From the Department of Surgery, Children's Hospital
and ¶ Department of Pathology, Brigham and Women's Hospital,
Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 7, 2002, and in revised form, November 6, 2002
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ABSTRACT |
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Mice deficient for the transcription factor
NFATc1 fail to form pulmonary and aortic valves, a defect reminiscent
of some types of congenital human heart disease. We examined the
mechanisms by which NFATc1 is activated and translocated to the nucleus
in human pulmonary valve endothelial cells to gain a better
understanding of its potential role(s) in post-natal valvular repair as
well as valve development. Herein we demonstrate that activation of NFATc1 in human pulmonary valve endothelial cells is specific to
vascular endothelial growth factor (VEGF) signaling through VEGF
receptor 2. VEGF-induced NFATc1 nuclear translocation was inhibited by
either cyclosporin A or a calcineurin-specific peptide inhibitor; these findings suggest that VEGF stimulates NFATc1 nuclear
import in human pulmonary valve endothelial cells by a calcineurin-dependent mechanism. Importantly, both
cyclosporin A and the calcineurin-specific peptide inhibitor reduced
VEGF-induced human pulmonary valve endothelial cell proliferation,
indicating a functional role for NFATc1 in endothelial growth. In
contrast, VEGF-induced proliferation of human dermal microvascular and
human umbilical vein endothelial cells was not sensitive to cyclosporin A. Finally, NFATc1 was detected in the endothelium of human pulmonary valve leaflets by immunohistochemistry. These results suggest VEGF-induced NFATc1 activation may be an important mechanism in cardiac
valve maintenance and function by enhancing endothelial proliferation.
Defects in heart development are the most common congenital
anomaly, occurring in 1% of all live births (1, 2). Some of the
defects, especially those that involve the aortic and pulmonary valves,
require surgical intervention, which can include replacement of a
defective valve. In the past decade, researchers have elucidated some
of the basic signaling interactions necessary for heart development. In
addition to initiating heart development, many of these pathways are
also required for optimal heart function in post-natal life (3).
However, comparatively little is known about the interactions that
control more specific events, for example, induction of cardiac valve
formation (4). In the mouse, primordial heart valves known as cardiac
cushions begin to form by embryonic day 9.5. These cushions appear as
swellings in the atrioventricular junction and outflow tract. As
development proceeds, the cushions contribute to chamber septation and
ultimately result in development of four adult valves.
Initiation of endocardial cushion formation involves a distinct subset
of endothelial cells in the cushion-forming area that undergo
endothelial to mesenchymal transdifferentiation
(EMT).1 It is believed that
the underlying myocardium sends inductive signals to the endocardial
cells, beginning the process of EMT (5). These newly formed mesenchymal
cells migrate into the underlying extracellular matrix, where further
remodeling transforms the cushions into fibrous leaflets. Studies have
demonstrated that transforming growth factor- For example, targeted gene deletion has demonstrated a specific
requirement for NFATc1, also known as NFAT2, in valve development. Knockout of NFATc1 in the mouse leads to defective aortic and pulmonary
valve development with subsequent death at embryonic days (E) 14-15
due to congestive heart failure (9, 10). The cardiac cushions in the
outflow tract of these mice are hypoplastic, suggesting that lack of
NFATc1 leads to dysregulation of an early step in cushion formation.
Knockout of other NFAT proteins, such as NFATc2, c3, and c4, has no
effect on valve development (11). Members of the NFAT family function
as mediators of the CsA-sensitive calcineurin-NFAT signaling pathway.
First discovered in the pathway of interleukin-2-mediated T-cell
activation and proliferation (12), NFAT (nuclear
factor in activated T cells)
signaling has since been shown to be crucial for neuronal guidance,
skeletal and cardiac muscle hypertrophy, and, as cited above, cardiac
valve development (13-15).
The upstream signaling events regulating NFATc1 activity in the valve
endothelium are unknown, although all known NFAT proteins are dependent
upon cytosolic Ca2+ flux for nuclear translocation.
Increased cytosolic Ca2+ leads to activation of calmodulin
and ultimately calcineurin, a serine/threonine phosphatase. When
activated, calcineurin dephosphorylates residues in the conserved
N-terminal region of various NFAT isoforms (16, 17). This
dephosphorylation prompts a conformational change in NFAT that exposes
a previously inaccessible nuclear localization sequence. NFAT is then
shuttled into the nucleus, where it interacts with other transcription
factors, including AP-1 and NF- Recent studies have demonstrated that VEGF, an endothelial mitogen
known to induce calcium mobilization upon receptor activation, may
regulate NFATp activity in endothelial cells (21). VEGF is a potent
stimulator of angiogenesis and vascular permeability with the ability
to induce endothelial proliferation, migration, and survival in
vitro (22). In human umbilical vein endothelial cells (HUVECs),
VEGF has been shown to induce nuclear localization of NFATc2, also
known as NFATp, by a CsA-sensitive mechanism, which resulted in
increased tissue factor expression (23). In a separate study,
administration of CsA prohibited VEGF from activating expression of
cylcooxygenase-2 and inhibited angiogenesis in a corneal
neovascularization model, again suggesting an important role for NFAT
in vascular endothelial cells (24).
