1 Division of Pulmonary, Sleep, and Critical Care Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island 02903; and 2 Department of Pathology, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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To determine whether atrial natriuretic peptide
(ANP) plays a physiological role in modulating pulmonary hypertensive
responses, we studied mice with gene-targeted disruption of the ANP
gene under normoxic and chronically hypoxic conditions. Right
ventricular peak pressure (RVPP), right ventricle weight- and left
ventricle plus septum weight-to-body weight ratios [RV/BW and
(LV+S)/BW, respectively], and muscularization of pulmonary
vessels were measured in wild-type mice (+/+) and in mice heterozygous
(+/) and homozygous (
/
) for a disrupted proANP
gene after 3 wk of normoxia or hypobaric hypoxia (0.5 atm). Under
normoxic conditions, homozygous mutants had higher RVPP (22 ± 2 vs.
15 ± 1 mmHg; P < 0.05) than
wild-type mice and greater RV/BW (1.22 ± 0.08 vs. 0.94 ± 0.07 and 0.76 ± 0.04 mg/g; P < 0.05)
and (LV+S)/BW (4.74 ± 0.42 vs. 3.53 ± 0.14 and 3.18 ± 0.18 mg/g; P < 0.05) than heterozygous or
wild-type mice, respectively. Three weeks of hypoxia increased RVPP in
heterozygous and wild-type mice and increased RV/BW and RV/(LV+S) in
all genotypes compared with their normoxic control animals but had no
effect on (LV+S)/BW. After 3 wk of hypoxia, homozygous mutants had
higher RVPP (29 ± 3 vs. 23 ± 1 and 22 ± 2 mmHg;
P < 0.05), RV/BW (2.03 ± 0.14 vs. 1.46 ± 0.04 and 1.33 ± 0.08 mg/g;
P < 0.05), and (LV+S)/BW (4.76 ± 0.23 vs. 3.82 ± 0.09 and 3.44 ± 0.14 mg/g;
P < 0.05) than heterozygous or
wild-type mice, respectively. The percent muscularization of peripheral
pulmonary vessels was greater in homozygous mutants than that in
heterozygous or wild-type mice under both normoxic and hypoxic
conditions. We conclude that endogenous ANP plays a physiological role
in modulating pulmonary arterial pressure, cardiac hypertrophy, and
pulmonary vascular remodeling under normoxic and hypoxic conditions.
anoxia; cardiac hypertrophy; pulmonary circulation
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INTRODUCTION |
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ATRIAL NATRIURETIC PEPTIDE (ANP) is a cardiac hormone with potent diuretic, vasorelaxant, and antimitogenic properties that helps to modulate intravascular volume and systemic hypertension in response to salt loading (9, 11). There is also considerable evidence to suggest that ANP plays an important role in modulating pulmonary hypertensive responses, particularly during exposure to hypoxia. ANP blunts pulmonary vasoconstriction in response to acute hypoxia in normoxic rats (1) and decreases basal pulmonary arterial pressure in hypoxia-adapted rats (15). During chronic hypoxia, right heart ANP synthesis increases (8, 29, 33) and pulmonary ANP clearance decreases (17), leading to a sustained elevation in circulating ANP levels (24). Exogenous ANP has been shown to attenuate the development of pulmonary hypertension, right ventricular (RV) hypertrophy, and pulmonary vascular remodeling during chronic hypoxia while increasing plasma and lung cGMP levels (14). Additional studies (13, 28) have shown aggravated pulmonary hypertensive responses to hypoxia in rats given monoclonal antibodies against ANP, suggesting that endogenous ANP may protect the heart against the development of hypoxic pulmonary hypertension. In one study (13), infusion of a monoclonal antibody against ANP resulted in a slight potentiation of pulmonary vasoconstriction after 6 h of hypoxia. In another study (28), pulmonary hypertension and RV hypertrophy were greater in rats given a monoclonal antibody against ANP than in rats given a monoclonal antibody against human apolipoprotein B during 3 wk of hypoxia. However, the effect of the anti-apolipoprotein B antibody was not controlled for in that study, and a pulmonary antihypertensive effect of the antibody could not be excluded (28). Thus additional studies are needed to confirm the potential role of ANP in modulating pulmonary hypertensive responses and in blunting hypoxic pulmonary hypertension.
