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Department of Physiology, Jefferson Medical College Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA; and
* Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130, USA
1Correspondence: Department of Physiology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107-6799, USA. E-mail: Rosario.Scalia{at}mail.tju.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: intravital microscopy • inflammation • neutrophil • microcirculation • mesentery
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Besides
its involvement in vascular development, VEGF has been shown to induce
endothelium-dependent vasodilation that is accompanied by a decrease in
mean arterial blood pressure and tachycardia (2)
. The VEGF-induced
vasodilation and its effect on vascular permeability are inhibited by
nitric oxide (NO) synthase inhibitors (3)
. Brock et al. (4)
demonstrated that VEGF increases cytosolic calcium, which is known to
promote calmodulin binding to the endothelial isoform of nitric oxide
synthase (NOS) and stimulate NO production (5)
. Similarly, VEGF has
been shown to increase NO release in bovine (6)
, rabbit (7)
, and human
endothelial cells (8)
. More recently, this effect of VEGF on NO
production has been associated with up-regulation of endothelial cell
nitric oxide synthase (ecNOS) activity (9)
.
A widely accepted feature of physiological concentrations of NO is its
protective role in several models of inflammation, including
ischemia-reperfusion and hypercholesterolemia (10
11
12
13)
. Several authors
have reported that impaired release of NO from ischemic-reperfused
vascular beds results in increased leukocyte–endothelium interaction
via up-regulation of endothelial cell adhesion molecules (14
15
16)
.
Similarly, systemic administration of NO donors to hypercholesterolemic
animals preserves endothelial function and attenuates pathological
interactions between circulating leukocytes and the vascular
endothelium (13)
. Under these conditions, numerous leukocytes adhere to
the vascular endothelium and some transmigrate, thus potentiating
endothelial dysfunction and tissue injury (17,
18)
.
Therefore, the purpose of this study was to examine the effect of VEGF
on leukocyte–endothelium interaction in vivo. Using
intravital microscopy of the rat mesenteric microcirculation, we
illustrated that systemic administration of VEGF inhibits
leukocyte–endothelium interaction induced by either thrombin or L-NAME
(NG-nitro L-arginine methyl ester).
This protective effect of VEGF was associated with enhanced release of
nitric oxide from the aortic endothelium. Furthermore, we showed a lack
of VEGF effect on leukocyte–endothelium interaction in mice missing
the gene codifying for ecNOS, thus demonstrating the essential role of
ecNOS-generated NO in mediating VEGF action. Taken together, our data
suggest a novel role for VEGF in acute inflammatory disorders of the
vasculature. These data also may explain the mechanism of the
protective effect of VEGF in acute vascular pathologies reported by
other investigators (19)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vascular endothelial growth factor
A nonglycosylated form of the human recombinant VEGF
(D9836AX-G143AB) was generously supplied by Genentech Inc. (South San
Francisco, California). Each animal received a single administration of
VEGF protein at a dose of 120 µg/kg, as an intravenous (i.v.) bolus.
Preliminary studies have shown that the i.v. infusion of 120 µg/kg
VEGF or injection of vehicle alone (400 µl 0.9% saline) does not
affect mean arterial blood pressure, heart rate, or cardiac output
(20)
.
Animal models and study protocols used for intravital microscopy
Rats
Male Sprague-Dawley rats, weighing 250–275 g, were anesthetized
with sodium pentobarbital (65 mg/kg) injected intraperitoneally (i.p.).
A tracheotomy was performed to maintain a patent airway throughout the
experiment. A PE-50 polyethylene catheter was inserted in the left
carotid artery for monitoring mean arterial blood pressure and infusion
of anesthetic. The abdominal cavity was opened via a midline laparotomy
and a loop of ileal mesentery was exteriorized for observation of the
mesenteric microcirculation via intravital microscopy. Rats were
randomly divided into one of five groups: 1) rats given VEGF
(120 µg·kg-1·i.v.
bolus-1) and superfused with Krebs-Henseleit
(K-H) buffer, 2) rats given 0.9% saline and superfused with
50 µM L-NAME, 3) rats given 0.9% saline and superfused
with 0.5 U/ml thrombin, 4) rats given VEGF and superfused
with 50 µM L-NAME, and 5) rats given VEGF and superfused
with 0.5 U/ml thrombin. Intravital microscopy experiments were
performed 2, 4, and 24 h after administration of VEGF. Therefore,
five experimental groups were completed for rats injected with VEGF 2,
4, or 24 h before intravital microscopy except the control group,
which was studied only at the 4 h time point. The sublingual vein
was used for VEGF administration in rats.