Studies of VEGF expression in transgenic mice provide evidence for its
role in cardiac valve development. LacZ-tagged VEGF knock-in
mice were found to express VEGF at E 9.0 in endocardial cells along the
entire heart tube (25). By E 9.5, VEGF expression was restricted to
endothelial cells of the outflow tract and atrioventricular valve area.
This expression pattern coincides precisely with the timing and
location of NFATc1 expression in the embryonic valve (9, 10). In a
different study, transgenic mice that overexpress VEGF in the embryonic
myocardium at E 10.5 demonstrated decreased EMT in the endocardial
cushions (26, 27). Although these studies suggest a role for VEGF in
heart valve development, little is known regarding the signaling
cascade downstream of VEGF in valvular endothelial cells. To study the
role of VEGF in NFATc1 signaling in endothelial cells, we performed
experiments on human pulmonary valve endothelial cells (HPVEC). Our
results demonstrate a unique pathway for VEGF-mediated proliferation of
these cells involving an NFATc1-dependent mechanism.
Cell Culture Conditions--
HPVEC were isolated from human
pulmonary valve leaflets obtained from patients undergoing
cardiovascular surgical procedures at Children's Hospital, Boston
under an institutional review board-approved protocol. Removal
of the pulmonary valve was a planned part of each procedure. Patients'
ages ranged from 5 months to 20 years. The time interval from surgical
excision to cell isolation was less than 1 h. Endothelial cells in
primary cultures of human pulmonic valve leaflets were isolated using
Ulex europaeus I-coated Dynabeads (28) as previously
described (8). HPVECs were cultured on 1% gelatin-coated tissue
culture plates in endothelial cell basal medium-2 (EBM-2, Clonetics)
supplemented with endothelial cell growth media-2 SingleQuots (human
VEGF, EGF, bFGF, insulin-like growth factor-1, ascorbic acid,
gentamicin, and heparin; Clonetics), 20% heat-inactivated FBS, and
glutamine/penicillin/streptomycin (GPS, Invitrogen). This
culture media will be referred to as EBM-2. Cells were maintained in
5% CO2 at 37 °C. Human umbilical vein endothelial cells
(HUVECs) were generously provided by Dr. F. W. Luscinskas at
Brigham and Women's Hospital, Boston. Human dermal microvascular
endothelial cells (HDMEC) were isolated and cultured as described
previously (29).
Endothelial Cell Characterization--
Immunofluorescence was
performed as previously described (8). Briefly, cells plated onto
gelatin-coated glass coverslips were fixed with Growth Factor Stimulation--
HPVEC were plated onto
gelatin-coated glass cover slips for 1-3 days and then switched from
EBM-2 medium to endothelial cell basal medium (EBM) containing 10% FBS
(without the additive growth factors described above) for at least
24 h. Cells were then stimulated for 30 min with either 50 ng/ml
VEGF165, bFGF (R&D Systems), or 1 µM calcium
ionophore A23187 (Sigma). 5 µM CsA or 1 µM
FK506 (Sigma) was added 2 h before VEGF stimulation. Cells were
also stimulated with receptor-selective variants of VEGF: KDR-sel2 was
shown to have wild-type affinity for KDR/VEGF-R2 but 2000-fold reduced
binding to Flt-1, whereas Flt-1-sel was shown to have wild-type
affinity for Flt-1 but 470-fold reduced binding to KDR/VEGF-R2 (30).
Drs. Bing Li and Abraham M. de Vos (Genentech, Inc., South San
Francisco, CA) kindly provided these receptor-selective variants, as
well as comparably produced wild-type VEGF (VEGFwt, amino acids 1-109). NFATc1 expression was analyzed by fixing cells in 4%
paraformaldehyde, permeabilizing with 0.5% Triton X-100, and
incubating with mouse anti-human NFATc1 mAb (7A6 from Santa Cruz
Biotechnology) diluted 1:500 followed by FITC-conjugated anti-mouse
IgG. An aliquot of the anti-human NFATc1 mAb for preliminary
experiments was kindly provided by Dr. Gerald Crabtree (Stanford
University). Cellular localizations of NFATc2, NFATc3, and NFATc4 were
analyzed as described above using monoclonal antibodies (mAbs)
purchased from Santa Cruz Biotechnology.
Western Blotting--
Cell lysates were prepared as described
previously (8) and subjected to SDS-PAGE using commercially available
gradient gels. Gels were transferred to nitrocellulose membrane
(Millipore). The membrane was rinsed in TBS-T solution (0.1% Tween 20 in TBS, pH 7.5) and incubated in blocking buffer (5% skim milk in
TBS-T) at the room temperature. The membranes were incubated with the mouse anti-human NFATc1 (7A6 from Santa Cruz Biotechnology), mouse anti-human NFATc2 (G1-D10 from Santa Cruz Biotechnology), mouse anti-human NFATc3 (F-1 from Santa Cruz Biotechnology), or
goat-anti-human NFATc4 (C-20 from Santa Cruz Biotechnology)
antibodies diluted 1:500 for overnight at 4 °C, followed by
horseradish peroxidase-labeled secondary antibodies diluted 1:2000 for
1 h at room temperature. The immunoreactive bands were visualized
using chemiluminescent reagent (LumiGLO, KPL).
