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1Departments of Pediatrics,2Pediatric Surgery,3Pharmacology and Toxicology, and4Cardiovascular Research Center, Medical College of Wisconsin, and5Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53226
Submitted 9 September 2002 ; accepted in final form 10 March 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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persistent pulmonary hypertension; ATP; newborn
Our laboratory's previous studies (15–18) demonstrated that the purine nucleotide ATP contributes to birth-related pulmonary vasodilation in fetal lambs. ATP causes pulmonary vasodilation in fetal lambs in part by stimulation of NO release (17). ATP stimulates NO release from cultured vascular endothelial cells (8, 28). However, the mechanism of NOS activation by ATP and its alteration in PPHN remain unknown.
Heat shock protein 90 (HSP90), a molecular chaperone, modulates the
endothelial NOS (eNOS) activity
(10,
12,
32) and balance of NO and
superoxide (
·) release
from eNOS (30) in response to
several physiological stimuli. However, the role of this constitutively
expressed chaperone in mediating vasodilation in the fetal pulmonary arteries
and its role in mediating the effects of ATP on eNOS is unknown. We proposed
the hypothesis that 1) association of HSP90 with eNOS facilitates the
release of NO and vasodilation when fetal pulmonary arteries are stimulated by
ATP and 2) dissociation of HSP90 from eNOS in PPHN impairs the
ability of NOS to release NO in response to stimulation by ATP. We
investigated the hypothesis in fetal lambs with pulmonary hypertension induced
by prenatal ligation of ductus arteriosus, an established model of PPHN. The
objectives of our study were to investigate the following: 1) the
role of HSP90-NOS association in the vasodilator response to ATP in isolated
intrapulmonary resistance arteries, 2) the HSP90-eNOS association in
isolated pulmonary arteries, and 3) the effects of ATP on the
HSP90-eNOS interaction and NO release from pulmonary artery endothelial
cells.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Ewes were obtained at 120 ± 2 days of gestation and were allowed to acclimatize to the facility for 1 wk before the surgical procedure. The ewes were fasted for 24 h and were given general anesthesia with 1–2% isoflurane and oxygen for the procedure. The fetus was approached via a midline laparotomy incision and a hysterotomy incision on the uterus close to the fetal head. The fetal left thorax was exteriorized, and a left lateral thoracotomy was done to identify and ligate the ductus arteriosus with the use of umbilical tape. Control fetal lambs had thoracotomy performed without ligation of the ductus arteriosus. The fetus was then returned to the uterus, and pregnancy was allowed to continue for 8 days. Previous studies (26, 43) have demonstrated that fetal lambs develop hemodynamic and structural alterations of PPHN with this procedure. Blood samples were obtained from fetal arterial catheter for measurement of blood pH and blood gas tensions with the use of a blood gas analyzer (model ABL30, Radiometer). These samples were obtained during surgery just before and 10 min after occlusion of ductus arteriosus and once on each postoperative day. Control fetal lambs had blood gas tension measurements at the same time points. In addition, the fetal mean systemic and pulmonary arterial pressures were measured at the same time points with the appropriate pressure transducers (model PT 100, Grass Instruments) and physiograph (model 7D, Grass), as described previously (19). After 8 days of ductal constriction, the ewe was euthanized and laparotomy and hysterotomy incisions were quickly opened. The fetal chest was opened and the heart and lungs were removed en bloc.
