Podokinesis in endothelial cell migration: role of nitric
oxide
Eisei
Noiri,
Eugene
Lee,
Jacqueline
Testa,
James
Quigley,
David
Colflesh,
Charles R.
Keese,
Ivar
Giaever, and
Michael S.
Goligorsky
Departments of Medicine and Physiology, State University of New
York, Stony Brook 11794-8152; and Rensselaer Polytechnic Institute,
Troy, New York 12180-3590
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ABSTRACT |
Previously, we demonstrated the role of nitric oxide (NO) in
transforming epithelial cells from a stationary to locomoting phenotype
[E. Noiri, T. Peresleni, N. Srivastava, P. Weber, W. F. Bahou, N. Peunova, and M. S. Goligorsky. Am. J. Physiol. 270 (Cell
Physiol. 39): C794-C802, 1996] and its
permissive function in endothelin-1-stimulated endothelial cell
migration (E. Noiri, Y. Hu, W. F. Bahou, C. Keese, I. Giaever, and M. S. Goligorsky. J. Biol. Chem. 272:
1747-1753, 1997). In the present study, the role of functional NO
synthase in executing the vascular endothelial growth factor
(VEGF)-guided program of endothelial cell migration and angiogenesis
was studied in two independent experimental settings. First, VEGF,
shown to stimulate NO release from simian virus 40-immortalized microvascular endothelial cells, induced endothelial cell transwell migration, whereas
NG-nitro-L-arginine methyl ester
(L-NAME) or antisense
oligonucleotides to endothelial NO synthase suppressed this effect of
VEGF. Second, in a series of experiments on endothelial cell wound
healing, the rate of VEGF-stimulated cell migration was significantly
blunted by the inhibition of NO synthesis. To gain insight into the
possible mode of NO action, we next addressed the possibility that NO
modulates cell matrix adhesion by performing impedance analysis of
endothelial cell monolayers subjected to NO. The data showed the
presence of spontaneous fluctuations of the resistance in ostensibly
stationary endothelial cells. Spontaneous oscillations were induced by
NO, which also inhibited cell matrix adhesion. This process we propose to term "podokinesis" to emphasize a scalar form of
micromotion that, in the presence of guidance cues, e.g., VEGF, is
transformed to a vectorial movement. In conclusion, execution of the
program for directional endothelial cell migration requires two
coexisting messages: NO-induced podokinesis (scalar motion) and
guidance cues, e.g., VEGF, which imparts a vectorial component to the
movement. Such a requirement for the dual signaling may explain a
mismatch in the demand and supply with newly formed vessels in
different pathological states accompanied by the inhibition of NO
synthase.
vascular endothelial growth factor; electrical impedance; chorioallantoic membrane; vectorial locomotion
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INTRODUCTION |
ANGIOGENESIS IS A well-recognized process of tissue
remodeling that is of vital importance for the execution of
developmental programs and physiological adaptations. The meander of
newly formed vessels is guided by a complex interplay of angiogenic and
angiostatic stimuli (4, 5, 25). Directed cell migration is a key
component of the angiogenic process. An array of growth and motility
factors induces endothelial cell sprouting and migration. Among those factors, vascular endothelial growth factor (VEGF) gained prominence due to its specificity for the endothelium. VEGF serves as a potent motogen for cultured endothelial cells and displays
angiogenic activity in vivo (7, 14, 15). It is secreted by mesenchymal and some other cells in response to stimuli as diverse as hypoxia, various hormones, cytokines, and growth factors (2, 15, 27) and acts on
cognate receptors expressed on endothelial cells (19, 21). However,
under pathological conditions such as atherosclerosis, heart failure,
or some forms of hypertension, among others, the angiogenic response is
deficient. Interestingly, these same pathological conditions are also
characterized by the diminished production of the endothelium-derived
relaxing factor, nitric oxide (NO) (3, 12, 16). We have recently
established the role of NO as a requisite component of epithelial cell
migration and wound healing (23). In epithelial cells, locomotion
stimulated by various agonists (hepatocyte growth factor, epidermal
growth factor, fibroblast growth factor 1, or insulin-like growth
factor I) was decelerated by inhibitors of NO synthase. Furthermore, in
rat microvascular endothelial cells and human umbilical vein
endothelial cells (HUVECs), we observed that inhibition of endothelial
NO synthase by L-arginine
derivatives or by antisense oligonucleotides attenuates
endothelin-induced cell migration (22). Collectively, these
observations prompted current investigation of a possible link between
directed migration stimulated by the endothelium-specific angiogenic
factor, VEGF, and NO production in endothelial cells. In two
independent experimental systems (transmigration in a Boyden chemotactic apparatus and wound healing), functional NO synthase was
obligatory for VEGF-stimulated endothelial cell migration. Analysis of
endothelial cell impedance showed that NO mediates spontaneous
micromotion (which we refer to as "podokinesis") even in
stationary cells. This scalar cell movement is transformed to vectorial
locomotion when VEGF guidance is present. The above model emphasizes
the need for the dual stimulatory input:
1) NO-induced switch from a
stationary to locomoting endothelial cell phenotype, which results in
scalar motion or podokinesis, and 2)
VEGF-provided guidance cues that impart a vectorial component to the
movement of endothelial cells.
