eNOS expression is not altered in pulmonary vascular
remodeling due to increased pulmonary blood flow
Allen D.
Everett1,
Timothy D.
Le Cras2,
Chun
Xue2, and
Roger A.
Johns2
Departments of 1 Pediatrics and
2 Anesthesiology, University
of Virginia, Charlottesville, Virginia 22908
 |
ABSTRACT |
Congenital heart
lesions resulting in increased pulmonary blood flow are common and if
unrepaired often lead to pulmonary hypertension and heart failure.
Therefore, we hypothesized that increased pulmonary blood flow without
changes in pressure would result in remodeling of the pulmonary
arterial wall. Furthermore, because the vasodilator nitric oxide is
produced by the lung, is regulated by flow in the systemic circulation,
and has been associated with the regulation of smooth muscle cell
proliferation, we hypothesized that increased pulmonary blood flow
would result in altered expression of endothelial nitric oxide synthase
(eNOS). To study this hypothesis, 42-day-old Sprague-Dawley
rats had creation of an aortocaval shunt to increase pulmonary blood
flow for 6 wk. The shunt resulted in a significant increase in the
heart- and lung-to-body weight ratios (>2-fold;
P < 0.05) without significant alteration of pulmonary or systemic blood pressures. Significant thickening of the pulmonary arterial medial wall developed, with increased muscularization of small (50-100 µm)- and medium
(101-200 µm)-sized arteries as evidenced by
-actin smooth
muscle staining. Proliferating cell nuclear antigen staining and
bromodeoxyuridine labeling did not detect proliferating smooth muscle
cells in the vascular wall. eNOS Western and Northern blot analyses and
immunohistochemical staining demonstrated that eNOS protein and mRNA
levels were not altered in the shunt lungs compared with sham controls.
Therefore, increased pulmonary flow without increased pressure resulted
in pulmonary artery medial thickening, without ongoing proliferation. Unlike chronic hypoxia-induced vascular remodeling, the pulmonary vascular remodeling resulting from increased pulmonary blood flow is
not associated with changes in eNOS.
shunt; proliferating cell nuclear antigen; congenital heart
disease; endothelial nitric oxide synthase
 |
INTRODUCTION |
INCREASED BLOOD FLOW without increased pressure to the
pulmonary arteries is a common scenario for a number of congenital heart defects, including the frequently occurring atrial septal defect
(1:1,000 live births; see Ref. 3). Although the pulmonary vasculature
is relatively tolerant to increased flow (reviewed in Ref. 18), if such
a defect is left unrepaired, chronic exposure of the pulmonary
vasculature to increased pulmonary blood flow can result in
irreversible pulmonary hypertension and right ventricular failure,
usually after the second decade of life (25, 34). Previously, Friedli
et al. (12) in 1975 observed that piglets exposed to increased
pulmonary blood flow via an arteriovenous fistula and pneumonectomy
began to develop thickening of the pulmonary arterial wall. To date,
pulmonary vascular remodeling that results from increased pulmonary
blood flow alone without the confounding variable of increased pressure
has been poorly described, and the mechanisms are unknown. One
potential mechanism is a flow-mediated alteration in the regulation of
endogenous pulmonary vasodilators.
Nitric oxide (NO) is a potent endogenous vasodilator produced in the
lung and other tissues by isoforms of the enzyme NO synthase (NOS). NO
is produced in pulmonary endothelial cells from the amino acid
L-arginine and molecular oxygen by endothelial NOS (eNOS). NO diffuses from the endothelium to adjacent smooth muscle cells and activates soluble guanylate cyclase to increase cGMP, resulting in vasodilation. The role of NO in the regulation of pulmonary vasculature basal tone and maintenance of low basal pulmonary
artery pressures remains controversial (2, 8, 11). Previously, we and
others have demonstrated that eNOS is increased in the hypertrophied
pulmonary vasculature of rats subjected to chronic hypoxic pulmonary
hypertension (22, 30, 32) and in humans with pulmonary hypertension
(31). This increase in eNOS precedes and then progresses with the
development of vascular remodeling (32). In vitro data suggest that
shear stress (i.e., viscous drag) exerted on the endothelial surface by
streaming of blood regulates eNOS expression (reviewed in Ref. 1),
although the role of increased shear stress versus hypoxia or
hypoxia-induced factors in the upregulation of eNOS is not clear.
