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INTRODUCTION |
The formation of blood vessels requires a series of events,
including differentiation of endothelial cells, tube formation, and
vascular maturation (1). Two processes termed vasculogenesis and
angiogenesis take place during the formation of a mature vascular network (2-4). Previous studies have revealed some of the molecular mechanisms involved, and two families of largely endothelial
cell-specific receptor tyrosine kinases are known to play crucial roles
in these processes. The vascular endothelial growth factor receptor
(VEGFR)1 family is composed
of Flt-1 (fms-like
tyrosine kinase-1; VEGFR-1) (5), Flk-1/KDR
(fetal liver
kinase/kinase domain-containing receptor; VEGFR-2) (6), and Flt-4
(fms-like
tyrosine kinase-4; VEGFR-3) (7). Targeted gene
disruption for Flk-1 in embryonic mice leads to loss of endothelial
cells and embryonic death at embryonic day 8.5 and thus indicates the
requisite role of this receptor in differentiation of hemangioblasts
into endothelial cells (8). Mice lacking Flt-1, despite the presence of
normal hematopoietic precursors and endothelial cells, also die at
embryonic day 8.5, and the absence of tube formation strongly suggests
a major role for this receptor in endothelial cell-cell or cell-matrix interactions (9). VEGF, a potent angiogenic factor and the common
ligand for these two receptors, is distinctive in that its mitogenic
effect is highly specific for endothelial cells (10) and its expression
is up-regulated by hypoglycemia (11) and by hypoxia (12, 13). It has
been suggested that VEGF is implicated not only in embryonic vascular
development, but also in both physiologic angiogenesis, such as in
female reproductive tissues (14), and pathologic angiogenesis,
including proliferative diabetic retinopathy (15) and solid tumor
growth (16). This is further substantiated by experiments in which
inhibition of either VEGF or Flk-1/KDR resulted in suppression of
pathologic angiogenesis (17, 18), which validates the hypothesis that the VEGF signal transduction system is a viable target for
antiangiogenic therapeutic intervention.
Another family of receptor tyrosine kinases, designated the Tie
(tyrosine kinase that contains
immunoglobulin-like loops and epidermal growth
factor-similar domains) family, has also been studied and found to be
expressed primarily on cells of endothelial lineage (19, 20). Recent
studies revealed that mice lacking either of these receptors, Tie1 or
Tie2, die later than do those lacking VEGF or VEGFRs, indicating that
this family exerts its effect in the later stages of embryonic blood
vessel formation (21, 22). The Tie1 signal has been implicated in the
control of fluid exchange and hemodynamic stress resistance (22, 23), even though its ligand remains unidentified. In contrast, Tie2 appears
to regulate the capability of endothelial cells to recruit stromal
cells around the endothelial tubes and stabilizes vascular integrity
(24). The phenotype of Tie2 knockout mice is distinct from that of mice
lacking VEGFRs. Endothelial cells are detected in normal numbers, and
tube formation occurs; but the distinction between large and small
vessels is obscure, and encapsulation by periendothelial cells is
absent (22). Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are newly
identified ligands for the Tie2 receptor, and both bind to Tie2
receptors with similar affinity (25-27). Ang1 induces
autophosphorylation of Tie2 and has a remarkable chemotactic effect on
endothelial cells, whereas Ang2 competitively inhibits this effect (26,
28). Moreover, Ang2-overexpressing transgenic mice mimic the phenotype
of knockout mice of Ang1 and Tie2, suggesting that Ang2 is a natural
antagonist for Tie2 (26). Ang1 has been reported to be down-regulated
by treatment with serum or several cytokines in human lung fibroblasts
(29, 30) and also by hypoxia in rat glioma cells (30). Regulation of Ang2 and Tie2 expression, however, has not yet been well characterized. This study addresses that both hypoxia and VEGF selectively up-regulate Ang2 expression in bovine endothelial cells despite the stable expression of Ang1 and Tie2 and that Ang2 expression is up-regulated in vivo in a mouse model of ischemia-induced retinal angiogenesis.
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EXPERIMENTAL PROCEDURES |
Cell Culture and VEGF Treatment--
Primary cultures of bovine
retinal endothelial cells (BRECs) were isolated by homogenization and a
series of filtration steps as described previously (31). Cells were
grown on fibronectin (Sigma)-coated dishes (Iwaki Glass, Tokyo, Japan)
containing Dulbecco's modified Eagle's medium with 5.5 mM
glucose, 10% platelet-derived horse serum (Wheaton, Pipersville, PA),
50 mg/ml heparin, and 50 units/ml endothelial cell growth factor (Roche
Molecular Biochemicals). Bovine aortic endothelial cells (BAECs) were
also isolated from bovine aorta and cultured in Dulbecco's modified
Eagle's medium containing 5% calf serum and 10% platelet-derived
horse serum. Cells were cultured in 5% CO2 at 37 °C,
and media were changed every 3 days. Cells were characterized for their
endothelial homogeneity by immunoreactivity for factor VIII antigen and
remained morphologically unchanged under these conditions, as confirmed
by light microscopy. Only cells from passages 4 to 7 were used for the
experiments. For the kinetic studies of VEGF treatment, cells were
incubated with VEGF (0-125 ng/ml; Genzyme, Cambridge, MA) for the
indicated time points. To determine the roles of tyrosine kinase,
protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) in VEGF-induced Ang2 mRNA expression, BRECs were pretreated with genistein (40 µM; LC Laboratories, Boston, MA), GF
109203X (5 µM; LC Laboratories), and PD 098059 (25 µM; Upstate Biotechnology, Inc., Lake Placid, NY),
respectively, followed by stimulation with 25 ng/ml VEGF. These drug
levels of the inhibitors have been shown to block each target
selectively and effectively in endothelial cells (32, 33).