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MATERIALS AND METHODS |
Cell culture.
Microvascular endothelial cells were previously established and
characterized by our laboratory; these simian virus 40 (SV40)-immortalized cells established from explant cultures of
microdissected rat renal resistance arteries express receptors for
acetylated low-density lipoprotein and immunodetectable von Willebrand
antigen and are capable of capillary tube formation (31). Cells were
grown in gelatin-coated dishes in M199 culture medium (Mediatech,
Washington, DC) supplemented with 5% fetal bovine serum (HyClone,
Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO
BRL, Gaithersburg, MD). HUVECs were obtained from Clonetics and
maintained in ECM-2 medium supplemented with 2% fetal calf serum.
Cell migration assay.
The migration assay was performed according to previously described
technique (24), with minor modifications, in a Boyden chemotaxis
apparatus (Neuroprobe, Cabin John, MD). Endothelial cells were lifted
with 0.05% trypsin-0.53 mM EDTA (GIBCO BRL) and washed, and
106 cells/ml
suspended in 25 µl of M199 with 0.1% bovine serum albumin were added
to the lower chamber of the Boyden apparatus. Polycarbonate filters
with 8-µm pores were coated with 10 µg/ml ProNectin F (Protein
Polymer Technologies, San Diego, CA), washed twice with phosphate-buffered saline, and positioned above the wells of the lower
chemotactic chamber, which contained cells. The top half of the chamber
was reattached, and the chamber was incubated in an inverted position
at 37°C in 95% air-5% CO2
for 2 h to allow a uniform cell attachment to the filter. In
experiments in which L-arginine
was added, an L-arginine-free
basal medium Eagle (BME; GIBCO) with 0.1% bovine serum albumin was
used. The test agents, L-arginine (0.1-1.0 mM),
NG-nitro-L-arginine methyl ester
(L-NAME; 400 µM to 2 mM),
S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100-500 µM; obtained from Molecular Probes, Eugene, OR), recombinant VEGF165 (1-10 ng/ml, obtained from Collaborative
Biomedical Products, Bedford, MA), or a vehicle suspended in 50 µl of
M199 or BME with 0.1% bovine serum albumin, were added to upper
chambers. The chambers were wrapped with Parafilm and incubated for an
additional 5 h in an upright position. After incubation, the filter was
removed from the apparatus and cells were fixed with methanol and
counterstained. The number of migrated cells on the upper surface of
the filter was counted in six randomly chosen fields under ×400
magnification and averaged. All experiments were performed in
quadruplicate, and each experiment was repeated at least three times.
Antisense oligodeoxynucleotide treatments.
