Rapid Communication |
From the Department of Ophthalmology and Visual Sciences, Kyoto (Japan) University Graduate School of Medicine.
Correspondence to Hitoshi Takagi, MD, PhD, Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto 606, Japan. E-mail hitoshi{at}kuhp.kyoto-u.ac.jp
Abstract
AbstractAngiotensin II (Ang II) plays a role in the development of many vascular diseases. In the present study, we have investigated the effect of Ang II on vascular endothelial growth factor (VEGF) receptor expression and VEGF-induced angiogenic activity in bovine retinal microcapillary endothelial cells (BRECs). Ang II induced a significant increase of kinase domaincontaining receptor/total liver kinase (KDR/Flk-1) mRNA in a time- and dose-dependent manner, with a maximal 4.3±0.8-fold increase after a 4-hour stimulation. Ang II increased the rate of KDR gene transcription by 5.4-fold, whereas the half-life of KDR mRNA was not increased significantly. The increase depended partially on new protein synthesis. The Ang IIinduced KDR mRNA increase was inhibited by either [Sar1,Ile8]angiotensin or angiotensin type 1 receptor antagonists but was not significantly altered by angiotensin type 2 receptor antagonists. The PKC inhibitor reduced Ang IIinduced KDR mRNA expression by 70±15%. The tyrosine kinase inhibitor reduced the Ang II and phorbol 12-myristate 13-acetateinduced KDR mRNA increases by 35±8% and 44±26%, respectively. Ang II increased by 3.1-fold the 35S-labeled KDR/Flk-1 immunoprecipitated by a specific antibody to KDR/Flk-1. Scatchard analysis demonstrated that Ang II induced a significant increase of binding sites without changing binding affinity. Ang II enhanced VEGF-induced cell growth and tube formation. Ang II itself had no effect on cell growth, tube formation, or mRNA levels of VEGF and tms-like tyrosine kinase (Flt-1) in BRECs. These findings suggest that Ang II might potentiate VEGF-induced angiogenic activity through an increase of the VEGF receptor KDR/Flk-1.
Key Words: diabetic retinopathy vascular endothelial growth factor KDR/Flk-1 angiotensin II angiogenesis
Diabetic retinopathy is one of the major complications of diabetes mellitus and often results in catastrophic loss of vision. Although the pathogenesis of this complication is not fully understood, emerging evidence strongly implicates VEGF, not only in the ischemic retinal neovascularization observed in proliferative retinopathy1 2 3 but also in early stages of diabetic retinopathy.4 5 VEGF is a potent angiogenic factor and vasopermeability factor6 7 whose expression is increased by hypoxia,8 which is one of the primary stimuli for ocular neovascularization. VEGF mediates its effects through endothelial cellspecific high-affinity phosphotyrosine kinase receptors: Flt-1 (VEGFR1)9 and KDR/Flk-1 (VEGFR2).10 These two receptors have been demonstrated to be different in function. KDR-expressing cells show changes in morphology, chemotaxis, and mitogenicity on VEGF stimulation, whereas Flt-1expressing cells lack such a response.11 Gene-knockout experiments also suggest differences of these receptor functions in the development of the vascular system.12 13 VEGF does not bind Flt-4 (VEGFR3), but a ligand for VEGFR3 has recently been cloned as VEGF-C, which binds KDR/Flk-1 as well.14
Ang II is well known to be a key factor in cardiovascular homeostasis and to exert many actions, such as controlling vascular tone, hormone secretion, and neuronal effects on the heart, vascular system, kidneys, adrenal glands, and central nervous system.15 From experimental data and clinical evidence, the RAS is thought to play an important role in many cardiovascular disorders. Ang II has been reported to regulate cell growth of vascular SMCs16 17 and to stimulate the induction of PDGF, basic FGF, IGF, and other autocrine growth factors in SMCs18 19 and the induction of ET-1 in endothelial cells.20 These effects have been linked to myocardial infarction,21 myointimal proliferation after vascular injury,22 essential hypertension,23 and diabetic nephropathy,24 and ACE inhibitors have been reported to be beneficial in these diseases.22 25 26
In diabetic retinopathy, intraocular and serum levels of ACE, prorenin, and Ang II have been reported to be correlated with the severity of retinopathy.27 28 29 ACE inhibitors have been reported to improve the blood-retina barrier in diabetic patients30 and to have a favorable effect on diabetic retinopathy.31 These reports suggest that Ang II may play a role in the development of diabetic retinopathy. How RAS is involved in the pathogenesis of diabetic retinopathy, however, has not been investigated in detail.
In the present study, we report that Ang II is a potent stimulant of VEGF-induced proliferation and tube formation in retinal microvascular endothelial cells through the induction of the VEGF receptor KDR/Flk-1. This Ang IIinduced KDR upregulation is transcriptionally regulated through AT1 receptors, with subsequent activation of both the PKC-dependent and tyrosine kinasedependent signaling pathways.
