
arterial tree. The hypothesis that this focality of atherosclerosis is
caused by local blood flow turbulence at vessel bifurcations and
curvatures has been firmly supported over the last
decades.1,2 Lowering of the shear stress on the vascular
endothelium at sites of flow turbulence, creating steep shear-stress
gradients,3 is now believed to be one of the initiating
factors in atherogenesis. Consequently, the ability of the
atheroprotective force of shear stress to modulate the transcription of
genes expressed by endothelial cells, denoted endothelial genes, has
triggered ample research effort on the identification of
shear-stress-regulated genes.2,4 In contrast to the
protective function of shear stress, the cytokine tumor necrosis
factor- Several studies have been aimed at the identification of endothelial
genes that are regulated by arterial levels of shear stress.7,8 These studies have typically focused on the
identification of genes that are selectively induced by laminar but not
by turbulent flow. Well-known examples include the transcriptional
regulation of various adhesion molecules like vascular cell adhesion
molecule-1 (VCAM-1) and intercellular adhesion molecule-1
(ICAM-1) and several atheroprotective genes involved in, for
instance, handling oxidative stresses; for example, superoxide
dismutases.7,9,10 Most of these genes can be considered
atheroprotective solely on the basis of their function and thus would
be significant in a physiological context. Their regulation patterns,
however, reveal that they are expressed in a variety of cell types and
are also induced in response to various cytokines believed to be
primarily atherogenic, including TNF- In this study, we took an alternative approach to identify genes that
are consistently expressed in endothelial cells exposed to prolonged
laminar flow and not in static cultures. Thus, we aimed at identifying
those genes that are truly discriminative for the physiological
long-term flow-exposed state of the endothelial cell and potentially
lie at the basis of the phenotypic changes induced by laminar flow.
This rationale is essentially based on the contrasting difference
between the in vivo protective effect of shear stress and the
atherogenic potential of inflammatory cytokines exclusively at sites of
flow turbulence in healthy and atherosclerotic tissue, respectively.
Our stringent selection scheme consisted of preselecting flow-regulated
candidate genes and focusing on genes that are still induced after
exposing endothelial cells to flow for 7 days. Absence of a
transcriptional response to a variety of atherosclerosis-related
stimuli was taken as a final criterion. Our results confirm the
existence of a set of highly flow-induced flow- and
endothelial-specific genes and demonstrate that the expression of one
such gene, the transcription factor lung Krüppel-like factor
(LKLF), occurs only with prolonged shear stress in vitro and in vivo.
Cell culture and shear-stress experiments
Cytokine stimulation
RNA amplification Total RNA (~4 µg) from shear-stress-exposed and static HUVEC cultures was amplified essentially as described.16 Double-stranded cDNA was prepared using the SuperScript Plasmid System for cDNA Synthesis (GIBCO-BRL) with a T7(dT)15 primer. The cDNA was transcribed using the AmpliScribe T7 Transcription Kit (Epicentre Technologies, Madison, WI). The yield and size distribution of the resulting amplified antisense RNA (aRNA) was determined by measuring A260 and by electrophoresis on a 1% (wt/vol) agarose gel.Probe synthesis and cDNA array hybridizations For the 4-hour flow experiments, probes labeled with -33P-dCTP (Amersham, Piscataway, NJ) were directly
reverse transcribed from 4 µg aRNA using 3 µg random hexamers.
Probes of the 24-hour flow experiments were transcribed from unlabeled
first-strand cDNA (transcribed from 4 µg aRNA using 3 µg random
hexamers) using 1 µg T7(dT)15 primer and SuperScript II
RT (GIBCO-BRL). These first- and second-strand cDNA probes were
hybridized for 60 hours to the filter-based Gene Discovery Array (GDA)
I Version 1.3 (Incyte Genomics, Palo Alto, CA), according to the
manufacturer's instructions. The filters were imaged on a Storm
PhosphorImager (Molecular Dynamics, Sunnyvale, CA), scanned with
50-micron resolution, and quantified at Incyte Genomics. Selected
differential clones were obtained from Incyte Genomics and
sequence verified.