Isolation of pulmonary arteries and investigation of the response to ATP. Pulmonary arteries were dissected into the lung parenchyma, with care taken not to damage the endothelium. Fifth- through seventh-generation intrapulmonary arterioles with an internal diameter of 80–120 µM were connected to two glass pipettes tapering to an outside diameter of 70 µM and were tied in place. Arterioles were superfused with Krebs buffer (37°C, equilibrated with 95% O2-5% CO2) and luminally pressurized to 25 mmHg. The pressure chosen reflects the transmural pressure for the resistance vessels in fetal lamb lungs (21, 31). Arterioles from control and PPHN lambs were studied at the same transmural pressure to normalize the conditions for these vessels. In addition, the increase in transmural pressure for resistance arterioles in PPHN is not expected to be >10 mmHg, on the basis of the 17-mmHg increase in pressure we observed in left pulmonary artery after ductal constriction and the expected 40% drop in pressure from conduit to resistance size arteries (31). Previous studies (6) have demonstrated an alteration in endothelium-dependent vasodilation when the transmural pressure was increased by twofold and not at smaller increases in pressure. The internal diameter of the arterioles was monitored by using a stereomicroscope (Zeiss), charge-coupled device television system camera (9KP 130 AU, Hitachi), a monitor (CVM 1271, Sony), and a calibrated video measuring system (Colorado Video). After an equilibration period of 30 min, reactivity of the vessel was confirmed by constriction with 30 mM KCl. The KCl-containing buffer was then removed, and arterioles were preconstricted with 10-6 M norepinephrine before the dilator responses were tested. The presence of intact endothelium was confirmed by the evaluation of vasodilator response to acetylcholine (10-5 M). Treatment of the vessels with 10-5 M geldanamycin, 10-4 M NG-nitro-L-arginine (L-NAME), and 10-2 M 4,5-dihydroxy-1,3-benzene-disulfonate (Tiron) was done with the superfusion stopped, and the vessels were allowed to incubate with each drug for 20 min. The response to intraluminal application of 10-8–10-3 M ATP was determined with or without pretreatment of vessels with these agents. Our pilot studies demonstrated no vasodilator response to ATP at 10-8 M and plateau of the vasodilator response at 10-3–10-2 M doses in control resistance pulmonary arteries. The doses we chose therefore are expected to reproduce the range of vasodilator responses of these arteries to ATP.
Western analysis for eNOS protein. Peripheral lung tissue and third- to fifth-generation pulmonary arteries were flash frozen in liquid nitrogen, pulverized, and placed in a cell lysis buffer (modified RIPA). The mixture was then homogenized, sonicated to break the cells, and the cell debris was removed by centrifugation. The sample was heated to 70°C for 10 min, and an aliquot of protein (50 µg) was removed and resolved by SDS-PAGE (10%). Separated proteins were transferred to nitrocellulose membranes and were blotted for eNOS with a specific antibody (9D10, Zymed) overnight at 4°C. Enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia) were used to visualize bands. The autoradiographs were imaged with Adobe Photoshop 5.5 software and relative band densities quantified by using NIH Image 1.62.
Isolation and culture of endothelial cells. Pulmonary arteries were dissected into lung parenchyma up to the third-generation branches, which were ligated and cut distally. The main pulmonary artery was detached from the right ventricle and ductus arteriosus was ligated. The endothelial cells were separated from pulmonary arteries with the use of 0.25% collagenase type A (Roche) and cells were grown to confluence in endothelial growth media (endothelial SFM, Life Technologies). Cells for individual experiments were grown to confluence in 6-well plates for NOS activity assays or 100-mm flasks for immunoprecipitation studies at passage 4.
Assay for nitrite. Endothelial cells from control and ductal ligation lambs were grown to confluence in 6-well plates. NOS activity was assessed by accumulation of nitrite in the supernatant, detected by ozone-chemiluminescence method as described previously (30). Briefly, confluent endothelial cells were washed clear of media and were incubated in Dulbecco's phosphate-buffered saline (PBS) for 30 min at 37°C in a tissue culture incubator. Cells in selected wells were treated with 10-4 M NG-monomethyl-L-arginine (L-NMMA), a NOS antagonist, during this period. We used L-NMMA and not L-NAME as a NOS antagonist for these assays because L-NAME contains a nitro group that is estimated as nitrite in ozone-chemiluminescence analyzer (30). The PBS in each well was aspirated and replaced with PBS containing 30 µM L-arginine with or without 10-6–10-5 M ATP. Cells were incubated for 15 min at 37°C, and the supernatant was aspirated for nitrite measurement by ozone chemiluminescence. The background nitrite levels in the solutions used for incubation of cells were also measured and subtracted from the values of test samples. Cell lysis buffer was added to each well and the protein concentration in each well was estimated by modified Bradford method to calculate the amount of nitrite per milligram of protein. The activity of NOS in PBS-treated wells was normalized to 100% and the percent change from this control was calculated for each treatment.