The antisense phosphorothioate derivative of oligodeoxynucleotides
(S-ODNs) to the human endothelial constitutive NO synthase (ecNOS)
(5'-AGT TGC CCA TGT TAC TGT GCG TCC GTC-3'), nucleotides 56-30 of human ecNOS cDNA (20), as well as the sense 5'-GAC GGA CGC ACA GTA ACA TGG GCA ACT-3' and scrambled 5'-CTG GGA
CCT GTT CGT ACA GGT CTC TTC-3' phosphorothioate sequences were
synthesized using an automated solid-phase DNA synthesizer (Applied
Biosystems, Foster City, CA). These sequences included the
5'-untranslated region of ecNOS cDNA and initiation codon. Thus
designed sequences showed no homology with other known mammalian
sequences deposited in the GenBank database. All the S-ODNs were
purified with oligonucleotide purification cartridges, dried down,
resuspended in tris(hydroxymethyl)aminomethane (Tris)-EDTA [10 mM
Tris (pH 7.4)-1 mM EDTA (pH 8.0)], and quantified spectrophotometrically.
Before migration assays, HUVECs were incubated with 10 µM S-ODNs for
12 h under serum-free condition. During typical experiments in a Boyden
chemotactic chamber (see above), S-ODNs were present in the medium. In
previously published experiments, HUVECs pretreated with 10 µM
antisense S-ODNs in the serum-free medium were unable to produce NO in
response to calcium-elevating stimuli and showed a significant
reduction in immunodetectable endothelial NO synthase (eNOS) (22).
Monitoring NO release.
Cells grown in gelatin-coated 35-mm dishes were preincubated in
Krebs-Ringer buffer of the following composition (in mM): 136 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, 1.0 NaH2PO4,
and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 1 g/l glucose, adjusted to pH 7.4, which was supplemented with
7.5 U/ml superoxide dismutase (Sigma, St. Louis, MO). NO release was
monitored with an NO-selective microprobe (Inter Medical, Nagoya,
Japan) as previously detailed (22, 23). The working electrode made of
Pt/Ir alloy was coated with a film containing KCl, NO-selective
nitrocellulose resin (pyroxyline lacquer), and a gas-permeable silicon
membrane (8). The counter electrode was made of carbon fiber. The redox
current was detected by a current-voltage converter circuit and
continuously recorded. Tip diameter of the probe (25 µm) permitted
the use of a micromanipulator (Zeiss-Eppendorff) attached to the stage
of an inverted microscope (Nikon Diaphot) and enclosed in a Faraday's
chamber to position the sensor 3-5 µm above the cell surface.
Calibration of the electrochemical sensor was performed using different
concentrations of a nitrosothiol donor (SNAP) as previously detailed
(8).
Cell adhesion, wound healing, and micromotion assays.
Electrode fabrication and the design of electric cell-substrate
impedance sensor (ECIS) have been reported previously (6, 17).
Electrodes were precoated with 10 µg/ml fibronectin. Endothelial cells at a density 2 × 105
were seeded on electrodes in the presence of different agents, as
detailed in RESULTS, and cell adhesion
was recorded as an increase in the electrical resistance. To study cell
detachment from the electrodes, confluent endothelial monolayers grown
on electrodes were exposed to different agonists. To generate
"wounds" on the surface of microelectrodes, confluent endothelial
monolayers were electropermeabilized with direct current from a 6-V
battery generating a local electrical current of ~0.12 mA. This
procedure resulted in a loss of cells from the surface of the
microelectrode, while all surrounding cells remained intact. The rate
of repopulation of the microelectrode with migrating cells was
monitored for 24 h as changes in resistance and capacitance.
Alternatively, cell adhesion was performed in 96-well clusters
precoated overnight with 20 µg/ml fibronectin, laminin, or collagens
I and IV. Endothelial cells (~20,000 cells/well) were seeded in the
presence or absence of 200 µM sodium nitroprusside (SNP) or 2 mM
L-NAME. After a 60-min
incubation, unattached cells were removed by washing with
phosphate-buffered saline and then cells were fixed with 2%
glutaraldehyde, air dried, and stained with 1% crystal violet in 0.2 M
boric acid (pH 9.0) for 20 min. The absorbance was measured at 590 nm
with a microplate reader. In addition, reflective confocal microscopy
(Noran Odyssey, Middleton, WI) was performed to obtain serial images of
focal contacts.
 |
RESULTS |
Effects of VEGF on NO production and cell migration.
Application of VEGF caused a dose-dependent increase in NO release from
SV40-immortalized renal microvascular endothelial cells (Fig.
1A).