Amplification of Human Ang1, Ang2, and Tie2 cDNAs Using
Reverse Transcriptase-Polymerase Chain Reaction (PCR)--
cDNA
templates for PCR were synthesized by reverse transcriptase (first
strand kit, Invitrogen, Carlsbad, CA) from human umbilical vein
endothelial cells (Kurabo, Osaka, Japan) according to the method
recommended by the manufacturer. For Ang1, Ang2, and Tie2 cDNAs, a
standard PCR was performed (PCR optimizer kit, Invitrogen) using
5'-AGA ACC ACA CGG CTA CCA TGC T-3' (Ang1 sense primer
corresponding to nucleotides +671 to +692),
5'-TGT GTC CAT CAG CTC CAG TTG C-3' (Ang1 antisense primer),
5'-AGC TGT GAT CTT GTC TTG GC-3' (Ang2 sense primer corresponding
to nucleotides +377 to +396),
5'-GTT CAA GTC TCG TGG TCT GA-3' (Ang2 antisense primer
corresponding to nucleotides +802 to +821),
5'-GCC TTA ATG AAC CAG CAC CAG G-3' (Tie2 sense primer
corresponding to nucleotides +335 to +356), and
5'-ACT TCT GGG CTT CAC ATC TCC G-3' (Tie2 antisense primer corresponding to nucleotides +773 to +794). These cDNAs were cloned by the reverse transcription-PCR method recommended by the
manufacturer. The PCR products were then subcloned into a vector
(pCRII, Invitrogen) and sequenced in their entirety, and comparison
with the published human sequences revealed complete sequence identity.
These cDNA probes were used for hybridization.
Northern Blot Analysis--
Total RNA was isolated using
acid-guanidium thiocyanate, and Northern blot analysis was performed as
described previously (34). Total RNA (20 µg) was electrophoresed
through 1% formaldehyde-agarose gels and then transferred to a nylon
membrane (BNRG3R, Pall BioSupport Division, East Hills, NY).
Radioactive cDNA probes were generated by use of labeling kits
(Megaprime DNA labeling systems, Amersham International,
Buckinghamshire, United Kingdom) and [32P]dCTP (NEG-513,
Bio-Rad). After ultraviolet cross-linking using a UV cross-linker
(FS-1500, Funakoshi, Tokyo), blots were prehybridized; hybridized with
the indicated cDNA probe; and washed in 0.5% SSC and 5% SDS at
65 °C, with changing of the solution four times over 1 h in a
rotating hybridization oven (Taitec, Saitama, Japan). All signals were
scanned and analyzed utilizing a densitometer (BAS-2000II, Fuji Film,
Tokyo), and lane loading differences were normalized by means of 36B4
control cDNA (35) (generously provided by Dr. Lloyd P. Aiello).
Nuclear Run-on Transcription Analysis--
BRECs were treated
with vehicle or VEGF (25 ng/ml) for 2 h. The cells were lysed in a
solubilization buffer (10 mM Tris-HCl, 10 mM
NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40), and
the nuclei were isolated. ATP, CTP, and GTP (50 mM each)
and 3.7 MBq of [32P]UTP (Amersham International) were
added to the nuclear suspension (100 µl) and incubated for 30 min.
The samples were extracted with phenol/chloroform and precipitated.
cDNA probes (Ang2 and 36B4, 10 µg each) were then
slot-blotted onto nitrocellulose filters (Schleicher & Schüll,
Dassel, Germany) and hybridized with the precipitated samples of equal
counts/min/ml in hybridization buffer at 45 °C for 48 h. The
filters were washed, and the radioactivity was measured using the
BAS-2000II densitometer. The level of Ang2 mRNA was normalized to
that of 36B4 mRNA.
Analysis of Ang2 mRNA Half-life--
BRECs were treated with
25 ng/ml VEGF for 2 h prior to mRNA stability experiments.
Thereafter, half of the plates were returned to Dulbecco's modified
Eagle's medium without VEGF, and actinomycin D (10 µg/ml; Wako,
Osaka) was added to all plates. Total RNA was isolated at 0, 3, and
6 h after the addition of actinomycin D, and Northern blot
analysis was performed.