MTT Assay--
1 × 104 cells/well were plated
onto gelatin-coated 48-well plates. One day later, cells were washed
with PBS and incubated with CsA at 1, 5, 10, and 20 µM in
low serum (2% FBS) or high serum (20% FBS) containing EBM-2 media for
48 h. Five hours before the end of the incubation, 50 µl of MTT
solution (5 mg/ml) was added into each well. Media was removed, and 500 µl of Me2SO was added to each well. Dissolved crystals
were transferred into 96-well plates, and absorbance at 550 nm was measured.
Cell Proliferation Assay--
Cell proliferation was assayed by
[3H]thymidine incorporation (31). Cells were plated onto
gelatin-coated 48-well plates at a density of 1 × 104
cells/well. The next day, cells were washed with PBS and incubated in
thymidine-free endothelial basal labeling medium (Clonetics) with 2% FBS and GPS (starvation medium) for 24 h. The quiescent cells were stimulated with fresh starvation medium containing 10 ng/ml
VEGF, KDR-sel2, or Flt-sel, with or without CsA. Approximately 16-24 h
after growth factor stimulation, 0.7 µCi of
[3H]thymidine (6.7 Ci/mmol, PerkinElmer Life Sciences)
was added to each well, and incubations were continued for another
4 h. [3H]Thymidine DNA incorporation in the cell
lysates was quantitated in a scintillation counter. Each condition was
tested in triplicate, and the mean ± S.D. of
[3H]thymidine DNA incorporation was calculated.
Statistical significance was determined by Student's t
test, with a p value of <0.05 considered statistically
significant. The -fold induction compared with cells with no VEGF
stimulation was also determined. Proliferation assays on retrovirally
transduced HPVEC were performed as above after the GFP-positive cells
were sorted by FACS.
Northern Blot Analysis--
Total RNA was isolated from human
endothelial cells using an RNeasy kit (Qiagen). Blots were hybridized
with 32P-labeled cDNA probes for KDR/VEGFR2,
Flt-1/VEGF-R1, and neuropilin-1.
Retroviral Transduction--
The retroviral expression vectors,
retro-EGFP, and retro-EGFP-VIVIT (contains an oligonucleotide coding
for MAGPHPVIVITGPHEE at the N terminus of the green
fluorescent protein (GFP)), were kindly provided by Dr. A. Blauvelt at
NCI, National Institutes of Health and have been previously described
(32). For viral production, 293 cells at 70% confluence were
transfected in Dulbecco's modified Eagle's medium with 2 µg of
retroviral expression vector and 2 µg of the packaging vector,
pCL-10A1 (33), using the Effectene transfection reagent (Qiagen)
according to the manufacturer's directions for a 100-mm dish. After
6 h, an equal volume of Dulbecco's modified Eagle's medium
containing 20% FBS, 1% GPS was added for a final concentration of
10% FBS. After 24 h, medium was replaced with 6 ml of EBM
containing 10% FBS, 1% GPS, and, after an additional 24 h,
supernatants were collected and filtered (0.45 µm). For retroviral
infection, HPVEC at 50% confluence were incubated twice for 4 h
with 5 ml of the viral supernatant containing 8 µg/ml Polybrene. 5 ml
of EBM-2 was added after the second infection, and, after 24 h,
the cells were washed and recovered in EBM-2. Cells positive for high
levels of GFP expression (fluorescence signal > 102)
were sorted by FACS and immediately plated onto a 48-well tissue culture dish for cell proliferation assays as described above. Similar
transduction efficiencies of 40-50% were obtained using either
retro-EGFP or retro-EGFP-VIVIT. To confirm that VIVIT was effectively
inhibiting NFATc1 nuclear translocation, GFP-positive HPVEC were also
plated in parallel onto gelatin-coated glass coverslips, cultured in
EBM supplemented with 10% FBS for at least 24 h, and then
stimulated with 50 ng/ml VEGF for 30 min. NFATc1 cellular localization
was detected with immunofluorescence using mouse anti-human NFATc1 at
1:500 dilution followed by biotinylated anti-mouse IgG (H+L) and Texas
Red Avidin D, both at 5 µg/ml.
Immunohistochemistry--
Normal human pulmonary valve leaflets
(n = 3) were obtained from autopsies with post-mortem
intervals ranging from 5 to 20 h according to a protocol approved
by the Human Research Committee at the Brigham and Women's Hospital,
Boston, MA. The mean age of the patients was 46.8 years. Formalin-fixed
paraffin-embedded human pulmonary valve tissues were sectioned in
5-µm slices. After antigen retrieval, sections were preincubated with
0.3% hydrogen peroxide to inhibit endogenous peroxidase activity and
then incubated with primary anti-human CD31 mAb, anti-human NFATc1 mAb,
or mouse IgG1 control mAb diluted in phosphate-buffered saline
supplemented with 4% of the species-respective normal serum. After
washing with phosphate-buffered saline, species-appropriate
biotinylated secondary antibodies were applied, followed by
avidin-peroxidase complex (Vectastain ABC kit, Vector Laboratories).
The reaction was visualized with 3-amino-9-ethyl carbazole substrate
(Sigma Chemical Co). Sections were counterstained with Gill's
hematoxylin solution (Sigma) and mounted.