The 50% effective concentration
(EC50) was ~8 ng/ml. VEGF
exhibited a similar potency in accelerating endothelial cell migration,
with an EC50 of ~10 ng/ml (Fig.
1B). In transwell migration
experiments, the above effect of VEGF was blunted in the presence of
the NO synthase inhibitor,
L-NAME (Fig.
1C).

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Fig. 1.
Effects of vascular endothelial growth factor (VEGF) on nitric oxide
(NO) production and endothelial cell migration.
A: representative recordings
(n = 4) of VEGF-induced release of NO
from cultured endothelial cells. Experiments were performed with an
NO-selective microelectrode, as detailed in MATERIALS
AND METHODS. Application of VEGF resulted in a
transient release of NO. The 50% effective concentration for
VEGF-induced NO production was 8 ng/ml. NO release could be blocked by
pretreatment with 2 mM
NG-nitro-L-arginine methyl ester
(L-NAME) (not shown).
Inset: calibrated NO concentration and
time scale. B: dose-response curve of
VEGF-induced endothelial cell migration in a Boyden apparatus.
* P < 0.05 vs. control
(n = 4).
C: VEGF-induced endothelial cell
migration requires functional NO synthase. VEGF was used at
concentration of 10 ng/ml, resulting in a more than 2-fold increase in
the number of migrated endothelial cells;
L-NAME (2 mM) abrogated the
effect of VEGF. These experiments were performed in medium depleted
of growth factors but containing
L-arginine. C, control.
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Effect of antisense oligonucleotides on VEGF-guided endothelial cell
migration.
To secure the highest possible selectivity of eNOS inhibition,
presently not achieved with the available
L-arginine derivatives, we next
utilized the antisense strategy to selectively knock down the enzyme.
Because the antisense construct was designed to target the initiation
codon of the human eNOS cDNA, HUVECs were utilized in these
experiments. After a 12-h incubation with 10 µM antisense, sense, or
scrambled S-ODNs in serum-free medium, cells were lifted and seeded
into wells of a Boyden chamber in the continuous presence of S-ODNs, as
detailed in MATERIALS AND METHODS. Two
hours later, when cells effectively adhered and spread, culture medium
containing 10 ng/ml VEGF was added to the opposite side of the
membrane. Transmigration assessed 7 h later showed that VEGF-induced
stimulation of HUVEC migration was virtually abrogated in cells
pretreated with the antisense construct, whereas effects of VEGF in the
presence of sense and scrambled S-ODNs were not significantly different from nonpretreated control cells (Fig. 2).
The efficacy of antisense treatment has been validated previously in
the same experimental setting (22). Furthermore, the toxicity of S-ODNs
at the concentrations used was undetectable, based on experiments with
ethidium homodimer staining of nuclei of damaged cells (not shown).
These findings, therefore, reconfirm the above data, which demonstrate
the requisite role of functional eNOS for VEGF-guided endothelial cell
migration.

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Fig. 2.
Effect of antisense oligonucleotides to endothelial NO synthase (eNOS)
on transmigration of human umbilical vein endothelial cells.
Experiments were performed in a Boyden apparatus, as detailed in
MATERIALS AND METHODS. Endothelial cells were pretreated
with antisense (AS), sense (S), or scrambled (SCR) phosphorothioated
oligonucleotides [all at 10 µg/ml, previously shown to be
nontoxic and sufficient for eNOS knockdown (22)].
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Wound healing experiments.
Endothelial cells were damaged by the application of a direct current
in the ECIS chamber. This resulted in a localized injury to the
endothelial cells occupying the electrode surface of 50,000 µm2 and denudation of the
electrode, as shown in Fig.
3A.
Consequently, the resistance of cell monolayers precipitously dropped
to 24 ± 3% of the basal level detected at confluence. The
resistance of the monolayers increased to 57 ± 4% of basal level
by 24 h after wounding, due to the migration of cells from wound edges (confirmed microscopically at the end of experiments, as exemplified in
Fig. 3A). The addition of VEGF
accelerated the process of wound healing (Fig.