Evidence exists for the regulation of eNOS by flow in the aorta of
exercising dogs (29). Recently, NO was also found to be associated with inhibition of smooth muscle cell proliferation and therefore may serve
to regulate vascular smooth muscle cell growth (19, 23, 27) during
vascular remodeling. Presently, it is unclear whether pulmonary
vascular eNOS is also regulated by flow or whether an alteration in
eNOS regulation could be responsible for mediating vascular remodeling
to increased flow. Therefore, we
hypothesize that increased pulmonary blood flow without changes in
pressure results in increased eNOS expression, possibly playing a role in pulmonary vascular remodeling.
In the present study, an abdominal aorta-to-inferior vena cava shunt
was created in rats to mimic the chronic increased pulmonary artery
flow state of an atrial septal defect. We demonstrated that increased
pulmonary blood flow resulting from the shunt induced medial thickening
of the pulmonary vascular wall without evidence of ongoing smooth
muscle cell proliferation. In this model, eNOS mRNA, protein levels,
and immunohistochemical staining pattern of the pulmonary vasculature
were unchanged in the lungs of the shunt animals.
 |
METHODS |
Aortocaval shunt. Forty-two-day old
Sprague-Dawley rats (Hilltop) underwent creation of an abdominal
aorta-inferior vena caval shunt superior to the renal arteries using
the technique described by Garcia and Diebold (14). In brief, animals
were anesthetized using pentobarbital sodium, the abdominal aorta and
inferior vena cava were isolated, and the fistula was created by
passing a 20 (large-shunt)- or 22 (small-shunt)-gauge sterile needle
through the inferior vena cava and into the aorta. The needle was
slowly withdrawn, allowing a small hematoma to form at the aortic and vena caval puncture. Super Glue (S. P. Richards, Atlanta, GA) was
applied as a sealant, and the needle was removed. Subsequently, the
abdominal wall was closed, and the animal was allowed to recover. Sham
animals were treated similarly without puncturing the aorta or vena
cava. To assay for changes in vascular smooth cell proliferation, on
the night before and the morning of the end of the study period, both
sham and shunt groups received a 10 mg/kg ip injection of the thymidine
analog bromodeoxyuridine (BrdU) as a marker of DNA replication (Sigma
Chemicals, St. Louis, MO). After 42 days, the animals were anesthetized
with pentobarbital sodium and ventilated with a respirator. A catheter
was placed in the right carotid artery, and arterial pressures were
recorded. The abdomen was exposed, and the aortocaval shunt was
inspected for size, the presence of a palpable thrill, and obvious
shunting of oxygenated blood into the inferior vena cava. Subsequently,
the chest was opened, and pulmonary artery pressure was measured
directly by main pulmonary artery puncture. The heart and lungs were
rapidly perfused with 20 ml of heparinized (1 U/ml) normal saline,
rapidly removed, and weighed. Approximately one-half of each lung was flash-frozen in liquid nitrogen and stored at
70°C for
subsequent Northern and Western blot analyses. At the same time, 1- to
2-mm-thick pieces of lung from different lobes were cut and immersed in
fixative (4% paraformaldehyde in phosphate-buffered saline) for 90 min on ice. The tissues were subsequently processed as previously described, with 2- to 4-µm sections cut for immunocytochemical analysis (32). After lung removal, the fistula was dissected open and
examined under an operating microscope to further confirm patency.
Northern blot. Total RNA from lungs
was extracted after the method of Chomczynski (6) using TriReagent
(10). Poly(A)+ RNA was isolated
from total RNA using oligo(dT) cellulose (5 Prime
3 Prime, Boulder,
CO). Twenty micrograms of poly(A)+
RNA per rat were denatured with glyoxal-DMSO, electrophoresed through a
0.01 M sodium phosphate-buffered agarose gel, and transferred to a
nylon membrane (Hybond-Nt; Amersham, Arlington Heights, IL) as
previously described (22). The membranes were hybridized with a
4,091-bp eNOS cDNA (a kind gift of Dr. W. C. Sessa) labeled by
random-prime labeling (22). Hybridization and washes were at 65°C
as previously described (10, 22, 32). To correct for the amount of RNA
loading, RNA transfer efficiency, and gene specific expression, all
blots were probed simultaneously with the cDNA for the control gene
-actin. eNOS mRNA signal was detected and quantified using a
phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale,
CA). Autoradiograms were also obtained by exposure to film
(Hyperfilm-MP; Amersham, Arlington Heights, IL).