Construction of custom microarrays A custom cardiovascular glass-based microarray was constructed containing a comprehensive set of prominent endothelial genes and general vascular genes from previous studies, for example, genes encoding various proteoglycans; factors involved in thrombosis/fibrinolysis, cell shape, and vesicular transport/transcytosis; and control genes for normalization purposes.6,7,17,18 Polymerase chain reaction (PCR)-amplified gene-specific fragments of these genes, deprived of any repetitive or highly homologous sequences, were spotted on glass microscope slides in triplicate and hybridized with fluorescent cDNA probes using the methods previously described by Brown and coworkers (detailed protocols taken from the web site http://cmgm.stanford.edu/pbrown/protocols/index.html).19 Arrays were scanned on a ScanArray 3000 microarray scanner (GSI Lumonics, Bedford, MA), and fluorescence signals were background corrected and normalized for glyceraldehyde phosphate dehydrogenase (GAPDH). All analyses were performed with the Eisen software package Scanalyze, Cluster, and TreeView, as described (http://rana.lbl.gov).20Semiquantitative real-time RT-PCR Reverse transcription of 3 µg of total RNA was performed with 1 µg (dT)12-18 primer (GIBCO-BRL) using SuperScript II. Real-time reverse transcriptase-polymerase chain reactions (RT-PCRs) were performed using the FastStart DNA Master SYBR Green I kit (Roche, Mannheim, Germany) in the LightCycler System (Roche). Primers were LKLF, (forward) 5'-GCACGCACACAGGTGAGAAG-3' and (reverse) 5'-ACCAGTCACAGTTTGGGAGGG-3'; CYP1B1, (forward) 5'-CTCCTCCTCTTCACCAGGTATCC-3' and (reverse) 5'-AACCACAGTGTCCTTGGGAATG-3'. The PCR efficiency, determined for each primer pair separately using a series of cDNA dilutions, was used to calculate relative differences between samples, which were expressed as ratios compared to the static controls.Nonradioactive mRNA in situ hybridization Vascular tissues were fixed and paraffin embedded as described,6 and 16 µm sections were mounted onto SuperFrost Plus microscope slides (Menzel-Gläser, Braunschweig, Germany). The in situ hybridizations were performed as described.21 Riboprobes were derived from the following cDNA fragments: 460-base pair (bp) BstNI-BstNI fragment of the LKLF cDNA (GenBank, H28611), entire 1350-bp insert of a human claudin-5 cDNA clone (GenBank, R60153), entire 1790-bp insert of a CYP1B1 cDNA clone (GenBank, N72909), 192-bp fragment of human von Willebrand factor cDNA 8239-8442 (GenBank, X04385). All cDNA clones were obtained from either Incyte Genomics or as IMAGE-consortium cDNA clones22 from the United Kingdom Human Genome Mapping Project Resource Centre (Cambridge, United Kingdom).
Identification of shear-stress-responsive endothelial genes We first set out to identify endothelial genes that are highly shear-stress-regulated. For that purpose we have performed transcriptional profiling using commercially available cDNA arrays containing approximately 18 000 cDNA clones. Primary HUVECs were subjected to laminar flow in a parallel plate-type perfusion chamber, generating a shear stress of 25 dyne/cm.2 For use as controls, HUVECs were cultured in parallel under static conditions. Cells were exposed to flow for 4 or 24 hours to evaluate early responses versus the effect of longer-term flow exposures. Subsequently, total RNA was isolated and amplified in one round of T7 RNA polymerase-based amplification yielding antisense RNA (aRNA).16 Reverse transcription of the aRNA was performed to obtain 33P-labeled cDNA probes, which were hybridized with the cDNA arrays. The hybridized arrays were quantified, background corrected, and normalized. A plot was constructed comparing the signal intensities of all spots in the shear-exposed situation to the static cultures (Figure 1A). This plot shows that the vast majority of the genes present on the array are only marginally modulated after a 24-hour flow exposure, and lie between the 5-fold up-down- regulation lines. Approximately 4,000 of 18 000 genes (~20%) were significantly expressed, of which about 230 (~5.7%) appeared more than 2-fold induced or repressed. Next, for each gene on the array the ratios were calculated of the hybridization signal intensities of the 4- and 24-hour shear experiments over their corresponding static controls. To score genes as significantly shear-stress-regulated, 2 stringent selection criteria were used. First, genes had to be at least 5-fold differentially expressed, that is, ratios > 5 or < 0.2. Second, background-corrected hybridization signals had to be above the selected intensity threshold in either the shear-exposed or static conditions, that is, > 1.5 · 104 for the 4-hour and > 5 · 103 for the 24-hour shear experiments (outside the gray area in Figure 1A). Of 18 000 GDA filter array clones, only 29 met these criteria. As the GDA arrays contain nonsequence verified clones, with an estimated 30% incorrect annotations, all 29 clones were ordered and their identity established by resequencing, and PCR fragments were analyzed by filter-based spot blot analysis. Thus, we could confirm shear responsiveness for 12 of these genes, of which the expression kinetics are shown in Figure 1B-D. Three different kinetic classes of gene regulation can be distinguished: genes that are quickly induced and reach a maximum response within 6 hours (Figure 1C), genes that have a delayed response and continue to increase in a linear fashion beyond 24 hours of shear-stress exposure (Figure 1B), and the down-regulated gene claudin-5 (Figure 1D).
