© 1999 American Heart Association, Inc.
Original Contribution |
Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration and Proliferation Depend on a Nitric Oxide–Mediated Decrease in Protein Kinase C
Activity
From the Cardiovascular Division, Department of Medicine (Y.S., S.T., R.Y., J.A.W.), and the Department of Molecular Pharmacology (J.A.W.), Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY.
Correspondence to J. Anthony Ware, MD, Albert Einstein College of Medicine, Forchheimer Bldg, G-46, 1300 Morris Park Ave, Bronx, NY 10461. E-mail jaware{at}aecom.yu.edu
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Abstract |
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Abstract—Vascular endothelial growth factor (VEGF) promotes angiogenesis and endothelial cell (EC) migration and proliferation by affecting intracellular mediators, only some of which are known, distal to its receptors. Protein kinase C (PKC) participates in the function of VEGF, but the role of individual PKC isoenzymes is unknown. In this study, we tested the importance of the activity of specific PKC isoenzymes in human EC migration and proliferation in response to VEGF. PKC
specific activity was depressed by the addition of VEGF (by
41±8% [P<0.05] at 24 hours) in human umbilical vein ECs (HUVECs)
and in a HUVEC-derived EC line, ECV, without changing the total amount
of either protein or mRNA encoding PKC. Neither basic fibroblast
growth factor (FGF-2) nor serum altered PKC specific
activity. The VEGF-induced decrease of PKC activity, which began at
8 hours after stimulation, was strongly blocked by pretreatment with
the nitric oxide (NO) synthase inhibitor
NG-monomethyl-L-arginine
in HUVECs; NO release peaked within 2 hours after stimulation. An
exogenous NO donor, sodium nitroprusside, also decreased PKC
activity. The inhibition by
NG-monomethyl-L-arginine
of VEGF-induced HUVEC migration and proliferation, but not that induced
by FGF-2 or serum, suggested that the decrease in PKC via NO pathway
is required for VEGF-induced EC migration and proliferation.
Overexpression of PKC in ECV cells specifically prevented EC
response to VEGF but not to FGF-2 or serum. Thus, we conclude that
suppression of PKC activity via a NO synthase mechanism is required
for VEGF-induced EC migration and proliferation, but not for that
induced by FGF-2 or serum.
Key Words: endothelium • growth factor • nitric oxide synthase • intracellular signaling • angiogenesis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vascular endothelial growth factor (VEGF) strongly induces both physiological and pathological angiogenesis1 2 and has been used clinically to promote angiogenesis in patients with limb ischemia.3 Physiological responses to VEGF that can contribute to angiogenesis include mitogenic and promigratory effects, chemotaxis, and release of proteolytic enzymes and their inhibitors.4,5 VEGF differs from other growth factors, including basic fibroblast growth factor (FGF-2), because it can increase vascular permeability, but its ability to induce capillary tube formation appears to be synergistic with FGF-2.4
The biological effects of VEGF on endothelial cells (ECs) are mediated by the specific membrane receptors KDR and flt-1, both of which are class III tyrosine kinase–linked receptors.6 7 The precise signaling pathways that mediate those responses have not yet been elucidated; however, phospholipase D8 and nitric oxide (NO) synthase9 have been identified as key enzymes activated by VEGF. These 2 enzymes have been shown to depend on protein kinase C (PKC) activity8 and increased binding of heat shock protein 90,10 respectively, to effect EC responses.
The PKC family consists of a large group of individual
phospholipid-dependent serine-threonine kinase isoenzymes involved in
cellular signaling.11 Inhibition of PKC activity with the
chemical inhibitor calphostin prevents VEGF-induced
vascular growth,12 but the role of specific isoenzymes in
the processes that constitute angiogenesis is unknown, because the
specificity of most PKC inhibitors for individual
isoenzymes has not been established. Overexpression or inhibition of
some of the individual PKC isoenzymes, including PKC
, PKC, and
PKC
, have diverse effects on EC migration and
proliferation.13 14 In particular, an increase in PKC
activity reduces serum-induced proliferation of ECs by slowing passage
through the S phase of the cell cycle in rat microvascular
ECs.13 In contrast, inhibition of PKC prevents
serum-induced EC proliferation and migration,14 whereas
enhancing PKC activity does not affect proliferation but promotes EC
migration.13 Thus, each PKC isoenzyme appears to have
specific substrates and activators and to mediate distinct
EC events that constitute angiogenesis.