3B), resulting in 69 ± 3%
(P < 0.05, n = 5) restoration of the resistance
(resistance at confluence was taken as 100%). This process was
dependent on the functional NO synthase because pretreatment of
endothelial cells with L-NAME
virtually abrogated wound healing [29 ± 5% of basal level
(Fig. 3B), whereas
D-NAME was ineffective (not
shown)].

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Fig. 3.
Endothelial wound healing is enhanced by VEGF in NO-dependent fashion.
A: micrographs of the
fibronectin-coated platinum electrode surface immediately after an
intact confluent cell monolayer was destroyed on the electrode surface
by a direct current pulse (note that cells are absent on the electrode,
whereas they are preserved around it) and 24 h later, when the
electrode surface has been repopulated by migrated endothelial cells.
Diameter of an electrode = 250 µm.
B: tracings of the electrical
impedance of endothelial cells. Initially, all monolayers had
resistances of >9,000 /cm2
(normalized resistance is shown) that fell to ~20-30% of
initial resistance after the application of direct current pulse. In
the absence of growth factors, endothelial cells slowly repopulated the
electrode surface, as judged by the increasing resistance. VEGF
accelerated the process of endothelial wound healing, whereas addition
of L-NAME, alone or in
combination with VEGF, abrogated endothelial cell migration. These data
are representative of 7 separate experiments performed with different
batches of endothelial cells.
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Role of NO in cell matrix adhesion.
To elucidate the cellular basis for the observed permissive role of NO
in VEGF-guided migration, endothelial cell monolayers were subjected to
electrical impedance analysis. Resting cells displayed spontaneous
fluctuations in electrical impedance that promptly disappeared after
addition of a fixative (Fig.
4A).
These data are consistent with the previously described micromotion in
ostensibly stationary cells (6). Furthermore, confluent endothelial
cell monolayers subjected to the NO donor, SNAP, displayed spontaneous
higher-amplitude fluctuations of impedance (at 100 µM) and a decrease
in the resistance of monolayers (at 500 µM), as shown in Fig. 4,
B and
C, respectively. To test the
possibility that NO interferes with cell matrix adhesion, the
attachment of endothelial cells to fibronectin-coated microelectrodes
was examined in the presence and absence of SNAP. When endothelial
cells were seeded on the surface of fibronectin-coated electrodes, cell
attachment and spreading (confirmed microscopically at 4 h) resulted in
a progressive increase of the resistance (Fig.
4D). The addition of SNAP resulted
in a dose-dependent inhibition of cell attachment [100 µM SNAP
resulted in 92.7 ± 0.87% of control (mean ± SE), n = 4, P > 0.05; 500 µM SNAP decreased
adhesion to 65.5 ± 3.57% of control,
n = 4, P < 0.005 vs. both control and 100 µM SNAP], thus supporting the notion that NO affects
endothelial cell matrix adhesion and implying observed spontaneous
fluctuations in cell resistance could be attributed to changes in
adhesive interactions.

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Fig. 4.
Real-time recordings of the electrical impedance of endothelial cells.
A: spontaneous fluctuations in the
resistance of confluent endothelial cell monolayers disappear after
fixation with 4% paraformaldehyde (PFA).
B and
C: NO donor
S-nitroso-N-acetyl-DL-penicillamine
(SNAP; arrow) increases the amplitude of spontaneous fluctuations in
the electrical resistance of confluent endothelial cells (at 100 µM
in B) and, at a higher concentration
(500 µM in C), decreases the
resistance. Note that the ordinate scale in
B and
C is different (to demonstrate
absolute and relative value of these miniature spontaneous fluctuations
detectable with high-resolution amplification).
D: real-time recordings of the
electrical impedance of freshly seeded endothelial cells, as an
indicator of their adhesion and spreading. Rate of endothelial cell
adhesion and spreading on fibronectin-coated microelectrode surface is
decreased in a dose-dependent fashion by the addition of the NO donor
SNAP. All tracings are representative of at least 4-5 experiments
performed on separate occasions using different passages of endothelial
cells.
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In addition to the above procedures, standard adhesion assays were
performed. Endothelial cells pretreated with either NO donor SNP or the
inhibitor of NO synthase L-NAME
were plated on fibronectin-, laminin-, or collagen-coated surfaces and
allowed to attach for 60 min. SNP treatment resulted in a significant blunting of endothelial cell adhesion to all matrices (Fig.