Gene transcription levels after long-term adaptation to flow Currently, no conclusive data are available on the time required for endothelial cells to fully adapt to flow in vitro. Life-long exposure of the endothelium to flow in vivo inhibits proliferation and lowers the metabolic rate, which is consistent with a resting phenotype.23 Therefore, sustained flow-regulated expression of the 12 flow-responsive and additional vascular genes was studied in an artificial capillary flow system using custom-made cardiovascular microarrays. This flow system allows culturing of endothelial cells under continuous unidirectional pulsatile laminar flow for long periods of time.13 In addition, cells are grown in a polar fashion on the luminal surface of permeable hollow fibers, which are in contact with a separate extracapillary (subendothelial) compartment. Thus, a more physiological model system is created compared to endothelial cells cultured on flat, solid supports. The time-dependent change of flow in the system was accurately measured and revealed a pulsatile, unidirectional flow profile generating a minimal, maximal, and mean wall shear stress of 8, 32, and 19 dynes/cm2 (19 ± 12 dynes/cm2), respectively (Figure 2). The capillaries were seeded with primary HUVECs and exposed to flow for 7 days after a 24-hour period of gradual flow increase, allowing cell attachment and growth. Next, total RNA was extracted and used in hybridization experiments with custom microarrays (construction and content is described in "Materials and methods" and footnotes of Table 1). These arrays comprise a selection of ~300 genes, facilitating a simultaneous quantitative analysis of the expression of the 12 shear-stress-responsive genes identified in this study and various additional genes/pathways that play prominent roles in endothelial cell biology. Table 1 shows the expression ratios calculated from 3 independent sustained-flow experiments and their control static cultures only for the 68 genes whose expression level exceeded 5% of the hybridization signal of GAPDH in at least the flow or static condition. Overall, the expression of 9 of the ~300 genes that are present on the array was increased at least 3-fold by sustained flow compared to static cultures. These expression analyses also show that 3 of the 12 genes, which were identified in this study after a 24-hour exposure to flow, were no longer significantly "differential" after chronic flow exposure. The induction levels of 6 genes had dropped to between 2- and 5-fold, but the expression of 3 genes remained elevated at least 5-fold after prolonged flow: lung Krüppel-like factor (LKLF/KLF2), cytochrome P450 1B1 (CYP1B1), and diaphorase 4 (DIA4/NQO1).
Endothelial cells differentially respond to a panel of atherosclerosis-related stimuli The qualification of the 3 genes KLF2, CYP1B1, and DIA4 as being highly responsive to prolonged shear stress prompted us to study the stimulus and cell-type specificity of their transcriptional regulation. Therefore, the custom microarrays were used to study the effect under static conditions of a set of established modulators of endothelial gene expression, including the cytokines/growth factors TNF-, IL-1 , TGF-, VEGF, as well
as thrombin, on the expression of the selected endothelial genes and
flow-responsive genes identified in this study. To that end, HUVECs
were cultured in the continuous presence of these agents for 2, 6, and
24 hours to allow identification of early- and late-induced genes,
whereas nonstimulated controls were taken at 0 and 24 hours. The human cell lines HL-60 (myelomonocytic) and HeLa (cervical carcinoma) were
included in the analysis to provide additional information on cell
type-specific expression. Total RNA was isolated from these cultures
and enriched for polyA+, which was then used to produce
Cy3-labeled cDNA probes that were hybridized to the arrays. The
fluorescence intensities were quantified for each individual gene under
the various conditions, and medians of the triplicates were used to
automatically exclude infrequent spotting artifacts, as medians were,
in most cases, close to the mean of the triplicate signals.