Materials and Methods
Cell Cultures
Primary cultures of BRECs were isolated by
homogenization and a series of filtration steps as
previously described.32 Primary BRECs were grown
on fibronectin (Sigma Chemical Co)coated dishes (Iwaki Glass Inc)
containing DMEM with 5.5 mmol/L glucose, 10% PDHS (Wheaton), 50
mg/L heparin, and 50 U/L endothelial cell growth factor
(Boehringer-Mannheim). BAECs were also isolated from bovine
aorta and cultured in DMEM containing 5% calf serum and 10% PDHS. The
cells were cultured in 5% CO2 at 37°C, and
media were changed every 3 days. Endothelial cell
homogeneity was confirmed by immunoreactivity with antifactor VIII
antibodies analyzed by confocal microscopy. After the cells
reached confluence, the medium was changed every 3 days, and only cells
from passages 7 to 11 were used for these experiments. For Ang II
receptor antagonist studies, confluent BRECs were
pretreated with 1 µmol/L of
[Sar1,Ile8]Ang II
(Sigma), DuP753 (Merck Research Laboratories Co), or PD123319 (Research
Biochemicals International Co) for 15 minutes, followed by stimulation
of 10 nmol/L Ang II for 4 hours. To determine the roles of PKC and
tyrosine kinase on Ang IIstimulated KDR mRNA expression, starved
confluent BRECs were pretreated with GFX (10 µmol/L,
Calbiochem-Novabiochem Co) or genistein (20 µmol/L, LC
Laboratories), followed by stimulation with 10 nmol/L Ang II or 160
nmol/L PMA (Sigma). These drug levels of the inhibitors
have been shown to be sufficient to selectively block each
target.33 34 35 36 37 38 39 40
Northern Blot Analysis
After serum starvation, confluent BRECs were treated with
stimulants or antagonists, and total RNA was isolated from
individual tissue culture plates using guanidinium thiocyanate.
Northern blot analysis was performed on 20 µg total RNA after
1% agarose/2 mol/L formaldehyde gel electrophoresis and subsequent
capillary transfer to Biodyne nylon membranes (Pall BioSupport) and
ultraviolet cross-linking using a FUNA-UV-LINKER (FS-1500, Funakoshi
Inc). Radioactive probes were generated using Amersham Megaprime
labeling kits and [32P]dATP (Amersham). Blots
were prehybridized, hybridized, and washed in 0.5x SSC and 5% SDS at
65°C with four changes over 1 hour in a rotating hybridization oven
(TAITEC). All signals were analyzed using a densitometer
(BAS-2000II, Fuji Photo Film), and lane loading differences were
normalized using a 36B4 cDNA probe, which hybridizes to acidic
ribosomal phosphoprotein PO.41 Human KDR cDNA was
used as a probe (generously provided by Dr Loyd P. Aiello, Boston,
Mass).
Analysis of KDR mRNA Half-Life
To determine whether the increase in KDR mRNA was caused by an
increase in transcription, BRECs were exposed to actinomycin D (4
µmol/L, Wako) after 4 hours of incubation with vehicle or Ang II (10
nmol/L). The total RNA was then extracted, and Northern blot
analyses were performed.
Nuclear Run-on Analysis
Confluent BRECs were serum-deprived for 18 hours in 0.1% BSA
DMEM, followed by treatment with vehicle or Ang II (10 nmol/L) for 4
hours. The cells were lysed in solubilizing buffer (10 mmol/L
Tris-HCl, 10 mmol/L NaCl, 3 mmol/L
MgCl2, and 0.5% NP-40), and the nuclei were
isolated. ATP, CTP, and GTP (500 mmol/L each) and 3.7 MBq of
32P-labeled UTP (Amersham) were added to the
nuclear suspension (100 µL) and incubated for 30 minutes. The samples
were extracted with phenol/chloroform and precipitated. cDNA probes
(KDR and 36B4, 10 µg each) were then slot-blotted onto nitrocellulose
filters (Schleicher & Schuell, Inc) and hybridized with the
precipitated samples of equal counts per minute per milliliter in
hybridization buffer at 45°C for 48 hours. The filters were washed,
and the radioactivity was measured using the densitometer (BAS-2000II,
Fuji Photo Film). The levels of KDR mRNA were normalized to 36B4 mRNA
expression.
Immunoprecipitation Analysis of KDR/Flk-1
Confluent BRECs were serum-deprived for 24 hours and treated
with 10 nmol/L Ang II or vehicle for 24 hours. The cells were then
incubated with [35S]methionine (100 µCi/mL,
Amersham) in methionine-free DMEM (Dainippon Pharmaceutical Co) for 4
hours and lysed in solubilizing buffer (50 mmol/L HEPES, pH 7.4,
10 mmol/L EDTA, 100 mmol/L NaF, 10 mmol/L sodium
pyrophosphate, 1% Triton X-100, 10 mmol/L
NaVO4, 20 µmol/L leupeptin, 1.5
µmol/L aprotinin, and 2 mmol/L phenylmethylsulfonyl
fluoride) at 4°C for 1 hour. To clear the protein extract,
protein A Sepharose (20 µL of 50% suspension, Pharmacia Biotech) was
added to the cell lysate and incubated for 1 hour, followed by
centrifugation and collection of the supernatant.
Protein concentrations were measured by a protein assay (BCA, Pierce).
Specific antibody to Flk-1 (50 ng/mL, Santa Cruz Biotechnology Inc) was
added to the protein samples (500 µg) and rocked at 4°C for 1.5
hours, and then 10 µg protein A Sepharose was added and rocked
another 1.5 hours at 4°C. Protein A Sepharose antigen-antibody
conjugates were separated by centrifugation, washed
five times, and boiled for 3 minutes in Laemmli sample buffer to
denature. The samples were separated by 7.5%
SDSpolyacrylamide gel (Bio-Rad Laboratories), and the gel was
vacuum-dried. Results were visualized and quantified by a BAS-2000II
densitometer (Fuji Photo Film).