Gene-targeted mice
Wild-type (C57BL/6) mice and ecNOS-deficient (ecNOS -/-) mice
(C57BL/6; Jackson Laboratories, Bar Harbor, Maine) were anesthetized
with sodium pentobarbital (120 mg/kg) injected i.p. The left carotid
artery was cannulated for continuous blood pressure monitoring.
Intravital microscopy was performed on mouse peri-intestinal venules
after exteriorization of a loop of ileal tissue via a midline
laparotomy. Mice were randomly divided into one of five groups:
1) wild-type mice given 120 µg/kg VEGF and superfused with
K-H buffer, 2) wild-type mice given 0.9% saline and
superfused with 50 µM L-NAME; 3) wild-type mice given 120
µg/kg i.v. VEGF and superfused with L-NAME, 4) ecNOS -/-
mice given 0.9% saline and superfused with K-H, or 5) ecNOS
-/- mice given 120-mg/(kg/i.v. bolus VEGF) and superfused with K-H.
Experiments in VEGF-injected mice were performed 4 h after
administration of VEGF. The mouse tail vein was used to administer VEGF
in mice.
Intravital microscopy recordings
Intravital microscopy of rat mesenteric tissue and mouse
peri-intestinal venules was performed according to a previously
described method (21)
. Briefly, the ileum and mesentery of anesthetized
animals were placed in a temperature-controlled Plexiglas chamber. A
modified K-H solution alone or a K-H solution containing either 50 µM
L-NAME or 0.5 U/ml thrombin was used to superfuse the rat mesentery and
the mouse intestine. A Microphot microscope (Nikon Corp., Tokyo, Japan)
was used to visualize both the mesenteric venules in the rat and
peri-intestinal venules in the mouse. The image was projected by a
high-resolution color video camera (DC-330, DAGE-MTI, Inc., Michigan
City, Ind.) onto a color Sony high-resolution video monitor (Multiscan
200-sf) and the image was recorded with a videocassette recorder. All
images were then analyzed using computerized imaging software (Phase 3
Image System, Media Cybernetics) on a Pentium-based IBM-compatible
computer (Micron Millenia Mxe, Micron Electronics Inc, Nampa, Idaho).
Red blood cell velocity was determined on-line using an optical Doppler
velocimeter (22)
. Red blood cell velocity (V) and venular diameter (D)
were used to calculate venular wall shear rate (g), using the formula
g = 8 (Vmean/D) (Vmean = Vrbc/1.6), where V=velocity and
D=diameter (23)
. Leukocytes were considered to be rolling if they were
moving at a velocity significantly slower than that of red blood cells.
A leukocyte was judged to be adherent if it remained stationary for
more than 30 s. Adherence is expressed as the number of leukocytes
adhering to the endothelium/100 µM of vessel length. The number of
extravasated leukocytes in the rat mesenteric tissue was counted in the
tissue area adjacent to the postcapillary venules and normalized with
respect to area (20 x 100 µM).
In vitro adherence of isolated neutrophils (PMNs) to
rat superior mesenteric artery endothelium
Immediately after intravital microscopy observations were
completed, the superior mesenteric artery (SMA) was rapidly removed
from three rats in each of the five experimental groups and placed into
warmed oxygenated K-H solution. Fat and connective tissue were removed
from SMA segments, which were cut into rings 2 mm in length. Each ring
was then opened and placed with the endothelial surface up in a cell
culture dish filled with 3 ml of oxygenated K-H solution at 37°C, pH
7.4. Neutrophils were isolated from donor rats according to the method
of Williams et al. (24)
using the hetastarch exchange transfusion
technique. Isolated PMNs (400,000/ml) were labeled with PKH2, a green
fluorescent dye (25)
, and added to the dishes. SMA segments were then
incubated for additional 30 min in a shaker culture bath at 37°C in
one of three different solutions: 1) K-H, 2) K-H
with thrombin (2 U/ml), or 3) K-H with thrombin (2 U/ml) and
the rat-specific anti-P-selectin antibody, CY1748 (20 µg/ml). After
the incubation period, the SMA segments were carefully removed, lightly
rinsed with K-H solution and placed onto glass microscope slides.