Isolation and Characterization of HPVEC--
The function of
NFATc1 in valvular endothelium was studied using HPVEC isolated from
human pulmonary valve leaflets. The cells uniformly expressed the
endothelial-specific markers von Willebrand factor (vWF), CD31/PECAM-1,
and LPS-inducible E-selectin, demonstrating that HPVECs retain
expression of endothelial-specific markers in culture (Fig.
1).
VEGF Stimulates NFATc1 Nuclear Translocation by a
KDR/VEGF-R2-specific and Calcineurin-dependent
Mechanism--
To identify extracellular ligands that stimulate NFATc1
activation in valvular endothelial cells, growth factors were tested for their ability to induce NFATc1 nuclear import in HPVEC. HPVEC were
stimulated with 50 ng/ml VEGF, KDR-sel2, Flt-sel, or bFGF and then
immunostained with an antibody specific for NFATc1. VEGF (50 ng/ml) was
shown previously to be sufficient for stimulating NFATc2 (also known as
NFATp) nuclear localization (23). VEGF stimulated the nuclear
translocation of NFATc1 (Fig.
2A, panel c)
compared with untreated HPVECs (Fig. 2A, panel
b), at concentrations of VEGF as low as 10 ng/ml (data not shown).
The pharmacological agents CsA (Fig. 2A, panel d)
and FK506 (data not shown) inhibited the VEGF-induced nuclear
translocation, suggesting a calcineurin-dependent mechanism. As expected, the calcium ionophore A23187 also stimulated NFATc1 nuclear import (data not shown). KDR-sel2 stimulated NFATc1 nuclear translocation (Fig. 2A, panel g), and, as
observed with VEGF, the translocation was inhibited when CsA was
included (Fig. 2A, panel h). In contrast,
Flt-1-sel did not induce translocation (Fig. 2A, panel
f), indicating that VEGF-induced NFATc1 translocation is mediated
by KDR/VEGF-R2 and not Flt-1/VEGF-R1. When cells were treated with
other endothelial mitogens such as bFGF, NFATc1 remained in the
cytosol, as evident by a diffuse pattern of immunofluorescence (Fig.
2A, panel e). Other growth factors, such as
insulin-like growth factor-1, EGF, and heparin-binding EGF, and
the endothelial factors angiopoietin-1 and -2, had no effect on NFATc1
localization (data not shown). These results demonstrate that the
mechanism of NFATc1 activation and nuclear translocation in HPVEC is
specific to VEGF signaling events mediated by KDR/VEGF-R2.
NFATc1 activation was further examined by Western blot (Fig.
2B). Dephosphorylation of NFATc1 results in increased
mobility on SDS-PAGE (20). As seen in Fig. 2B, three
variants of NFATc1, with apparent molecular masses between 90 and 140 kDa, were detected with anti-human NFATc1 mAb (Fig.
2B, lane 1). Removal of growth factors from the
culture medium for 24 h had no effect on migration of the NFATc1
isoforms (lane 2). Treatment with VEGF for 15 min (lane 3) or for 24 h (lane 5) resulted in an
increase in electrophoretic mobility of all three isoforms. This
apparent dephosphorylation at 15 min and 24 h was inhibited in the
presence of 5 µm CsA (lanes 4 and 6). Tubulin
expression in each cell lysate is shown as a control (Fig.
2B, lower panel).
NFATc2 and NFATc3 Are Expressed in HPVEC but Not Translocated into
the Nucleus in Response to VEGF--
VEGF-induced translocation of
NFATc2, NFATc3, and NFATc4 was examined as described in Fig.
2A to determine which NFAT family members are activated by
VEGF. NFAT family members c1, c2, and c3, but not c4, were detected in
the cytoplasm of HPVEC in non-stimulated cells by indirect
immunofluorescence using isoform-specific mAbs (Fig. 2C,
panels a-d). However, only NFATc1 was translocated into the
nucleus when HPVEC were treated with 50 ng/ml VEGF for 30 min (Fig.
2C, panels e-h). In Fig. 2D, protein
expression of NFATc1, c2, and c3 was analyzed by Western blot of HPVEC
(lane 3), HUVEC (lane 4), and HDMEC (lane
5) cell lysates. Human lymphoma cell lines served as positive
controls (lanes 1 and 2). As seen in Fig.
2D, NFATc1, c2, and c3 were detected in all three human
endothelial cultures. NFATc4 was not detected by Western blot (data not
shown). NFATc2 expression was highest in HUVEC, compared with NFATc1
and NFATc3, consistent with previous studies showing NFATc2
(i.e. NFATp) in HUVECs. Also consistent with Armesilla
et al. (23), NFATc2 was translocated into the nucleus of
HUVECs in response to VEGF (data not shown). These results suggest that
VEGF specifically induces translocation of NFATc1, but not NFATc2 or
NFATc3, in valvular endothelial cells.
VEGF Induces Proliferation by a CsA-sensitive Mechanism in HPVEC
but Not in HDMEC or HUVEC--
We next sought to investigate the
cellular effects of preventing VEGF-induced NFATc1 nuclear import.