Hypoxic Treatment--
BRECs or BAECs were exposed to hypoxic
conditions of 1% oxygen using an advanced computer-controlled infrared
water-jacketed multigas incubator (Model BL-M10, Jujikagaku, Tokyo).
All cells were maintained at 37 °C in a constant 5% CO2
atmosphere with oxygen deficit induced by nitrogen replacement. Cells
maintained under these conditions for periods exceeding 24 h
showed no morphologic changes by light microscopy and could
subsequently be passaged normally. Cells incubated under standard
normoxic conditions (95% air and 5% CO2) from the same
batch and passage were used as controls. To study the effect of
hypoxia-induced VEGF on Ang2 expression, BRECs were incubated under
hypoxic or normoxic conditions for 2 h with or without anti-VEGF
neutralizing antibody (10 µg/ml; R&D Systems, Minneapolis, MN).
Immunoprecipitation Analysis of Ang2--
BRECs were treated
with 25 ng/ml VEGF for the VEGF study or subjected to hypoxic
conditions for the hypoxic study for 12 h in serum-free,
methionine-free medium with 35S (100 µCi/ml; Amersham
International). Cells were washed three times with cold
phosphate-buffered saline and lysed in a solubilization buffer (50 mM Hepes, pH 7.4, 10 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1% Triton
X-100, 10 mM NaVO4, 20 µM
leupeptin, 1.5 µM aprotinin, and 2 mM
phenylmethylsulfonyl fluoride) at 4 °C for 1 h. To clear the
protein extract, protein A-Sepharose (20 µl of a 50% suspension;
Pharmacia Biotech, Uppsala, Sweden) was added to the cell lysates,
after which they were incubated for 1 h, followed by
centrifugation and collection of the supernatant. Protein
concentrations were measured by a protein assay (BCA protein assay,
Pierce). A specific goat anti-human Ang2 antibody (5 µg; Santa Cruz
Biotechnology, Santa Cruz, CA) was added and rocked with the protein
sample (500 µg) at 4 °C for 1.5 h; 10 µg of protein A-Sepharose was then added, and the sample was rocked for another 1.5 h at 4 °C. For denaturation, protein A-Sepharose
antigen-antibody conjugates were separated by centrifugation, washed
five times, and boiled for 3 min in Laemmli sample buffer. The samples
were separated on 7.5% SDS-polyacrylamide gel (Bio-Rad), and the gel was vacuum-dried. Results were visualized and analyzed by densitometric scanning (BAS-2000II).
Mouse Model of Ischemia-induced Retinal
Neovascularization--
The well established mouse model of
ischemia-induced retinal neovascularization was created as described
previously (36). Briefly, litters of 7-day-old (postnatal day 7)
C57BL/6J mice were exposed to 75 ± 2% oxygen for 5 days and then
returned to room air at postnatal day 12 to produce retinal
neovascularization. Mice of the same age maintained in room air served
as controls. For in situ hybridization studies, mice at
different time points during the induction of neovascularization were
deeply and intraperitoneally anesthetized with sodium pentobarbital and
killed by perfusion through the left ventricle with 4%
paraformaldehyde in phosphate-buffered saline. Eyes were enucleated,
fixed in 4% paraformaldehyde at 4 °C overnight, and embedded in
paraffin. Serial 5-µm sections of the whole eyes were placed on
microscope slides.
In Situ Hybridization of Ang2 mRNA Expression--
Slides
were treated with 0.2 N HCl for 20 min, followed by washing
in phosphate-buffered saline containing 0.01% diethyl pyrocarbonate, digestion with 20 µg/ml proteinase K at 37 °C for 10 min, and fixation in 4% paraformaldehyde for 5 min. Blocking was performed in
phosphate-buffered saline containing 50% formamide and 2× SSC at room
temperature for 1 h. Sense and antisense Ang2 cRNA probes were
generated from the same plasmid used for Northern hybridization and
labeled with digoxigenin-dUTP (DIG RNA labeling kit, Roche Molecular
Biochemicals) as recommended by the manufacturer. The efficiency of
labeling was confirmed by agarose gel electrophoresis. The probe was
used at a concentration of 50 ng/section. Hybridization was performed
at 45 °C for 16 h. After extensive sequential washings in 2×,
1×, and 0.5× SSC, the unhybridized probe was digested with ribonuclease (Promega, Madison, WI) in 0.5× SSC. The hybridization product was detected after incubation with an alkaline
phosphatase-conjugated anti-digoxigenin antibody (1:500 dilution; Roche
Molecular Biochemicals) overnight at 4 °C, followed by development
in 4-tetrazolium chloride (1:50 dilution; Roche Molecular
Biochemicals) overnight at room temperature.
Statistical Analysis--
All determinations were performed in
triplicate, and results are expressed as means ± S.D. One-way
analysis of variance, followed by Fisher's t test, was used
to evaluate significant differences, and p < 0.05 was
selected as the statistically significant value.
