Departments of 1 Anesthesiology and 2 Surgery, University of Virginia, Charlottesville, Virginia 22906; and 3 Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan 48702
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ABSTRACT |
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Pulmonary hypertension is
characterized by structural and morphological changes to the lung
vasculature. To determine the potential role of nitric oxide in the
vascular remodeling induced by hypoxia, we exposed wild-type
[WT(+/+)] and endothelial nitric oxide synthase (eNOS)-deficient
[(/
)] mice to normoxia or hypoxia (10% O2) for 2, 4, and 6 days or for 3 wk. Smooth muscle
-actin and von Willebrand
factor immunohistochemistry revealed significantly less muscularization
of small vessels in hypoxic eNOS(
/
) mouse lungs than in WT(+/+)
mouse lungs at early time points, a finding that correlated with
decreases in proliferating vascular cells (5-bromo-2'-deoxyuridine
positive) at 4 and 6 days of hypoxia in the eNOS(
/
) mice. After 3 wk of hypoxia, both mouse types exhibited similar percentages of
muscularized small vessels; however, only the WT(+/+) mice exhibited an
increase in the percentage of fully muscularized vessels and increased
vessel wall thickness. eNOS protein expression was increased in hypoxic
WT(+/+) mouse lung homogenates at all time points examined, with
significantly increased percentages of small vessels expressing eNOS
protein after 3 wk. These results indicate that eNOS deficiency causes decreased muscularization of small pulmonary vessels in hypoxia, likely
attributable to the decrease in vascular cell proliferation observed in
these mice.
lung; hypertension; endothelial nitric oxide synthase; muscularization
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INTRODUCTION |
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VASCULAR REMODELING, a dynamic process that occurs in response to a variety of stimuli, including shear stress and hypoxia, is one of the characteristics of pulmonary hypertension. The remodeling process of the pulmonary vasculature in response to hypoxia-induced pulmonary hypertension is known to involve structural alterations that extend to the periphery of the vascular tree and includes thickening of the smooth muscle (SM) layer down to the precapillary vessels (15). This muscularization of existing vessels involves both hypertrophy and hyperplasia of SM cells of the tunica media. Additionally, proliferation, differentiation, and migration of SM precursor cells such as pericytes and fibroblasts may contribute to this phenomenon (15).
Nitric oxide (NO) is a highly reactive gas that produces a variety of physiological and pathophysiological effects (26, 28). NO is an important modulator of blood pressure due to its vasodilatory capabilities as an endothelial cell-derived relaxing factor (EDRF) (11, 26, 33). In addition, many studies (2, 8, 9, 29) have demonstrated that NO is involved in modulation of cell proliferation, differentiation, and apoptosis in the vasculature. NO has been shown to exert both proliferative and antiproliferative effects on vascular SM (1, 6, 23, 29, 35, 46). The site of origin of NO, its concentration, and the stage of remodeling are all factors that may influence which action NO shows.
Many studies (7, 18, 39, 40, 45) have led to conflicting
data about the roles of NO in the development of the structural and
pathological changes seen in the vasculature of hypoxia-induced pulmonary hypertensive lungs. Although some studies (18,
45) have implicated alterations in the NO signaling pathway in
the pathogenesis of pulmonary hypertension, others (12, 13,
20) have yielded conflicting results that indicate a protective
and antiproliferative role for NO in the pulmonary vasculature. In vivo
experiments performed in our laboratory revealed an upregulation of
both the constitutive endothelial (eNOS) and inducible (iNOS) isoforms
of NO synthase (NOS) and NO production in the lung vasculature of rats
exposed to chronic hypoxia (18, 45). Elevations in NOS
expression correlated temporally with the onset and progression of
remodeling of these vessels (45). Meanwhile, the mRNA,
protein, and enzyme activities of the NO receptor protein-soluble
guanylate cyclase are all increased in rat lungs exposed to chronic
hypoxia (21). Recent studies that used mice with
homozygous-targeted disruptions of the eNOS gene [eNOS deficient
(/
)] have yielded insight into the potential role of
endothelium-derived NO in the etiology of pulmonary hypertension and
vessel remodeling. eNOS(
/
) mice exhibit both systemic and
pulmonary hypertension. However, in eNOS(
/
) mice, there
is increased pulmonary vascular remodeling in hypoxia-induced pulmonary
hypertension (7, 37, 39).