Adherent PMNs were counted using epifluorescence microscopy (Nikon,
Tokyo, Japan) on five fields from each vessel segment and expressed as
numbers of adherent PMNs/mm2 of endothelial
surface area (26)
.
Effect of VEGF on NO released from isolated rat aortic segments
We used freshly isolated rat aortic rings as the source of
primary endothelial cells. After completion of intravital microscopy
recordings, thoracic aortae were rapidly isolated from three rats in
each of the five experimental groups and immersed in warm oxygenated
K-H solution. Aortas were cleaned of adherent fat and connective
tissue, and rings 6–7 mm in length were carefully prepared. Rings were
subsequently cut and opened from randomly selected areas of the aorta
and fixed by small pins with the endothelial surface facing up in
24-well culture dishes containing 1 ml K-H solution. After
equilibration at 37°C, NO released into the K-H solution was
measured. NO was measured according to the method of Guo et al. (27)
.
Briefly, a NO meter (Iso-NO, World Precision Instruments, Inc.,
Sarasota, Fla.) connected to a polarographic NO electrode was used. The
model of the NO electrode we used was internally shielded to minimize
external electrical interference. Calibration of the NO electrode was
performed daily before each experimental protocol. Standard calibration
curve was obtained by graded concentrations of
KNO2 at 0, 5, 10, 25, 50, 100, 250, and 500 nM
(final concentrations) into a calibration solution containing 0.1 M KI
and 0.1 M H2SO4.
Data analysis
All data are presented as means ± SE. Data
were compared by analysis of variance (ANOVA) using post hoc analysis
with Fisher's corrected t test. All data on leukocyte
rolling, adherence, transmigration, arterial blood pressure, and shear
rates were analyzed by ANOVA, incorporating repeated measurements.
Probabilities of 0.05 or less were considered statistically
significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
. We examined mesenteric venules ranging from
38 ± 4 to 42 ± 3 µM in diameter, and there was no
difference in venular diameter among any of the groups studied. The
venular shear rates were also very similar in all groups, ranging from
587 ± 28 to 613 ± 32 s-1. Similarly,
there was no significant difference in initial mean arterial blood
pressures among all groups of rats after all surgical procedures were
completed. Mean arterial blood pressures ranged between 145 ± 8
and 154 ± 10 mmHg over the 2 h observation period. In
addition, no significant systemic effect was recorded after the
exposure of the rat mesentery to either thrombin or L-NAME. These
findings clearly indicate that the adhesive interactions observed
between circulating leukocytes and the microvascular endothelium were
not due to changes in hemodynamics brought about by the superfusion
with thrombin or L-NAME or by systemic administration of 120 µg/kg
recombinant VEGF protein (20)
.
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Thrombin (0.5 U/ml) markedly increased leukocyte rolling and adherence
60–120 min after superfusion (Fig. 1
). However, both leukocyte rolling and adherence were virtually
abolished in thrombin-treated rats given VEGF at 4 and 24 h
postinjection (Fig. 1)
. Similarly, leukocyte transmigration was
significantly increased at 90–120 min after thrombin stimulation of
the mesentery, and this response was attenuated by VEGF treatment at 4
and 24 h postinjection (Fig. 3)
. In contrast, administration of
VEGF 2 h postinjection failed to inhibit thrombin-stimulated
leukocyte rolling, adherence, and transmigration (Fig. 1
and Fig. 3
).
In parallel experimental studies, we investigated the effects of VEGF
treatment on leukocyte rolling, adherence, and transmigration induced
by superfusion of the rat mesentery with L-NAME (Fig. 2
and Fig. 3
). Superfusion of the mesenteric tissue with 50 µM L-NAME increased
leukocyte rolling fivefold, leukocyte adherence sevenfold, and
leukocyte transmigration sevenfold at 120 min. Systemic administration
of VEGF also significantly inhibited the L-NAME-induced increase in
leukocyte–endothelium interactions to a degree comparable to its
effects on thrombin-stimulated mesenteries. In particular, 4 h
after injection of VEGF, leukocyte rolling, adherence, and
transmigration induced by L-NAME superfusion were attenuated to 10 ± 4, 2 ± 1 and 4 ± 1, respectively. In addition,
L-NAME-induced leukocyte–endothelium interactions were not inhibited
at 2 h postinjection, thus confirming that a 4 h lag time is
required in vivo for initiating the antiinflammatory
activity of VEGF. Therefore, systemic administration of VEGF to the rat
is able to inhibit leukocyte–endothelium interaction after exposure of
the mesenteric microvasculature to both inflammatory stimuli or nitric
oxide synthase inhibitors.