Western blotting. Western blotting was
performed as previously described (22, 32). Briefly, lung homogenates
(150 µg/rat) were separated under denaturing conditions in a 7.5%
SDS-PAGE gel, followed by blotting of the proteins to nitrocellulose
(Bio-Rad, Burlingame, CA). Blots were blocked at room temperature for 1 h in 50 mM Tris · HCl, pH 7.4, 0.15 M NaCl, 2% BSA,
and 0.1% Tween 20. Subsequently, blots were incubated with a mouse
anti-eNOS monoclonal antibody (dilution 1:500; Transduction
Laboratories, Lexington, KY) for 1 h at room temperature. Subsequently,
membranes were washed at room temperature and incubated with an
anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad) for 1 h
at room temperature. eNOS immunoreactive protein was detected with enhanced chemiluminescence (ECL; Amersham) and exposure to film (Hyperfilm-ECL; Amersham). Signal bands were quantified by densitometry (Personal Densitometer, ImageQuant; Molecular Dynamics).
Immunocytochemistry.
Immunohistochemical analysis was performed as previously described (32)
in groups of 10 animals each (4 sham, 6 shunt). Separate lung sections
were incubated with one of the following antibodies:
1) monoclonal
-smooth muscle actin antibody (dilution 1:500; Sigma; see Ref. 4);
2) a monoclonal antibody against
BrdU (dilution 1:200; Vector Laboratories, Burlingame, CA; see Ref. 9);
or 3) a monoclonal antibody against
proliferating cell nuclear antigen (PCNA, dilution 1:100; Vector; see
Ref. 20) or the eNOS antibody (32) described above. Controls were
slides incubated with nonspecific mouse IgG (Vector) at the same
concentration as the primary antibody. Briefly, sections were
deparaffinized with xylene and decreasing concentrations of alcohols
and finally washed in PBS (10 mM
NaPO4-0.9% NaCl, pH 7.5) in
preparation for immunodetection with the exception of sections for eNOS
staining, which were further treated by boiling for 25 min in 10 mM
citric acid, pH 6, as recommended by the manufacturer. Sections were blocked for 1 h at room temperature with horse serum followed by
incubation for 1 h with the primary antibody. After the sections were
washed for 30 min with PBS plus 0.5% Tween 20, immunodetection was
observed using the avidin-biotin complex method (Vector
Laboratories) with diaminobenzidine (brown) as a color substrate.
Sections were not counterstained with the exception of eNOS-stained
sections, which were counterstained with Gill's hematoxylin (Sigma).
All slides were mounted and examined using an Olympus Vanox AHBS3 microscope for bright-field microscopy.
Pulmonary vascular histology was described according to the method of
Meyrick and Reid (24). Pulmonary veins were differentiated from
arteries by veins having thinner media, less medial elastic material,
and a larger lumen compared with arteries accompanying a bronchiole.
Approximately 30 arteries per animal were randomly evaluated in a
blinded manner. Using an Olympus-BHS microscope coupled to an MTI color
video camera (DAGE-MTI, Michigan City, IN) and I Cube video grabber
board, lung images were captured, and arterial diameter and medial wall
area were measured using Image Pro Plus software (Media Cybernetics,
Silver Spring, MD) after calibration with an Olympus 0.01-mm microscope
calibration slide. Artery diameters for two vessel sizes (50-100
and 101-200 µm) were determined by measuring the length of the
internal elastic lamina. Medial wall area
(µm2) was calculated as the
area between the internal elastic lamina and the adventitia.
Statistics. All data are expressed as
means ± SE. Statistical comparisons of groups were performed by
one-way ANOVA by ranks using the Kruskal-Wallis statistic (Sigmastat;
Jandel Scientific, San Rafael, CA). A value of
P < 0.05 was considered
statistically significant.
 |
RESULTS |
Physiological measurements. Creation
of an arteriovenous fistula (shunt) between the abdominal aorta
superior to the renal arteries and the inferior vena cava as described
by Garcia and Diebold (14) is a well-tolerated surgical procedure with
high animal survival. Of the 24 animals that had a shunt created, all but 4 survived. Mortality in all cases was in the first hours of shunt
creation. There were no late deaths, with the remaining animals
surviving to the end of the study period (6 wk), and both groups
demonstrated a similar weight gain (Table
1). Despite the creation of a large
systemic arteriovenous shunt, neither systemic nor pulmonary artery
blood pressures were altered in the shunted animals. As shown in Table
1, heart weight, heart-to-body weight ratio, and lung-to-body weight
ratio were all significantly increased (>2-fold;
P < 0.05) in the shunt animal group
compared with the sham control. Therefore, increased venous return to
the right heart, with a subsequent increase in pulmonary blood flow, resulted in a compensatory increase in cardiac and pulmonary mass without an alteration in systemic or pulmonary blood pressures.