Subsequently, stringent data-filtering was applied by including only
those genes in the analysis that had hybridization signals above 10%
of the GAPDH signal with at least one of the stimuli used. Filtered
expression data were median centered, and genes were arranged using a
self-organizing map.20 Finally, these results were
subjected to complete linkage clustering with uncentered correlation to
produce a hierarchical tree (Figure
3A).20
Thus, the comprehensive series of expression data obtained by the
microarray analyses organizes into various clusters, of which 3 reveal
a particularly interesting trend, that is, they reveal genes
predominantly induced by shear stress (Figure 3B), genes induced by
TNF- Kinetics of LKLF and CYP1B1 induction by flow and
inverse regulation by TNF- stimulations were
performed on HUVECs for 2, 4, 6, 12, and 24 hours, with static
nonstimulated controls taken at 0, 6, and 24 hours. In addition, HUVECs
were exposed to a steady laminar flow for 2, 6, and 24 hours (25 dyne/cm2). Relative expression levels of LKLF and
CYP1B1 were determined using real-time RT-PCR and expressed
as ratios over the controls (Figure 4).
LKLF and CYP1B1 were induced by flow to maximum levels within 6 hours and 12 hours, respectively. Interestingly, compared to
steady laminar flow, pulsatile flow resulted in an additional 3-fold
increase in LKLF expression. These expression levels were sustained
well beyond 24 hours of flow exposure. In contrast, both LKLF and
CYP1B1 were significantly down-regulated by TNF- within 6 hours. LKLF expression remained at levels that were still 2.5-fold
lower than in static cultures, whereas repression of CYP1B1
by TNF- was sustained.
Human vascular expression of LKLF, cytochrome P450 1B1, and claudin-5 The microarray data have aided us in limiting the extensive (endothelial) gene collection to a set of 3 genes with interesting vascular cell-type specificity and patterns of transcriptional regulation by various stimuli. In contrast to all other genes tested, up-regulation of both LKLF and CYP1B1 was largely endothelial specific, whereas LKLF was exclusively expressed in endothelial cells under flow. In contrast, claudin-5 (CLDN5) was the only gene found to be considerably down-regulated after 24 hours of exposure to laminar flow, but its expression was induced by TNF- (Figures 1D, 3D). We therefore studied the expression of these genes in human vascular tissue. Nonradioactive mRNA in situ
hybridizations were performed on various sections from our human
vascular tissue collection.6,18 Figure
5 shows the results obtained with the thoracic aorta taken from a 13-year-old female donor. In this tissue,
no neointima was present, and inflammatory processes in the vessel wall
were absent. Expression of LKLF was restricted to the endothelium and
continuous throughout all the endothelial cells of the aorta itself
(Figure 5A-B). In the small branch, however, substantial differences in
LKLF expression were observed (Figure 5D-E). Whereas the endothelial
LKLF expression at the opposite wall of the branching point was
comparable to that in the aorta (Figure 5D), no LKLF mRNA was detected
in the endothelium covering the area where the branch physically
disconnects from the larger aorta (Figure 5E). In contrast, expression
of claudin-5 also was restricted to the endothelium but was not
significantly differential at this branching point, in accordance with
the observation that in vitro, its transient down-regulation at 24 hours (Figure 1D) returns to baseline levels after 7 days' (Table 1)
exposure to pulsatile shear (Figure 5H). Remarkably, in contrast to its high level of expression under flow in vitro (150% of GAPDH), the
level of CYP1B1 mRNA detected in vivo was not significantly above background (Figure 5I), confirming earlier histochemical reports
on the tissue distribution of CYP1B1
expression.24-26 As a positive control for the integrity
of both the endothelial cell lining and the mRNA at this site, in situ
hybridizations for von Willebrand factor (VWF) mRNA were performed on
consecutive sections (Figure 5C,F-G). There was a strong and uniform
signal for VWF, demonstrating the presence of a continuous layer of
endothelium and the good RNA quality in these tissues, as well as
confirming the endothelium-specific expression of LKLF and
claudin-5.
