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Division of Endocrinology, Veterans Affairs Medical Center, Long Beach, California 90822; and Departments of Medicine (A.P., M.R., E.R.L.) and Pharmacology (E.R.L.), University of California, Irvine, California 92717
Address all correspondence and requests for reprints to: Ellis R. Levin, M.D., Medical Service (11/111-I), Long Beach Veterans Affairs Medical Center, 5901 East 7th Street, Long Beach, California 90822. E-mail: ellis.levin{at}med.va.gov
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Flk receptors in particular phosphorylate and activate membrane-associated kinases, such as Src and phosphoinositol 3-kinase (4, 6). Complexes of signal molecules/adapter proteins/GTP exchange factors, such as Shc-Grb2-Nck, assemble in response to the ligation of Flk and transmit activating signals to the mitogen-activated protein (MAP) kinase extracellular signal-regulated protein kinase (ERK), in part through the generation of nitric oxide (9). This leads to ERK- dependent proliferation in cells expressing transfected KDR (10). VEGF activation of c-Jun N-terminal kinase (JNK) has also been demonstrated, and it has been recently described that VEGF stimulation of ERK activity is necessary for the subsequent activation of JNK in EC (11). This occurred when VEGF-induced ERK led to the upstream activation of SEK-1 (JNK kinase), which resulted in JNK activation. JNK was shown to be the final effector of the ability of VEGF to induce cell cycle progression (11).
Relevant to a discussion of modulators of vascular biology, the natriuretic peptides (NP) are a family of small proteins that modulate salt and water balance, as well as vascular tone (reviewed in Ref. 12). Atrial and brain natriuretic peptides (ANP and BNP) are produced predominantly in the heart, whereas C-type NP (CNP) is synthesized by the endothelial cell. Both circulating and locally produced NP have been shown to inhibit vascular cell growth as well as regulate vessel tone. These peptides act through binding two classes of receptors. Most biological functions of the NP occur after binding the guanylate cyclase A (reactive to ANP or BNP) or B (CNP-specific) receptors (13, 14). This stimulates the production of cGMP and subsequent activation of protein kinase G (PKG), to stimulate target genes or the modulation of K+ channels (14). Alternatively, a second class of NP receptors is the clearance receptor (NPRC) (15), and this protein may modulate other aspects of vascular biology. It has been reported that the NP act to inhibit cardiomyocyte, vascular EC, or astrocyte proliferation in part through this receptor (16, 17). We have recently shown that NP inhibit VEGF transcription and protein production in cultured human vascular smooth muscle cells via the NPRC (18). Overall, the ability of the NP to impede cardiac or vascular remodeling may limit the cellular response to chronic cardiovascular insult.
The ability to modulate VEGF-induced angiogenesis would theoretically provide a tool to influence wound healing, tumor propagation, or diabetic retinopathy. This could result from modulating VEGF synthesis or VEGF receptor signaling function. Vascular endocrine peptides such as angiotensin II, ANP, or endothelin, regulate cardiovascular remodeling and could also play important roles as angiogenesis-modulating factors (19, 20). In this study we examined the ability of natriuretic peptides to inhibit VEGF- induced signaling to MAP kinases and the subsequent effects of JNK activation on the angiogenesis program.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell preparation
Primary cultures of bovine aortic EC were prepared and used as
previously described (11, 18). In transfection studies, EC
were used in passages 4–5, because this greatly increases the
transfection efficiency of these cells.
Kinase activity assays
For ERK and JNK activity assays, the cells were synchronized for
24 h in serum- and growth factor-free medium. The cells were then
exposed to VEGF (10–20 ng/ml) for 10 (ERK) or 15 (JNK) min with or
without additional substances or peptides, as previously described
(11). Immunoprecipitated kinases were then added to the
proteins myelin basic protein (for ERK) or
glutathione-S-transferase-c-Jun-(1–79) (for JNK) for
in vitro kinase assays. In addition, the VEGF-induced
phosphorylations of ERK, JNK, and p38 MAP kinases were determined as
indexes of activation. From each experimental plate of cells, 100 µl
lysate were dissolved in SDS sample buffer, boiled, separated, then
transferred to nitrocellulose. Phosphorylated kinase proteins were
detected using phospho-specific monoclonal MAP kinase antibodies
(Santa Cruz Biotechnology, Inc.) and the enhanced
chemiluminescence Western blot kit. Equal samples from the above plates
of cells were processed similarly, and immunoblots of the precipitated
kinase protein from each experimental condition were determined to show
equal loading on the gel of total MAP kinase protein. For
cyclin-dependent kianse (Cdk) activity, EC were cultured with
VEGF for 8 h. Cell lysate was added to protein
A-Sepharose-conjugated Cdk4 antibody, then added to in vitro
kinase activity tubes containing
glutathione-S-transferase-pRB as substrate. Samples from
each condition were assayed for Cdk4 protein by immunoblot. All
experiments were repeated two or three times.