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RESULTS |
VEGF Stimulates Ang2 (but Not Ang1 or Tie2) mRNA
Expression--
To investigate the effect of VEGF treatment on Ang1,
Ang2, and Tie2 expression, BRECs were exposed to VEGF (25 ng/ml), and Northern blot analysis was performed. Increased Ang2 mRNA
expression was observed after 1 h of stimulation and was
time-dependent, with a maximal response of a 4.6 ± 0.7-fold (p = 0.0031) increase after 2 h of
stimulation (Fig. 1A). In
contrast, both Ang1 and Tie2 mRNA expression remained stable. To
analyze dose dependence, cells were treated with various concentrations
of VEGF for 2 h. A dose-dependent increase in Ang2
mRNA was observed with an EC50 of
12.5 ng/ml and
peaked at 25 ng/ml (p < 0.0001) (Fig. 1B). These data suggest that VEGF increases Ang2 mRNA expression in both
a time- and dose-dependent manner. Since expression of
angiopoietins has been reported to be cell-type dependent (28) and to
confirm a similar effect of VEGF on macrovascular endothelial cells, we also tested the effect of VEGF on BAECs. The resultant response revealed a 2.6 ± 0.3-fold (p = 0.0026) increase
in Ang2 mRNA expression after 2 h of VEGF stimulation (25 ng/ml) (Fig. 1C). As in BRECs, VEGF had no significant
effects on Ang1 or Tie2 mRNA expression in BAECs.

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Fig. 1.
Kinetic studies of gene expression of the
angiopoietin-Tie2 system in response to VEGF treatment.
A, time course of Ang1, Ang2, and Tie2 mRNA expression
stimulated by VEGF in BRECs. Total RNA was isolated at the indicated
time points after the cells were stimulated with VEGF (25 ng/ml).
B, concentration dependence of mRNA regulation by VEGF
stimulation. BRECs were treated with the indicated concentrations of
VEGF for 2 h and harvested to prepare total RNA. C,
time course of Ang1, Ang2, and Tie2 mRNA expression stimulated by
VEGF in BAECs. Cells were stimulated with VEGF (25 ng/ml) and harvested
at the indicated time points to prepare total RNA. Northern blot
analysis (20 µg of total RNA/lane) was performed with the indicated
[32P]dCTP-labeled cDNA probe. Experiments were
performed in triplicates, and the representative blots of three
independent experiments are shown (upper panels). Results
were quantified by densitometric analysis of the autoradiograms derived
from the upper panels after normalization to the 36B4
control cDNA signals. Values are presented as a percentage of the
control and are expressed as means ± S.D. (lower
panels). Black, hatched, and white
bars indicate Ang1, Ang2, and Tie2 mRNA levels, respectively.
kb, kilobases.
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VEGF Increases the Rate of Ang2 mRNA Transcription--
We
investigated whether the VEGF-induced increase in Ang2 mRNA is
derived from up-regulation of transcription or from increased mRNA
stability. Nuclear run-on transcription analysis was employed to
determine whether VEGF leads to an increase in the transcription initiation rate. Nuclei prepared from cells treated with VEGF (25 ng/ml) or vehicle were evaluated. VEGF treatment increased the rate of
Ang2 gene transcription by 3.8-fold compared with that of controls
(Fig. 2).

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Fig. 2.
Effect of VEGF on the transcription rate of
Ang2. BRECs were treated with 25 ng/ml VEGF or vehicle (control
(CTR)) for 2 h. Nuclei were isolated and incubated with
ATP, CTP, and GTP, and 32P-labeled RNA probes were
hybridized to nitrocellulose filters on which Ang2 and 36B4 cDNAs
had been blotted. To normalize the difference of the loading RNA, the
radioactivity of Ang2 was normalized to that of 36B4. Data are shown as
a percentage of the control. Representative blots of three independent
experiments are shown.
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VEGF Does Not Increase the Half-life of Ang2 mRNA--
To
determine whether VEGF affects the stability of Ang2 mRNA, we
evaluated the half-life of Ang2 mRNA with the aid of actinomycin D
to inhibit de novo gene transcription. The half-life of Ang2 mRNA was 4.2 h when treated with VEGF and 3.8 h in
unstimulated controls (Fig. 3). No
significant difference was observed. These findings, together with the
data from nuclear run-on transcription analysis, clearly demonstrate
that the VEGF-induced increase in Ang2 mRNA was derived mainly from
an increase in the transcription rate.

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Fig. 3.
Decay of Ang2 mRNA in the presence of
actinomycin D in BRECs. Cells were preincubated with VEGF (25 ng/ml) for 2 h. Half of the plates were returned to Dulbecco's
modified Eagle's medium without VEGF, and 10 µg/ml actinomycin D
(ACD) was added to all plates. Total RNA was isolated at the
indicated time points after administration of actinomycin D, and
Northern blot analysis was performed. Ang2 mRNA levels were
normalized to those of 36B4 mRNA for correction of the loading
differences. , control cells; , cells in the presence of VEGF.
