From the Department of Vascular Biology and
Thrombosis Research, Vienna International Research Cooperation Center,
University of Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria and the
¶ Department of Molecular Cell Biology, Max Planck Institute for
Physiological and Clinical Research,
D-61231 Bad Nauheim, Germany
Received for publication, May 20, 2002, and in revised form, October 4, 2002
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ABSTRACT |
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In this study we have investigated the role of a
specific corepressor of EGR-1, NAB2, to down-regulate vascular
endothelial growth factor (VEGF)-induced gene expression in endothelial
cells and to inhibit angiogenesis. Firstly, we show a reciprocal
regulation of EGR-1 and NAB2 following VEGF treatment. During the
initial phase EGR-1 is rapidly induced and NAB2 levels are
down-regulated. This is followed by a reduction of EGR-1 and a
concomitant increase of NAB2. Secondly, using the tissue factor gene as
a readout for VEGF-induced and EGR-1-regulated gene expression we
demonstrate that NAB2 can completely block VEGF-induced tissue factor
reporter gene activity. Thirdly, by adenovirus-mediated expression we
show that NAB2 inhibits up-regulation of tissue factor, VEGF
receptor-1, and urokinase plasminogen activator mRNAs even when a
combination of VEGF and bFGF is used for induction. In addition, NAB2
overexpression significantly reduced tubule and sprout formation in two
different in vitro angiogenesis assays and largely
prevented the invasion of cells and formation of vessel-like
structures in the murine Matrigel model. These data suggest that NAB2
regulation represents a mechanism to guarantee transient EGR-1 activity
following exposure of endothelial cells to VEGF and that NAB2
overexpression could be used to inhibit signals involved in the early
phase of angiogenesis.
Vascular endothelial growth factor,
VEGF,1 has a predominant role
in vasculogenesis as well as in physiological and pathological angiogenesis (1-3). Major signals induced by VEGF via VEGFR-2 in
endothelial cells include activation of the phosphoinositol 3-kinase/PKB and phospholipase C- Comparable to some other transactivators, EGR-1 associates with
corepressor proteins that can modulate transcription of
EGR-dependent genes. Two corepressors of EGR-1, NAB1 and
NAB2, have been identified using yeast two-hybrid screening (15, 16).
These factors bind to EGR-1 by direct protein-protein interactions with
a conserved R1 region found in several members of the EGR family
(EGR-1, -2 and -3), thus inhibiting the transactivating potential of
EGR-1. Whereas NAB1 is constitutively expressed in most tissues and
appears to be a general transcriptional regulator (15), NAB2 may
function as an important inducible regulator of gene expression (16). Initially, the physiologic activities of NAB2 have been analyzed in
nerve cells where the EGR-1-mediated differentiation process stimulated
by nerve growth factor was blocked by the corepressor NAB2 (9).
Furthermore, an inhibition of EGR-1-dependent transcription and growth factor production in smooth muscle cells with implications for tissue repair and angiogenesis was recently reported (17, 18). In
general, gene regulation mediated by the interplay of EGR-1 and NAB2
might be a unifying principle in different invasive processes such as
neurite outgrowth, wound healing, angiogenesis, and tumor invasion
(19).
Based on our previous finding that the transcription factor EGR-1 is
decisively involved in the up-regulation of the TF gene by VEGF in
endothelial cells (7, 8), we have here analyzed to what extent NAB2 can
down-modulate expression of several different genes induced by
angiogenic growth factors and thus plays a direct role in the control
of angiogenesis-related responses of endothelial cells. In this respect
NAB2 gene transfer to endothelial cells by recombinant adenoviruses was
further evaluated as a potential approach to inhibit angiogenesis. Our
results show that NAB2 can strongly inhibit VEGF- and bFGF-induced
expression of the TF, VEGFR-1, and uPA genes. Furthermore, the
adenovirus-mediated overexpression of NAB2 led to significant
inhibition of migration, sprouting, and tubule formation in
angiogenesis models in vitro and in vivo without
obvious cytotoxic side effects.
Cell Culture and Materials--
Human umbilical vein endothelial
cells (HUVEC) and human uterine microvascular endothelial cells (HUMEC)
were isolated and cultured in medium 199 with 20% SCS or a 1:1 mixture
of SCS and fetal calf serum (HyClone, Logan, UT), respectively,
supplemented with 1 unit/ml heparin, 50 µg/ml endothelial cell
growth supplement (Technoclone, Vienna, Austria), 2 mM
glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin as
described in more detail in Refs. 8 and 20. Human lung microvascular
cells (HLMEC) were obtained from Bio-Whittaker (Walkersville, MD) and
cultured as described in the protocol provided. Short-starved cells
were obtained by starving with 1% serum for 5 h. Recombinant
human VEGF165 and bFGF was obtained from PromoCell
(Heidelberg, Germany). Polyclonal anti-EGR-1 and anti-Sp1 antibodies
were from Santa Cruz Biotechnology (Santa Cruz, CA) and polyclonal
anti-GFP antibodies were from New England Biolabs (Beverly, MA).