Recently, a lineage of ANP-deficient mice has been generated by targeted disruption of the ANP gene (16). Mice homozygous for the disrupted ANP gene (homozygous mutants) are incapable of synthesizing ANP (16). In the present study, we hypothesized that if ANP is a physiological modulator of pulmonary hypertension, then mice with absent ANP expression would develop more severe pulmonary hypertension in response to chronic hypoxia than their heterozygous or wild-type littermates and that heterozygotes might also have greater pulmonary hypertension than wild-type mice. To test this hypothesis, we examined RV pressure, RV and left ventricular (LV) weight, and pulmonary vascular morphology in all three genotypes after 3 wk of exposure to normoxia or hypobaric hypoxia.
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METHODS |
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Mice. Targeted disruption of the
proANP gene has previously been reported in mice (16). Briefly, a
mutated construct of the mouse natriuretic peptide precursor type A
(Nppa) gene that codes for proANP
(27) was created in mouse embryonic stem cells by deleting 11 bp of
exon 2 and replacing it with the neomycin resistance gene. Chimeras
generated from one of the targeted strain 129 embryonic stem cell lines
transmitted the mutated Nppa gene to
their offspring. These mice were then mated with strain C57BL/6J (B6)
mice to produce 129 × B6 F1
animals. Mice heterozygous for the mutated
Nppa gene were healthy, fertile, and
overtly indistinct from wild-type mice. Mating pairs of
F1 heterozygotes produced all
three genotypes: wild-type (+/+), heterozygous (+/), and homozygous (
/
) mutants in Mendelian proportions (1:2:1).
Wild-type mice have two functional
Nppa genes and normal levels of atrial ANP; heterozygotes have one functional
Nppa gene and atrial levels of ANP
that are half normal; homozygous mutants have no functional copies of
Nppa and no detectable ANP (16).
Multiple litters from the breeding pairs were studied between 3 and 6 mo of age. Because we were uncertain whether ANP-deficient mice would
tolerate chronic hypoxia, twice as many mice were exposed to hypoxia as were kept under normoxic conditions. Genotyping by Southern blot was
done after all measurements had been obtained. The wild-type and
mutated Nppa genes were identified as
single bands migrating at ~7.0 and 8.8 kb, respectively. Both bands
were clearly visible in heterozygotes.
Hypoxic exposure. Mice were placed in hypobaric chambers (0.5 atm) for 3 wk. An air intake valve was adjusted to maintain intrachamber pressure at 380 mmHg (0.5 atm) while allowing adequate airflow through the chamber (10-15 l/min) to prevent accumulation of CO2 and NH3. Intrachamber pressure was monitored via a pressure gauge in the wall of the chamber. Chambers were opened three times weekly to clean animal cages and to replace food and water. All mice were fed standard rat chow (0.5% NaCl) and allowed to take food and water ad libitum. Normoxic control animals were kept in identical cages adjacent to the hypoxic chambers.
Hemodynamic measurements. After 3 wk of hypoxia or normoxia, mice were weighed and anesthetized under normoxic conditions with ketamine (60 mg/kg im) and pentobarbital sodium (20 mg/kg ip). The trachea was cannulated with a blunted 20-gauge needle, and the lungs were ventilated with room air with an inspiratory pressure of 9 cmH2O and positive end-expiratory pressure of 2 cmH2O. RV peak pressure (RVPP) was measured by inserting a 26-gauge needle with 12 in. of P-50 tubing connected to a Statham P23G pressure transducer into the right ventricle as described previously (20, 32). Continuous measurement of RV pressure was recorded on a polygraph, and RVPP was measured as the top of the pressure recording over a 1- to 3-min period during which pressure recordings remained stable.
After completion of hemodynamic measurements, blood was collected from the inferior vena cava (IVC) for determination of hematocrit. Mice were then killed by exsanguination, and the heart and lungs were removed en bloc. The heart was dissected into right and left atria, RV and LV free walls, and the interventricular septum (IVS). Each section of the heart was blotted dry on sterile gauze to remove excess blood and weighed. Wet weight measurements were normalized to body weight (BW; in mg/g) and expressed in the following ratios: right atrial (RA) weight to BW (RA/BW), left atrial (LA) weight to BW (LA/BW), RV free wall weight to BW (RV/BW), LV free wall weight to BW (LV/BW), IVS weight to BW (IVS/BW), and LV free wall plus septum (LV+S) weight to BW (LV+S)/BW.