In the studies presented here, we attempted to elucidate the roles of
endothelium-derived NO in hypoxia-induced pulmonary vascular
remodeling and, in particular, during the early phase of
remodeling when hyperplasia and vascular cell proliferation are
most prominent. To accomplish this, we exposed both wild-type [WT(+/+)] and eNOS(/
) mice to a time course of normoxia or
chronic hypoxia. We then examined the pulmonary vasculature of these
mice to determine the kinetics of their remodeling response to hypoxia. In addition, we examined whether proliferation of vascular cells plays
a mechanistic role in this remodeling and what effect the deletion of
eNOS produces on proliferative indexes and the vascular remodeling
response to hypoxia. We found that remodeling of the small pulmonary
vessels begins as early as 2 days of hypoxia, with cell proliferation
in the pulmonary vasculature distinctly upregulated by 4 days of
exposure. In contrast to previous reports (7, 40), we
observed that eNOS(
/
) mice exhibit decreased remodeling of the
small pulmonary vessels after exposure to chronic hypoxia compared with
that in WT(+/+) mice. Furthermore, the underlying mechanism for this
remodeling deficit is a decrease in vascular cell proliferation in the
small vessels of the eNOS(
/
) mice compared with that in the WT(+/+) mice.
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MATERIALS AND METHODS |
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Animal exposures.
WT(+/+) C57BL/6J (Jackson Laboratories, Bar Harbor, ME) or eNOS(/
)
mice aged 8-12 wk (n = 5-8 total/group) were
generated as previously described (20) and backcrossed at
least seven generations to the parental C57BL/6J strain. The mice were
exposed to either normoxia or hypoxia (10% O2) for 2, 4, and 6 days or for 3 wk according to published methods (18, 21,
42). Forty-eight and twenty-four hours before tissue harvest,
all animals were injected intraperitoneally with 100 mg/kg body wt of
5-bromo-2'-deoxyuridine (BrdU; Boehringer Mannheim, Indianapolis, IN).
All animals were fed standard mouse chow and water ad libitum and were
treated humanely in accordance with institutional and federal guidelines.
Tissue processing.
At the indicated time points, each mouse was anesthetized with
methoxyflurane, the chest cavity was opened, and the lungs were
perfused with 2-4 ml of heparinized saline through the pulmonary artery until they appeared white. The lungs were removed and slightly inflated through the trachea with Ca2+- and
Mg2+-free PBS (GIBCO BRL, Life Technologies, Grand Island,
NY). The left lobe was sectioned into 1- to 2-mm sections, fixed in 4% paraformaldehyde in PBS, and processed for paraffin-embedded and frozen
sections as previously described (45). The right lung lobe
was flash-frozen in liquid nitrogen and stored at 70°C for Western
blot analysis.
Immunohistochemical techniques: muscularization of pulmonary
vessels.
Frozen sections (3-5 µm) were immunolabeled with antibodies
specific for von Willebrand factor (vWF) for endothelium and SM -actin for SM cells to differentiate these tissue types, as
previously described (45). In brief, tissue sections were
incubated overnight at 4°C with a rabbit polyclonal and a monoclonal
antibody for vWF (DAKO, Carpinteria, CA) and SM
-actin (Sigma, St.
Louis, MO), respectively. The sections were subsequently incubated with horseradish peroxidase-labeled goat anti-rabbit (Bio-Rad, Hercules, CA)
and alkaline phosphatase-labeled rabbit anti-mouse (Sigma) secondary
antibodies. vWF staining was visualized with the 3'-diaminobenzidine (Vector, Burlingame, CA) substrate, which produces a brown/black color.
SM
-actin staining was accomplished with the New Fuchsin (Dako)
substrate, which produces a red color. Sections were then briefly
counterstained with hematoxylin before they were mounted.