Transient transfections
Bovine aortic endothelial cells (BAEC; passages 4–5) were grown
to 40–50% confluence and then transiently transfected with 1.5–10
µg fusion plasmids depending on the plate size and the amount of
cells. Plasmids included constituitively active, wild-type JNK-1
(pcDNA3Flag-JNK-1), dominant negative JNK-1 (pcDNA3 Flag-JNK-1
APF) (11), or control pcDNA3, and transfection was
carried out using Lipofectamine. Cells were incubated with liposome-DNA
complexes at 37 C for 5 h, followed by overnight recovery in DMEM
containing 10% FBS. Then, before experimental treatment cells were
synchronized for 24 h in serum-free DMEM and treated with VEGF.
The efficiency of transfection of all constructs ranged from 50–65%
based upon cotransfection of green fluorescent protein-pcDNA3.
Cyclin D1 synthesis
EC were synchronized by serum deprivation for 48 h, then
incubated in methionine-free DMEM with dialyzed 10% FBS for 1 h
before experimentation, as previously described (21). The
culture plates were then incubated in the absence of serum or unlabeled
methionine, but with 250 µCi [35S]methionine
in the presence or absence of VEGF for 16 h. The cells were lysed
and precleared, and specifically labeled cyclin D1 protein was
immunoprecipitated using polyclonal antibody. The immunoprecipitated
proteins were solubilized and denatured in SDS reducing buffer,
electrophoretically resolved by PAGE, and subjected to fluorography,
followed by autoradiography for 1 day. Each translation experiment was
performed at least three times.
EC proliferation
Nontransfected or transfected EC were synchronized overnight in
the absence of serum, then subjected to incubation with VEGF with or
without NP for 72 h. The cells were then exposed to trypsin,
scraped, resuspended, and counted using a hemocytometer, as triplicate
field counts per condition. The experiment was repeated, and the data
were combined.
EC migration assay
Nontransfected or transfected EC were grown to confluence on
six-well plates in DMEM with 10% serum. The cells were synchronized
for 24 h in the absence of serum, and a wound was created by
scraping the monolayer with a single edge razor blade. Selected
reagents were added to the wounded BAEC for 24 h at 37 C. The
cells were then fixed in 3.7% formaldehyde and assessed for migration.
BAEC migration was measured using an image analyzer system composed of
an inverted microscope and a 20- to 24-in. digitizing board (Jandel
Scientific, Corte Madera, CA) attached to an IBM computer (IBM
Corp.). The SigmaScan program (Jandel) was used for analysis of
measurements of the distance traveled by the cells within the
calibrated area adjacent to the wound. Five measurements per well were
taken, and results from three separate experiments contributed to the
bar graph.
Microvascular capillary tube formation
Human dermal microvascular EC were plated on growth
factor-reduced Matrigel in the presence or absence of VEGF with or
without NP and maintained for 6 h at 37 C. The cells were fixed at
6 h (maximum tube formation), stained with Diff-Quik, and
photographed and assessed at x10 magnification using phase microscopy.
Five random fields per condition were examined, the number of cords per
tubes was counted in each, and mean values were determined. The
experiment was repeated twice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, left. We found that
HS-142–1, a specific inhibitor of only the NP GC receptor, prevented
the action of ANP (lane 7), but had an insignificant effect on the
action of C-ANP-(4–23) (lane 8). Supporting the idea that the GC
receptor and the generation of cGMP are involved, addition of
8-bromo-cGMP to the VEGF-incubated EC in the absence of ANP also
significantly prevented ERK activation (lane 9). Using antibodies to
the phosphorylated form of ERK, we found that NP inhibited VEGF-induced
ERK phosphorylation in a dose-related fashion (70% maximally at 100
nM; Fig. 1A, right).
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, left). Again, HS-142–1
addition to the ANP-incubated EC prevented the inhibition of
VEGF-induced JNK activation. However, this compound had little effect
on the action of C-ANP-(4–23). Further, incubation of the EC with VEGF
plus 8-bromo-cGMP (without ANP) significantly inhibited VEGF-induced
JNK. The NP were able to accomplish this in a dose-related fashion,
showing a maximal 69% decrease in VEGF-induced JNK phosphorylation
(Fig. 1B, right). The NP could also block platelet-derived
growth factor (PDGF)-induced JNK phosphorylation (Fig. 1B, right).