Lung histology. Lungs were fixed by intratracheal infusion of normal buffered Formalin at a pressure of 23 cmH2O. After fixation, lungs were embedded in paraffin, sectioned, and stained with trichrome. All slides were reviewed simultaneously by two investigators in a blinded fashion. Pulmonary vessels > 100 µm in diameter or fully muscularized vessels associated with bronchi were excluded because these vessels are uniformly muscularized. Each vessel was categorized as being nonmuscular (no evidence of any muscularization of vessel wall), partially muscularized (smooth muscle identifiable in less than three-fourths of the vessel circumference), or fully muscularized (muscularization in more than three-fourths of vessel circumference) as previously described (19). The percent muscularization of pulmonary vessels was determined by dividing the number of vessels in each category by the total number counted for that experimental group. At least 35 vessels were examined in each group of normoxic mice, and at least 75 vessels were examined in each group of hypoxic mice.
ANP measurements. Right atria were
homogenized in Chomczynski-Sacchi RNA extraction buffer (6) and frozen
at 70°C until needed. All tissue samples were assayed within
6 mo of collection. Tissue samples were thawed on ice and centrifuged
to remove particulate matter. Samples were then diluted 1:50 in assay
buffer and assayed directly in duplicate with a commercially available
ELISA (Cayman Chemical, Ann Arbor, MI) and rat
-ANP as a standard.
Interassay variability with this immunoassay is ~10% in our laboratory.
Southern blots. Total DNA was isolated
from mouse tails and stored at 20°C until needed. For
Southern blot analysis, 3 µl of the DNA extract were digested
overnight at 37°C with 1 µl of EcoR I (10,000 U/ml;
New England Biolabs, Beverly, MA) in a total volume of 20 µl. The
entire digest was then electrophoresed on a 0.8% agarose gel and
passively transferred to Hybond-C nitrocellulose membranes (Amersham
Life Sciences, Cleveland, OH). The probe used was an 850-bp cDNA
segment extending from exon 1 to exon 3 of the
Nppa gene. It was generated by PCR
with the following primers: 5'-CTG TCC AAC ACA GAT CTG
ATG-3' and 3'-GG CTG TTA TCT TCG GTA CTA C-5'.
Statistics. Mean values were calculated for each group of mice and compared by two-way analysis of variance (ANOVA). Where significance was found between genotypes or between normoxic and hypoxia-adapted mice, pairwise multiple comparisons were done with Fisher's probable least significant difference test. Differences in percent muscularization of pulmonary vessels were analyzed by proportion comparison and Yates correction. Differences were considered significant at P < 0.05.
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RESULTS |
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Effects of hypoxia and ANP deficiency on BW,
hematocrit, and RA ANP levels. The number of mice with
each genotype in the normoxic and hypoxia-adapted groups is shown in
Table 1. Gene-targeted disruption of ANP
did not affect BW or hematocrit, but homozygous mutants
had RA ANP levels that were at least 20-fold lower than those in
wild-type mice under both normoxic and chronically hypoxic conditions
(Table 1). Exposure to 3 wk of hypoxia increased hematocrit in all
genotypes but had no effect on BW or RA ANP content.
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Effects of hypoxia and ANP deficiency on
RVPP. Under normoxic conditions, mice with absent ANP
expression (/
) had higher RVPP than mice with normal
(+/+) ANP expression (Fig. 1). In
hypoxia-adapted mice, RVPP was higher in homozygous mutants than in
heterozygous and wild-type mice. There were no differences in RVPP
between heterozygous and wild-type mice. In hypoxia-adapted mice, RVPP was higher than in normoxic control animals for each genotype, but the
absolute increase in mean RVPP was no greater in homozygous mutants
than that in wild-type mice (7.0 vs. 6.8 mmHg).
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Effect of hypoxia and ANP deficiency on ventricular
weight. Mice homozygous for the disrupted ANP gene had
higher RV/BW and (LV+S)/BW values than heterozygous and wild-type mice
under normoxic and chronically hypoxic conditions (Fig.