Because VEGF is a mitogen of endothelial cells (21) and NFAT signaling
can increase proliferation in other cell types (16), we used CsA to
test whether VEGF induced valvular endothelial proliferation by an
NFATc1-dependent mechanism. First, the potential
cytotoxicity of CsA (34) was addressed. MTT assays were performed on
HPVEC, HDMEC, and HUVEC at 1, 5, 10, and 20 µM CsA
in the presence of 2 and 20% FBS (2% FBS was used in the growth
factor-induced proliferation assays). Cytotoxicity was not detected at
1, 5, or 10 µM CsA in these three human endothelial cell
cultures in either 2% or 20% FBS (data not shown). Quiescent HPVEC
were stimulated with VEGF, VEGFwt, KDR-sel2, or Flt-sel (30), in the
absence or presence of 5 µM CsA (Fig.
3A). Cell proliferation was
assayed by [3H]thymidine incorporation. We found that
VEGF, VEGFwt, and KDR-sel 2 induced HPVEC proliferation by 4- to 6-fold
in the absence of CsA (Fig. 3A, open bars), but
the proliferation was attenuated significantly by CsA (Fig.
3A, black bars). CsA inhibited VEGF-induced proliferation by 30% (p = 0.032), VEGFwt-induced
proliferation by 64% (p = 0.031), and KDR-sel2-induced
proliferation by 37% (p = 0.004). A similar inhibitory
effect was observed with FK506 (data not shown). Flt-sel did not induce
proliferation compared with control cells. Thus, VEGF stimulates HPVEC
proliferation, via KDR/VEGF-R2, by a calcineurin-dependent
mechanism that may be dependent upon the dephosphorylation and nuclear
translocation of NFATc1. This result was observed in cultures of HPVEC
isolated from the pulmonary valve leaflets of three different
patients.
The CsA-mediated inhibition of VEGF-induced proliferation appears to be
specific to the valve endothelium, because proliferation of HDMEC and
HUVEC was not inhibited by CsA-mediated NFATc1 inactivation (Fig.
3B). 5 µM CsA was sufficient to inhibit
VEGF-induced NFATc1 nuclear translocation in HDMEC and HUVEC (data not
shown). It is unlikely that this cell-specific CsA effect is due to
differences in VEGF receptor expression, because mRNA expression
levels for KDR/VEGF-R2, neuropilin-1, and Flt-1/VEGF-R1 did not vary
more than 2-fold among the three types of human endothelial cells
tested (data not shown). It is possible that the interaction of
NFATc1 with valve-specific nuclear factors accounts for the
CsA-sensitive proliferation response to VEGF observed in HPVECs.
The NFAT-specific Inhibitor, VIVIT, Inhibits VEGF-induced
Proliferation of HPVEC--
Because CsA and FK506 can potentially
disrupt calcineurin-dependent pathways other than the
activation of NFAT, we used the synthetic peptide VIVIT to inhibit NFAT
activation selectively without disrupting other
calcineurin-dependent pathways (20). We demonstrated that
VIVIT inhibits VEGF-induced NFATc1 nuclear translocation and that this
results in inhibition of VEGF-mediated HPVEC proliferation (Fig.
4). HPVEC retrovirally transduced with GFP-VIVIT (an oligonucleotide coding for VIVIT-containing peptide fused
to the N terminus of the green fluorescent protein) did not import
NFATc1 to the nucleus upon VEGF stimulation (Fig. 4, C and
D). HPVEC expressing GFP alone did show VEGF-induced NFATc1 nuclear localization (Fig. 4, A and B),
indicating that the inhibition of NFATc1 nuclear import is specific to
the VIVIT peptide and that NFATc1 nuclear shuttling was not disrupted
by retroviral transduction. VEGF-induced proliferation was inhibited by
35% (p = 0.022), whereas KDR-sel2-induced
proliferation was inhibited 62% (p = 0.001) (Fig.
4E). Although VIVIT peptide could potentially inhibit NFATc2
and c3, our data show that these two family members are not
translocated to the nucleus in response to VEGF (Fig. 2C)
and therefore would not be functionally disrupted in this experiment.
These data provide strong evidence that nuclear translocation of NFATc1
is required for maximal VEGF-induced proliferation of HPVEC. The
smaller -fold induction observed in this experiment compared with Fig.
3 was likely due to the sequential retroviral transduction, FACS
sorting, and 24 h serum starvation prior to measuring VEGF-induced
proliferation.
NFATc1 Expression in Adult Human Pulmonary Valve Leaflets--
In
the mouse, expression patterns of NFATc1 in the developing heart have
been examined thoroughly to gain insights into its role in
valvulogenesis. NFATc1 can be detected in developing murine hearts
beginning at embryonic day E7.5, becomes increasingly restricted to
nascent valvular structures by E11.5, but is then undetectable by
either reverse transcription-PCR or immunohistochemistry, after E13.5 (9, 10). NFATc1 has been reported undetectable in newborn or
adult murine valves as well (9). Despite the lack of reported evidence
for NFATc1 expression in post-natal murine valves, we examined NFATc1
protein expression in human adult pulmonary valve leaflets, because our
experiments in cultured HPVEC show that NFATc1 is expressed in adult
HPVECs and suggest that NFATc1 may play an important physiological role
in post-natal valve leaflets. Paraffin sections from human pulmonary
and aortic valve leaflets were stained with anti-human NFATc1 or
anti-human CD31/PECAM-1 mAbs. Fig. 5
shows representative immunohistochemical staining seen in human
pulmonary valve leaflet tissue (n = 3). NFATc1 was detected in a nuclear-localized pattern along the surface of the leaflets (Fig. 5A), coinciding with CD31-positive
endothelial cells in an adjacent tissue section (Fig. 5B).