Monoclonal anti-NAB2 antibodies 1C4 (21) were a gift of Dr. Judith
Johnson (Institute of Immunology, University of Munich, Munich,
Germany). Peroxidase-conjugated donkey anti-rabbit immunoglobulin G
(IgG) and sheep anti-mouse IgG were purchased from Amersham
Biosciences), and goat anti-rat IgG was from Serotec (Oxford, UK).
Real-time PCR--
RNA was extracted from endothelial cells with
TRIzol Reagent (Invitrogen). 2 µg of total RNA was
reverse-transcribed using SuperScriptTM II enzyme using
oligo-dT primers as specified by Invitrogen. Real-time PCR including
SYBR Green PCR reagent was performed on a Light CyclerTM
instrument (Hoffmann-La Roche) according to instructions provided by
the manufacturer (22). Oligonucleotides used were TF-forward: ccgaacagttaaccggaaga, TF-reverse: tcagtggggagttctccttc; EGR-1-forward: cagcaccttcaaccctcag, EGR-1-reverse: cacaaggtgttgccactgtt; NAB2-forward: acatcctgcagcagacactg, NAB2-reverse: ctccactttcacgctgctc;
VEGFR-1-forward: tgctcagctgtctgcttctc, VEGFR-1-reverse:
ccatttcaggcaaagaccat; and uPA-forward: tgaggtggaaaacctcatcc,
uPA-reverse: ggcaggcagatggtctgtat.
Western Blot Analysis--
Cells were washed twice with PBS,
lysed in 100 µl of Laemmli buffer, scraped, and heated for 5 min at
95 °C. Total cell lysates were separated by SDS-PAGE and transferred
to Immobilon-P membrane (Millipore, Bedford, MA). The membrane was
blocked for 30 min with PBS containing 0.1% Tween 20 and 3% skim milk
and incubated for 1 h at room temperature with the primary
antibody diluted in blocking buffer. Then the membrane was washed three
times for 5 min with PBS containing 0.1% Tween 20 and incubated with
peroxidase-conjugated secondary antibodies for 1 h at room
temperature. After a washing step, the membrane was incubated for 1 min
with ECL reagent (Amersham Biosciences) and exposed to film. For
reprobing with another antibody, the membrane was washed twice in PBS,
stripped for 30 min at 55 °C with stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM
2-mercaptoethanol), and washed three times for 5 min with PBS at room temperature.
Transient Transfections--
The TF reporter gene construct
containing the TF promoter from Recombinant Adenoviral Constructs and
Infection--
Construction of recombinant adenoviruses was done as
previously described (25, 26). NAB2 and NAB2.AS cDNAs (16) were first subcloned into the XmaI site of the pBluescript II SK
+/
For infection HUVEC were incubated in PBS complete for 30 min at a
m.o.i. of 100. Thereafter, cells were washed and cultured in normal
medium 199.
In Vitro Angiogenesis Assays--
The formation of capillary
tube-like structures by HUVEC was analyzed on tumor-derived
extracellular membrane matrix (Matrigel; Becton Dickinson, Franklin
Lakes, NJ). 48-well culture dishes (Costar, Cambridge, MA) were coated
with 100 µl/well Matrigel, and the gel was allowed to solidify. Cells
were starved for 4 h in medium 199 with 1% SCS, seeded on the
polymerized Matrigel (3 × 104 cells/well), further
incubated for 16 h, and then fixed in PBS containing 3%
formaldehyde and 2% sucrose (29). Images of the network formed were
taken on a phase contrast microscope (Nikon Diaphot TMD) using a cooled
charge-coupled device camera (Kappa DX30, Kappa GmbH, Gleichen,
Germany). The total length of the tube-like structures formed was then
determined with the help of the Analysis Software (Softimaging System,
Munster, Germany).
An endothelial sprouting angiogenesis assay was performed using HLMEC
or HUMEC according to a modification of the method used by Nehls
et al. (30). Briefly, microcarrier beads coated with denatured collagen (Cytodex 3; Sigma) were seeded with the infected cells, the cells were grown overnight on the beads in medium 199, and
the beads then embedded in fibrin gels in 12-well plates (Costar). To
prepare the fibrin gel, human fibrinogen (Sigma) was dissolved in PBS
complete at a concentration of 2 mg/ml and aprotinin (Bayer, Leverkusen, Germany) was added at a concentration of 200 KIU/ml. The
fibrinogen solution was then supplemented with 50 ng/ml VEGF. The
solutions were transferred to 12-well plates together with the beads
covered by cells at a density of about 200 beads/well, and fibrin
formation was induced by addition of 1.2 units/ml thrombin (Sigma).
Fibrin gels were equilibrated with serum-free medium containing
aprotinin (200 KIU/ml) for 1 h and then incubated with M199 medium
supplemented with 20% FCS and growth factors as indicated. After 1 to
2 days the number of capillary-like sprouts formed was counted in the
microscope. Only sprouts with a minimal length of ~150 µm were
counted. To visualize cell nuclei, cells were fixed with 3.7%
formaldehyde and 2% sucrose in PBS and permeabilized with 0.5% Triton
X-100 in PBS. Then Hoechst 333258 was added to the cells at 500 ng/ml
for 30 min. The cytoskeleton was stained with rhodamine-phalloidin
(Molecular Probes, Eugene, OR) for 1 h in the dark. Cells were
analyzed by phase contrast microscopy and images taken using a CCD camera.