Which, if any, of the specific PKC isoenzymes is critical for
VEGF-induced EC function is not known. We chose to investigate the role
of PKC in VEGF-induced EC proliferation and migration, because NO,
which is a key mediator generated by VEGF, reduces PKC activity in
peritoneal macrophages,15 and because of the
inhibitory effect of enhanced PKC expression on
proliferation and cell cycle progression in rat microvascular
cells.13 To determine the specificity of these effects for
VEGF signaling, we compared the importance of PKC and NO synthase in
EC function in response to another angiogenic growth factor, FGF-2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Cultures, Growth Factors, and Other Materials
Human umbilical vein ECs (HUVECs) were cultured in M199 medium containing 20% newborn calf serum (NCS, GIBCO-BRL), 5% human serum (Gemini Bio-Products, Inc), 50 µg/mL ascorbic acid, 1.6 mmol/L L-glutamine, 5 µg/mL bovine brain extract (Clonetec Corp), 7.5 µg/mL endothelial growth supplement (Sigma), 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 U/mL heparin. HUVECs of the third to fifth passage were used for all experiments. Subconfluent HUVECs were synchronized with the above-mentioned media with reduced NCS (10%) and lacking endothelial growth supplements. After 24 hours of synchronization, VEGF (a gift from Dr Michael Simons, Beth Israel Deaconess Medical Center, Boston, Mass) in a final concentration of 20 ng/mL, FGF-2 (Sigma, 20 ng/mL), or 20% NCS was added to the cell media depending on the experimental protocol. In the experiments in which a NO synthase inhibitor was used, NG-monomethyl-L-arginine (L-NMMA, Sigma) was added to the media at a final concentration of 500 µmol/L. In experiments to investigate effects of suppression of total PKC activity on EC migration and proliferation, 100 nmol/L phorbol 12-myristate 13-acetate (PMA; Sigma) was added to media 24 hours before the experimental protocols to downregulate PKC activity. In experiments in which a NO donor was used, sodium nitroprusside (SNP; Sigma) at the final concentration of 500 µmol/L to 1 mmol/L was added at the time that the protocols began.
Immortalized human ECs (ECV cells, obtained from American Type Culture Collection, Manassas, VA) were cultured in M199 (GIBCO-BRL) supplemented with 10% FBS (GIBCO-BRL) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). Subconfluent cells were synchronized for 24 hours in serum-free media before the addition of growth factors. Growth factors were added as described above, except that 10% FBS was used instead of NCS for serum stimulation.
ECV cells were used in experiments in which PKC was overexpressed.
The full-length cDNA encoding PKC was cloned into pcDNA3 mammalian
expression vector (Invitrogen) using the EcoRI restriction
endonucleotide enzyme site. The construct was transfected
into ECs using the lipofectin method (GIBCO-BRL). Cells that stably
overexpressed either the vector alone or PKC were selected by
resistance to neomycin.
Immunoblot Analysis
Cell lysates were prepared by addition of 2 mL of lysis buffer
(PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.57 mol/L
phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, and
4.2 µmol/L leupeptin) per 1x107 cells.
The immunoblot was performed using an anti-PKC–specific
monoclonal antibody (Transduction Laboratories) as previously
described.14
Northern Transfer Analysis
Subconfluent monolayers of ECs in 100-mm plates were treated
with PBS twice and then treated with Trizol reagent (GIBCO-BRL). Twenty
micrograms of total RNA extracted was subjected to electrophoresis on a
1.5% formaldehyde-agarose gel and transferred to a nylon-based
transfer membrane (GeneScreen Plus) according to the
manufacturer's recommendation. The blot was hybridized with a
random-primed cDNA probe at 65°C for 3 hours in a rapid Northern
hybridization solution (Quick-Hyb, Stratagene, Inc, La Jolla, Calif).