Morphological analysis,
-smooth muscle actin, PCNA,
and BrdU labeling of shunt lungs. Examination of the
lungs from the shunt and sham animals revealed that the basic
architecture of the lung respiratory apparatus (i.e., alveoli and
bronchioles) appeared normal in both study groups. The most striking
feature was the development of medial thickening in pulmonary
arterioles (Fig. 1). As shown in Fig. 1,
the medial layer of pulmonary arterioles was increased relative to the
sham animals, whereas the intimal layer was unchanged. This is
similar to the Heath-Edwards type 1 classification of vascular
arteriopathy (17).
-Smooth muscle actin immunostaining demonstrated
specific localization of
-smooth muscle actin to the pulmonary
vascular wall and muscular portion of the airway. Importantly, as shown
in Fig. 2, there is an increase in
-smooth muscle
actin and thus muscularity of very small (<50 µM) arterioles of the
shunt lungs compared with the sham controls. The presence of pulmonary
artery thickening was confirmed by measuring the arterial medial wall
area (Fig. 3). As shown (Fig. 3), there was a
significant increase (180%; P < 0.05) in the medial area of arteries 50-100 and 101-200 µm
in diameter from the large-shunt group. To determine whether the
increase in medial wall area was a result of ongoing smooth muscle
hyperplasia, lung sections were stained for PCNA, an endogenous marker
for cells entering cell division and for the presence of BrdU
incorporation into cells replicating their DNA. As shown in Fig.
4, no PCNA-positive cells were detected in the media or
intimal portions of the arteriolar wall. However, PCNA-positive cells
in the pulmonary epithelium could be readily identified and served as a
positive control for PCNA staining (Fig. 4) in each section. Similarly,
BrdU-positive cells could not be detected in the arteriolar wall,
indicating a lack of accelerated smooth muscle cell replication in the
arteriolar wall. Positive BrdU labeling was identified in pulmonary
epithelial cells and served as a positive control in each section (Fig.
4).

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Fig. 1.
Histological appearance of pulmonary arteries from sham and large-shunt
lungs. Representative histological sections of sham and large-shunt
lungs are shown demonstrating hypertrophy of the arterial medial wall.
A, pulmonary arteriole; B, bronchiole; arrowhead, location of the
internal elastic lamina. Scale bar = 25 µm.
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Fig. 2.
-Smooth muscle actin staining of sham
(A) and large-shunt
(B) lungs. Representative sections
of sham and large-shunt lung are shown, with increased staining for
-smooth muscle actin in the small arterioles (arrows) of the shunt
compared with the sham lung.
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Fig. 3.
Measurement of pulmonary arterial medial wall area in sham and shunt
lungs. Graph demonstrates arterial medial area
(µm2) of arteries 50-100
and 101-200 µm in diameter. As shown, the area of the arterial
wall medial layer was significantly increased in the large-shunt group,
* P < 0.05.
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Fig. 4.
Proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine
(BrdU) staining of large-shunt lungs. As shown in representative
arterioles, no PCNA staining or BrdU-labeled proliferating cells could
be detected in the pulmonary arteries of the shunt lungs. Arrows
indicate PCNA- or BrdU-positive respiratory epithelial cells, which
served as a positive control.
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eNOS expression in the shunt lung. To
determine whether increased pulmonary blood flow to the lung results in
changes in eNOS expression, lung eNOS mRNA and protein levels were
determined in shunt and sham animals. As shown in Fig.
5,
A and
B, Western blot analysis of whole lung
homogenates demonstrated no significant differences in eNOS protein
levels in the lungs of animals with both small and large shunts
compared with the sham controls. Similarly, eNOS mRNA levels (Fig.
6, A and
B) as determined by Northern blot analysis were not significantly altered in the shunt lungs, whether shunts were small or large.
-Actin hybridization indicated similar RNA loading and transfer. To examine whether there could be local changes in eNOS expression in the lungs of the shunt animals, eNOS
immunostaining of lung sections was performed (Fig.
7). As expected, endothelium in shunt and
sham lungs was positive for eNOS immunostaining. As shown in Fig. 7,
pulmonary artery endothelium was positive for eNOS, although no
differences in vascular localization or pattern of eNOS immunostaining
were observed. Occasional weak staining of respiratory epithelial cells
was also observed but was unchanged between sham and shunt groups.