BrdU incorporation. BrdU is a nonradioactive thymidine analog incorporated into the DNA of actively proliferating cells only. Immunohistochemical staining of paraffin sections was performed as follows. Sections (3- to 5-µm) were baked onto slides at 60°C. Lung tissue was then digested with 0.1% trypsin (Sigma) before denaturation of DNA with 4 M hydrochloric acid to expose the antigen. After neutralization of the acid with PBS, the sections were incubated overnight at 4°C with an alkaline phosphatase-labeled monoclonal anti-BrdU antibody (Boehringer Mannheim). Identical procedures for the vWF immunolabeling mentioned in Immunohistochemical techniques: muscularization of pulmonary vessels were performed the next day. BrdU localization within proliferating cells was visualized with Vector Blue substrate (Vector Laboratories), and sections were counterstained with Nuclear Fast Red (Vector Laboratories) before they were mounted.
Vessel morphometry and cell proliferation.
All analyses of vessel morphometry and cell proliferation were
performed with the use of a blind code. The degree of muscularization of small (80 µm), medium (
81-149 µm), and large (
150
µm) vessels was determined in a minimum of 200 small, 50 medium, and
25 large vessels per animal. Vessels were classified as nonmuscular,
partly muscular, or fully muscular by SM
-actin staining. Partially muscular vessels were defined as those exhibiting at least one SM cell
but no continuous media. Fully muscularized vessels had a continuous SM
layer. The number of vessels exhibiting at least one SM cell divided by
the total number of vessels yielded the percentage of muscularized
vessels. Muscularized vessels were then classified as partly or fully
muscularized by the criteria described above. The mean of repeated
measurements from the endothelium to the outer edge of the SM layer in
fully muscularized vessels was used in the formula (2 × wall
thickness × 100)/external diameter to give percent vessel wall
thickness (%VWT) or, for the three sizes of vessels, to analyze
increases in VWT vascular remodeling.
Western blot analysis and immunohistochemistry for eNOS protein. The details of Western blotting and immunohistochemistry have been published previously (18, 45). Briefly, the right lobe from each mouse lung was homogenized in homogenization buffer and centrifuged, and the protein content was analyzed by the method of Bradford (4). One hundred micrograms of protein were electrophoresed in a 7.5% SDS-polyacrylamide gel by the method of Laemmli (17) and transferred to nitrocellulose. The blot was incubated with a polyclonal anti-eNOS primary antibody (1:1,000 dilution; Transduction Laboratories, Lexington, KY) and a goat anti-rabbit horseradish peroxidase-labeled secondary antibody (1:3,000 dilution; Bio-Rad). eNOS protein signal was detected with enhanced chemiluminescence (ECL; Amersham) reagent. For immunohistochemistry, paraffin sections were processed as described in BrdU incorporation, then incubated overnight at 4°C with either a monoclonal anti-eNOS antibody (1:500 dilution; Transduction Laboratories) or a nonspecific mouse IgG (negative control). The sections were then incubated with a biotinylated anti-mouse secondary antibody (1:500 dilution; Vector Laboratories) before treatment with avidin-biotin reagent (Vector Laboratories) and visualization of the eNOS protein signal with 3'-diaminobenzidine and counterstaining with hematoxylin.
Statistical analysis.
Data were analyzed for significance with multiple comparisons ANOVA.
P 0.05 was considered significant.
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RESULTS |
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Muscularization of pulmonary vessels.
As depicted in Fig.
1A, WT(+/+) and
eNOS(/
) mice exposed to hypoxia exhibited significant increases in
muscularization of small pulmonary vessels over normoxic control mice
as early as 2 days of exposure. This increase in
muscularization is reflected in the percentage of total vessels
showing at least one SM cell. WT(+/+) mice exposed to hypoxia showed a
roughly twofold increase in muscularized small vessels by 4-6 days
of exposure compared with normoxic animals; however, the
eNOS(
/
) animals exhibited a significantly lower percentage of
muscularized small vessels in hypoxia compared with WT(+/+) mice at
both time points. No significant differences between WT(+/+) and
eNOS(
/
) mice were found with regard to percentage of fully
muscularized small vessels at early time points (2,4, or 6 days) in
normoxia or hypoxia or in the degree of muscularization of medium- and
large-sized vessels (data not shown).
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Cell proliferation and BrdU labeling.
As depicted in Fig.