In Vivo Matrigel Assay--
Matrigel solution (BD Biosciences)
was supplemented with 1.5 × 108 pfu/ml of recombinant
adenoviruses and 300 ng/ml VEGF and injected subcutaneously into the
flank of C57BL/6 mice (31). On day 6 post injection the mice were
sacrificed, and the Matrigel plug was removed and embedded in paraffin.
Freeze sections of the plug were prepared and stained with hematoxylin
(Merck), DAPI (Vector Laboratories, Burlingame, CA), or rat anti-mouse
CD31 antibodies (Oxford, UK) as described in Ref. 31. Pictures were
taken on an AX-70 Olympus microscope (Olympus Optical Co.) using an
Optronics DEI-750D CCD camera (Optronics, Muskogee, OK). The number of
cells in the Matrigel plugs were quantitated on pictures displaying largely complete sections of the plugs, which were established from
serial images taken from the individual sections. The circular images
of the sections were divided into 10-degree segments, and the number of
the cells within six segments were counted for each section.
Regulation of EGR-1 and NAB2 Expression by VEGF--
The
transcription factor EGR-1 has been previously shown by us to be
induced by VEGF in endothelial cells with the kinetics typical for an
immediate-early gene product showing maximal levels ~60 min after
stimulation (7). Here we tested the regulation of NAB2 mRNA and
protein expression in endothelial cells following VEGF treatment in
comparison to EGR-1. HUVEC were short-starved for 5 h in
medium containing 1% serum and then treated with VEGF (1.25 nM) for various time periods up to 6 h, and the amount
of EGR-1 and NAB2 mRNA and protein was determined by real-time
reverse transcription-PCR and Western blotting, respectively. A
transient induction of EGR-1 mRNA was observed with maximal
expression at about 30 min (Fig.
1A), which was followed by a
transient increase in NAB2 mRNA displaying highest values at 120 min. EGR-1 protein levels decreased during starvation (data not shown),
were very low in 5 h serum-starved endothelial cells, and reached
maximal values 60 min after VEGF treatment (Fig. 1B). NAB2
levels also decreased during starvation reaching a constant basal level
within 5 h. In contrast to EGR-1, VEGF treatment resulted first in
a further 4- to 5-fold decrease of NAB2 within 60 min, the time period
when EGR-1 levels reached highest values. Thereafter, concomitant with
a decrease in EGR-1, NAB2 protein increased again about 10-fold reaching levels at least 2- to 3-fold over initial values. Thus, NAB2
and EGR-1 expression are regulated in a reciprocal way by VEGF. In
accordance with important roles of EGR-1 and NAB2 for TF gene
transcription, TF mRNA levels reached highest levels at 60 min
(Fig. 1C) at a time period when maximal EGR-1 and lowest NAB2 levels were observed.
VEGF-mediated Induction of the TF Promoter Is Blocked by
NAB2--
We have previously shown (7, 8) that EGR-1 plays an
essential role in the activation of the TF gene by VEGF. Therefore, overexpression of NAB2 was tested for its ability to repress
VEGF-induced TF promoter activity in reporter gene assays in comparison
to EGR-1-triggered activation. Indeed, overexpression of NAB2 resulted in a dose-dependent complete inhibition of VEGF-induced TF
promoter activity (Fig. 2), which was
comparable to the inhibition of EGR-1-triggered promoter activity.
NAB2.AS, a truncated variant of NAB2, which lacks the C-terminal part
interacting with EGR-1 (16) and does not localize to the
nucleus,2 did not block VEGF-
and EGR-1-mediated transactivation. These results demonstrate that the
transcriptional corepressor NAB2 is able to block VEGF-induced gene
regulation mediated by EGR-1 in endothelial cells.
NAB2 Overexpression by Recombinant Adenoviruses Inhibits Inducible
Expression of TF, VEGFR-1, and uPA mRNAs--
To test the effect
of overexpression of NAB2 on VEGF-inducible responses of endothelial
cells that are linked to and regulated by EGR-1, we constructed a
recombinant adenovirus expressing NAB2 and infected human endothelial
cells with 107 pfu/105 cells. Infected HUVEC
showed increasing levels of NAB2 expression from day 1 to day 3 following infection that persisted for over 7 days (Fig.
3). These expression levels were
significantly higher than physiological levels in endothelial cells as
indicated by the fact that the endogenous NAB2 is not visible on images
of short Western blot exposures displaying strong bands of
adenovirus-expressed NAB2.