In vitro adherence of PMNs to SMA endothelium
Thrombin stimulation of isolated SMA segments isolated from
control rats resulted in a marked increase in the adhesion of
unstimulated rat PMNs to the SMA endothelium (Fig. 4
). The number of adhered PMNs/mm2 increased by
sevenfold in thrombin-stimulated SMA endothelium as compared to control
SMA endothelium (Fig. 4)
. However, the adhesion of neutrophils was
significantly lower in SMA endothelium obtained from both 4 and 24 h VEGF-injected rats (Fig. 4)
. VEGF reduced PMN adhesion to
thrombin-stimulated SMA segments by 40% (P<0.05 vs.
thrombin alone). Comparable results were obtained by coincubation of
thrombin-stimulated SMA segments with 20 µg/ml of an anti-P-selectin
monoclonal antibody (Fig. 4)
. Thus, systemic administration of VEGF
significantly attenuated the adhesion of rat PMNs to
thrombin-stimulated superior mesenteric vascular endothelium via a
P-selectin mechanism. This clearly demonstrates that systemic
administration of VEGF to the rat results in attenuation of
leukocyte–endothelium interaction at both the macrovascular and
microvascular level.
|
Effect of VEGF administration on NO release from isolated rat
aortic segments
We detected a small basal level of NO release in the range of
35 ± 6 pmol/µg of tissue in aortic rings isolated from control
rats (Fig. 5
). This basal release of NO was not different in aortic tissue obtained
2 h after VEGF administration to the rats. However, 4 and 24 h after rats were given the bolus dose of VEGF, the basal release of NO
measured in aortic rings increased from 35 ± 6 to 110 ± 15
pmol/mg tissue (P<0.001). Moreover, the addition of a
maximal concentration of L-NAME (i.e., 100 µM) inhibited NO release
in aortic rings obtained from both control rats and VEGF-treated rats
(Fig. 5)
. Therefore, systemic administration of recombinant VEGF
protein to the rats increases endothelium-derived nitric oxide release
in the aorta. This effect begins 4 h after a bolus ejection of
VEGF and lasts at least up to 24 h postinjection. In addition,
VEGF-induced NO release is likely mediated via stimulation of nitric
oxide synthase on the vascular endothelium, as confirmed by its
blockade by the NOS-inhibitor L-NAME.
|
Mouse peri-intestinal venules
To test our hypothesis that ecNOS mediates the attenuation of
leukocyte–endothelium interaction in the mesenteric vasculature during
acute inflammation, we performed similar experiments in wild-type mice
and ecNOS-deficient mice. Compared with C57BL/6 wild-type mice,
ecNOS-deficient mice exhibited a higher number of rolling (Fig. 6
, upper panel) and adherent leukocytes (Fig. 6
, lower panel) in
peri-intestinal venules. Superfusion of the ileal tissue of wild-type
mice with 50 µM L-NAME increased the number of rolling and adhering
leukocytes to 51 ± 4 cells/min and 12 ± 1 cells/100 µM,
respectively (P<0.001 vs. control wild-type mice superfused
with K-H buffer). These values were comparable to those observed in
ecNOS-deficient mice superfused with only K-H buffer. At 4 h
postinjection, VEGF (120 µg/kg) consistently attenuated
L-NAME-induced leukocyte rolling and adherence in peri-intestinal
venules of wild-type mice (data not shown) while failing to inhibit the
abnormal increase in baseline leukocyte rolling (Fig. 6
, upper panel)
and adherence (Fig. 6
, lower panel) observed in ecNOS-deficient mice.