Finally we determined whether the NP could also block p38 activation by
VEGF (Fig. 1C). In a dose-related fashion, the NP were capable of a
maximal 72% inhibition of VEGF-induced p38
phosphorylation/activation.
VEGF-induced cyclin D1 protein synthesis is inhibited by
NP
We previously showed that VEGF stimulates cyclin D1 synthesis
through a JNK-related mechanism, an important stimulus to
G1/S stage progression. Here we found that VEGF
induces cyclin D1 synthesis more than 2-fold and that ANP significantly
prevents this by 58% (Fig. 2).
C-ANP-(4–23) was less potent than ANP in this regard, perhaps
identifying differential roles for the ANP GC receptor and NPRC
proteins. We also determined whether the NP could directly interfere
with JNK induction of cyclin D1 synthesis. To do this, we expressed a
mildly constituitively active JNK-1 protein in the transfected EC and
found that this could significantly stimulate cyclin D1 activity (Fig. 2, lane 10). However, ANP and C-ANP-(4–23) could not block this
particular action of JNK.
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, lane 4). ANP
blocked this effect of VEGF by 50% (lanes 5 and 6) and also blocked
the activation of Cdk4 by constituitively active JNK (lanes 10–12).
C-ANP-(4–23) less potently simulated the actions of ANP.
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). Each NP significantly prevented
this, with ANP causing a 53% inhibition. To further support the idea
that e NP work primarily through blocking VEGF-induced JNK activation,
we expressed a mildly constituitively active JNK-1 (Flag-JNK1) in this
setting. As a result, the effects of NP to act as antimitogens were
significantly prevented. The ability of VEGF to stimulate EC
proliferation was partially, but significantly, inhibited by dominant
negative JNK1 (APF), whereas expression of active JNK1 in the absence
of VEGF stimulated EC proliferation. The involvement of JNK in this
action of VEGF is probably underestimated, because the efficiency of
transfecting/expressing the dominant negative JNK is at best 65%, as
previously determined.
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, left, A vs. B and bar graph). This did
not reflect VEGF-induced proliferation, because increased cell number
in response to this growth factor was not seen at 24 h (data not
shown). In contrast, when the cells were coincubated with VEGF and ANP
(Fig. 5C) or C-ANP-(4–23) (Fig. 5D), there was a significant
inhibition of VEGF-induced cell migration, whereas NP had no effect on
basal migration (Fig. 5, E and F). VEGF comparably stimulated the
migration of EC transfected to express the empty vector, pcDNA3 (Fig. 5, right, A vs. B). This was substantially
prevented by expression of the dominant negative JNK-1 (Fig. 5C). To
further support the idea that NP act through the inhibition of
VEGF-induced JNK activity, we overexpressed the constituitively active
JNK construct. We found that this completely reversed the inhibition of
EC migration by the NP (Fig. 5, D and E). Transfection/expression of
JNK-1 in the absence of VEGF independently stimulated EC migration by
6-fold (Fig. 5F), compared with that in the control (empty vector).
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, A vs. B and bar
graph). In the presence of ANP or C-ANP-(4–23), VEGF was
significantly less effective (Fig. 5, C and D), forming 53% fewer
capillary cords in the presence of ANP for the same number of plated
EC. NP had no effect on basal tube formation (Fig. 5, D and
E).
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, H vs. I).
We also found that the ability of ANP or C-ANP to inhibit VEGF-induced
capillary formation was significantly reversed by expression of the
mildly constituitively active Jnk-1 protein (Flag-JNK1; Fig. 5, J and K
vs. G). In the absence of VEGF, the expression of JNK-1
induced a smaller, but significant, increase in capillary formation
compared with the control value (Fig. 5, G vs. L). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although we define a role for JNK, other investigators have implicated protein kinase C, ERK, phosphatidylinositol 3OH- kinase (PI3K), and p38 in the ability of VEGF to promote EC proliferation or migration in vitro (10, 25, 26, 27). VEGF activation of p38 has been noted by several laboratories and has been proposed to significantly contribute to the migration of EC in response to this growth factor (28, 29). We found that the NP significantly prevents VEGF-induced p38 phosphorylation, and therefore, this probably contributes to the overall inhibition by ANP of migration seen here. In addition to stimulating new blood vessel formation, VEGF serves as a survival factor, operating through PI3K and protein kinase B, via the Flk-1 tyrosine kinase receptor on EC (30).