2, A and
B). The greater (LV+S)/BW value in
homozygous mutants was not the result of greater weight in the RV
portion of the IVS as demonstrated by the same effect of ANP genotype
on LV/BW and IVS/BW (Table 2). The effect of ANP genotype on RV and LV weight was similar in normoxic and hypoxia-adapted mice. Compared with wild-type mice, RV/BW
was 60 and 53% greater and (LV+S)/BW was 49 and 38% greater in
homozygous mutants kept in normoxic and chronically hypoxic conditions,
respectively.
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Under normoxic conditions, no differences in RV/(LV+S) were seen between genotypes (Fig. 2C), demonstrating that the increase in ventricular mass associated with absent expression of the ANP gene was similar in the right and left ventricles. However, in hypoxia-adapted mice, absence of ANP deficiency was associated with a slightly greater degree of hypertrophy in the right ventricle than in the left as indicated by a greater RV/(LV+S) in homozygous mutants than in heterozygous and wild-type mice. The RA/BW and LA/BW values were also greater in homozygous mutants than in heterozygous or wild-type mice (Table 2). As with the ventricles, the effect of ANP genotype on RA and LA weight was not affected by the level of oxygenation to which the mice were exposed. Adaptation to hypoxia increased RV/BW and RV/(LV+S) in all genotypes but had no effect on LV/BW or (LV+S)/BW (Fig. 2 and Table 2). The IVS/BW in heterozygotes was lower in hypoxia-adapted mice than in normoxic mice. The interaction between hypoxia and genotype as assessed by two-way ANOVA was not significant for any of the measurements of atrial or ventricular size.
Effects of ANP deficiency on muscularization of
pulmonary vessels. As shown in Fig.
3, no difference in percent muscularization of peripheral pulmonary vessels was seen between wild-type and heterozygous mice under normoxic or hypoxic
conditions. However, both normoxic and
hypoxia-adapted homozygous mutants had a higher percent
muscularization of pulmonary vessels than their wild-type or
heterozygous cohorts. Homozygous mutants had a greater percentage of
partially muscularized vessels under normoxic conditions and a greater
percentage of fully muscularized vessels under hypoxic conditions.
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DISCUSSION |
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In the present study, increased RV pressure, RV weight, and muscularization of peripheral pulmonary vessels was present in mice homozygous for the disrupted ANP gene compared with their wild-type and heterozygous littermates under both normoxic and chronically hypoxic conditions. These findings demonstrate that elimination of endogenous ANP results in pulmonary hypertension and strongly support the hypothesis that endogenous ANP plays an important role in modulating pulmonary vascular tone, RV weight, and pulmonary vascular remodeling. Although mice homozygous for the disrupted ANP gene developed more severe hypoxic pulmonary hypertension than mice with partial or normal ANP gene expression, the hypoxia-induced increases in RVPP and RV weight were similar in all ANP genotypes when measured as the percent increase from normoxic controls. Thus we did not find definitive evidence of an inhibitory effect of endogenous ANP on the development of hypoxic pulmonary hypertension. On the other hand, there was some evidence to suggest that mice with absent ANP expression had greater pulmonary hypertensive responses to hypoxia. The RV/(LV+S) was greater in homozygous mutants than that in heterozygous or wild-type mice under hypoxic but not normoxic conditions, suggesting that absent ANP expression resulted in greater hypertrophy of the right ventricle than of the left ventricle plus septum in hypoxia-adapted mice. Previous studies (13, 28) that used monoclonal antibodies against ANP have also suggested that inhibition of ANP activity potentiates the severity of pulmonary hypertension and RV hypertrophy. Unfortunately, the genotype-related differences in baseline values make it difficult to draw firm conclusions as to whether absence of ANP expression aggravates hypoxic pulmonary hypertension. To truly test this hypothesis would require disruption of ANP expression at the onset of hypoxic exposure so that baseline conditions would be comparable. An inducible construct that disrupts ANP expression is not yet available, but in the future, it could supply important information regarding the physiological role of ANP in mitigating hypoxic pulmonary hypertension.