The values shown represent the percentage of initial Ang2 mRNA
signal remaining under the specified conditions and are plotted in
logarithmic scale. Representative data of three independent experiments
are shown.
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Effects of Tyrosine Kinase, PKC, and MAPK Inhibition on
VEGF-induced Ang2 mRNA Expression--
Since previous reports have
shown that tyrosine kinase and PKC (32) and MAPK (37) have significant
roles in VEGF-induced intracellular signaling pathways, we determined
whether these molecules could have effects on VEGF-induced Ang2
mRNA expression. BRECs were treated with 25 ng/ml VEGF for 2 h
after pretreatment with a protein kinase inhibitor: genistein, a
tyrosine kinase inhibitor (40 µM); GF 109203X, a
PKC-specific inhibitor (5 µM); or PD 098059, a MAPK
kinase inhibitor (25 µM) (Fig.
4). The addition of these agents
abolished the VEGF-induced increase in Ang2 mRNA expression by
84.6 ± 13.1% (p = 0.0009), 65.4 ± 16.8%
(p = 0.0029), and 92.4 ± 8.5% (p = 0.0005), respectively. The 0.1% Me2SO carrier used to
solubilize these inhibitors did not significantly alter Ang2 mRNA
expression (data not shown). These data indicate that MAPK and tyrosine
phosphorylation have a predominant role in VEGF-induced Ang2 mRNA
expression and that the PKC-dependent pathway makes a more
minor contribution.

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Fig. 4.
Role of tyrosine kinase, PKC, and MAPK.
BRECs were pretreated with genistein (40 µM), GF 109203X
(5 µM), or PD 098059 (25 µM), followed by
stimulation with VEGF (25 ng/ml) for 2 h. Total RNA was isolated,
and Northern blot analysis was performed. Ang2 mRNA levels were
normalized to those of 36B4 mRNA. Representative blots of three
independent experiments are shown (upper panel). Results are
presented as a percentage of the VEGF-induced increase in Ang2 mRNA
levels obtained without inhibitors and are expressed as means ± S.D. (lower panel). kb, kilobases.
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Hypoxia Increases Ang2 mRNA Expression--
To investigate the
effects of hypoxia on the angiopoietin-Tie2 system, BRECs were exposed
to hypoxic conditions. Ang2 mRNA expression revealed a
time-dependent increase that peaked at 2 h (3.6 ± 0.09-fold, p < 0.0001) and returned to the basal
level after 4 h of stimulation (Fig.
5A). In contrast, Ang1 and
Tie2 mRNA expression remained stable despite hypoxia. As in the
VEGF stimulation study, we found a similar response in BAECs. Two hours of hypoxia induced a 2.1 ± 0.1-fold (p = 0.0327)
increase in Ang2 mRNA expression, whereas no significant effect on
Ang1 or Tie2 mRNA expression was observed (Fig. 5B).
Since hypoxia is the major stimulus for VEGF induction and our result
showed that VEGF increases Ang2 expression, we investigated whether
hypoxia-induced VEGF is involved in the observed hypoxic regulation of
Ang2 in BRECs. The anti-VEGF neutralizing antibody (10 µg/ml)
exhibited no significant effect on Ang2 mRNA under not only
normoxic, but also hypoxic conditions (Fig.
6). These results suggest that the
increase in Ang2 expression under hypoxic conditions is the direct
effect of hypoxia and is not mediated by VEGF induction.

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Fig. 5.
Hypoxic regulation of Ang1, Ang2, and Tie2
gene expression. Shown is the time course of Ang1, Ang2, and Tie2
mRNA expression in BRECs (A) and BAECs (B)
subjected to hypoxia. Total RNA was isolated at the indicated time
points of hypoxia, and Northern blot analysis was performed. Three
triplicate experiments were performed, and the representative blots are
shown (upper panels). Results were quantified by
densitometric analysis of the autoradiograms derived from the
upper panels after normalization to the 36B4 control
cDNA signals. Values are presented as a percentage of the control
and are expressed as means ± S.D. (lower panels).
Black, hatched, and white bars
indicate Ang1, Ang2, and Tie2 mRNA levels, respectively.
kb, kilobases.
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Fig. 6.
Effect of anti-VEGF neutralizing antibody on
hypoxia-induced Ang2 mRNA expression in BRECs. Total RNA was
isolated at 2 h after stimulation by hypoxia or normoxia with or
without 10 µg/ml anti-VEGF antibody, and Northern blot analysis was
performed. Three triplicate experiments were performed, and the
representative blots are shown (upper panel). Results were
quantified by densitometric analysis of the autoradiogram derived from
the upper panel after normalization to the 36B4 control
cDNA signals. Values are presented as a percentage of the control
and are expressed as means ± S.D. (lower panel).
kb, kilobases.