We first evaluated the effect of adenovirus-mediated NAB2 expression on
the induction of mRNAs for TF, uPA, and VEGFR-1 by VEGF (data not
shown) and a combination of VEGF and bFGF (Fig. 4). uPA and VEGFR-1 were chosen in
addition to TF since EGR-1 has been reported to be involved in the
up-regulation of the respective genes (12, 13). Furthermore, several
lines of evidence support an important role of uPA for migration and
invasion and of VEGFR-1 for pathological angiogenesis, respectively (2,
14). A combination of VEGF and bFGF was tested because both factors
induce EGR-1, have been described to be present in the Matrigel
preparations used for the angiogenesis assays described below, and
contribute to tumor angiogenesis in vivo (2, 32). When
endothelial cells were infected with the NAB2-expressing virus and
induced with the growth factors 2 days thereafter, the normally
observed induction of all three mRNAs was inhibited to a large
degree (Fig. 4). This shows that by blocking EGR-1 activity NAB2 can
inhibit the expression of several genes induced by angiogenic growth
factors and involved in angiogenesis.
Effects of NAB2 Overexpression in the in Vitro Angiogenesis
Models--
Next we have tested whether this inhibition of gene
up-regulation by NAB2 would have consequences for the cellular
angiogenic responses of endothelial cells in two different in
vitro angiogenesis assays. In one assay we have evaluated the
potential of Ad.NAB2 to inhibit migration and tubular network formation
after plating of endothelial cells on Matrigel. In the second assay the
capacity of endothelial cells to form sprouts and migrate into fibrin
gels was investigated. In both cases cells infected with Ad.NAB2 were compared with cells infected with control virus for 24 h.
Infection with Ad.NAB2 reduced by 52 ± 14% the tubular network
established by HUVEC 16 h after seeding on Matrigel (Fig.
5). Parallel cultures seeded on
gelatin-coated plates did not display any cytotoxicity caused by the
virus infection (data not shown). For the sprouting assay HLMEC or
HUMEC were seeded on microcarrier beads, and the beads incorporated
into fibrin gels. In this assay the sprouting of the microvascular
endothelial cells from the beads into the fibrin gel was dependent to a
large degree on the presence of VEGF in the medium (Fig.
6). Individual sprouts contained usually between 1 to 3 cells in a string and displayed thin protrusions into
the fibrin gel. Also in this assay the capacity of the VEGF-induced cells to form sprouts and to migrate into the fibrin gel was
significantly reduced (45 ± 18%, Fig. 6B).
Effects of NAB2 Overexpression in the in Vivo Matrigel
Model--
Finally, we have evaluated to which degree
Ad.NAB2 would inhibit the invasion of cells and the formation of
vessel-like structures in the murine Matrigel model in vivo
(31). For this purpose GFP- or NAB2-expressing adenoviruses were mixed
into Matrigel solution without or supplemented with VEGF. These
mixtures were injected subcutaneously into mice and analyzed 6 days
thereafter. We have first tested whether adenoviruses would efficiently
infect cells invading the Matrigel plug by staining sections of Ad.GFP- and VEGF-containing plugs with anti-GFP antibodies and DAPI. By comparing the number of GFP-expressing cells inside the plug (Fig. 7, picture 1) with the number
of nuclei stained with DAPI (Fig. 7, picture 2), it was
evident that the cells inside the Matrigel plug were almost completely
infected. This is best displayed in the overlay of both stainings in
Fig. 7, picture 3. Even the multiple cell
layers that had formed around the Matrigel plug were infected to a
large degree. Then we analyzed the total number of cells in the
sections by hematoxylin staining and displayed endothelial cells by
staining with anti-CD31. As seen in the hematoxylin staining of
sections (Fig. 8A), a high
number of cells invaded the Matrigel plug when VEGF was present,
whereas only few cells could be detected in the Matrigel without added
VEGF. In both cases, without and in the presence of added VEGF,
multiple cell layers had formed on the outside of the Matrigel plug. A
fraction of the invading cells were endothelial cells as shown by
staining with anti-CD31 antibodies (Fig. 8B). Some of these
endothelial cells formed strings, and several vessel-like structures
were observed. Significant numbers of invading endothelial cells in the
plug were again only observed in Matrigel supplemented with VEGF. A
quantification of cells in the Matrigel plugs demonstrated that the
presence of Ad.NAB2 in the Matrigel reduced the number of total cells
in sections of the plugs supplemented with VEGF to the low level seen
in sections of plugs without added VEGF (Fig. 8C).
Furthermore, no vessel-like structures formed by endothelial cells
could be observed inside the Matrigel in Ad.NAB2-containing plugs.
These inhibitory effects observed for Ad.NAB2 support an important role of EGR-1-mediated gene regulation for the in vivo
processes.
VEGF is the most critical driver of vascular formation since it is
required to initiate the formation of vessels by vasculogenesis and
angiogenic sprouting (1, 2, 33, 34). It can trigger several different
cellular responses involved in angiogenesis such as proliferation,
survival, sprouting, migration, and increased vascular permeability.