Presently, the only cell types known to exhibit a physiologically
significant response to flow are endothelial cells and
osteocytes.27 Adaptation of these cells to continuous flow
during their entire life span is crucial for correct functioning in
their respective physiological contexts. A change of this biomechanical
force likely leads to dysfunction of those processes that are specific
for these cell types and are specifically under the control of flow. Studies with novel screening techniques such as DNA microarray hybridizations and differential display have shown that TNF- The kinetics of steady laminar flow-induced genes identified in this
study can be divided into 2 different classes: genes that are
up-regulated to steady levels within 6 hours and genes that have a
delayed response increasing beyond 24 hours of flow exposure. In
contrast to the immediate-early induced genes, the induction of the
late genes likely requires de novo protein synthesis, for
example, flow-induced transcription/translation of transcription factors. Up until now, a panel of transcription factors has been demonstrated to be modestly regulated by flow, and their response elements have been identified.28-30 However, the wide
patterns of expression of the transcription factors, early growth
response-1 (Egr-1), nuclear factor- The long-term exposure of endothelial cells to flow in this study shows that the regulation of most of the shear-stress-responsive genes identified in this study and by others is transient. Although still elevated at 24 hours, they return to basal levels after a 7-day exposure to flow. CYP1B1 proved to be one of the few highly flow-specific and nontransiently expressed endothelial genes involved in metabolic processes. The high CYP1B1 levels found in the HL-60 cell line confirm reports that this is the major cytochrome P450 isoform in human blood monocytes.24 Also, the expression in HeLa cells is in agreement with the high CYP1B1 expression levels that are observed in many human tumors.25 The lack of expression in healthy human vascular tissue suggests that its strong transcriptional induction by flow, also noted by others,8 seems restricted to the conditions in in vitro model systems, whereas its expression in vivo is regulated in a more complex manner. It can be assumed that transient effects of flow on gene expression are more generally observed for a wide variety of stimuli and are therefore general stress responses. The continuous, life-long exposure of the endothelium to flow in vivo raises the question whether in vitro general stress-responsive, and in particular, transiently flow-modulated genes would indeed be induced in a physiological context. In this view, the transient flow responsiveness of claudin-5 and connexin-40, which was found in this study, demonstrates its possible in vivo triviality, although a plausible rationale is at hand. Both claudin-5 and connexin-40 are involved in mediating endothelial cell-to-cell contacts in tight junctions and gap junctions, respectively. The process of flow-induced reorganization of endothelial cells to their stretched shape might require the transient transcriptional modulation of these genes because of the temporary relief of cell-to-cell contacts.38 In addition, in situ hybridization for claudin-5 indeed showed its continuous presence in the endothelial lining of all human vessels tested so far. Our results with both claudin-5 and CYP1B1 stress the fact that in vitro data, especially when dealing with the transient application of a normally continuous force or activator, need to be backed up with in vivo confirmation. The most notable panel from our clustering analysis identifies the
small set of genes whose expression seems almost exclusively regulated
by flow in vitro. The most remarkable exponent of this set is lung
Krüppel-like factor (KLF2). Also, the difference in
transcriptional response of LKLF to steady laminar versus
unidirectional pulsatile flow is noteworthy. Exposure of HUVECs to
pulsatile flow for 24 hours up to 7 days induced LKLF expression
continuously as much as 20-fold, whereas steady flow induced LKLF
less than 5-fold with a peak around 4 hours. In contrast to its
induction by flow, the expression of LKLF in vitro was actually
repressed by TNF- In conclusion, this study on the transcriptional regulation of an extensive collection of atherosclerosis-related genes by a variety of (anti-) atherogenic stimuli demonstrates that only a very limited set of endothelial genes is more or less exclusively regulated by flow and is cell-type specific. Furthermore, the induction of the majority of flow-regulated endothelial genes both by flow and by cytokines likely results from potential cross-talk between flow- and inflammatory-mediated downstream signaling mechanisms. The endothelial-specific transcription factor LKLF was identified as a promising target to aid the further elucidation of the molecular basis of the flow-mediated vascular protection from atherosclerosis.
Submitted January 7, 2002; accepted April 12, 2002.
Prepublished online as Blood First Edition Paper, May 13, 2002; DOI 10.1182/blood-2002-01-0046.
Supported by the Netherlands Heart Foundation, the Hague, by grants NHS96.094 and NHS97.209; and the Molecular Cardiology Program grant M93.007.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Anton J. G. Horrevoets, Department of Biochemistry, Academic Medical Center K1-161, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands; e-mail: a.j.horrevoets{at}amc.uva.nl.
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Top
Abstract
Introduction
5 and +10/
) or unidirectional pulsatile laminar flow
generating a shear stress of 12 ± 7 dyne/cm2 (
)
for various time intervals. Static HUVECs cultured in parallel were
treated with 50 ng/mL TNF-
). Ratios of the relative expression
levels for LKLF (A) and CYP1B1 (B) determined by
semiquantitative real-time RT-PCR over static cultures were calculated
and expressed as fold induction or fold repression.
B (NF-
/