Blots were washed under high-stringency conditions and subjected to
autoradiography.
Kinase Activity Assay
Subconfluent monolayers of ECs (in 100-mm plates) were treated
with trypsin, counted, and then washed with PBS. HUVECs
(4.0x106) or ECV cells
(2.0x106) were resuspended in 1 mL of ice-cold
lysis buffer for 10 minutes and were then homogenized by
repeated aspiration through a 21-gauge needle. Cell debris was removed
by centrifugation at 3500 rpm at 4°C for 15
minutes.
To determine the specific activity of PKC, the PKC was
immunoprecipitated using an anti-PKC–specific monoclonal antibody
(Transduction Laboratories) from the whole-cell lysate. The kinase
assay was carried out according to the methods described
previously.14 The reaction mixture did not contain
additional calcium acetate for this assay. The presence of PKC was
confirmed by immunoblotting with an anti-PKC
antibody. PKC activity was normalized to the cell number and expressed
as the percentage of PKC activity measured simultaneously
in unstimulated ECs that were cultured in the absence of serum and
growth factors for 2 days. In the study, PKC specific activity was
measured, cell lysates were immunoprecipitated with a polyclonal
antibody against PKC (Santa Cruz Biotechnology Inc), and the
specific activity was measured with the method described above, except
the reaction buffer contained 3 mmol/L calcium acetate.
RT-PCR of VEGF Receptors
RT-PCR was performed with the SuperScript One-Step RT-PCR System
(GIBCO-BRL) according to the manufacturer's instructions. A PCR cycle
(94°C for 1 minute, 50°C for 1.5 minutes, and 72°C for 1 minute)
was repeated for 35 cycles. To detect the VEGF receptor 1 (flt-1) and
VEGF receptor 2 (KDR/flk-1), the following primers were used,
respectively, as previously reported: sense primer
5'-CAGCGGCTTTTGTGGAAGACTCAC-3' and antisense primer
5'-ACTTCTCGGTGTCACTTCTTGGAC-3' (corresponding to 735 bp), and sense
primer 5'-CAACAAAGTCGGGAGAGGAG-3' and antisense primer
5'-ATGACGATGGACAAGTAGCC-3' (corresponding to 819 bp).16 As
a positive control, mRNA of GAPDH was amplified by using sense
primer 5'-TGAAGGTCGGAGTCAACGGATTTG-3' and antisense primer
5'-CATGTGGGCCATGAGGTCCACCAC-3' (corresponding to 983
bp).16
Measurement of Total NO Release
The ECs were cultured in 6-well plates (Corning Glass Works).
After subconfluence was achieved, ECs were synchronized for 24 hours as
previously described, and then medium was replaced with medium without
phenol red in the presence and absence of VEGF. After incubation in 30
minutes to 24 hours, ECs were washed twice with PBS and replaced with
fresh medium. After a 30-minute incubation with fresh medium, this
medium was collected and total NO was measured with a nitrate/nitrite
colorimetric assay kit (Cayman Chemical) according to
the manufacturer's instruction. The absorbency at 540 nm was measured
with a plate reader. The measured amount (nmol) was divided by 30 and
normalized to the number of ECs in the well from which the medium was
collected.
Cell Proliferation Analysis
EC growth was determined by counting the cells with a
hemocytometer under x50 magnification. Subconfluent cells were seeded
at 2.0x104 per 16-mm plate in 1 mL of medium and
synchronized for 24 hours before the addition of a growth factor. After
the indicated periods, the ECs were washed with PBS, treated with
trypsin, and suspended in medium for counting.