The inset in Fig. 5A shows staining with mouse
IgG1 as a negative control. Regions of NFATc1-positive endothelial
cells were seen intermittently along the leaflet surface, suggesting
NFATc1 is expressed in a subset of endothelial cells in adult valves.
This observation, combined with the expression of NFATc1 in HPVEC
isolated from post-natal pulmonary valve leaflets, obtained from
patients ranging in age from 5 months to 20 years of age, indicates
that NFATc1 can be expressed focally along the endothelium of
post-natal valves.
We show here that HPVEC require nuclear translocation of NFATc1
for maximal VEGF-induced proliferation. Furthermore, NFATc1 nuclear
translocation and resulting proliferation is mediated by KDR/VEGF-R2.
These results are specific to HPVEC, because addition of CsA did not
affect VEGF-induced proliferation of HUVEC and HDMEC (Fig. 3). The
expression of other NFAT family members, specifically NFATc2 and
NFATc3, was detected in HPVEC, but these proteins were not translocated
into the nucleus in response to VEGF (Fig. 2, C and
D). This specificity suggests that VEGF and NFATc1 signaling interact to activate a proliferative pathway unique to cardiac valvular
endothelial cells.
Our results show a functional relationship between VEGF and
NFATc1 in post-natal valvular endothelial cells. Several reports in the
literature suggest that a similar pathway may also operate in
endocardial cells during cardiac valve formation in the developing mouse. During embryogenesis, NFATc1 is initially expressed in the
endocardium by day E8.5. By E11.5-E13.5, NFATc1 is expressed in the
lining of the pulmonic, aortic, and atrioventricular valves (9, 10).
Similarly, VEGF is expressed in the endocardial cells lining the
endocardial cushions at day E8.5 as well as in the myocardium from E9.5
to E13.5 (25). Interestingly, endocardial cells that have undergone
transdifferentiation to mesenchymal cells are negative for both NFATc1
and VEGF expression (9, 10, 25), suggesting that NFATc1 and VEGF play
pivotal roles both spatially and temporally in development of the
endocardial cushion.
Given our results that VEGF stimulation leads to strong HPVEC
proliferation in vitro, one might hypothesize that
overexpression of VEGF would result in hyperplastic cardiac valves.
However, the opposite result is observed in mice. Premature induction
of VEGF expression in developing mouse embryos results in decreased endocardial cushion formation (27). This is phenotypically the same
result observed with the NFATc1 murine knockout (9, 10). To explain
these findings, we propose that VEGF-mediated NFAT signaling influences
a cascade of events, including proliferation, migration, and
differentiation, that is critical for valve development and post-natal
valvular endothelial function (Fig. 6). In this model, VEGF induces
proliferation of valvular endothelial cells and at the same time may
influence the TGF- We postulate that this model may apply to heart valve
regeneration and repair. Because our experiments were carried out in postnatal cells, we hypothesize that the signaling pathways used during
development are re-established in mature valve leaflets to replenish
endothelial and interstitial cells as needed throughout adult life. The
focal expression of NFATc1 in mature valve leaflets (Fig. 5) is
consistent with this hypothesis. The source of VEGF that would activate
NFATc1 expressed in the leaflet endothelium is unknown. Possible
sources of VEGF include the endothelial cells, the mesenchymal
interstitial cells, or release of VEGF from circulating cells in the
blood. Also consistent with the repair concept, Paranya et
al. (8) demonstrated that clonal populations of valve endothelial cells isolated from post-natal valves can undergo EMT in a manner similar to what occurs in fetal valve development. Therefore, TGF- Our results demonstrate a unique signaling pathway for
endothelial cell proliferation. Previous studies have demonstrated that
VEGF signal transduction in endothelial cells is dominated by
inositol 3,4,5-phosphate/diacylglycerol and extracellular-related kinase (ERK)/mitogen-activated proliferation kinase (MAPK) pathways (35), the latter being dependent on Ras activation (36). Activation of
inositol 3,4,5-phosphate leads to mobilization of cytosolic Ca2+, which may ultimately stimulate calcineurin to
dephosphorylate NFATc1. Because [3H]thymidine
incorporation in HPVECs was reduced ~30-60% after administration of
CsA (Fig. 3), calcineurin appears to be an important mediator for VEGF
stimulation of HPVEC proliferation. We used the VIVIT peptide to
dissect this signaling pathway further and to circumvent potential
endothelial toxicity that has been observed with CsA (34). This
approach revealed that expression of VIVIT in valvular endothelial
cells reduced HPVEC proliferation by 30-60%. A role for ERK and MAPK
signaling in HPVECs remains to be defined but may represent the
remaining proliferative activity of HPVECs after addition of CsA or
VIVIT peptide.