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Both VEGF and Hypoxia Increase Ang2 Protein Synthesis--
To
determine whether the increase in Ang2 mRNA was accompanied by an
increase in new protein synthesis, we precipitated the 35S-labeled cell lysates with a specific goat anti-human
Ang2 antibody. The molecular mass of Ang2 protein has been reported to
range from 55 to 70 kDa, due to glycosylation (27). The detected size of Ang2 protein was ~55 kDa, and the expression level increased 5.3-fold after VEGF treatment (25 ng/ml) and 4.3-fold after hypoxic exposure (Fig. 7).

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Fig. 7.
Immunoprecipitation analysis of de
novo Ang2 protein synthesis. Confluent BRECs were
either treated with VEGF (25 ng/ml) or shifted to hypoxic conditions
for 12 h and then labeled with [35S]methionine. The
cell lysates were incubated with a specific goat anti-human Ang2
antibody and then immunoprecipitated with protein A-Sepharose. The
conjugates were removed by centrifugation and washing, denatured by
boiling, and size-fractionated on 7.5% SDS-polyacrylamide gel. Labeled
protein signals were analyzed by densitometric scanning. Representative
results of three independent experiments are shown. Lane
1, control; lane 2, VEGF treatment; lane
3, hypoxic exposure.
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Ang2 mRNA Expression Is Increased in the Retina of the Mouse
Model of Ischemia-induced Retinal Neovascularization--
To determine
in vivo whether Ang2 expression is up-regulated in response
to hypoxia, the well established mouse model of ischemia-induced retinal neovascularization was employed. In this model, proliferative retinal neovascularization peaks at 5 days after hypoxia (postnatal day
17) and regresses by postnatal day 26. Hybridization with an antisense
probe showed a basal level of signal located in the ganglion cell layer
and the inner nuclear layer at postnatal day 12, just prior to removal
of the animal from oxygen, in both hypoxic retinas and age-matched
nonhypoxic control retinas (Fig. 8,
A and B). After 12 h of relative hypoxia at
postnatal day 12, when the expression of VEGF has been shown to peak
(38), a mild elevation of signal level was detected in the ganglion
cell layer, and an intense signal was observed in the inner nuclear
layer (Fig. 8C). Of note, the signal of vascular cells in
the inner nuclear layer was also up-regulated. In contrast, control
retinas did not show a marked change (Fig. 8D). At postnatal
day 17, the time at which retinal neovascularization has been shown to
be most prominent (38), an intense signal was detected in neovascular
tufts (Fig. 8E), which have been reported to develop
preferentially in the mid-peripheral retina at the junction of perfused
and nonperfused retinas. Control retinas of the same age did not show
significant change (Fig. 8F). At postnatal day 21, when
neovascular vessels began to regress, the difference between hypoxic
and control retinas had diminished, although stronger signals were
still detected in both the ganglion cell layer and the inner nuclear
layer of the hypoxic retinas (Fig. 8, G and
H).

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Fig. 8.
In situ hybridization analysis of
Ang2 mRNA expression during development of retinal
neovascularization. Sections from the mouse model of
ischemia-induced proliferative retinopathy (A, C,
E, and G) and normal age-matched controls
(B, D, F, and H) were
hybridized with digoxigenin-labeled Ang2 cRNA probes. The ganglion cell
layer (G), inner nuclear layer (I), outer nuclear
layer (O), and retinal pigment epithelium (R) are
indicated in A. A, postnatal day 12, just prior
to removal from oxygen; B, age-matched control of
A; C, postnatal day 12, after 12 h of
hypoxia (V indicates retinal vessels); D,
age-matched control of C; E, postnatal day 17, after 5 days of hypoxia (arrowheads indicate neovascular
tufts); F, age-matched control of E;
G, postnatal day 21, after 9 days of hypoxia; H,
age-matched control of G.
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DISCUSSION |
Ischemia or hypoxia is well known to be a primary inducer of
neovascularization in a variety of conditions, such as tumor angiogenesis (12), collateral vessel formation in cardiovascular diseases (39), and proliferative retinal neovascularization (40, 41),
and VEGF has been shown to be a potent mediator of these
ischemia-induced neovascularizations (18, 42, 43). This study is the
first demonstration that both hypoxia and VEGF selectively enhance Ang2
expression, with Ang1 and Tie2 remaining stable in retinal
microvascular endothelial cells, and that Ang2 is up-regulated in
hypoxic retinas and neovascular vessels in vivo. Since Ang2
has been verified to be a natural antagonist for Tie2, the angiogenic
stimuli of hypoxia and VEGF might well deteriorate the integrity of the
vasculature by suppressing Ang1 activation of Tie2.