VEGF Binding Analysis
Monolayers of confluent BRECs grown in 12-well dishes (Iwaki
Glass) were incubated with Ang II for 12 to 48 hours and then placed on
ice and washed three times with ice-cold PBS containing calcium and
magnesium. 125I-labeled VEGF was added, along
with increasing amounts of unlabeled VEGF, and binding was carried out
by rocking for 4 hours at 4°C. Binding was terminated by washing each
well three times with ice-cold PBS containing 0.1% BSA. The cells were
lysed in 1 mL of 0.1% SDS and counted in a gamma counter (ARC-600,
Aloka).
[3H]Thymidine Incorporation Assay
Subconfluent BRECs (grown on a 24-well dish) that were
serum-deprived for 24 hours in DMEM with 0.1% BSA were pretreated with
Ang II (Sigma) at 1, 10, or 100 nmol/L or with vehicle for 12 hours
before the addition of 0.6 nmol/L (25 ng/mL) VEGF (recombinant human
VEGF, Genzyme) or vehicle. The cells were treated with vehicle or VEGF
for 18 hours and then labeled with 1 µCi/mL of
[3H]thymidine (Amersham) for 4 hours. The
labeled cells were washed with ice-cold PBS, fixed in ice-cold 10%
trichloroacetic acid, and then lysed with 0.5N NaOH. The incorporated
[3H]thymidine was extracted by filtration
(Whatman GF/C filter) and measured in a liquid scintillation counter
(Aloka).
Tube Formation Assay
Vitrogen 100 (Celtrix), 0.2N NaOH, and 200 mmol/L HEPES
(8:1:1 [vol/vol/vol]) and 10x RPMI medium (GIBCO BRL) were made to
400 µL and added to 24-well plates. After polymerization of the gels,
1.0x105 BRECs were seeded and incubated with
DMEM containing 20% PDHS for 24 hours at 37°C. The medium was
removed, and additional collagen gel was introduced on the cell layer.
Ang II (10 nmol/L) or vehicle was added in the medium and incubated for
24 hours, and then the cells were stimulated with 0.6 nmol/L (25 ng/mL)
VEGF. Five days later, five different fields (x10 objective) were
chosen, and total tubelike structures were measured using Adobe
Photoshop (Adobe Systems Inc).
Statistical Analysis
Determinations were performed in triplicate, and experiments
were performed at least three times. Results were expressed as
mean±SE, unless otherwise indicated. For multiple treatment groups, a
factorial ANOVA followed by Fisher's least significant difference test
was performed. Statistical significance was accepted at
P<.05.
Results
Ang II stimulates KDR/Flk-1 mRNA Expression in BRECs and
BAECs
To investigate the effect of Ang II on VEGF receptor expression.
BRECs were treated with 10 nmol/L Ang II for the indicated times, and
Northern blot analysis was performed on 20 µg/lane of total
RNA (Fig 1A
). After 1 hour of stimulation
with 10 nmol/L Ang II, an increase in KDR mRNA was observed.
Furthermore, this increase was time dependent, with a maximal
4.3±0.8-fold (P<.05) increase at 4 hours (Fig 1A
). To
investigate dose dependence of the Ang II effect, BRECs were stimulated
with various concentrations of Ang II for 4 hours, and KDR mRNA
expression was examined by Northern blot analysis (Fig 1B
). We
found that Ang II stimulates KDR mRNA expression in a dose-dependent
fashion, with an EC50 of
3 nmol/L and a
maximal 4.4±1.1-fold (P<.05) increase at 10 nmol/L (Fig 1B
). These data suggest that Ang II increases the expression of mRNA in
BRECs for the VEGF receptor KDR/Flk-1 in a time- and dose-dependent
manner.
|
To determine whether a similar response occurred in macrovascular
endothelial cells, BAECs were stimulated with 10 nmol/L
Ang II for 4 hours. Again, Ang II increased mRNA levels of KDR by
2.5±0.7-fold in BAECs (Fig 1B
).
Although KDR, rather than Flt-1, was predominantly expressed in BRECs,42 we also examined Ang II effect on expression of the other VEGF receptor, Flt-1. Expression of Flt-1 was not observed in either Ang IIstimulated or nonstimulated BRECs using similar Northern blot analysis (data not shown). Thus, subsequent studies evaluated only KDR/Flk-1 expression in BRECs.
Ang II Does Not Increase the Half-Life of KDR/Flk-1 mRNA
We investigated whether the Ang IIinduced increase of KDR mRNA
level is mediated through regulation of transcription or mRNA
stability. To determine whether Ang II affects the half-life of KDR
mRNA, we examined the effect of inhibition of de novo gene
transcription. Northern blot analyses were performed after the
administration of actinomycin D (4 µmol/L) with or without 10
nmol/L Ang II. The half-life of KDR mRNA was 1.3 hours in unstimulated
controls and 1.5 hours after treatment with 10 nmol/L Ang II. No
significantly difference was observed (Fig 2
).
|
Ang II Increases the Rate of Transcription of KDR mRNA in
BRECs
To determine whether Ang II affects the transcriptional rate of
KDR, we performed nuclear run-on analysis in the presence or
absence of Ang II. To correct for differences in loading of the RNA
probe, the rate of KDR mRNA transcription was compared with that of
36B4 mRNA, which was constitutively expressed. Treatment with 10 nmol/L
Ang II increased the rate of KDR gene transcription 5.4-fold compared
with that of control (Fig 3
). These data
clearly demonstrate that increased expression of KDR mRNA induced by
Ang II was not through mRNA stability but through an increase in
transcriptional rate.