In conclusion, our data provide evidence that VEGF and the receptor
KDR/VEGF-R2 are upstream mediators of NFATc1 activation and nuclear
translocation in HPVEC, which may lead to the expression of
endothelial-specific genes required for valvular endothelial cell
proliferation. To elucidate fully the function of NFATc1 in heart valve
formation and function, it will be essential to identify additional
upstream regulators of NFATc1 activity and downstream valve-specific
target genes whose expression is mediated by NFATc1. We speculate that
expression of NFATc1 in subsets of endothelium along the native valve
leaflets from human adult pulmonary and aortic valve specimens may be a
repair mechanism for replacing damaged endothelial cells. Although we
have demonstrated an important role for NFATc1 in mediating valvular
endothelial proliferation in cultured HPVEC, its functional role in
mature heart valves in vivo must be investigated.
Understanding the signaling mechanisms controlling valve endothelium
proliferation and differentiation will provide insights on heart valve
disease and for creating tissue-engineered valves that mimic normal
valve function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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(TGF-
) signaling
(6) and the type III TGF-
receptor are required for EMT in avian
models (7). Indeed, TGF-
-mediated EMT has been shown to occur in post-natal ovine aortic valve endothelial cells (8). More recently, the
phenotypes of mice and zebrafish in which specific genes have been
"knocked out" or mutated have provided new insight into the genes
required for normal valve development.
B, to alter gene transcription (18).
Although Ca2+ flux across the membrane is a ubiquitous
signaling mechanism, specificity can be accomplished by differential
expression of NFAT isoforms or use of specific ligand-receptor
complexes that alter the pattern of calcium flux. Inhibitors of NFAT
activation include the pharmacological agents CsA, FK506, and the
synthetic peptide VIVIT. CsA and FK506 are immunosuppressant drugs
that, in combination with immunophilins, bind to the catalytic subunit of calcineurin and inhibit its enzymatic activity (17). VIVIT is a
hydrophobic 16-amino acid peptide that mimics the calcineurin-docking motif of NFAT proteins and thereby inhibits calcineurin-mediated activation of NFATc1 (20).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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20 °C methanol and
incubated with primary antibody diluted 1:1000 followed by FITC- or
Texas Red-conjugated secondary antibody at 5 µg/ml. von Willebrand
factor (vWF) was detected using rabbit anti-human vWF (Dako) and Texas
Red anti-rabbit IgG. CD31 (also known as platelet endothelial cell
adhesion molecule, PECAM-1) was detected using goat anti-human CD31
(Santa Cruz Biotechnology) and Texas Red anti-goat IgG. To detect
lipopolysaccharide (LPS)-induced E-selectin, cells were treated with 1 µg/ml LPS for 3 h prior to staining with mouse anti-human
E-selectin (29) and FITC-anti-mouse IgG.
RESULTS
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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View larger version (128K):
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Fig. 1.
HPVECs express endothelial-specific
markers. HPVEC were stained by indirect immunofluorescence using
rabbit control IgG (A), rabbit anti-human von Willebrand
Factor (vWF) (B), goat anti-human CD31/PECAM-1
(C), or mouse anti-human E-selectin (D). In
panel D, HPVEC were stimulated with 1 µg/ml LPS for 3 h to induce E-selectin expression. Photographs were taken at 630×
magnification.
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[in a new window]
Fig. 2.
VEGF stimulates NFATc1 nuclear translocation
by a calcineurin-dependent mechanism. A,
HPVEC were stimulated for 30 min with no growth factor (a
and b), 50 ng/ml VEGF (c and d), 50 ng/ml bFGF (e), or 50 ng/ml Flt-1sel (f) or 50 ng/ml KDR-sel-2 (g and h). HPVECs were incubated
with a mouse isotyped-matched control IgG1 (a) or with mouse
anti-human NFATc1 (b-h), followed by anti-mouse-conjugated
FITC. In panels d and h, HPVEC were preincubated
with 5 µM CsA for 2 h prior to addition of VEGF and
KDR-sel-2, respectively. Photographs were taken at 630× magnification.
In B, cell lysates from HPVEC in EBM-2 with growth factors
(lane 1), EBM-2 without growth factors for 24 h
(lanes 2-6) were analyzed for expression of NFATc1 by
Western blot. Cells were stimulated with VEGF for 15 min (lanes
3 and 4) or 24 h (lanes 5 and
6) in the absence (lanes 3 and 5) or
presence (lanes 4 and 6) of 5 µm CsA.
-Tubulin levels in each cell lysate are shown as a control.
C, expression of NFATc1, NFATc2, NFATc3, and NFATc4 in
HPVECs treated without (panels a-d) or with (panels
d-h) VEGF was analyzed as described in A using family
member-specific mAbs. D, cell lysates from human lymphoma
cell lines (lanes 1 and 2), HPVEC (lane
3), HUVEC (lane 4), and HDMEC (lane 5) were
analyzed by Western blot using family member-specific mAbs against
NFATc1, NFATc2, and NFATc3.
-Tubulin levels in the cell lysates are
shown as a control.
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Fig. 3.
VEGF induces proliferation by a
cyclosporin-sensitive mechanism in HPVEC but not in HDMEC or HUVEC.
A, quiescent HPVEC were stimulated with 10 ng/ml VEGF or
with 10 ng/ml of the VEGF receptor-selective variants, VEGFwt,
KDR-sel-2, or Flt-sel, in the absence (open bars) or
presence (black bars) of 5 µM CsA. Cell
proliferation was assayed by [3H]thymidine incorporation.
Data represent mean ± S.D. of a representative experiment
(n = 3), each performed in triplicate.