Possible mechanisms by which impaired ANP expression causes pulmonary hypertension include increases in vascular tone or remodeling. Previous studies (28) have demonstrated that ANP antiserum raises pulmonary arterial pressure in hypoxia-adapted rats without affecting cardiac output and potentiates the pressor response to acute hypoxia in isolated perfused lungs obtained from hypoxia-adapted rats (25). However, neither ANP (1) nor a monoclonal antibody against ANP (13, 28) had any acute effect on pulmonary arterial pressure under normoxic conditions. Thus increased pulmonary vascular tone may not be the mechanism that increases RVPP in normoxic mice with absent ANP expression. An alternative mechanism may be greater pulmonary vascular remodeling as evidenced by increased muscularization of pulmonary vessels that we observed in normoxic homozygous mutants compared with that in wild-type mice. This possible mechanism is supported by a study (14) showing that exogenous ANP reduces muscularization of the pulmonary vascular bed during exposure to chronic hypoxia in vivo. Furthermore, studies showing antihypertrophic and antimitogenic effects of ANP on cultured vascular smooth muscle cells from systemic (12) and pulmonary (2) vessels demonstrate that ANP is capable of directly inhibiting proliferation of pulmonary vascular smooth muscle independent of hemodynamic effects. Although inhibition of endogenous ANP by monoclonal antibody did not affect pulmonary vascular remodeling in hypoxia-adapted rats in a previous study (28), it is possible that monoclonal antibodies only partially antagonized the effect of ANP on pulmonary vascular remodeling.
In addition to the effect on pulmonary vascular smooth muscle, we found evidence that ANP genotype had a strong influence on cardiac size. Homozygous mutants had greater LV weight than heterozygous or wild-type mice under both normoxic and hypoxic conditions. Under normoxic conditions, the increase in LV weight in homozygous mutants compared with that in wild-type mice was proportional to the increase in RV weight. Lacking measurements of systemic blood pressure, we cannot be certain about the possible contribution of systemic hypertension to the differences in LV weight. However, a previous study (16) in these mice found only a slight increase in systemic blood pressure (8 mmHg greater in homozygous mutants than in wild-type mice). Thus the marked RV and LV hypertrophy in homozygous mutants appears to be out of proportion to the observed increases in pulmonary and systemic arterial pressures. In a previous study (18), our laboratory found that transgenic mice with overexpression of ANP have 31% less RV weight than nontransgenic littermates despite similar RV pressures. These mice also had a greater inverse correlation between plasma ANP levels and RV weight than between plasma ANP levels and RVPP. Thus, in the absence of large hemodynamic differences, the inverse relationship between cardiac mass and ANP expression suggests that endogenous ANP has a direct inhibitory effect on cardiac growth. An in vitro study (4) showing that natriuretic peptides, including ANP, suppress thymidine incorporation and cell proliferation of cardiac fibroblasts further supports this hypothesis.
The induction of ANP gene expression in the hypertrophied ventricle
has not previously been considered a physiological response to pressure
overload. Chien et al. (5) have interpreted reactivation of ventricular
ANP expression as a "highly conserved and a cardinal feature of
ventricular hypertrophy" that occurs as part of the reexpression of
a program of fetal isogenes such as skeletal -actin and myosin light
chain-2. Our findings, however, suggest that reactivation of
ventricular ANP expression serves not only as a marker of hypertrophy
but also as a physiological mitigator of cardiac hypertrophic responses.
The primary signaling pathway for the biological actions of ANP consists of a membrane-bound guanylyl cyclase receptor (GC-A) that increases intracellular cGMP levels. GC-A is believed to mediate ANP-induced increases in cGMP in systemic vascular endothelial and smooth muscle cells (21) and is likely to be the receptor by which ANP increases pulmonary cGMP levels in hypoxia-adapted rats (25). Recent work in our laboratory (19) demonstrated that infusion of brain natriuretic peptide, like ANP, attenuates the development of hypoxic pulmonary hypertension. Because GC-A is the primary receptor for both ANP and brain natriuretic peptide, it is likely to be the common pathway by which these natriuretic peptides modulate pulmonary hypertensive responses to hypoxia. Findings of increased cardiac mass in GC-A-deficient mice (26) suggest that this receptor is also involved in the inhibitory effect of ANP on cardiac growth.