In this study, we used cells of endothelial lineage to delineate
regulation of the angiopoietin-Tie2 system in response to VEGF
stimulation and hypoxic exposure. Among the receptor and its two
relevant ligands, Ang2 was up-regulated selectively in both a time- and
concentration-dependent manner by VEGF treatment. The
increased Ang2 mRNA expression peaked at 2 h and returned to
almost base-line levels after 24 h of stimulation. This rapid reaction seems to suggest that de novo protein synthesis is
not required for the VEGF-induced increase of Ang2 mRNA expression. The dose-response study revealed an EC50 of
12.5 ng/ml,
which approximates the range of the previously reported VEGF
concentrations in vitreous fluid from patients with active neovascular
ocular diseases (15), and thus suggests a clinical implication of the VEGF-induced Ang2 expression in these patients. For further elucidation of the mechanisms underlying the VEGF-induced increase of Ang2 mRNA
expression, we performed run-on transcription assays and mRNA
stability analyses. A 3-fold increase in the Ang2 mRNA
transcription rate was detected, whereas only a minimal stabilizing
effect of Ang2 mRNA was obtained by VEGF treatment. These data,
together with the quick response revealed by the time course study of
this gene, indicate that up-regulation of Ang2 mRNA is derived
primarily from transcriptional activation.
Previous studies have revealed that VEGF promotes activation of several
signaling molecules, including phospholipase C
, phospholipase D,
PKC, phosphatidylinositol 3-kinase, GTPase-activating protein, Nck,
and MAPK (32, 37, 44, 45). We performed further studies to explore the
signaling pathway underlying the VEGF-induced Ang2 mRNA expression.
Blockage of tyrosine kinase by genistein abrogated
90% of the
increase in Ang2 mRNA stimulated by VEGF. Recent studies revealed
the critical roles of VEGF-induced MAPK activation in diverse
biological responses of endothelial cells, including mitogenesis (46,
47) and actin reorganization and cell migration (33). We further tested
the role of the MAPK-dependent signaling pathway using PD
098059, a specific MAPK kinase inhibitor, in the VEGF-induced increase
in Ang2 mRNA expression. Our results revealed that PD 098059 abrogated the increase in Ang2 mRNA expression by >90%, which
suggests a predominant role of this signaling molecule in VEGF-induced
Ang2 expression. PKC has also been reported to mediate both the
mitogenic effect on endothelial cells and increased vascular permeability in response to VEGF (32, 48). The PKC inhibitor GF 109203X
caused a significant decrease (by 65%) in Ang2 expression, suggesting
also a crucial role of PKC-dependent signaling. The observed inhibition by GF 109203X was less than that induced by the
MAPK kinase inhibitor. The difference in the inhibitory effect of PD
098059 and GF 109203X on VEGF-stimulated Ang2 expression suggests a
contribution of the PKC-independent pathway to MAPK activation
following VEGF stimulation, such as the Ras-dependent pathway (49) and the nitric oxide-dependent cascade
(46).
Hypoxia is the major stimulus that leads to ischemia-induced
angiogenesis and has been reported to up-regulate various genes that
encode erythropoietin (50), the platelet-derived growth factor B chain
(50), fibroblast growth factor (51), and VEGF (12, 16). In this study,
we demonstrated that hypoxia induces both mRNA expression and
protein synthesis of Ang2. Since the response peaked at as soon as
2 h after hypoxic exposure, it is probably not mediated by
induction of other growth factors, such as VEGF; rather, it may be
regulated directly by hypoxia. This hypothesis is further confirmed by
the experiment showing that anti-VEGF neutralizing antibody had no
significant effect on hypoxic Ang2 induction. Transcriptional
regulation by hypoxia-inducible factor (52) and stabilization of
mRNA by the AUUUA motif of the 3'-untranslated region (53) have
been reported to underlie hypoxic gene regulation of VEGF and
erythropoietin. Since the observed response of Ang2 to hypoxia is much
more rapid and transient in comparison with these genes (54, 55), it is
possible that a different molecular mechanism underlies this response.
Ang1 expression has generally been detected in nonendothelial cells
surrounding blood vessels in vivo (27), which suggests a
paracrine role of this ligand. Our results from Northern blot analysis,
however, demonstrated that Ang1 mRNA was also detectable in both
BRECs and BAECs and thus indicate that, in addition to its paracrine
role, Ang1 can act in an autocrine manner, as recently evidenced in a
leukemia cell line and cutaneous fat pad endothelial cells (28, 56).
Ang1 has been reported to be down-regulated not only by hypoxia in rat
glioma cells, but also by treatment with serum or several cytokines,
such as platelet-derived growth factor and tumor growth factor-
, in
human lung fibroblasts (30). In contrast, this study demonstrated that
Ang1 mRNA expression in BRECs remained essentially unchanged in
response to both VEGF stimulation and hypoxia. The observed response is
consistent with the informative histologic study showing that Ang1
exhibits uniform expression, irrespective of both developmental stage
and VEGF expression, in ovaries undergoing the reproductive cycle
(26).