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Fig. 5.
Western blot analysis of endothelial nitric oxide synthase (eNOS)
protein expression in sham and shunt lungs.
A: representative Western blot of 150 µg of whole lung homogenate protein from sham and small- and
large-shunt animals was analyzed. Monoclonal antibody detected eNOS
protein as a single 135-kDa band. B:
densitometric analysis of eNOS protein signal from Western blots of
sham and shunt lung homogenates. Results are means ± SE.
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Fig. 6.
Northern blot analysis of eNOS mRNA levels in sham and shunt lungs.
A: Northern blot showing 20 µg
poly(A)+ mRNA from sham and small-
and large-shunt lungs and bovine arterial endothelial cells (BAEC,
positive control) analyzed for eNOS mRNA content.
B: densitometric analysis of eNOS mRNA
signal from Northern blots of RNA from sham and shunt lungs. Results
are expressed as means ± SE. As shown, eNOS mRNA levels were not
significantly different in the sham and shunt groups.
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Fig. 7.
eNOS immunostaining of sham and shunt pulmonary arteries. Sham
(B), small-shunt
(C), and large-shunt
(D) lung eNOS immunostaining was
positive in the endothelium (arrow) of the pulmonary arterioles.
A: representative control section (no
primary antibody) from large-shunt animal. eNOS immunostaining was
unchanged in the shunt vs. sham lung arterioles.
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 |
DISCUSSION |
The pulmonary arterial bed is tolerant to increases in pulmonary blood
flow (reviewed in Ref. 15). This is clearly demonstrated in the case of
humans with atrial septal defects in which high levels of elevated
pulmonary blood flow are well tolerated for decades (16, 25, 34).
However, if unrepaired, this high flow state can eventually result in
pulmonary hypertension and right heart failure (16, 34). The mechanisms
whereby increased pulmonary blood flow alone results in an eventual
detrimental remodeling of the pulmonary vasculature are unknown. The
hemodynamic consequences of an atrial septal defect can be mimicked by
the creation of systemic arteriovenous connections (i.e., aortocaval shunts). As a consequence of the reduced vascular resistance in the
systemic venous system compared with the arterial system, a substantial
left-to-right shunt occurs, with increased volume load on the right
ventricle and increased pulmonary blood flow at low pressures (5,
13-15, 28). The development of a rodent model of cardiac and
pulmonary flow overload by the creation of an aortocaval shunt has
allowed the determination of the fate of prolonged volume overload on
heart and lung growth and physiology (5, 14, 28).
The present study demonstrates that, in the rat, the pulmonary arterial
bed also has features of flow tolerance. As demonstrated, even with a
large shunt that results in increased heart and lung weights, pulmonary
artery pressures were unchanged. Increased pulmonary blood flow alone,
without an associated increase in pressure, appeared to be a
significant stimulus for medial thickening of the pulmonary vascular
wall and muscularization of small arterioles. These changes in the
pulmonary vasculature are similar to the changes described by Heath and
Edwards (17) as a type 1 lesion. Similarly, Friedli et al. (12)
reported that piglets with an arteriovenous (a-v) fistula and a late
pneumonectomy similarly developed occasional hypertrophy of small
arterioles, whereas an a-v fistula alone was insufficient to alter the
pulmonary architecture. The addition of the pneumonectomy also resulted
in an increase in pulmonary artery pressure. Unfortunately, this prior
study was composed of a small number of animals, and it is unclear
whether significant vascular hypertrophy could have developed in the
a-v fistula group alone. In addition, age may also play a factor
because the piglets in that study were newborn, whereas the rats in our study were 42 days of age at the time of shunting. Significant elevations in pulmonary artery pressures were not seen in our study,
suggesting that pulmonary vascular resistance was not increased. Similarly, Fullerton et al. (13) did not observe an increase in
pulmonary artery pressure in dogs 5 mo after creation of a femoral
artery-vein fistula, with a sustained 3:1 left-to-right shunt.
Importantly, lambs with an a-v fistula for 6 wk and a shunt of 2.4:1
did not have increased pulmonary vascular resistance or altered
vascular compliance. Combined, these studies indicate that at least 6 wk of increased pulmonary blood flow has little effect on pulmonary
vascular resistance. Whether vascular shear forces are also altered in
this model is unclear and requires further study.