Endothelial Migration Assay
ECs were cultured on Corning 6-well plates in complete medium
until confluent. After a 24-hour synchronization, the monolayer was
wounded by scraping 
800 µm with a 200-µL pipette tip
(Continental Laboratory Products, Inc). The distance of the gap was
measured as previously described.14
Statistical Analysis
All data are presented as mean±SE. Comparisons of the
effects among the different growth factors, time points, and cell lines
were performed by ANOVA. Multigroup comparison was carried out using
Bonferroni-modified t tests. P<0.05 was accepted
as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PKC
Specific Activity After Growth Factor Stimulation in
HUVECsIn experiments to test whether the activities of PKC
were
changed by serum or growth factors in HUVECs, VEGF decreased PKC
specific activity to 58.8±7.5% of the control value (n=9,
P<0.05). Neither FGF-2 nor serum, on the other hand,
influences the PKC specific activity significantly (Figure 1A
). Similar experiments were conducted
in the presence of the NO synthase inhibitor L-NMMA in
HUVECs. Suppression of PKC activity by VEGF was completely blocked
by L-NMMA. PKC activity with FGF-2 or serum stimulation was not
affected by NO inhibition (Figure 1B). Thus, the suppression of
PKC activity by VEGF required NO synthase. In contrast to its
effects in PKC, VEGF increased PKC specific activity
significantly after 24-hour stimulation (136.1±10.8% [n=4,
P<0.05 versus unstimulated controls in HUVECs] and
123.3±5.5% [n=4, P<0.05 versus unstimulated controls in
ECV cells]) as previously reported.17
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Time Course of NO Release and PKC Suppression With VEGF in
HUVECs and ECV Cells
In HUVECs, PKC specific activity started to decline 8 hours
after VEGF stimulation after a peak NO within 2 hours after VEGF
stimulation. The suppression of PKC activity reached its maximum at
16 hours of treatment (Figure 2A). A
similar relation between PKC activity and NO release was seen in ECV
cells (Figure 2B). In ECV cells, PKC activity seemed to
decrease somewhat faster than that in HUVECs and reached a steady state
at 8 hours of treatment with VEGF. These results demonstrate that NO
release peaks before the decrease in PKC activity with VEGF in both
HUVECs and ECV cells and that PKC specific activity decreases
significantly in 8 hours of treatment.
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denotes measurement with VEGF;
•, measurement with VEGF in the presence of L-NMMA; and 
,
unstimulated cells. *P<0.05 vs unstimulated cells;
§P<0.05 vs VEGF with L-NMMA. Data are from 6 to 9
experiments.
PKC Specific Activity After SNP Treatment
To assess whether an exogenous NO donor can suppress PKC
specific activity, SNP at 500 µmol/L to 1 mmol/L was added
to the medium and incubated for 24 hours. PKC specific activity of
HUVECs decreased significantly with 1 mmol/L of SNP in HUVECs
(84.5±2.0% of unstimulated cells, n=6, P<0.05). A
significant decrease in PKC specific activity was seen also in ECV
cells with 500 µmol/L (Figure 3).
This result further supports the finding that release of NO decreases
PKC activity.
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PKC Protein and mRNA After Growth Factor Stimulation With
VEGF Stimulation
Possibilities for the decrease in PKC enzymatic activity after
VEGF stimulation include a VEGF-mediated reduction in the levels of
mRNA or protein corresponding to PKC, or posttranslational
mechanisms. Immunoblot analysis failed to detect
changes in the amount of PKC protein in HUVECs (Figure 4A) and ECV cells (Figure 4B).
Similarly, Northern transfer analysis did not show a change in
mRNA encoding PKC in ECs after stimulation with VEGF in either
HUVECs (Figure 4A) or ECV cells (Figure 4B). Thus, the
decrease in PKC activity after VEGF stimulation was not modulated by
a change of translation or transcription of PKC, which suggests that
the change in enzymatic activity occurred at the posttranslation
level.