Unlike VEGFR-1 and VEGF-2, whose regulation has been well documented by
recent findings that stimuli such as hypoxia (37, 57, 58) and several
cytokines, including VEGF, tumor necrosis factor-
, and
interleukin-1
, can exert significant effects on their expression
(29, 59), little is known about the regulation of Tie2. A recent report
suggests that Tie2 is present in both quiescent and angiogenic adult
tissue and that up-regulation of Tie2 is observed in skin wounds (60);
however, the possibility that increased numbers of blood vessels
per se during wound healing lead to the increased amount of
Tie2 protein expression was not excluded in that study. Our data
demonstrating that Tie2 remains stable in response to both VEGF
stimulation and hypoxic exposure, together with a recent report showing
that proinflammatory stimuli such as interleukin-1
and tumor
necrosis factor-
also have no effect on the expression of this
receptor (29), might suggest that the potential role of the
angiopoietin-Tie2 system in pathologic angiogenesis or under
proinflammatory conditions is derived mainly from altered ligand
expression rather than from changes in Tie2 receptor expression.
As in retinal microvascular endothelial cells, we have demonstrated
that Ang2 is up-regulated by hypoxia and VEGF stimulation in bovine
aortic endothelial cells as well, with Ang1 and Tie2 expression
remaining stable. In the macrovascular milieu, VEGF has been suggested
to play a role in vascular injury such as atherosclerosis (61).
Although the significance of the angiopoietin-Tie2 system remains
unknown in such lesions, VEGF might decrease vascular integrity by
up-regulating Ang2, thereby facilitating endothelial damage and intima
formation. Further studies are required to address this point.
Our in situ hybridization studies using the mouse model of
retinal neovascularization demonstrated that Ang2 mRNA is produced in the ganglion cell layer and inner nuclear layer of the retina. These
portions of retina are consistent with the locations where transcripts
of VEGF have been detected (38). As in the case of VEGF, the
up-regulation of Ang2 mRNA expression precedes the development of
neovascularization and parallels the temporal and spatial changes of
neovascularization development, which suggests that Ang2 plays a
critical role in retinal neovascularization. In addition to these
layers, we also detected a substantial signal in intraretinal vessels
and neovascular tufts that grew into the vitreous cavity through the
inner limiting membrane of the retina. Since pericytes have not been
shown to be associated with neovascular tufts or neovascular vessels
(36), the prominent up-regulation of Ang2 message in these retinal
components might be attributed to endothelial cells and thus
further supports the results of our in vitro study.
Interestingly, in ovaries undergoing the reproductive cycle, Ang2
expression appears to increase from the stage of the developing corpus
luteum, when VEGF shows remarkable expression (26), thus suggesting a
critical role of VEGF in in vivo Ang2 induction. Although
our in vitro data suggested that autocrine VEGF production
has little effect on hypoxic induction of Ang2, it is possible that
paracrine VEGF production in vivo under hypoxic conditions
might have a role in the observed hypoxia-induced Ang2 expression.
These findings, together with our results of in situ hybridization, suggest that hypoxia per se, hypoxic
induction of VEGF, or both in concert might play a major role in at
least the early stage of Ang2 induction in vivo.
Recent in vitro findings regarding the bioactivity of Ang1
have demonstrated that this ligand can induce potent chemotaxis, weak
but positive mitogenesis, and capillary sprouts in endothelial cells,
which confirms its critical role in angiogenesis (28, 62). When
coadministrated with VEGF in vivo, Ang1 can potentiate vascular network maturation, whereas Ang2 can contribute substantially to initiation of neovascularization (63). In light of the latter finding, the observed up-regulation of Ang2 in this study might implicate this ligand in the initial step of pathologic angiogenesis, where VEGF expression is abundant. Several studies have shown that
blocking the Tie2 signal using soluble Tie2 receptor can reduce
tumorigenic vascular growth (64, 65). Since neither Ang1 nor Ang2
alone, although contradictory to in vitro studies (28, 62),
can significantly promote in vivo neovascularization (63),
the decreased vascular growth might be attributed at least partly to
suppression of Ang2 induced by angiogenic stimuli such as hypoxia and
VEGF, as is evidenced in this study. The angiogenic effect of Ang2
might possibly be explained by destabilization of the preexisting
vascular tube structure to set up a favorable environment for
endothelial cells to migrate or to contact with additional angiogenic
cytokines. Alternatively, since communication between endothelial cells
and surrounding mesenchymal cells has been reported to have an
inhibitory effect on endothelial cell growth by modulating the
bioactivity of tumor growth factor-
(66, 67), destabilization of
blood vessels by Ang2 may diminish this inhibition and facilitate
subsequent neovascularization. In light of recent studies showing the
differential effects of Ang2 on endothelial cells and nonendothelial
cells (26, 28) and by virtue of the fact that endothelial progenitor
cells, for which the resultant effects of angiopoietin-Tie2 signaling
still remain unknown, also contribute to angiogenesis (68), further investigations are required to fully elucidate the detailed roles of
the increased Ang2 expression in angiogenesis.