3A, significant
elevations (2.0- to 3.5-fold) in the percentage of small pulmonary
vessels containing BrdU-positive cells were detected at 4 days of
hypoxic exposure in both WT(+/+) and eNOS(/
) mice, and the
percentage remained elevated at 6 days compared with that in normoxic
control mice. Elevations in the percentage of small vessels with
BrdU-positive cells in eNOS(
/
) mice in hypoxia were significantly
less than those observed in the WT(+/+) animals at both 4 and 6 days.
Additionally, the number of BrdU-positive cells per positive small
vessel increased in hypoxia versus normoxia at 4 and 6 days (Fig.
3B) in both the WT(+/+) and eNOS(
/
) mice; again the
increases in the eNOS(
/
) mice were less than those observed in the
WT(+/+) mice. In medium-sized vessels, increases in BrdU labeling in
hypoxia were detected as early as 2 days of exposure, with increases in
the number of BrdU-positive cells per positive vessel significantly
elevated in both mouse types. These increases persisted through the
day 6 time point, with less proliferation detected in the
hypoxic eNOS(
/
) mice compared with WT(+/+) mice at 6 days (Fig.
3C). Large vessels exhibited increases in the average number
of BrdU-positive cells per positive vessel at 4 and 6 days of hypoxia
for both WT(+/+) and eNOS(
/
) animals, with significantly less
proliferation detected in the eNOS(
/
) mice at 6 days of hypoxia
(Fig. 3D). No differences in proliferative indexes were
detected at 3 wk of exposure between normoxia and hypoxia or between
the mouse types (data not shown). Examples of immunohistochemical
staining for BrdU-positive cells in the pulmonary vasculature in
normoxia and hypoxia are provided in Fig.
4.
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Western blot analysis and immunohistochemistry for eNOS protein.
As depicted in Fig. 5, A and
B, exposure of WT(+/+) mice to hypoxia resulted in a
significant upregulation of eNOS protein in whole lung homogenates
compared with normoxic mice at all time points examined (P 0.05). Peak increases in eNOS protein expression were found at 6 days of hypoxic exposure, and expression remained elevated after 3 wk.
No evidence of eNOS protein was detectable in lung homogenates from
eNOS(
/
) mice in either normoxia or hypoxia (data not shown) as
previously described (37). Immunostaining for eNOS protein
in lung sections of WT(+/+) mice revealed a significant upregulation of
eNOS in the small pulmonary vessels of hypoxia-exposed mice compared
with normoxic mice after 3 wk of exposure (Fig. 5C). In
contrast to findings in the rat (45), ~50% of small vessels in the mouse exhibited positive eNOS staining in normoxia. Examples of eNOS immunohistochemistry are provided in Fig.
6.
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DISCUSSION |
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The underlying cellular and molecular mechanisms of
hypoxia-induced pulmonary hypertension have been the subject of intense scrutiny. Various studies (7, 13, 14, 39-41, 45, 46) have provided conflicting data regarding the potential role of NO in SM
cell proliferation in the pulmonary vascular remodeling induced by
hypoxia. In a rat model of chronic hypoxia, upregulation of eNOS in the
small vessels of the lung precedes and progresses with the time course
of muscularization of these vessels (45). The development
of a mouse model of pulmonary vascular remodeling and the use of
eNOS(/
) mice to determine whether eNOS-generated NO is a mediator
of vascular remodeling induced by hypoxia has been an area of intense
recent interest. Our results demonstrate that lack of eNOS protein and
interruption of endothelium-derived NO produce a defect in the
remodeling response to chronic hypoxia in the small resistance vessels
in the pulmonary vasculature. This deficiency in remodeling manifests
itself early in the hypoxic time course and results in decreases in SM
cell proliferation and in the percentage of small vessels showing
muscularization compared with those in hypoxic WT(+/+) mice. Recent
studies lent further support to the concept of a role for NO in
vascular remodeling. Left common carotid artery ligation in rats
produced elevations in expression of eNOS protein in the right carotid,
which correlates with remodeling of this vessel (24).