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Expression of VEGF Receptors in HUVECs and ECV Cells
Next, we asked whether these effects of VEGF could be attributed
to a specific receptor tyrosine kinase that interacts with VEGF. We
found that HUVECs expressed both flt-1 and KDR/flk-1 receptor as
previously described (Reference 1818 , Figure 5A); however, ECV cells expressed flt-1
but not KDR/flk-1 (Figure 5B). The similar modulation of PKC
by VEGF in both HUVECs and ECV cells suggests that KDR/flk-1 receptor
is not required for the decrease in PKC activity by VEGF and for
VEGF-induced EC migration and proliferation.
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Effects of NO Synthase Inhibition on VEGF- or FGF-2–Mediated EC
Migration and Proliferation in HUVECs
As seen in Figure 6, addition
of VEGF, FGF-2, or serum for 12 hours stimulates HUVECs migration, as
measured in an EC wound healing assay. In Figure 6A, the NO
synthase inhibitor L-NMMA reduced the VEGF-induced HUVEC
migration to the level of that seen in unstimulated cells at 12 hours.
L-NMMA did not significantly reduce migration meditated by either FGF-2
or serum (Figure 6B and 6C).
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VEGF, FGF-2, and serum increased the number of HUVECs significantly
(Figure 6D
through 6F). The increase in HUVECs by VEGF was
blocked by L-NMMA. The mitogenic effect of FGF-2 or serum,
on the other hand, was not suppressed by L-NMMA (Figure 6E and 6F). Thus, HUVEC proliferation induced by VEGF, but not that induced by
FGF-2 or serum, required the activation of NO synthase.
Response of ECs to PKC Downregulation by PMA
PMA (100 nmol/L) was added to media of HUVECs at 24 hours
before migration and proliferation were measured to investigate whether
suppression of total PKC effects on growth factor induced migration and
proliferation of HUVECs. PMA downregulates total PKC activity
to <8% in HUVECs treated for 16 hours.19
Migration induced by VEGF was significantly blunted (Figure 7A); however, HUVEC migration induced by
FGF-2 or serum was not altered (Figure 7B and 7C).
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The effect of total PKC downregulation on growth factor-induced HUVEC
proliferation is shown in Figure 7D through 7F. Proliferation of
HUVECs by VEGF, FGF-2, or serum was almost abolished. These findings
suggest that the migratory responses of HUVECs to growth factors were
less PKC dependent than were proliferation responses to growth factors,
except for those that induced by VEGF.
Response of PKC-Overexpressing ECs to VEGF, FGF-2, and
Serum
To determine whether VEGF-mediated PKC suppression was required
for VEGF-induced EC migration and proliferation, we prevented such
suppression by overexpression of PKC in stable clones of ECs. VEGF
did not significantly increase either migration (Figure 8A) or proliferation (Figure 8D)
of the PKC-overexpressing ECs, in contrast to the case of those ECs
that overexpressed vector alone. PKC did not inhibit unstimulated EC
migration or proliferation. Interestingly, PKC-overexpressing ECs,
rather than showing inhibited migration and proliferation to FGF-2,
actually demonstrated somewhat enhanced migration to this growth factor
(Figure 8B) and no effect on serum-induced EC migration and
proliferation (Figure 8C and 8F), which suggests that the
prevention of proliferation and migration to VEGF was not a nonspecific
effect of PKC overexpression on EC function. Thus, suppression of
PKC activity appears to be required for VEGF-induced EC migration
and proliferation, but not that induced by FGF-2 or serum.
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) growth factor stimulation.
Circles denote PKC |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The major findings of this study are that the enzymatic activity of a specific PKC isoenzyme, PKC
, is decreased by VEGF and that this
decrease is mediated by the activation of NO synthase in ECs. This
process does not require the KDR/flk-1 receptor for VEGF.