Coprecipitation studies for eNOS and HSP90. Confluent endothelial cells in 100-mm flasks were treated with either PBS or 10-5 M ATP in PBS for 10 min. The PBS was then aspirated and cells were lysed in modified RIPA buffer (12). The samples were sonicated, and cell debris was removed by centrifugation (14,000 g at 4°C for 10 min). A 500-µg aliquot of protein was used for immunoprecipitation (30) with H32 antibody for eNOS (BioMol). The immunoprecipitated proteins were separated by SDS-PAGE (7.5–15%) and transblotted to nitrocellulose membrane. The membranes were blocked with 5% nonfat milk in TBS-Tween (0.1%) and then immunoblotted for eNOS, phosphorylated-eNOS, and HSP90 with the use of appropriate antibodies [9D10-Zymed (Ser1177) Cell Signaling.com and (H38220 [GenBank] ) Transduction Laboratories, respectively]. The bands were visualized with the appropriate anti-immunoglobulin horseradish peroxidase conjugate (Sigma), ECL reagents (Amersham Pharmacia), and Kodak X-OMAT film. Autoradiograms were imaged with Adobe PhotoShop version 5.5 software, and the relative band densities were quantified with the use of NIH Image software version 1.62.
Third- to fifth-generation pulmonary arteries were also harvested from both groups of fetal lambs. The arteries were dissected clear of surrounding parenchyma, flash frozen in liquid nitrogen, pulverized, and placed in modified RIPA buffer (12). The mixture was then homogenized, sonicated to break the cells, and cell debris was removed by centrifugation. An aliquot (1 mg) of the protein was removed for immunoprecipitation with H32 antibody for eNOS (Biomol). The immunoprecipitated proteins were separated by SDS-PAGE and transblotted to nitrocellulose membrane. Immunoblotting for eNOS and HSP90 were performed as described above.
Statistical analysis. Data are shown as means ± SD. Comparison of baseline data with those obtained at different doses of ATP was done by single-factor ANOVA for repeated measures. Comparison of control data with those obtained from PPHN animals at each dose of ATP was done by two-way ANOVA (44). The two factors affecting the outcome were assumed to be the dose of ATP and presence or absence of pulmonary hypertension. When a significant difference (P < 0.05) was found, Duncan's multiple-range test was used to determine which means were different. Comparison of densitometric data for eNOS and HSP90 from control and pulmonary hypertension groups was done by unpaired t-tests.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Constriction of ductus arteriosus in fetal lambs was accompanied by an increase of 17 ± 4 mmHg in pulmonary artery pressure and no significant change in systemic pressure. The increased gradient between systemic and pulmonary artery pressures in the fetus was maintained for the duration of ductal constriction (Table 1). The arterial pH, PCO2, and PaO2 did not change immediately after constriction of the ductus arteriosus (Table 1). Serial measurements of arterial blood gas values during the 8-day period of ductal constriction demonstrated a 20% decrease in PaO2 over time, accompanied by an increase in pH and no change in PaCO2 (Table 1). The control fetal lambs also had an increase in pH, 24 h after procedure, but no changes in PaO2 and PaCO2 after the procedure (Table 1).
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ATP caused a dose-dependent vasodilation of intrapulmonary resistance
arteries harvested from control lambs (Fig.
1). The maximum increase in luminal diameter was observed at
10-3 M dose of ATP. Both L-NAME and
geldanamycin attenuated the relaxation response to ATP, with a decrease in
maximal response to ATP. The attenuation of the vasodilator response to ATP by
geldanamycin was similar to the inhibition caused by NOS antagonist
L-NAME (Fig. 1). The
· scavenger Tiron did
not affect the vasodilator response to ATP in control studies, but improved
the response to ATP in geldanamycin-treated vessels
(Fig. 1). These data suggest
that release of NO plays a significant role in mediating the vasodilator
response to ATP in normal pulmonary arteries. Geldanamycin, an inhibitor of
HSP90-eNOS interactions appears to cause
·-dependent impairment
of vasodilator response to ATP in the intrapulmonary resistance
arterioles.
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The vasodilator response to ATP was attenuated in the intrapulmonary
resistance arterioles isolated from fetal lambs with ductal ligation
(Fig. 2). The vasodilator
response to acetylcholine was also attenuated in the PPHN arteries, as
reported by Steinhorn et al.
(36) (data not shown).
Pretreatment of these vessels with the NOS antagonist L-NAME or the
· scavenger Tiron
improved the response to ATP (Fig.