5). The adhesion of
L-NAME-treated cells was not
different from control cell population. Increasing the concentration of
L-NAME up to 10 mM did not
increase cell matrix adhesion over control levels (not shown). This
lack of L-NAME effect in
unstimulated cells indicates that HUVECs in culture express minimal NO
synthase activity and only when cells are stimulated with VEGF is it
possible to elicit an inhibitory effect of
L-NAME. Collectively, these data further support the above findings on NO interference with endothelial cell adhesion.

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Fig. 5.
Effect of modulating NO availability on endothelial cell matrix
adhesion. Endothelial cells pretreated with 200 µM sodium
nitroprusside (SNP) or 2 mM
L-NAME were plated into wells
coated with the indicated matrix proteins. After 60-min adhesion
period, reaction was stopped, cells were stained, and cell density was
determined by measuring the absorbance (optical density units). LN,
laminin; Col I, collagen I; Col IV, collagen IV; FN, fibronectin.
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To explore the possibility that observed spontaneous fluctuations in
cell resistance could be attributed to NO-induced changes in adhesive
interactions, mathematical analysis of impedance and capacitance of
endothelial cells subjected to NO donor SNAP or pretreated with NO
synthase inhibitor L-NAME was
performed according to the previously published equations (6) aimed to
distinguish between changes in the resistance of the paracellular
pathway or to changes in the distance between the ventral cell surface and the electrode. The data showed that the addition of the NO donor
SNAP resulted in a statistically significant (Wilcoxon's signed rank
test, P < 0.05, n = 5) increase in the subendothelial space, thus accounting for the observed decrease in the electrical resistance (Fig.
6A).
Pretreatment of endothelial cells with
L-NAME did not appreciably
affect this parameter (Fig. 6B).
Neither treatment was associated with significant changes in the
paracellular resistance (not shown).

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Fig. 6.
Analysis of changes in resistivity of endothelial cell monolayers after
application of 500 µM SNAP (A) or
pretreatment with 1 mM L-NAME
(B).
Top: scheme of endothelial cell
monolayer considered as a series of resistors, with the electrical
current passing either paracellularly or subcellularly underneath the
ventral cell surface (h space).
Parameter equals the product of cell radius
(r) and square root of a ratio
between a constant (resistance of the culture medium) and
subendothelial space (h).
A and
B: changes in the before and after
specified treatments, based on results of individual experiments.
Calculations were performed using an assumption that cell radius did
not change after the treatments. * Significant difference
(P < 0.05) between pre-
and posttreatment data.
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The results of these calculations were further challenged using
reflective confocal microscopy. Endothelial cells were seeded on a
fibronectin-coated glass coverslip and allowed to attach for 30 min,
unattached cells were removed by changing the medium, and images were
recorded at 5-min intervals. During the 30-min period of cell
attachment, focal contacts gained in prominence. Addition of 100 µM
SNP, a short-acting NO donor, resulted in the disappearance of some
focal contacts (Fig. 7). This effect was temporal (images obtained at 35-45 min), suggesting that the
immediate effect of NO is the decreased attachment of endothelial cells to the matrix.

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Fig. 7.
Serial images of focal adhesions in endothelial cells (reflective
confocal microscopy). Endothelial cells were seeded on
fibronectin-coated glass coverslips and allowed to attach for 30 min.
Unattached cells were removed by changing the medium, and images were
recorded at 5-min interval. During the recorded 30-min period of cell
attachment (images obtained at 0-30 min), focal contacts,
visualized as dark streaks using this technique, gained in prominence
and eventually stabilized (images obtained at 10-30 min). Addition
of 100 µM SNP, a short-acting NO donor, resulted in the transient
disappearance of some focal contacts (images obtained at 35-45
min). Quantitative analysis of these changes was performed using
Universal Imaging software program. Histograms of light intensity
(right) correspond to the flanking
images and graphically illustrate the shift in intensity occurring
after the addition of SNP (detected as the right shift toward brighter
pixels in images obtained at 35 and 40 min).