Overexpression of PKC blunted the ability of VEGF to induce EC
migration and proliferation, which suggests that the decrease in PKC
activity is necessary for VEGF to induce those events. Our study
confirms and extends the recent observation9 that
VEGF-induced EC migration and proliferation are blocked effectively by
NO synthase inhibition. The present investigation suggests that NO
works at a posttranslational level to decrease VEGF-induced EC
migration and proliferation, at least in part by reducing PKC
activity.
The dependence of VEGF on this PKC-NO mechanism is not shown by all
EC mitogens; FGF-2–induced EC proliferation and migration were not
influenced by either NO inhibition or PKC overexpression in our
study. The lack of effect on FGF-2 and serum provides a useful control
as well; the ability of PKC overexpression to prevent VEGF-induced
EC migration and proliferation is unlikely to reflect a nonspecific
interference with the EC cell cycle or migratory mechanisms, because a
similar effect after FGF-2 and serum would be expected under those
circumstances. Our results demonstrate that ECs in which PKC was
overexpressed had responses to FGF-2 or serum that equaled or exceeded
the responses of control ECs. Therefore, it appears that PKC
suppression was required for VEGF to induce EC migration and
proliferation but not for FGF-2 or serum to induce similar effects.
Thus, the present study provides additional evidence that VEGF and
FGF-2 use different intracellular mediators to effect EC functions
linked to angiogenesis. Previously, 2 distinct pathways to angiogenesis
were identified that used different integrin receptors for
vitronectin, as follows: FGF-2 and tumor necrosis
factor- trigger a pathway that requires interaction with the
integrin Vß3; VEGF, on
the other hand, uses a pathway that depends on integrin
Vß5.12 We
speculate that the requirement for NO and PKC suppression for VEGF,
but not for FGF-2, may reflect differing control mechanisms for these 2
vitronectin receptors. Our finding that enhanced EC
function by FGF-2 or serum did not depend on either the activation of
NO synthase or a reduction in PKC activity suggests that the
downstream signals between VEGF and FGF-2 can be clearly separated and
supports distinctive intracellular pathways for each growth factor.
PKC overexpression did not alter the serum-induced proliferation of
ECV cells, which differed from the results obtained previously in rat
microvascular ECs. Several variations in the cellular physiology
between large vessel–derived ECs, such as HUVECs and microvascular
ECs, have been reported. Such differences include surface adhesion
molecule expression,21 prostanoid
production,22 and cytokines such as
interleukin-1 and tumor necrosis factor-.23 Our finding
suggests that signaling of serum-induced EC proliferation may also
differ between HUVECs and rat microvascular ECs. Also, it is possible
that the response to PKC-overexpressing ECs may be attenuated to
certain growth factors, such as VEGF, and responses to another growth
factor, such as FGF-2, are enhanced as a compensatory mechanism.
Stimulation by serum reflects the combined effects of the multiple
growth stimulants present in serum and thus may represent
conflicting effects of individual growth factors.
Our results suggest that VEGF-induced EC migration and proliferation
are totally dependent on NO synthase activation, in agreement with a
recent study.9 The present study demonstrates that the
suppression of PKC activity after VEGF stimulation also depends on
NO synthase activation. Exactly how reduction of PKC activity
facilitates VEGF-induced EC proliferation and migration is unknown. It
cannot be ruled out that NO limits VEGF-induced function by means other
than reducing PKC activity, although it appears that such reduction
is necessary. NO has been shown to alter cell cycle progression induced
by a number of agents. NO inhibits proliferation of vascular smooth
muscle cells by inhibiting cyclin-dependent kinase 2, which is required
for cell cycle progression,24 but whether this occurs via
a reduction in PKC activity is not clear, nor is it known whether a
similar or inverse relationship exists between NO and cyclin-dependent
kinase 2 in ECs. Another possibility is that PKC affects EC function
by altering events downstream of Ras. PKC activates
Ras-dependent signal transduction; however, the inability of
concomitant overexpression of Ras to alter the inhibitory
effect of PKC in NIH3T3 cells25 suggests that
additional effectors for PKC outside of the Ras-dependent pathway
are important for the suppression of cell growth.