These responses are likely linked to distinct, although partially
overlapping, signaling pathways connected to various gene expression
programs. VEGF can bind to two different receptors on endothelial
cells, VEGFR-1/Flt-1 and VEGFR-2/Flk-1, but primarily VEGFR-2 seems to
mediate the initial responses leading to angiogenesis and increased
permeability (35). Over the last years, signal transduction pathways
activated by VEGF via VEGFR-2 have been studied intensively. Important
signals induced by VEGF include phospholipase C- An important association with growth and development has been suggested
by several reports for EGR-1 (9, 10). Furthermore, a number of data
support a major role of EGR-1 in the acute response to various kinds of
stress such as physical and ischemic injury (39). EGR-1 was suggested
to contribute to restenosis and atherosclerotic disease through the
up-regulation of a number of pathophysiologically relevant genes
including PDGF A/B chains, TNF- According to our previous data, EGR-1 is prominently induced in
endothelial cells by VEGF and this induction is mediated by the
PKC/MEK/ERK cascade and followed by the up-regulation of the TF gene
(7, 8). Since EGR-1-mediated transcription is negatively controlled by
a corepressor, NAB2 (16), we analyzed NAB2 expression in endothelial
cells. The obtained data support an inhibitory role of NAB2 for
VEGF-induced transcription. However, whereas in nerve PC12 cells
stimulated by nerve growth factor (9) or in bovine aortic smooth
muscle cells induced by phorbol 12-myristate 13-acetate (17) NAB2
protein levels were initially very low and peaked at 2 h after
exposure to stimuli; in endothelial cells significant basal NAB2 levels
were present and declined rapidly following serum starvation.
Furthermore, VEGF-regulation included a further down-regulation phase
that was followed by an up-regulation beginning 60 min after VEGF
exposure. These kinetics suggest that the down-modulation of NAB2
allows full EGR-1 activity and that the following up-regulation phase
ensures that EGR-1 activity will be transient and turned off again.
We have previously shown that TF is an example of a protein that is
induced by VEGF mainly via EGR-1 (7). TF has been proposed to possess,
in addition to its role as initiator of the coagulation cascade,
important functions for cell adhesion and signaling (41) and has been
reported to be expressed on tumor endothelium (42). Although the
potential contribution of endothelial TF expression to angiogenesis has
so far not been clarified, induced TF expression can be used as a
readout for modulations on the level of transcriptional activity
mediated by EGR-1. Therefore we have tested whether overexpression of
NAB2 can modulate VEGF-triggered TF promoter activity. The results show
that NAB2 is able to completely inhibit TF reporter gene activity
triggered by VEGF. Since the used This is in line with findings indicating an important function of EGR-1
in the regulation of growth and differentiation by growth factors.
PKC-dependent EGR-1 activation has been implicated in some
other cell types in differentiation processes. Examples are the
nerve growth factor-induced differentiation of nerve cells and
the IL-3 and GM-CSF-triggered differentiation of hematopoietic progenitor cells (9, 10). In regard to angiogenesis, it is possible
that sprouting and tube formation starting from mature endothelial
cells include regulatory mechanisms similar to angioblast differentiation (2), and there is further evidence that the recruitment
of angioblasts from the circulation and their differentiation plays a
role for vessel formation in the adult. VEGF-induced and EGR-1-mediated
gene transcription could be one of the mechanisms important for both processes.
However, the absence of an abnormal vascular phenotype in unchallenged
EGR-1 null mice (45) suggests that efficient back-up mechanisms are
available or get activated in knock-out animals. In this respect a role
for EGR-2 and EGR-3 appears possible. Since the regulatory R1 domain,
which is involved in the binding of NAB2, is also present in EGR-2 and
EGR-3, we have tested expression of EGR-2 in HUVEC, but it was almost
undetectable by Western blotting and not inducible by VEGF. In the case
of EGR-3 significant levels were detected but did not change following
VEGF treatment.2 Therefore it appears that
at least in cell culture assays using normal endothelial cells mainly
EGR-1 can be considered as a mediator of VEGF-induced gene transcription.
To further substantiate the role of EGR-1 and NAB2 for angiogenic
responses of endothelial cells, we have prepared adenoviruses overexpressing NAB2. In line with a more general role of EGR-1 for
VEGF-regulated genes Ad.NAB2 strongly inhibited, in addition to the
induction of TF mRNA, also the up-regulation of mRNAs for VEGFR-1 and uPA. Both genes have previously been described to contain
functional EGR-1 binding sites in their promoters (12, 13) and to
fulfill important roles during angiogenesis. Although VEGFR-1 has been
proposed initially to function mainly as an inert decoy by regulating
availability of VEGF, more recent data demonstrate an important
contribution to angiogenesis (46). The responsiveness of endothelial
cells to VEGF is amplified by up-regulating PlGF and VEGFR-1 in many
pathological disorders. The uPA/uPAR system contributes to cell
migration through the activation of signaling pathways, extracellular
proteolysis, cell adhesion, and chemotaxis (14). Pericellular
proteolysis and plasmin generation is an important component of the
ability of the endothelial cell to cleave linkages to the extracellular
matrix and other cells, to degrade basement membrane barriers, and thus
to invade surrounding tissue and fibrin clots. As such Flt-1 and uPA
are likely contributors to the migration and invasion of endothelial
cells in the angiogenesis models used in this study. By inhibiting the
up-regulation of at least three genes with importance for processes
essential for pathological angiogenesis, NAB2 seems to fulfill the
criteria for a key negative regulator of angiogenesis.