|
Role of Ang II Receptor Subtypes AT1 and
AT2 in the Ang IIStimulated KDR/Flk-1 mRNA Expression
in BRECs
To characterize the Ang II receptor subtype that is responsible
for KDR induction, Northern blot analyses were performed using
total RNA from BRECs pretreated with the nonsubtype-specific Ang II
antagonist saralasin, the AT1
antagonist DuP753, or the AT2
antagonist PD123319 for 15 minutes before Ang II
stimulation. Saralasin inhibited the KDR mRNA expression to the control
level by 98.6±1.6% (Fig 4
), and the
AT1 antagonist inhibited the Ang
IIinduced KDR mRNA expression significantly by 92.7±3.6%
(P<.05). In contrast, the AT2
antagonist inhibited the Ang IIinduced KDR mRNA
expression by only 20.3±10.3% (Fig 4
). These data suggest that Ang
IIinduced KDR expression is mediated mainly through the
AT1 receptor. We also examined the role of
AT1 and AT2 in VEGF-induced
BREC growth by thymidine incorporation. When pretreated with the
AT1 antagonist before the addition of
Ang II, VEGF-induced cell growth was inhibited significantly
(P<.05), by 46±26%, whereas the AT2
antagonist had no inhibitory effects (Fig 8
).
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Role of PKC and Tyrosine Kinase in Ang IIInduced KDR mRNA
Expression
Previous reports have shown that PKC and tyrosine kinase have a
role in Ang IIstimulated signaling
pathways.33 43 44 45 To determine the role of PKC
and tyrosine kinase in Ang IIinduced KDR mRNA expression, BRECs were
pretreated with a highly selective PKC inhibitor,
bisindolylmaleimide (GFX), or a specific tyrosine kinase
inhibitor, genistein, followed by treatment with Ang II and
PMA, a direct PKC stimulator. PMA increased the expression of KDR mRNA
1.9±0.2-fold after 2 hours of stimulation compared with unstimulated
control (data not shown), and the effect was completely inhibited by
10 µmol/L GFX (Fig 5
). The same
concentration of GFX reduced Ang IIinduced KDR mRNA expression by
70±15% (Fig 5
). The role of tyrosine phosphorylation
in Ang IIstimulated and PMA-stimulated KDR expression was also
examined. Treatment with 20 µmol/L genistein reduced the Ang
IIinduced and PMA-induced KDR mRNA expression by 35±8% and
44±26%, respectively (Fig 5
). When both inhibitors were
applied simultaneously, KDR increase was inhibited by
90±10%, which was more than when each component was applied
separately (Fig 5
). The 0.1% (vol/vol) dimethyl sulfoxide carrier used
to solubilize these inhibitors did not significantly alter
KDR mRNA expression (data not shown). These data indicate that PKC has
a predominant role in the pathway of Ang IIstimulated KDR mRNA
expression and that tyrosine phosphorylation may
contribute to both the PKC-dependent and -independent mechanisms of Ang
IIstimulated KDR mRNA expression in BRECs.
|
Ang II Increases KDR Protein Synthesis and Cell Surface
Binding Sites
To determine whether the increase in KDR mRNA expression was
accompanied by an increase of new protein synthesis, we precipitated
the 35 S-labeled cell lysates of BRECs with
anti-KDR antibody. A single band at
205 kD was detected by
immunoprecipitation of a rabbit anti-human KDR antibody, and the level
was increased by 3.1-fold with Ang II stimulation at 10 nmol/L (Fig 6
).
|
To determine whether the number or affinity of VEGF binding sites
at the cell surface was changed in BRECs by Ang II stimulation, we
performed 125I-VEGF binding analysis.
Time-course study indicated that specific VEGF binding increased after
12 hours, and maximal increase was observed after 24 hours (data not
shown). Scatchard analysis was performed after 24 hours and
demonstrated a significant increase of binding sites
(5.8±0.3x104/cell to
9.9±0.2x104/cell, P=.004) without
changes of binding affinity (0.27±0.02 to 0.29±0.03 nmol/L,
P=.5) after 24 hours of stimulation (Fig 7
).
|
These results suggest that Ang II increased KDR protein synthesis and cell surface VEGF binding sites in BRECs.
Ang II Accelerates the VEGF-Induced Cell Growth in BRECs
To investigate the effect of Ang II on VEGF-induced angiogenesis,
we measured [3H]thymidine incorporation in
BRECs. Stimulation with 0.6 nmol/L (25 ng/mL) VEGF increased thymidine
incorporation 1.3±0.1-fold (P<.01) compared with the
unstimulated control, and treatment with 10 nmol/L of Ang II alone did
not affect thymidine incorporation in BRECs (Fig 8
). When BRECs were pretreated with Ang
II (10 nmol/L) followed by VEGF stimulation, thymidine incorporation
was enhanced in a dose-dependent manner, with a maximum 2.1±0.1-fold
(P<.01) increase at 10 nmol/L (Fig 8
). These data show that
Ang II enhances the effect of VEGF on cell proliferation of BRECs.