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DISCUSSION |
In the present study, the absolute requirement for functional NO
synthase in executing the VEGF-guided program of endothelial cell
migration and wound healing was demonstrated in two independent experimental settings. First, VEGF, shown to stimulate NO release from
endothelial cells, induced endothelial cell migration (75% stimulation), whereas L-NAME or
antisense oligonucleotides targeting eNOS completely suppressed
VEGF-stimulated migration in a Boyden apparatus. Second, in a series of
experiments on endothelial cell wound healing, the rate of
VEGF-stimulated cell migration was significantly blunted by the
inhibition of NO synthesis. The above findings led us to conclude that
VEGF-induced endothelial cell locomotion is mediated in part via NO
production.
To gain insight into the possible mode of NO action, impedance analysis
of endothelial cell monolayers subjected to NO was performed. Using
this impedance bioprobe, we have previously demonstrated micromotion,
i.e., spontaneous fluctuations in the resistance, in quiescent cells
(6). In the present study, we show that the micromotion in ostensibly
stationary endothelial cells is elicited in part by NO. We proposed to
term this process podokinesis to emphasize a scalar form of cell
locomotion occurring in nonmigrating endothelial cells, which, in the
presence of guidance cues, e.g., VEGF, may be transformed to a
vectorial movement. Thus the execution of the program for a directional
endothelial cell migration requires two simultaneous messages: scalar
NO-induced podokinesis and guidance cues provided by motogens like VEGF
(the present study), epidermal growth factor (23), or endothelin-1
(22).
The above data on the role of NO in mediating motogenic effects of VEGF
are in concert with the findings of substance P-stimulated cell
migration. Recently, it has been shown that the angiogenic effect of
substance P is mediated by NO (33). NO synthase inhibitors abolished
substance P-induced migration of microvascular endothelial cells,
whereas SNP potentiated cell migration. Leibovich et al. (13) have
observed angiogenic activity produced by stimulated human monocytes in
the presence of L-arginine, but
not in its absence, and suppressed angiogenesis in the presence of
inhibitors of NO synthase. Because these manipulations of the NO
pathway did not affect the release of several cytokines (tumor necrosis factor-
and interleukin-8), it is conceivable that the angiogenic activity in question was NO per se. Similarly, we have recently demonstrated that epithelial cell migration and wound healing are NO
dependent: NO serves as a switch from a stationary to locomoting phenotype in BS-C-1 cells (23). Furthermore, exogenous NO has been
shown to elicit chemotaxis of neutrophils in vitro (1). Most recently,
we have presented experimental evidence that NO plays a permissive role
in endothelin-1-elicited locomotion of rat microvascular and of HUVECs
(22). Inhibition of endothelial NO synthase with either
L-NAME or antisense S-ODNs
prevented endothelin-induced locomotion. A similar effect was observed
in Chinese hamster ovary cells expressing endothelin B receptor and NO
synthase (22). When the present study was in preparation, Morbidelli et
al. (20) reported that VEGF-induced proliferation of coronary venular
endothelial cells is mediated by NO and NO synthase inhibition
attenuates this effect. Indeed, previous studies in canine coronary
arteries have strongly suggested that VEGF-mediated vasorelaxation is
dependent on the production of endothelium-derived relaxing factor,
thus suggesting that this growth factor stimulates generation of NO (10). In the present study, through the use of an NO-selective microelectrode, the VEGF-stimulated release of NO was documented directly (Fig. 1A). Furthermore,
the role of cell migration, with lesser impact of cell proliferation,
is emphasized in experiments presented herein, since the time course of
experiments and incubation of cells in low-serum medium were designed
so as to minimize the mitogenic effects of VEGF.
It should be realized that NO effects on locomotion may be cell
specific and dose dependent. For instance, after balloon injury of rat
carotid artery endothelium, in vivo gene transfection of the
endothelial NO synthase into vascular smooth muscle cells abolished
neointimal formation (32). Similar results have been reported by Marks
et al. (18), demonstrating that a protein adduct of NO prevented
neointimal formation. Although the observed prevention by NO of
neointimal formation could be secondary to the inhibition of smooth
muscle cell proliferation, there was a biphasic effect of protein
adduct of NO on smooth muscle cell migration in a Boyden apparatus: low
concentrations of the protein enhanced it, whereas high concentrations
suppressed it (18).