The inhibition of VEGFR-1 and uPA expression was observed even when a
combination of VEGF and bFGF was used. Similar to VEGF, bFGF has
previously been described to be a strong inducer of EGR-1 (32). We have
used bFGF in addition to VEGF in certain experiments since this factor
is a known potent inducer of endothelial cell proliferation, migration,
and angiogenesis in vitro and in vivo (47) and
contributes to tumor angiogenesis (48). Inhibition of VEGF-and
bFGF-triggered induction indicates a broad activity of NAB2
irrespective of the angiogenic inducer.
In line with an inhibition of genes and processes important for
angiogenesis, infection with Ad.NAB2 inhibited tubule formation on
Matrigel and sprouting in fibrin gels. Whereas tubule formation on
Matrigel is observed without added growth factors, since several growth
factors including bFGF are present in the Matrigel, sprouting of
endothelial cells in fibrin gels was dependent on the addition of
growth factors. Our data show that AdNAB2 inhibited the number of
sprouts specifically induced by VEGF. These observations suggest a
significant contribution of EGR-1 to the angiogenic response of
endothelial cells.
Finally, we have evaluated the NAB2 adenovirus in the
murine Matrigel model, which is a widely used model to evaluate
migration, invasion, and formation of vessel-like structures in
vivo (31, 49). This model evaluates the more complex effects of
NAB2 expression in several cell types. In accordance with an important
role of transcriptional processes regulated by EGR-1 and NAB2 for
invasion and angiogenesis in vivo, we observed that the
invasion of cells was strongly inhibited when Ad.NAB2 was included in
the Matrigel, and the occurrence of vessel-like structures, as observed
in control VEGF-containing plugs, was largely prevented. Generally the
system is characterized by the formation of a fibroblast cell-like wall around the plug. A massive invasion of cells into the plug is observed
only when the Matrigel is supplemented with growth factors. Part of the
invading cells are endothelial cells, which build rod-like and
unordered vessel-like structures as has been previously described (31).
The cell invasion is likely triggered indirectly by VEGF-stimulating
endothelial and possibly monocytic cells to produce additional factors
with activities on several cell types and by direct stimulation of
migration of endothelial cells. In addition, VEGF diffusing out of the
gel may induce endothelial cells of the vessels in the outer cell wall
to become leaky and to produce uPA and proteases facilitating cellular
invasion. The inhibition observed in vivo was more strongly
pronounced than the effects in the in vitro models
supporting that NAB2 exerts its effects on several mechanisms in
different cell types. Although we can not exclude that NAB2 might
interact with and also inhibit other transcription factors, these data
support the view that the EGR-1 pathway contributes at multiple steps
to invasion and angiogenesis.
One of these steps could be, as previously reported, the
EGR-1-dependent production of angiogenic growth factors by
smooth muscle cells, which can be inhibited by NAB2 (18). Whereas our work is the first report to show a direct inhibition of early angiogenic mechanisms of endothelial cells in vitro, it is
likely that interference at several levels contribute to inhibition
in vivo. In summary, our results suggest that modulation of
EGR-1 activity by NAB2 is important for physiological and pathological angiogenesis and, based on the preliminary observation that NAB2 may
have little pathological consequences, could be a promising modality
for gene therapies of diseases with excess angiogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/PKC pathways (4, 5). Whereas
activation of PKB has been primarily implicated in cell survival (6),
recent in vitro studies have shown that VEGF treatment of
endothelial cells leads to a PKC-dependent activation of
the MEK/ERK module of MAP kinases resulting in a rapid up-regulation of
the transcription factor EGR-1 (7, 8), which has been associated with growth and differentiation of various cell types (9,
10). Furthermore, EGR-1 is critically involved in the up-regulation of
genes such as tissue factor (TF) (7, 8), VEGF receptor-1 (VEGFR-1) (11,
12), and urokinase-type plasminogen activator (uPA) (13). These genes
have been proposed to fulfill important functions for different aspects
of vasculogenesis and angiogenesis (1, 2, 14).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
330 to +118 bp in a luciferase
expression vector was previously described (7, 23). The coding region
of the human EGR-1 gene including a single intron was obtained from the
PAC clone E13873Q3 (library number 704, RZPD, Berlin, Germany) by PCR
amplification. The resulting 2.3-kb DNA product was subcloned into
HindIII-EcoRI-digested pACCMVplpASR+ vector (24).
The expression constructs for the human full-length NAB2 (pCMVNAB2) and
the alternatively spliced NAB2.AS (pCMVNAB2.AS) (16) were kindly
provided by J. Milbrandt and J. Svaren (Departments of Pathology and
Internal Medicine, Washington University, St. Louis, MO). Transient
transfections of HUVEC were carried out by using the LipofectAMINE
PLUSTM reagent (Invitrogen) as previously described (8). 24 h
prior to transfection, HUVEC were seeded in six-well tissue culture
plates to reach 70-90% confluency the next morning. Cells were
incubated with transfection mixtures containing a total of 1.5 µg of
DNA (0.5 µg of TF promoter/luciferase reporter, 0.5 µg of a
CMV-
-galactosidase construct as internal control and various amounts
of NAB2, NAB2.AS, and EGR-1 expression plasmids or empty control
vector), 6 µl of PLUS reagent, and 4 µl of LipofectAMINE in a total
volume of 1 ml of medium 199 per well for 2 h. All experimental
values were determined from triplicate wells.
vector and then transferred to the BamHI site of the
vector pACCMVpLpASR+ (24). The obtained constructs were verified by
sequencing and cotransfected with pJM17, a plasmid containing the
adenoviral genome with a deletion in the E1 region (27), into 293 cells. Clones were tested for protein expression by Western blots.