The precise mechanism of how VEGF decreases PKC activity via NO is
not known. In murine peritoneal macrophages,
lipopolysaccharide with interferon-
results in a significant
increase of NO release that is associated with suppression of mRNA
encoding PKC.15 In our study, we found that activation
of NO synthase was associated with a reduction in PKC specific
activity after VEGF stimulation in ECs, but we did not observe changes
in the level of either protein or mRNA encoding PKC, in contrast to
the results in macrophages. Only the activity of the specific
PKC isoenzyme was altered by VEGF and could be correlated with EC
function, rather than transcription or translation of the PKC
isoenzyme. Many PKC isoenzymes can be regulated by serine-threonine
phosphorylation26 ; thus, it is possible
that cyclic GMP-dependent protein kinase, which is activated by
NO and can regulate overall PKC activity,27 might be
involved in reduction of PKC activity after VEGF stimulation. In
addition, PKC has a novel feature, in that it can become
phosphorylated on a tyrosine residue in the catalytic
domain.28 One could speculate that the selective effect of
VEGF on PKC activity might be related to its ability to alter
tyrosine phosphorylation of this isoenzyme.
Nonspecific downregulation of all phorbol ester–sensitive isoenzymes
of PKC blocked the induction of migration induced by VEGF and
proliferation induced by VEGF, FGF-2, or serum in our study. These
findings indicate that downregulation of some isoenzymes of PKC other
than PKC inhibit the migratory and mitogenic effects of
VEGF, in contrast to the enhancement of VEGF-induced migration and
proliferation caused by downregulation of PKC. With VEGF
stimulation, the activity of those yet-unidentified isoenzymes might be
increased or unchanged. It is also possible that such isoenzymes might
be involved in the proliferation induced by other growth factors such
as FGF-2 or serum. Therefore, previous results that have been based on
the use of nonspecific inhibitors or activators
of PKC must be interpreted cautiously, because changes in the activity
of individual PKC isoenzymes might be obscured.
In this study, the activity of PKC isoenzymes is assessed by direct
enzymatic assay. In a previous report,17 VEGF up-regulates
PKC activity as shown by translocation of PKC in HUVECs. Although
translocation of PKC by immunoblot analysis was
shown, translocation of PKC did not occur. In our study, the
enzymatic assay used to detect the decrease in PKC activity also
showed the increase in PKC in HUVECs and ECV cells stimulated with
VEGF for 24 hours, which suggests that the enzymatic assay of
individual PKC isoenzymes may reflect at least one aspect of PKC
activation more sensitively than does translocation by
immunoblotting.
Our results suggest that KDR/flk-1 receptor is not required for the
decrease in PKC activity. When VEGF receptors were overexpressed in
porcine ECs, which have no intrinsic VEGF receptors, the KDR/flk-1
receptor can mediate VEGF-induced proliferation.29 A
recent report, however, suggests that flt-1 receptor mediates
VEGF-induced NO release in both human trophoblasts and
HUVECs.30 Taken together with our data, it would seem that
the KDR/flk-1 receptor, although perhaps sufficient, is not necessary
for the VEGF-induced response in ECs. It is also possible that a VEGF
receptor as yet unidentified may mediate the VEGF-induced NO
release.
In conclusion, we have demonstrated that VEGF-induced EC migration and
proliferation depends on the reduction of PKC activity, an effect
that requires the activation of NO synthase. The KDR/flk-1 receptor for
VEGF is not required for this process. In contrast, the ability of
FGF-2 or serum to induce these effects did not depend on either PKC
suppression or NO synthase. These findings highlight the differences in
signal transduction between different angiogenic factors, which
suggests that a therapeutic strategy based on activation or inhibition
of intracellular mediators could provide an additional control over the
angiogenic response.
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Acknowledgments |
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This study was supported by grants (HL 51043 and HL 47032) from the National Heart, Lung, and Blood Institute.
Received March 26, 1999; accepted May 10, 1999.
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References |
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