Ang II Enhances VEGF-Induced Tube Formation in BRECs
BRECs were seeded in three-dimensional gels and pretreated with
Ang II (10 nmol/L) or vehicle for 24 hours, followed by treatment with
VEGF to evaluate tube formation. The lengths of five different fields
per well were measured and compared. From three independent
experiments, Ang II alone had little effect on tube formation of BRECs
(Fig 9
). VEGF, however, induced tube
formation that was 7.5±0.5-fold (P<.01) greater than that
seen in controls (Fig 9
), and pretreatment with Ang II further promoted
the VEGF-induced tube formation in BRECs by 1.6±0.1-fold compared with
VEGF stimulation alone (P<.01, Fig 9
). These data suggest
that Ang II potentiates VEGF-induced angiogenic activity in retinal
microvascular endothelial cells and that Ang II alone
has no significant effect on retinal endothelial tube
formation and cell growth.
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Discussion
In diabetic retinopathy, development of retinal vascular nonperfusion followed by pathological angiogenesis often leads to vision loss. VEGF has been suggested to mediate such an ischemia-induced retinal neovascularization. Indeed, suppression of VEGF has been shown to inhibit neovascularization in animal models of retinal ischemia,2 and VEGF levels are elevated in patients with proliferative retinopathy and decrease after successful laser treatment.1 3 In addition, VEGF itself is sufficient to produce many of the vascular abnormalities common to diabetic retinopathy,4 and an increase of VEGF expression is observed in the retinas of diabetic patients with little or no retinopathy,5 suggesting a role of VEGF in the initiation and early stages of diabetic retinopathy. From this evidence, VEGF-induced angiogenesis in retinal microvascular cells might be a predominant pathological change in the development of diabetic retinopathy.
RAS has also been suggested to play a role in the development of
diabetic retinopathy, indicated by the correlation of
increased levels of serum and intraocular RAS with the clinical
progression of retinopathy27 28 29
and by the favorable results produced by the ACE inhibitor
on diabetic retinopathy.30 31
However, it remains unclear what mechanism underlies the interaction of
RAS and the development of diabetic retinopathy. In the
present study, we investigated whether Ang II affects VEGF/VEGF
receptor expression and VEGF-induced angiogenic activity, which is
thought to be a major pathological change in retinal microvascular
cells in diabetic retinopathy. We have demonstrated
that Ang II increases the expression of the VEGF receptor KDR/Flk-1 and
potentiates VEGF-dependent cell growth (Fig 8
) and tube formation (Fig 9
) in cultured BRECs. In addition, Ang II itself had no effect on cell
growth, tube formation, or mRNA levels of VEGF and Flt-1 in BRECs.
These data suggest that Ang II potentiation of VEGF-induced angiogenic
activity probably results from an Ang IIinduced increase in the
expression of the VEGF receptor KDR/Flk-1 in BRECs.
Since VEGF exerts its biological effects through binding to two
high-affinity tyrosine kinase receptors,
KDR/Flk-110 and Flt-1,9 we
sought to determine whether Ang II upregulates VEGF receptor expression
as a mechanism of potentiating angiogenic effects. We performed
Northern blot analysis and showed that Ang II stimulates KDR
mRNA expression in BRECs in a dose- and time-dependent manner (Fig 1
).
In contrast, as previously reported,42 we could
not detect Flt-1 mRNA in nonstimulated BRECs, and Flt-1 mRNA was not
observed even in Ang IIstimulated BRECs studied by similar Northern
blot analysis using total RNA. Although quantitative polymerase
chain reaction analysis or RNA protection assay might detect
the upregulation of Flt-1 mRNA, the effect of VEGF is probably
negligible in BRECs. Thus, we focused on KDR expression for further
analysis. The dose-response study demonstrated an
EC50 of
3 nmol/L and a maximal 4.4±1.1-fold
increase at 10 nmol/L Ang II stimulation. Although the concentrations
are considerably higher than those in the plasma and vitreous fluid of
diabetic patients,29 they are similar to or lower
than others that have been reported.18 20 33 46
Moreover, it is likely that local concentrations of Ang II in retinal
microvasculature are much higher than serum and vitreous levels, since
an autocrine paracrine production system of Ang II is
present in ocular tissues.29 48
Two major angiotensin receptor subtypes have been defined:
AT1 and
AT2.49 50 Most of the
actions of angiotensin are mediated by the
AT1 receptor, whereas actions of the
AT2 receptor are not well
understood.15 The expression of
AT2 receptors is reported to be regulated by cell
types and the developmental stage of tissues and is speculated to be
involved in tissue growth and
differentiation.35 49 51 The growth-promoting
effect of Ang II in SMCs has been reported to be through the
AT1 receptor,52 and
recently, the AT2 receptor was reported to
mediate antigrowth effects on AT2-overexpressed
vascular SMCs and coronary endothelial
cells.36 37 In our experiments using
AT1- and AT2-specific
receptor antagonists, the receptor subtypes involved in
both the regulation of KDR and the potentiation of VEGF
mitogenic effects in Ang IIstimulated BRECs indicated
that most of these effects were via AT1 receptors
(Figs 4
and 8
). The observed responses are well correlated with the
concept that AT1 mediates proliferative effects
and AT2 elicits antiproliferative responses.