Purification of large batches of the recombinant adenoviruses was done
by two consecutive cesium chloride centrifugations (28). Adenoviruses without cDNA inserts and viruses expressing GFP (26) were grown in
parallel and used as controls.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
EGR-1 and NAB2 expression is regulated by
VEGF in endothelial cells. A, real-time PCR analysis of
EGR-1 and NAB2 mRNA. RNA was isolated from 5-h short-starved
unstimulated cells ("0" time point) and cells exposed for 30-300
min to VEGF (1.25 nM). Real-time PCR was performed as
described under "Materials and Methods." Data are displayed as fold
induction of the value obtained with RNA from unstimulated cells.
B, Western blot analysis of total cell extracts from
short-starved unstimulated cells and short-starved cells exposed to
VEGF (1.25 nM). Samples were harvested, and the proteins in
the lysates separated by SDS-PAGE and subjected to Western blot
analysis. Membranes were probed with anti-NAB2 antibodies followed by
reprobing with anti-EGR-1 antibodies. An arrow indicates a
nonspecific (ns) band displaying equal loading of the
samples. NAB2 protein levels in unstarved cells (us) are
compared with short-starved cells (ss) in the right part of
the panel. C, real-time PCR analysis of TF mRNA. The RNA
samples described under A were tested for TF mRNA.
Results representative for two (A and C) or three
separate experiments (B) are shown.
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Fig. 2.
Activation of the TF promoter by VEGF and
EGR-1 is equally inhibited by NAB2. HUVEC were cotransfected with
a TF promoter/luciferase reporter gene, a CMV
promoter/ -galactosidase construct and increasing amounts of NAB2 or
NAB2.AS (1.6 and 3.2 µg, respectively) expression vectors as
indicated. 20-h post transfection cells were treated with VEGF for an
additional 6 h, harvested, and analyzed. When indicated, an EGR-1
expression plasmid (1.6 µg) was cotransfected instead of VEGF
induction. Results are displayed as relative light units
(RLU) luciferase normalized to
-galactosidase
concentration. Results are shown as mean values ± S.D. Data are
representative of three independent experiments performed with
triplicate wells.
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Fig. 3.
Kinetics of adenovirus-mediated
overexpression of NAB2. A recombinant adenovirus expressing NAB2
cDNA under the control of the CMV promoter was produced and used to
infect HUVEC at a m.o.i. of 100. Cells were harvested 1-3 days post
infection, and cellular lysates subjected to Western blotting using
anti-NAB2 antibodies. The same blot was subsequently reprobed with
anti-Sp1 antibodies to ensure equal loading of samples.
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Fig. 4.
Adenovirus-mediated overexpression of NAB2
inhibits induction of TF, VEGFR-1, and uPA mRNAs. HUMEC cells
were infected with control virus or the NAB2 expressing adenovirus at a
m.o.i. of 100. Two days following infection cells were treated with a
combination of VEGF (50 ng/ml) and bFGF (50 ng/ml) for 1-3 h, and
total RNA was isolated and used for real-time PCR analysis.
Oligonucleotides specific for TF (A), VEGFR-1 (B) and uPA mRNA
(C) as given under "Materials and Methods" were used.
Results are displayed as fold induction of the corresponding values
obtained with uninduced samples. The figure displays an experiment
representative of two performed with duplicate values.
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Fig. 5.
NAB2 overexpression inhibits tubule formation
on Matrigel. HUVEC cultures were infected with Ad.NAB2 or control
virus. 24 h following infection cells were short-starved for
5 h in 4% serum-containing medium and then trypsinized, and
samples were seeded in parallel on Matrigel in 48-well tissue culture
plates to analyze tubule formation. Cells were photographed using a CCD
camera 16 h after seeding, and the total length of the tubule-like
network formed in the well was established as described under
"Materials and Methods." A, displays the strong
inhibition of tubule formation by Ad.NAB2 in a representative
experiment. B, shows a quantitation of the reduction of
tube-length obtained with Ad.NAB2 in comparison to cells infected with
control virus. Results are displayed as mean values ± S.D.
obtained from three independent experiments performed with duplicate
wells.
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Fig. 6.