It appears, then, that a number of signaling pathways contribute to the ability of VEGF to promote various aspects of angiogenesis. It is likely that cross-talk and/or convergence between signaling systems exist for the actions of the EC growth/survival factor. This is comparable to other vascular growth factors, such as basic fibroblast growth factor, insulin-like growth factor I, and PDGF, that activate multiple signaling pathways leading to growth, differentiation, and survival functions in cardiovascular cells (31). For instance, PDGF or activation of the mast cell kit tyrosine kinase receptor can activate JNK through a pathway dependent on PI3K activation (32, 33). In this respect, we previously showed that activation of JNK by basic fibroblast growth factor, PDGF, angiotensin II, and thrombin all depended on the earlier activation of ERK in EC (11). Our overall results indicate that JNK activation by VEGF contributes to multiple steps in the angiogenesis pathway, culminating in new capillary tube formation.
As we show here, the effects of NP on VEGF-induced angiogenesis result from binding either class of NP receptor. The GC receptor modulates most of the in vivo effects of ANP reported to date (34) Upon binding GC receptors, NP stimulate the generation of cGMP, and this underlies the inhibition of VEGF-induced signaling and angiogenesis. Involvement of the GC receptor was shown, in that a specific GC antagonist, HS-142–1 (35, 36), significantly prevented the effects of ANP, but not those of the NPRC-specific ligand, C-ANP-(4–23). Further, in limited studies, 8-bromo-cGMP simulated the actions of ANP. However, we also implicate the NPRC in NP action, in that the inhibition of ANP action by HS-142–1 was never complete. C-ANP-(4–23) is a synthetic truncated form of ANP and serves as a specific ligand for the NPRC, whose in vivo function remains poorly defined, except to clear NP from serum (15). We previously reported that this receptor mediates the ability of various NP to inhibit VEGF synthesis in vitro (18) and now report that the NPRC contributes to inhibit VEGF-induced signaling to angiogenesis.
Both GC and NPRC are found extensively on vascular smooth muscle cells and EC, and therefore are potentially relevant to the in vivo vascular actions of the NP. Many of the effects of cGMP (and ANP) are mediated through the activation of PKG (37), but it is not well defined what targets downstream from PKG might mediate the actions of ANP in this setting. The signaling mechanisms for NPRC are even less understood, but perhaps involve the inhibition of cAMP generation (reviewed in Ref. 12). We speculate that there is a common final pathway downstream of the GC receptor and the NPRC that can be saturated by either receptor. Hence, in general, there is no substantial additional inhibition of VEGF signaling by ANP binding both receptors, compared with only one receptor (the response to C-ANP). This EC model can be used to further understand the cross-talk of GC and NPRC signaling with VEGF-induced ERK activation. In some cells, inhibition of cAMP (as perhaps occurs via the NPRC) can inhibit ERK activation (38). This would provide a partial mechanism for the findings of past studies that implicate the NPRC in preventing EC proliferation (16).
In animals with GC or NPRC genetic inactivation, no obvious abnormalities of blood vessel formation have been noticed (39, 40). Thus, one might conclude that VEGF- induced angiogenesis during embryonic development is not modulated by NP. However, there is much redundancy in the NP system, with three receptors and at least four peptides, and double or triple knockouts have not yet been reported. During severe congestive heart failure, ANP (and BNP) plasma levels are 10-fold elevated compared with those in normal humans (41). In this disease, ANP may play a role to prevent further deleterious cardiac remodeling or fibrosis (24). This idea is supported by the finding that GC-A knockout mice develop cardiac hypertrophy (42). However, prevention of angiogenesis by ANP in this setting could impair compensatory new blood vessel formation and long-term recovery from ischemia-induced heart disease. On the other hand, the inhibition of VEGF synthesis and action by ANP provides a potential therapeutic approach to undesirable angiogenesis (8, 22). These proposals can only be tested in vivo, and such studies are underway in our laboratory.
In summary, VEGF-induced angiogenesis is partially dependent on signaling through JNK activation, and this serves as a mechanism that the NP use to inhibit this critical process. The actions of ANP result from activation of both GC and NPRC, and the NP represent one of the first described endogenous inhibitors of both VEGF synthesis and function.
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Acknowledgments |
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Footnotes |
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Received September 11, 2000.
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and the
phosphatidylinositol 3-kinase signaling pathways of the receptor.
Biochem Biophys Res Commun 261:641–645[CrossRef][Medline]
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