Strengthening this hypothesis, N-nitro-L-arginine methyl ester
(L-NAME) treatment diminishes vascular remodeling in
rabbits after creation of an arteriovenous fistula (43),
and studies utilizing mice with targeted disruption of the eNOS locus
have shown that the absence of eNOS induces elevations in pulmonary
pressure (39) and abnormal remodeling of the carotid
artery in response to decreased flow (34). It is also
apparent from our results that other compensatory mechanisms exist in
the lung vasculature to counterbalance the lack of eNOS-derived NO as
indicated by our data showing that by 3 wk of hypoxic exposure, eNOS(
/
) mice exhibited elevations in the percentage of muscularized small vessels similar to those observed for the WT(+/+) mice. In a
previous study from our laboratory (32), we observed
upregulation of iNOS protein expression in lung homogenates of
eNOS(
/
) mice exposed to both normoxia and chronic hypoxia. This
enhanced expression of an alternative isoform of NOS in eNOS(
/
)
animals may compensate, in part, for the lack of NO produced by
deletion of the endothelial isoform.
An interesting observation from this study is that after long-term
hypoxic exposure (3 wk), deletion of eNOS seemed primarily to inhibit
progression from a partly muscularized pulmonary vessel to a fully
muscularized one and also prevented thickening of the SM wall in fully
muscularized small vessels. Our results are in contrast to recent
studies (7, 40) of hypoxia-induced pulmonary hypertension
that detected increases in vascular remodeling in eNOS(/
) mice
compared with WT(+/+) mice. However, both of these studies evaluated
the time response of remodeling (6 wk of hypoxia exposure) and utilized
eNOS(
/
) mice of a different genetic background (C57BL/6-SV129) than
those used in our experiments. They also examined either a single WT
control strain (40) or nonbackcrossed hybrid WT animals
(7). Studies have demonstrated that controlling for
genetic background is essential in vascular research, including gene
deletion. It has been very well documented that there are strain
differences in resting levels of arterial blood pressure (36), in susceptibility to atherosclerosis (31,
38), and in angiogenesis (42). The eNOS(
/
) mice
used in our study were backcrossed extensively (seven generations) to
the parental C57BL/6 strain to minimize potential variability
introduced by the use of mice of mixed genetic background. Our control
mice more extensively remodeled the pulmonary vasculature in hypoxia
than the control mice used by Steudel et al. (40) and
Fagan et al. (7), which accounts, in part, for the
discrepancies in results. Additionally, the study by Fagan et al. used
a hypobaric hypoxic exposure protocol, which may have had an impact on
their results. It is also possible that different strains of mice and,
more importantly, the effect of mixing genetically different strains
may produce varying levels of growth factors, receptors, or other
physiologically important molecules that could alter the extent of
remodeling in response to hypoxia. Clearly, this is an area that
deserves further study.
Our study is novel and distinct from previous studies in the
examination of the role of cell proliferation in pulmonary vascular remodeling in all sizes of vessels and of how eNOS deletion affects proliferative indexes in vivo. Analysis of data from the early time
points indicates the importance of vascular cell proliferation in
remodeling of the mouse pulmonary vasculature in response to more acute
hypoxic exposures. Our findings suggest that the levels of NO generated
by eNOS in vivo under hypoxic conditions may be proproliferative for
vascular SM cells or precursors. This hypothesis is reinforced by
the pronounced elevations in indexes of cell proliferation
observed in the small pulmonary vessels at 4 and 6 days of exposure and
the diminished proliferation in the vasculature of eNOS(/
)
mice in hypoxia at these early time points compared with those in
WT(+/+) mice. The decreased vascular cell proliferation correlates with
less muscularization of small vessels in this mouse type than that
observed in the WT(+/+) animals and provides a mechanistic explanation
for the decreased remodeling. These results differ from previous
studies (25, 34) that examined vascular cell proliferation
in eNOS(
/
) mice. However, these studies focused on large systemic
arteries and utilized carotid artery ligation (34) and
femoral cuff (25) models, both of which are significantly
different from the methodology used in our study, making direct
comparison of results problematic. A recent study by Ulibarri and
coworkers (44) showed that NO concentration may influence
its pro- versus antiproliferative response. They found that low levels
(5-10 µM) of sodium nitroprusside and
S-nitroso-N-acetylpenicillamine stimulated
myoblast proliferation and that a high level (50 µM) of
S-nitroso-N-acetylpenicillamine inhibited
proliferation. The exact mechanism for the role of NO induction of cell
proliferation is currently unknown. Reports from other studies have
implicated NO as a mediator of cell proliferation during angiogenesis.