Sporadic fluctuations in endothelial resistance and their enhancement
by NO, as resolved due to unique amplification of miniature changes in
impedance of cells grown on the surface of gold microelectrodes in ECIS
(Fig. 5), are due at least in part to changes in cell matrix adhesion.
Teleologically, they may represent the state of cellular
"vigilance" (preparedness to migrate when the integrity of
endothelial layer becomes compromised or motogenic guidance cues are
applied). The observed spontaneous changes in cell matrix adhesion,
referred to as podokinesis, may represent an essential step in
transforming the scalar motion of nonmigrating endothelial cells to the
vectorial movement on application of VEGF or endothelin. Experiments
with SNAP-induced stimulation of podokinesis implicate NO in its
generation. Furthermore, NO attenuated endothelial cell adhesion to
several matrix proteins, including fibronectin, and, at a higher
concentration, mediated cell detachment, indicating that NO affects
cell matrix adhesion. In this context, observations by Oliver et al.
(Ref. 24 and reviewed in Ref. 28) that the rate of locomotion has a
biphasic dependence on the tightness of cell-substratum adhesion may
explain the general phenomenon of NO-mediated motility. Somewhat
similar antiadhesive effects of NO have been described in leukocytes.
Adhesion of leukocytes to the venular endothelium in a superfused cat
mesenteric preparation is inhibited by
L-arginine and enhanced by
inhibitors of NO synthase (11). Analogous inhibition of leukocyte
adhesion to type I collagen was observed with SNAP or 8-bromoguanosine
3',5'-cyclic monophosphate (29). Thus it appears that NO
affects cell adhesion and migration in a broad range of cells.
Experiments conducted in collaboration with Zachary and Abedi in HUVECs
further confirm the effect of NO on formation and stability of focal
adhesions with reference to different matrix proteins (M. S. Goligorsky, H. Abedi, and I. Zachary, unpublished observations). The
proposed hypothesis on NO-driven changes in cell matrix adhesion,
resulting in podokinesis, may provide the explanation for these diverse
observations. Our model predicts the requirement for two coexisting
inputs: first, NO-dependent podokinetic scalar movements and, second,
presentation of guidance cues, e.g., VEGF or endothelin, to execute the
transition from a stationary phenotype to vectorial locomotion
resulting in angiogenesis (as schematically depicted in Fig.
8).

View larger version (28K):
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|
Fig. 8.
Hypothetical scheme of the dual-signal requirement (NO-driven
podokinesis and guidance cues) for directed migration of endothelial
cells in vitro and angiogenesis in vivo with the emphasis on the role
of functional NO synthase in these processes.
|
|
The implications of the above findings are twofold. First, in
situations in which angiogenesis is undesirable, i.e., tumor angiogenesis, a targeted inhibition of NO production by endothelial cells may provide a tool to halt neovascularization. In this vein, some
tumors express enhanced activity of NO synthases (9), in addition to
the production of various stimulators of angiogenesis. This may explain
in part the enhanced angiogenic meander toward such foci (5). Second,
the above findings may shed light on the reasons for inadequate
angiogenesis, despite the presence of appropriate gradients of
angiogenic stimuli, in the states of impaired endothelial NO
production, i.e., atherosclerosis (3, 12, 16). Under these
circumstances, supplying NO to the endothelium at ischemic sites may
improve angiogenesis. Application of the above model predicting the
requirement for two messengers, NO and VEGF, for execution of a program
for the directional locomotion should provide a theoretical basis for
the design of future angiogenic and angiostatic therapies.
 |
ACKNOWLEDGEMENTS |
These studies were supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grants DK-41573, DK-45695, and
DK-52783 (to M. S. Goligorsky). E. Noiri was supported by a National
Kidney Foundation fellowship award.
 |
FOOTNOTES |
Address for reprint requests: M. S. Goligorsky, Dept. of Medicine,
State Univ. of New York, Stony Brook, NY 11794-8152.
Received 15 April 1997; accepted in final form 8 October 1997.
 |
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