Overexpression of NAB2 reduces
sprouting in fibrin gels. Cultures of HLMEC or HUMEC were infected
with Ad.NAB2 or control virus. After 24 h cells were trypsinized,
seeded onto microcarrier beads and further cultivated overnight. On the
following day the microcarrier beads covered by a dense monolayer of
cells were incorporated into fibrin gels in 24-well tissue culture
plates and incubated with growth medium without added growth factors or
supplemented with VEGF. 24 to 48 h after incorporation into fibrin
gels and incubation with the growth factor cells migrated and formed
sprouts into the fibrin gel. A, shows an example of sprouts
formed by HLMEC in the presence of VEGF. The cytoskeleton of the cells
and the nuclei were stained with rhodamine-phalloidin and Hoechst
333258, respectively. An overlay of the stained cytoskeleton
(red) and nuclei (blue) is displayed. Typically
between 0.5 and 3 sprouts per bead were observed for growth
factor-treated cells. B, shows the analysis of the
inhibition in the number of sprouts observed in VEGF-containing culture
when Ad.NAB2 infected cells were used in comparison to control
virus-infected cells. The mean value ± S.D. calculated from three
independent experiments performed with duplicate wells is given.
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Fig. 7.
Efficient infection of cells in the
murine Matrigel assay. GFP adenoviruses (1.5 × 108 pfu/ml) and VEGF (300 ng/ml) were added to the Matrigel
solution and injected into the abdominal subcutaneous tissue of C57BL/6
mice. On day 6 plugs were excised and freeze sections prepared.
Staining of the sections was performed with GFP antibodies
(picture 1) or DAPI to reveal cell nuclei (picture
2). Picture 3 displays an overlay of the staining with
GFP antibodies and DAPI confirming that a majority of the cells was
infected. The left upper parts of the pictures correspond to
the cell layers formed around the Matrigel plug from which cells invade
toward the center of the plug located in the direction of the
right lower corner.
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Fig. 8.
The NAB2-expressing adenovirus
inhibits invasion and tubule formation by endothelial cells in the
murine Matrigel assay. GFP- and NAB2-expressing adenoviruses
(1.5 × 108 pfu/ml) were added to Matrigel solution
containing VEGF (300 ng/ml) or without added growth factor and injected
subcutaneously into mice. After 6 days plugs were removed and processed
to prepare freeze sections as described under "Materials and
Methods." Individual sections were either stained with hematoxylin
(A) to detect total invading cells or with anti-CD31 and
DAPI (B) to visualize endothelial cells and total number of cell
nuclei, respectively. Overlays of the CD31 staining (green)
and DAPI staining (blue) are shown. Pictures 1 of
A and B display sections of a Matrigel plug
containing Ad.GFP without VEGF, pictures 2 of A
and B containing Ad.GFP and VEGF and pictures
3 containing Ad.NAB2 and VEGF. The cell layers formed around
the plug are on the left side, the center of the plug is
located toward the right or right upper side of
the pictures. Significant invasion of endothelial cells is only seen in
VEGF and Ad.GFP containing Matrigel. Picture 2 of
B displays an area with a high number of invaded cells close
to the outer cell layer on the left border. C,
total cells in sections of the Matrigel plugs were quantitated as
described under "Materials and Methods." Results obtained from two
experiments with duplicate plugs are displayed as mean values ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, MAP kinases,
phosphoinositol 3-kinase, Akt/protein kinase B, and focal adhesion
kinase (4, 36-38). However, the key transcription factors and
regulatory genes activated by these signals have not yet been
unequivocally identified. In this context, we evaluated the
physiological role of EGR-1 and of NAB2, a specific corepressor of
EGR-1, in VEGF-triggered responses in endothelial cells.
, TGF-
, and TF and was found to be
itself induced by factors such as bFGF (32, 40).
330-bp TF promoter fragment
includes binding sites for NF
B, NFAT, AP-1, and Sp1 transcription factors (23, 43, 44), these data underline the importance
of the EGR-1/NAB2 balance for VEGF-mediated TF reporter gene induction.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Jeffrey Milbrandt and John Svaren for a gift of NAB2 and NAB2.AS expression constructs. We thank Judith Johnson for providing anti-NAB2 antibodies and Rainer deMartin for GFP-expressing adenoviruses. We also thank our colleagues from the Department of Vascular Biology for helpful discussions and critical comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by Austrian Science Fund Grant SFB05-10, the Interdisciplinary Cooperation Project Molecular Medicine Program of the Austrian Federal Ministry for Education, Science and Culture, and the 5th Framework Program of the European Commission Grant QLK3-CT-2002-02059.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Novartis Research Institute, Brunnerstrasse 59, A-1235 Vienna, Austria.
To whom correspondence should be addressed. Tel.:
0043-1-4277-62553; Fax: 0043-1-4277-62550; E-mail:
erhard.hofer@univie.ac.at.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M204937200
2 D. Mechtcheriakova, M. Lucerna, and E. Hofer, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: VEGF, vascular endothelial growth factor; PKB, protein kinase B; PKC, protein kinase C; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; TF, tissue factor; uPA, urokinase-type plasminogen activator; EGR, early growth response protein; HUVEC, human umbilical vein endothelial cells; HUMEC, human uterine microvascular endothelial cells; SCS, supplemented calf serum; HLMEC, human lung microvascular endothelial cells; GFP, green fluorescent protein; PBS, phosphate-buffered saline; CMV, cytomegalovirus; KIU, kallikrein-inactivating unit; pfu, plaque-forming unit; CCD, charge-coupled device; m.o.i., multiplicity of infection; DAPI, 4',6-diamidino-2-phenylindole.
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