The vascular lesion induced by the increased pulmonary blood flow in
this study was medial thickening, as intimal expansion was not
observed. Ongoing hyperplasia was not obvious in the thickened arteries, as confirmed by PCNA staining and BrdU labeling of
proliferating cells, both of which were negative in vascular smooth
muscle cells. This confirms the findings of Heath and Edwards (17) that
the initial lesion in the spectrum of pulmonary vascular obstructive disease is medial hypertrophy alone. Furthermore, it also explains why
a type 1 lesion is able to regress after surgical repair of a shunt
lesion (17, 25).
Because the mechanisms by which flow regulates pulmonary vascular
remodeling are largely unknown, we examined the potential role of eNOS
in this process. As shown in the present study, eNOS protein, mRNA, and
immunohistochemical localization were not changed in the lungs of the
shunt animals. eNOS could play two potential roles in modulating the
pulmonary vascular response to increased pulmonary blood flow: first,
increased expression in small arteries to facilitate vascular
recruitment and maintain flow tolerance (i.e., increased flow without
alteration in pulmonary vascular resistance) or second, increased
expression to regulate smooth muscle cell growth (19, 23, 27). However,
the findings of the present study suggest that, in this model,
increased blood flow alone is not a significant regulator of pulmonary
eNOS expression and that alterations in eNOS are not responsible for
mediating the arterial remodeling observed. This hypothesis is
supported by the recent report that a twofold increase in pulmonary
artery blood flow as a result of contralateral pulmonary artery
stenosis also does not alter lung eNOS expression (21). There are,
however, data to suggest that blood flow regulation of eNOS is
different between the aorta and pulmonary arteries. The eNOS gene does
appear to be regulated by increases in blood flow in the aorta of the dog where Sessa et al. (29) demonstrated that exercise for 10 days
resulted in increased aortic endothelial eNOS mRNA and increased NOS
activity in epicardial coronary arteries. Similarly, Nadaud et al. (26)
demonstrated that aortic eNOS mRNA and protein were increased in rats
after creation of an a-v shunt. It is
unclear whether this represents tissue-specific regulation of eNOS or whether this effect is specific to large-conduit arteries. Support for
pulmonary-specific regulation of vasodilators is suggested by Fullerton
et al. (13) where increased pulmonary blood flow resulted in decreased
endothelium-dependent cGMP-mediated relaxation of pulmonary artery
rings with acetylcholine after 2 wk and further after 5 mo of increased
flow. The lack of endothelium-dependent cGMP-mediated relaxation may be
related to progressive thickening of the arterial wall, rendering the
pulmonary artery less responsive. Unfortunately, eNOS mRNA, protein, or
vasculature morphology was not assessed in their study. In addition, it
is unclear as to what effect the increased pulmonary arterial oxygen
content produced by the creation of the shunt could have on eNOS
regulation, as previously we have demonstrated that chronic hypoxia can
increase eNOS protein and gene expression in the rat lung (22,
32).
The current study in combination with the report by Le Cras et al. (21)
strongly suggests that the upregulation of eNOS in the chronic hypoxia
model is related to hypoxia per se and not increased flow.
Collectively, these data would suggest that pulmonary and peripheral
arterial eNOS are regulated differently by flow. It is difficult to
implicate eNOS in the regulation of the smooth muscle cell hypertrophy
in the current model because eNOS levels were unchanged.
In summary, chronic increased pulmonary blood flow in the shunted rat
model results in pulmonary arteriole medial thickening without changes
in eNOS protein or mRNA expression. Therefore, we speculate that eNOS
does not play a significant role in mediating pulmonary vascular
remodeling in this model of increased pulmonary blood flow.
 |
ACKNOWLEDGEMENTS |
We are grateful to Nan Zhou and Audrey Fisher for technical
assistance.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Clinical
Investigator Development Award K08HL-02937 and Grants RO1-HL-39706 and
RO1-GM-49111 (R. A. Johns), March of Dimes Basil O'Connor Scholar
Award 5-FY94-0930 (A. D. Everett), and by two research awards from
the American Heart Association-Virginia Affiliate (C. Xue and T. D. Le
Cras).
Present addresses: T. D. LeCras, Dept. of Pediatrics, University of
Colorado, Box C218, 4200 E Ninth Ave., Denver, CO 80262; C. Xue,
Novartis, Pharmaceuticals Division, 1L-125.10.16, Basel, Switzerland
CH-4002.
Address for reprint requests: A. D. Everett, Univ. of Virginia,
Pediatric Cardiology, MR4 Bldg., Box 14, Rm. 3033, Charlottesville, VA
22908.
Received 13 May 1997; accepted in final form 9 March 1998.
 |
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