This clearly demonstrates that up-regulation of ecNOs activity is the
predominant, if not the only, mechanism by which VEGF attenuates
leukocyte–endothelium interaction in acute model of inflammation. In
addition, we found no significant systemic effects on hemodynamics
after administration of VEGF to either wild-type or ecNOS-deficient
mice. When venular shear rates were calculated in the five experimental
groups of mice, no significant difference was recorded despite the
higher mean arterial blood pressure values observed in ecNOS-deficient
mice as compared to control wild-type mice (MABP 155 ± 15 and
110 ± 8 mmHg, respectively; P<0.05). Initial shear
rates values were 606 ± 41, 560 ± 31, 555 ± 39,
608 ± 30, and 597 ± 29 s-1 for the
five groups of mice. These values were not different from each other
and did not change over the 60 min observation period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The widespread expression and organ-specific distribution of VEGF
messenger RNA and protein in normal rat tissue support the concept that
VEGF may play a multifunctional role in the maintenance of normal
vascular function in addition to mediating vascular growth (28)
. Since
Ku and co-workers (29)
demonstrated that VEGF stimulates the release of
endothelial-derived nitric oxide in canine coronary arteries, several
others have shown that VEGF is able to attenuate tissue injury after
ischemia-reperfusion of the heart (30)
, restore endothelial function in
balloon-injured arteries (31)
, and promote recovery of normal
endothelial reactivity of ischemic large arteries (32)
. Our results are
the first to explain the mechanism of the protective effect of VEGF
against inflammatory states of the large conduit arteries, as well as
of the microcirculation.
It is well established that NO is a potent modulator of leukocyte
adhesion to the vascular endothelium (15,
33)
. Previous studies have
clearly demonstrated antineutrophil properties of NO in vivo
as well as in vitro (11,
15,
34)
. In fact, administration of
authentic NO (12)
, NO donors (11)
, or the NO precursor L-arginine (35)
have all proved to be highly beneficial in a number of inflammatory
disease states, including ischemia-reperfusion and hypercholesterolemia
(13)
. Furthermore, we have recently shown that NO donation inhibits
leukocyte–endothelium interactions induced by activation of the
microvascular endothelium with both L-NAME (14)
or thrombin (18)
. In
addition, De Caterina et al. (36)
have shown that that nitric oxide
inhibits the expression of cell adhesion molecules on the vascular
endothelium. In this regard, inhibition of adhesion molecule expression
is likely to be an essential mechanism of the antileukocytic actions of
NO.
Here we provide strong evidence that systemic administration of
VEGF is able to attenuate enhanced leukocyte–endothelium interaction
induced by inflammatory stimuli via increased release of NO from the
vascular endothelium. This result is consistent with earlier findings
showing that VEGF up-regulates ecNOS enzyme, thus eliciting a biphasic
stimulation of endothelial NO production (9)
. Due to this
NO-potentiating property of VEGF, it is likely that after
administration of VEGF, increased NO release is responsible for
down-regulation of leukocyte–endothelium interactions. Nevertheless,
the precise mechanism of this immunomodulatory function of VEGF and how
it relates to cell adhesion molecule expression in vivo
remain to be determined.
We also obtained evidence that VEGF did not inhibit
leukocyte–endothelium interaction in mice lacking the gene encoding
for ecNOS enzyme. Experiments performed in the mouse model indicate
that ecNOS activity is essential for mediating VEGF actions. In this
regard, Murohara et al. (37)
have recently shown that in
ecNOS-deficient mice angiogenesis is not induced by either recombinant
VEGF protein administration or adenovirus-mediated VEGF gene transfer.
In summary, we have demonstrated that a single i.v. dose of VEGF markedly inhibits pathological leukocyte–endothelium interaction 4 and 24 h later in a well-established experimental model of inflammation. In particular, VEGF inhibited leukocyte rolling, adhesion, and transmigration within rat mesenteric venules, attenuated adherence of rat PMNs to isolated rat SMA endothelium, and increased release of nitric oxide from the aortic endothelium. In addition, no VEGF action on leukocyte–endothelium interaction was observed in the absence of ecNOS, as assessed in ecNOS-deficient mice. To our knowledge, this is the first in vivo evidence showing that potentiation of NO release via systemic administration of VEGF results in attenuation of pathological leukocyte recruitment in inflamed vascular areas. Since inhibition of leukocyte–endothelium interaction has been found to be beneficial in several pathophysiological states of the cardiovascular system, this effect may be important in understanding the role of VEGF therapy in cardiovascular diseases, including ischemia-reperfusion injury and atherosclerosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication December 9, 1998.
Revision received January 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), ecNOS -/- mice
(
), and ecNOS -/- mice (
) given VEGF protein. Bar heights show
number of transmigrated leukocytes for the three experimental groups of
mice. All values are means ± SE observed at 0, 15,
30, 45, and 60 min for six mice in each group. The VEGF protein was
administered to the rats at a dose of 120 µg/kg, as an i.v. bolus.
**P<0.01 from ecNOS -/- mice.