AT1 blockade did not completely block the effects
Ang II on VEGF-induced cell growth, whereas it blocked KDR induction
almost completely. There is a possibility that Ang II might affect
VEGF-elicited signal transduction or posttranscriptional regulation of
KDR. Stoll et al36 have reported that in
coronary endothelial cells,
AT2 mediates antiproliferative effects and
offsets the growth-promoting effects mediated by
AT1, whereas Ang II stimulates the growth of
quiescent vascular SMCs that express only AT1. In
our experiments, Ang II alone had no significant effects on cell
proliferation, suggesting that both AT1 and
AT2 receptors probably exist in BRECs, similar to
coronary endothelial cells.
AT2 inhibition did not affect VEGF-induced growth
nor KDR expression significantly, suggesting that
AT2 receptor mediation is probably not involved
in the regulation of such cell responses in BRECs (Fig 8
).
We made further analyses to delineate the signal transduction
pathway responsible for the effect of Ang II on increases of the KDR
gene. The AT1 receptor is a G proteincoupled
receptor and activates phospholipase C, which is known to
induce the hydrolysis of phosphoinositol and the
activation of PKC.15 Ang IIinduced increases of
several growth factors, such as PDGF A-chain, TGF-ß, basic FGF, IGF
I, and ET-1, are reported to be mediated through
AT1 with activation of
PKC.18 19 20 46 53 In BRECs, KDR induction by Ang
II appeared to be mediated predominantly by a PKC-dependent pathway.
Tyrosine phosphorylation has also been reported to be
elicited by Ang II and PMA in vascular SMCs, glomerular
mesangial cells, and microvessel
endothelial cells.33 43 44 45
Experiments using genistein revealed that tyrosine
phosphorylation is required in both Ang IIstimulated
and PMA-stimulated expression of KDR. Feener et
al33 reported that Ang IIinduced and
PMA-induced plasminogen activator
inhibitor-2 mRNA expression is inhibited by >70% with the
same concentration of genistein in rat epididymal fatderived
microvascular endothelial cells. Since the
inhibitory effect of genistein in BRECs is not so potent,
the tyrosine phosphorylation pathway is probably not so
much involved in the induction of the KDR gene in BRECs as it is in
plasminogen activator inhibitor-2
expression in rat microvascular endothelial cells. The
PKC-independent pathway also contributed to Ang IIinduced KDR mRNA
expression, which is defined as the Ang IIinduced increase in the KDR
mRNA level in the presence of the same concentration of GFX that could
completely reverse PMA-induced change. This pathway accounts for
30% of the total effect of Ang II. The inhibition of this component
by genistein suggests that this pathway also involves tyrosine
phosphorylation.
The time-course study demonstrated that the Ang IIinduced increase of
KDR mRNA was rapid and peaked at 4 hours (Fig 1A
). Nuclear run-on
assays and experiments using actinomycin D to inhibit RNA synthesis
indicate that the effect of Ang II is primarily to increase
transcription of the KDR gene. Moreover, new protein synthesis was
necessary for complete upregulation of KDR mRNA by Ang II (data not
shown). These data suggest that transcriptional regulation of this gene
is mediated through a transacting transcription factor. Similar
transcriptional regulation was observed in tumor necrosis
factor-
induced downregulation of KDR
expression.54 Recent analyses
demonstrated that the 5' flanking region of KDR or the Flk-1 gene
contains several potential binding sites for a transacting
transcription factor, such as activator protein-2, nuclear
factor-
B, or stimulatory protein-1.55 The
PKC-dependent signaling pathway might stimulate transcription of the
KDR gene through some transcriptional factor, such as nuclear
factor-
B, which is activated by the PKC-dependent pathway.
Further studies are to be performed to elucidate the detailed mechanism
of transcriptional regulation of the KDR gene by Ang II.
The increase of KDR mRNA expression was accompanied by new protein synthesis of the receptor. Scatchard analyses further demonstrated an increase of cell surface binding sites for VEGF. We detected no significant effect of Ang II on the affinity of the receptor. Ang II has been reported to regulate several receptors, such as those for IGF I, ET-1, and low density lipoprotein, but regulation of receptor affinities has not been reported.56 57 58 The magnitudes of the Ang IIinduced increases in KDR/Flk-1 mRNA, protein synthesis, and VEGF binding sites correlated well with each other. Although we have not investigated posttranscriptional regulation of KDR by Ang II, such as recycling of the receptor, the observed upregulation of the cell surface receptors probably results from an increase of KDR gene expression induced by Ang II.
Ang II has been reported to regulate cell growth through induction of
several autocrine growth factors, such as PDGF A-chain, TGF-ß, basic
FGF, and IGF I in vascular SMCs18 19 46 53 and
through ET-1 in cardiomyocytes.46 In
vascular SMCs, Ang II is reported to increase the gene expression of
VEGF.34 We examined VEGF mRNA induction by Ang II
to determine whether Ang II induces the endogenous
production of VEGF in BRECs. We did not find any increase in
VEGF mRNA levels (data not shown). Ang II alone had no significant
effect on cell proliferation or tube formation in retinal microvascular
endothelial cells (Figs 8
and 9
). These findings
suggest that Ang II potentiation of VEGF-induced angiogenic activity is
probably not through induction of autocrine growth factors but through
the induction of KDR/Flk-1. However, we cannot exclude the possibility
that a growth factor such as FGF, which has synergism with VEGF in
angiogenic effect,47 might be induced by Ang II
but that its effects are suppressed by antiproliferative growth factor
(such as TGF-ß) induction.53 On VEGF
stimulation, such growth factors might stimulate BRECs more potently
than does Ang II stimulation alone.