ANP may also alter the synthesis of other mediators that contribute to the development of hypoxic pulmonary hypertension. Previous studies have shown that hypoxia increases pulmonary endothelin (ET)-1 and ET-receptor expression (22, 31) and that acute hypoxic pulmonary hypertension can be abolished by blocking the ETA receptor (3, 7). Because ANP inhibits ET-1 synthesis and secretion from endothelial cells (10), it is possible that impaired expression of the ANP gene may aggravate hypoxic pulmonary hypertension by permitting unopposed hypoxia-induced increases in ET-1 synthesis and secretion. Alternatively, ANP deficiency may result in an enhanced vasoconstrictor effect of ET-1 on the pulmonary circulation. Other investigators (34) have demonstrated that ANP inhibits the release of angiotensin II, which is a potent pulmonary vasoconstrictor (23) and cardiac hypertrophic agent (30) that could contribute to the development of pulmonary hypertension and RV hypertrophy during chronic hypoxia.
The use of animals with gene-targeted disruption of ANP synthesis
avoids many of the limitations of ANP antibodies used in prior studies,
including partial rather than complete inhibition of ANP activity and
possible biological actions of control antibodies (28). Mice homozygous
for the disrupted ANP gene produce no ANP under normoxic conditions
(16), and disruption of the ANP gene prevents the increase in ANP
synthesis that normally accompanies exposure to chronic hypoxia (24,
29, 33). Although we found small quantities of RA ANP in homozygous
mutants ( that of wild-type mice), it is likely that the
ELISA used in the present study detected low levels of something other
than ANP. The absence of any detectable ANP in mice homozygous for the
disrupted ANP gene has previously been reported (16) and recently
confirmed by HPLC (S. John, personal communication). On the other hand, one of the limitations of experiments in gene-targeted animals is that
the absence of the targeted gene may have developmental effects that
result in different phenotypes before experimentation. For example, in
the present study, homozygous mutants had pulmonary hypertension
compared with wild-type mice before hypoxic exposure. Because both
genotypes developed hypoxic pulmonary hypertension, it is possible that
absence of ANP expression affects cardiac mass and pulmonary
vasculature only during developmental life.
Mice heterozygous for the disrupted ANP gene did not demonstrate aggravation of hypoxic pulmonary hypertension. No significant differences in RVPP, RV/BW, RV/(LV+S), or percentage of muscularized pulmonary vessels were observed between hypoxia-adapted heterozygous and wild-type mice, although each of these values tended to be higher in heterozygotes. Heterozygotes have previously been shown to have the same systemic blood pressure as wild-type mice under normoxic conditions when fed normal (0.5% NaCl)- and intermediate-salt (2% NaCl) diets (16). However, they develop higher systemic pressure than wild-type mice when challenged with a high-salt (8% NaCl) diet, suggesting that partial ANP deficiency predisposes mice to salt-sensitive hypertension. The findings from this study suggest that partial ANP expression, as evidenced by a heterozygous genotype on Southern blot, does not alter RVPP, cardiac weight, or muscularization of pulmonary vessels.
In summary, our findings offer some of the strongest evidence to date that endogenous ANP plays a physiological role in regulating RV pressure and muscularization of the pulmonary vessels as well as the overall weight of the atria and ventricles. However, because of differences in baseline values of RVPP and RV weight, we have been unable to definitively determine whether the absence of ANP expression aggravates hypoxic pulmonary hypertension or merely produces normoxic pulmonary hypertension that is then amplified by hypoxia. To what extent the changes in cardiac weight and pulmonary muscularization seen in this study were due to hemodynamic or antimitogenic effects of ANP remains to be determined. Finally, the effect of ANP deficiency on the expression and bioactivity of other vasoactive mediators and growth factors, including the other natriuretic peptides, requires further study.
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ACKNOWLEDGEMENTS |
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We thank Deborah Britt for technical assistance and Dr. M. W. Simon John for review of the manuscript.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-02613 (to J. R. Klinger) and HL-45050 (to N. S. Hill).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. R. Klinger, Div. of Pulmonary, Sleep, and Critical Care Medicine, SWP Rm. 420, Rhode Island Hospital, Providence, RI 02903 (E-mail: jklinger{at}lifespan.org).
Received 17 August 1998; accepted in final form 29 January 1999.
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