There is reduced angiogenesis in response to tissue ischemia
(27), impaired wound healing, and angiogenesis
(19) in eNOS(
/
) mice, implying that NO may act through
vascular endothelial growth factor (10, 30) and activation
of mitogen-activated protein kinase (16).
Although the role of NO in modulating proliferation of vascular cells is controversial (6, 23, 35, 46), most studies showing that NO is antiproliferative in vascular SM cells in vitro made use of cells at high passage (22), in which cGMP-dependent protein kinase, a major downstream effector of NO signaling, is downregulated. Decreased cGMP-dependent protein kinase expression correlates with a dedifferentiated phenotype, and stimulation of these cells with NO results in cGMP-mediated activation of the cAMP-dependent protein kinase pathway and inhibition of mitogenesis (3, 5). Furthermore, exposure of primary cultures of aortic SM cells to NO donors enhances the mitogenicity of basic fibroblast growth factor (14), implying that NO is a proproliferative molecule in fully differentiated vascular cells.
An intriguing result of our study is that muscularization of small lung vessels begins as early as 2 days of exposure to hypoxia, with no accompanying increase in vascular cell proliferation. This finding indicates the possibility of two distinct processes initiated sequentially in the pulmonary vasculature as it adapts to hypoxic conditions. The early acute phase may involve differentiation and/or migration (15) of SM cell precursors such as pericytes or fibroblasts, which provide the initial muscularization of previously nonmuscular small vessels. The later phase, which begins between 2 and 4 days of hypoxia, involves induction of vascular cell proliferation to further muscularize these resistance vessels. Also of note is the finding that elevations in proliferating SM cells were detected in both medium and large vessels at 4 and 6 days of hypoxia compared with those in normoxic control mice. However, these classes of vessels showed no corresponding increases in muscularization, likely due to counteracting mechanisms such as apoptosis, which prevents increases in muscularity of SM walls. This topic is presently under investigation in our laboratory. Our results suggest that endogenous eNOS activity is critical for the proliferation of vascular SM cells in hypoxia-induced pulmonary hypertension. It is possible, however, that the amount of NO delivered, local levels of NO achieved, and/or temporal requirements may be different from endogenous synthesis when NO donors are used to compensate for a deficiency of authentic NO.
In conclusion, our studies indicate that NO derived from eNOS is a
mediator of vascular cell proliferation and muscularization of small
pulmonary vessels in response to hypoxia and support previous
observations from our laboratory and others (18, 34, 45) regarding the role of eNOS in vascular remodeling. Lack of
eNOS leads to decreased hyperplastic potential and muscularization of
small vessels during more acute exposures. Furthermore, NO deficiency
inhibits progression from partly to fully muscularized vessels and
abrogates the thickening of SM walls seen in WT(+/+) mice exposed to
prolonged hypoxic conditions. However, pulmonary vessels in eNOS(/
)
mice do undergo remodeling, including proliferation and
muscularization, in response to hypoxia, indicating that alternative mechanisms such as iNOS upregulation exist in these mice, enabling them
to adapt to the lack of NO and to conditions of low oxygen tension.
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ACKNOWLEDGEMENTS |
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We thank Oliver Smithies for his kind gift of the original endothelial nitric oxide synthase-deficient mice used in this study and Mary Davison for assistance in the preparation of this manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute National Research Service Award 1-F32-HL-09797-01 to T. R. Quinlan and National Heart, Lung, and Blood Institute Grant R01-HL-39706 and National Institute of General Medical Sciences Grant GM-49111 to R. A. Johns.
Address for reprint requests and other correspondence: R. A. Johns, Johns Hopkins Univ. School of Medicine, Dept. of Anesthesiology and Critical Care Medicine, 600 N. Wolfe St., Blalock 1415, Baltimore, MD 21287-4965 (E-mail: rajohns{at}jhmi.edu).
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.
Received 30 June 1999; accepted in final form 1 May 2000.
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