Regulation of blood pressure and ocular blood flow by Ang II and regulation of Na+,K+-ATPase by ACE have been suggested as functions of the RAS in the progression of diabetic retinopathy.59 60 Our results suggest a new hypothesis: Ang II potentiates the progression of diabetic retinopathy by stimulating VEGF-induced retinal neovascularization through an increase of VEGF receptor KDR/Flk-1. Besides angiogenic effects, VEGF has been reported to increase vasopermeability and to generate a procoagulant state by the induction of von Willebrand factor and tissue factor.61 62 The upregulation of KDR/Flk-1 expression probably potentiates these functions of VEGF as well as the angiogenic effect in retinal microvascular cells, which might worsen the retinal vascular embolization, exudation, and macular edema in diabetic retinopathy. In the macrovascular milieu, we have demonstrated that Ang II upregulates KDR mRNA expression in BAECs. In addition, Williams et al34 have reported that Ang II increases VEGF mRNA expression in vascular SMCs. These observations suggest upregulation of the VEGF paracrine system in large vessel walls, which probably plays a prominent role in vascular injury such as atherosclerosis.
From a clinical standpoint, our data suggest that inhibition of RAS is beneficial for the treatment of diabetic retinopathy. An ACE inhibitor, captopril, was recently reported to suppress neovascularization by directly inhibiting migration of vascular endothelial cells.63 Such drugs and an AT1 blocker might be proven to be effective in the prevention of diabetic retinopathy.
Selected Abbreviations and Acronyms
|
Acknowledgments
This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of the Japanese Government and by the Japan Association for Inhibition of Blindness. The authors thank MSD (Merck Sharp & Dohme Research Laboratories Japan) for providing DuP735. We also thank Dr George L. King for his helpful discussion.
Received November 17, 1997; accepted January 29, 1998.
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R. A. de Boer, Y. M. Pinto, A. J.H. Suurmeijer, S. Pokharel, E. Scholtens, M. Humler, J. M. Saavedra, F. Boomsma, W. H. van Gilst, and D. J. van Veldhuisen Increased expression of cardiac angiotensin II type 1 (AT1) receptors decreases myocardial microvessel density after experimental myocardial infarction Cardiovasc Res, February 1, 2003; 57(2): 434 - 442. [Abstract] [Full Text] [PDF] |
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H. Takagi, S. Koyama, H. Seike, H. Oh, A. Otani, M. Matsumura, and Y. Honda Potential Role of the Angiopoietin/Tie2 System in Ischemia-Induced Retinal Neovascularization Invest. Ophthalmol. Vis. Sci., January 1, 2003; 44(1): 393 - 402. [Abstract] [Full Text] [PDF] |
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W D. Strain and N. Chaturvedi Review: The renin-angiotensin-aldosterone system and the eye in diabetes Journal of Renin-Angiotensin-Aldosterone System, December 1, 2002; 3(4): 243 - 246. [Abstract] [PDF] |
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X. Zhao, X. Lu, and Q. Feng Deficiency in endothelial nitric oxide synthase impairs myocardial angiogenesis Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2371 - H2378. [Abstract] [Full Text] [PDF] |
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S. Jesmin, Y. Hattori, I. Sakuma, C. N. Mowa, and A. Kitabatake Role of ANG II in coronary capillary angiogenesis at the insulin-resistant stage of a NIDDM rat model Am J Physiol Heart Circ Physiol, October 1, 2002; 283 (4): H1387 - H1397. [Abstract] [Full Text] [PDF] |
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F Mori, T Hikichi, T Nagaoka, J Takahashi, N Kitaya, and A Yoshida Inhibitory effect of losartan, an AT1 angiotensin II receptor antagonist, on increased leucocyte entrapment in retinal microcirculation of diabetic rats Br J Ophthalmol, October 1, 2002; 86(10): 1172 - 1174. [Abstract] [Full Text] [PDF] |
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T. Takagi, Y. Nakano, S. Takekoshi, T. Inagami, and M. Tamura Hemizygous mice for the angiotensin II type 2 receptor gene have attenuated susceptibility to azoxymethane-induced colon tumorigenesis Carcinogenesis, July 1, 2002; 23(7): 1235 - 1241. [Abstract] [Full Text] [PDF] |
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H. Ando, T. Nagasaka, M. Nomura, S.-I. Tsukahara, Y. Kotani, S. Toda, Y. Murata, A. Itakura, and S. Mizutani Premenstrual Disappearance of Aminopeptidase A in Endometrial Stromal Cells around Endometrial Spiral Arteries/Arterioles during the Decidual Change J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2303 - 2309. [Abstract] [Full Text] [PDF] |
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H Funatsu, H Yamashita, Y Nakanishi, and S Hori Angiotensin II and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy Br J Ophthalmol, March 1, 2002; 86(3): 311 - 315. [Abstract] [Full Text] [PDF] |
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R. Tamarat, J.-S. Silvestre, N. Kubis, J. Benessiano, M. Duriez, M. deGasparo, D. Henrion, and B. I. Levy Endothelial Nitric Oxide Synthase Lies Downstream From Angiotensin II-Induced Angiogenesis in Ischemic Hindlimb Hypertension, March 1, 2002; 39(3): 830 - 835. [Abstract] [Full Text] [PDF] |