Asterisks denote a statistically significant
(p < 0.05) difference. B, HDMEC and HUVEC
were stimulated with 10 ng/ml VEGF in the absence (open
bars) or presence (black bars) of 5 µM
CsA. Data are plotted as -fold induction. The change in VEGF-mediated
proliferation of these cell types after addition of CsA was not
statistically significant.
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Fig. 4.
VIVIT inhibits VEGF-induced proliferation of
HPVEC. HPVEC were retrovirally transduced with GFP or GFP-VIVIT.
The brightest GFP-positive cells (fluorescence signal > 102) were sorted for analysis of NFATc1 localization in
GFP-positive (A and B) and GFP-VIVIT-positive
(C and D) cells and for proliferation assays
(E). GFP-positive and GFP-VIVIT-positive sorted cells are
shown in A and C, respectively. For indirect
immunofluorescence, the same cells were stimulated with 10 ng/ml VEGF
for 30 min followed by staining with anti-human NFATc1 mAb, followed by
a Texas Red-conjugated anti-mouse IgG (B and D).
Photographs were taken at 630× magnification. For proliferation assays
(E), quiescent HPVEC were stimulated with 10 ng/ml VEGF or
KDR-sel-2 for 24 h and assayed for [3H]thymidine
incorporation. Data represent mean ± S.D. of a representative
experiment (n = 3), each performed in triplicate.
Asterisks denote a statistically significant
(p < 0.05) difference.
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Fig. 5.
Expression and localization of NFATc1 in
human pulmonary valve leaflets. Serial sections of formalin-fixed
paraffin-embedded sections human pulmonic valve leaflets were
immunostained with anti-human NFATc1 mAb (A) or with
anti-human CD31 mAb (B), and endothelial marker. The
inset in A shows a section stained with an
isotype-matched control mouse IgG1.
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Fig. 6.
Model for VEGF-induced proliferation in
cardiac valve endothelium. These results and previous studies (8)
demonstrate that two pathways present during valvulogenesis are also
active in post-natal heart valves. In this study, we show that a
VEGF-mediated pathway, signaling through KDR/VEGF-R2, results in
intranuclear NFATc1 and valvular endothelial cell proliferation. A
TGF- -mediated pathway induces differentiation to a mesenchymal
phenotype (8). Whether or not cross-talk between these two pathways, as
has been shown in the mouse (27), occurs in post-natal valves and
affects postnatal valve function or repair warrants
investigation.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-induced differentiation to a mesenchymal
phenotype. Inhibition of VEGF signaling, either by genetic ablation of
NFATc1 or by addition of CsA, would reduce the number of endothelial
cells available to undergo EMT.
-mediated EMT could be a common mechanism in both developing and
adult heart valves, whereas VEGF-mediated proliferation may be a
mechanism for repopulation of the valvular endothelium. VEGF may also
elicit other endothelial responses in the valve. Further studies will
be required to determine the full spectrum of VEGF-induced events in
cardiac valve endothelium.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Bing Li and Abraham M. de Vos at Genentech Inc., South San Francisco, CA for generously providing the receptor-selective variants of VEGF. We also thank Dr. Andrew. Blauvelt at NCI, National Institutes of Health, for the retroviral expression vectors and Drs. D. Friedman and S. Soker at Children's Hospital, Boston, for providing the retroviral packaging vector and cDNA probes, respectively. We also thank Dr. Gerald Crabtree at Stanford University for providing an aliquot of the anti-human NFATc1 (clone 7A6) for pilot experiments. Finally, we thank Alan Flint in the Genetics Division at Children's Hospital for FACS of GFP-positive cells.
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FOOTNOTES |
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* This work was supported by the National Institute of Health (Grant RO1-HL60490 to J. B.), a gift from the Harvey-Wolff family, and a stipend from the Academic Societies for Student Research at Harvard Medical School (to E. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
Current address: Dept. of Cardiac Surgery, Johns Hopkins
Hospital, Baltimore, MD 21205.
** To whom correspondence should be addressed: Dept. of Surgery, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-7865; Fax: 617-566-6467; E-mail: joyce.bischoff@tch.harvard.edu.
Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M210250200
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ABBREVIATIONS |
---|
The abbreviations used are:
EMT, endothelial to
mesenchymal transdifferentiation;
TGF-, transforming growth
factor-
;
CsA, cyclosporin A;
HUVEC, human umbilical vein endothelial
cell;
HPVEC, human pulmonary valve endothelial cell;
HDMEC, human
dermal microvascular endothelial cell;
mAbs, monoclonal antibodies;
GFP, green fluorescent protein;
EGFP, enhanced GFP;
E, embryonic day;
EGF, epidermal growth factor;
VEGF, vascular endothelial growth factor;
EBM-2, endothelial cell basal medium-2;
FBS, fetal bovine serum;
bFGF, basic fibroblast growth factor;
GPS, glutamine/penicillin/streptomycin;
FITC, fluorescein isothiocyanate;
vWF, von Willebrand factor;
PECAM-1, platelet endothelial cell adhesion molecule 1;
LPS, lipopolysaccharide;
wt, wild-type;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
FACS, fluorescence-activated cell sorting;
ERK, extracellular-related
kinase;
MAPK, mitogen-activated proliferation kinase;
KDR, kinase
domain region.
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