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J. L. Segar, G. B. Dalshaug, K. A. Bedell, O. M. Smith, and T. D. Scholz Angiotensin II in cardiac pressure-overload hypertrophy in fetal sheep Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R2037 - R2047. [Abstract] [Full Text] [PDF] |
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S. L. Amaral, P. E. Papanek, and A. S. Greene Angiotensin II and VEGF are involved in angiogenesis induced by short-term exercise training Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1163 - H1169. [Abstract] [Full Text] [PDF] |
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A. Otani, H. Takagi, H. Oh, S. Koyama, and Y. Honda Angiotensin II Induces Expression of the Tie2 Receptor Ligand, Angiopoietin-2, in Bovine Retinal Endothelial Cells Diabetes, April 1, 2001; 50(4): 867 - 875. [Abstract] [Full Text] |
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M. Lonchampt, L. Pennel, and J. Duhault Hyperoxia/Normoxia-Driven Retinal Angiogenesis in Mice: A Role for Angiotensin II Invest. Ophthalmol. Vis. Sci., February 1, 2001; 42(2): 429 - 432. [Abstract] [Full Text] |
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I. Suzuma, Y. Hata, A. Clermont, F. Pokras, S. L. Rook, K. Suzuma, E. P. Feener, and L. P. Aiello Cyclic Stretch and Hypertension Induce Retinal Expression of Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor--2: Potential Mechanisms for Exacerbation of Diabetic Retinopathy by Hypertension Diabetes, February 1, 2001; 50(2): 444 - 454. [Abstract] [Full Text] |
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O. Saijonmaa, T. Nyman, R. Kosonen, and F. Fyhrquist Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H885 - H891. [Abstract] [Full Text] [PDF] |
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S. Fujiyama, H. Matsubara, Y. Nozawa, K. Maruyama, Y. Mori, Y. Tsutsumi, H. Masaki, Y. Uchiyama, Y. Koyama, A. Nose, et al. Angiotensin AT1 and AT2 Receptors Differentially Regulate Angiopoietin-2 and Vascular Endothelial Growth Factor Expression and Angiogenesis by Modulating Heparin Binding-Epidermal Growth Factor (EGF)-Mediated EGF Receptor Transactivation Circ. Res., January 19, 2001; 88(1): 22 - 29. [Abstract] [Full Text] [PDF] |
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C. J. Moravski, D. J. Kelly, M. E. Cooper, R. E. Gilbert, J. F. Bertram, S. Shahinfar, S. L. Skinner, and J. L. Wilkinson-Berka Retinal Neovascularization Is Prevented by Blockade of the Renin-Angiotensin System Hypertension, December 1, 2000; 36(6): 1099 - 1104. [Abstract] [Full Text] [PDF] |
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P. L. SINN and C. D. SIGMUND Identification of three human renin mRNA isoforms from alternative tissue-specific transcriptional initiation Physiol Genomics, June 29, 2000; 3(1): 25 - 31. [Abstract] [Full Text] [PDF] |
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T. P. Gavin, D. A. Spector, H. Wagner, E. C. Breen, and P. D. Wagner Effect of captopril on skeletal muscle angiogenic growth factor responses to exercise J Appl Physiol, May 1, 2000; 88(5): 1690 - 1697. [Abstract] [Full Text] [PDF] |
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A. Otani, H. Takagi, H. Oh, K. Suzuma, M. Matsumura, E. Ikeda, and Y. Honda Angiotensin II-Stimulated Vascular Endothelial Growth Factor Expression in Bovine Retinal Pericytes Invest. Ophthalmol. Vis. Sci., April 1, 2000; 41(5): 1192 - 1199. [Abstract] [Full Text] |
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S. Kim and H. Iwao Molecular and Cellular Mechanisms of Angiotensin II-Mediated Cardiovascular and Renal Diseases Pharmacol. Rev., March 1, 2000; 52(1): 11 - 34. [Abstract] [Full Text] [PDF] |
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M. Muramatsu, J. Katada, I. Hayashi, and M. Majima Chymase as a Proangiogenic Factor. A POSSIBLE INVOLVEMENT OF CHYMASE-ANGIOTENSIN-DEPENDENT PATHWAY IN THE HAMSTER SPONGE ANGIOGENESIS MODEL J. Biol. Chem., February 25, 2000; 275(8): 5545 - 5552. [Abstract] [Full Text] [PDF] |
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H. Oh, H. Takagi, K. Suzuma, A. Otani, M. Matsumura, and Y. Honda Hypoxia and Vascular Endothelial Growth Factor Selectively Up-regulate Angiopoietin-2 in Bovine Microvascular Endothelial Cells J. Biol. Chem., May 28, 1999; 274(22): 15732 - 15739. [Abstract] [Full Text] [PDF] |
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M. Thibonnier, D. M. Conarty, J. A. Preston, C. L. Plesnicher, R. A. Dweik, and S. C. Erzurum Human Vascular Endothelial Cells Express Oxytocin Receptors Endocrinology, March 1, 1999; 140(3): 1301 - 1309. [Abstract] [Full Text] |
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F. C. Luft, E. Mervaala, D. N. Muller, V. Gross, F. Schmidt, J. K. Park, C. Schmitz, A. Lippoldt, V. Breu, R. Dechend, et al. Hypertension-Induced End-Organ Damage : A New Transgenic Approach to an Old Problem Hypertension, January 1, 1999; 33(1): 212 - 218. [Abstract] [Full Text] [PDF] |
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