2). These data suggest that a dysfunctional NOS and an increased
release of ·
contribute to the impaired vasodilator response to ATP in pulmonary
hypertension.
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Pulmonary arteries isolated from lambs with ductal ligation had decreased HSP90 in association with eNOS compared with control pulmonary arteries (Fig. 3). Western blots for eNOS in peripheral lung tissue and in pulmonary arteries demonstrated a 50% decrease in eNOS protein levels in ductal ligation lambs (Fig. 4), consistent with decreased expression of eNOS previously reported in this model (5, 35, 41).
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ATP increased the nitrite levels in the supernatant of control pulmonary artery endothelial cells (Fig. 5). Pretreatment of endothelial cells with the NOS antagonist, L-NMMA prevented the increase in nitrite levels in response to ATP, suggesting that increased NOS activity was the source of increased nitrites in the supernatant (Fig. 5). ATP failed to increase nitrite release from the hypertensive endothelial cells (Fig. 5).
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ATP increased the Ser1177 phosphorylation of eNOS and the association of HSP90 with eNOS in control endothelial cells (Fig. 6). Exposure of the pulmonary artery endothelial cells from ductal ligation lambs to ATP resulted in an increase in phosphorylation of the enzyme, but a decrease in the association of HSP90 with eNOS (Fig. 7).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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·
(30), contributing to impaired
vasodilator response in this model of pulmonary hypertension. To the best of
our knowledge, this is the first report of an alteration in HSP90-NOS
signaling in PPHN.
PPHN affects 
0.2% of full-term newborn infants during their transition
to postnatal life (42). The
affected infants have persistent elevation of pulmonary vascular resistance,
resulting in a failure to establish oxygenation during early postnatal life
(3). Although the
pathophysiology of PPHN remains unclear, the constriction of ductus arteriosus
in the fetus is known to be associated with this disease. Thus PPHN has been
reported (22,
29) in infants born to mothers
that had exposure to indomethacin and aspirin, cyclooxygenase inhibitors that
cause constriction of fetal ductus arteriosus. Administration of nonsteroidal
anti-inflammatory drugs to pregnant animals causes PPHN in the offspring
(13). We recently reported
(4) a high incidence of
exposure to nonsteroidal anti-inflammatory drugs in infants with PPHN.
Although prenatal ductal constriction may not occur in all infants with PPHN,
it has been used in animal models because it reproduces the hemodynamic and
structural alterations of PPHN
(3,
26,
43). After an 8-day period of
ductal constriction, a decrease or loss of postnatal pulmonary vasodilation is
seen in fetal lambs (3,
26). Lambs delivered at term
after a 10-day period of prenatal ductal ligation have persistence of high
PVR, hypoxemia, and structural changes in pulmonary arteries that mimic those
seen in infants with severe PPHN
(26,
43). A significant impairment
of vasodilator response to several physiological and pharmacological stimuli
occurs after ductal ligation in fetal lambs
(36,
38). The altered reactivity
includes a rapid and sustained loss of vasodilator response to oxygen, a major
stimulus for birth-related pulmonary vasodilation
(24,
38). However, a potential
alteration in the response of pulmonary arteries to ATP in the ductal ligation
model of PPHN was previously unknown.
Constriction of ductus arteriosus leads to an initial increase in the pulmonary flow and a more sustained increase in pressure and resistance in the fetal pulmonary circulation (1, 24). We found that the increase in pulmonary artery pressure is maintained for the duration of ductal constriction in our model. In addition, we observed a 20% decrease in PaO2 in the fetal lambs with ductal constriction. We speculate that the decrease in PaO2 was due to increased right to left shunting of blood across foramen ovale in response to the increase in pulmonary artery pressure. This admixture of blood and hypoxemia were previously reported (3, 26) in lambs with prenatal ligation of ductus arteriosus. The elevated pressure and hypoxemia are associated with impaired release of NO (24, 27, 35, 38), a major regulator of pulmonary vascular tone. Previous studies (5, 35, 41) have demonstrated attenuated NO-dependent vasodilation and decreased NOS activity and expression in the lung in this model. Though a decrease in eNOS protein levels alone partly account for the decreased NO release, impaired NOS activity also occurs in PPHN. An increase in fetal pulmonary artery pressure is associated with rapid decreases in eNOS activity and NO-dependent vasodilation in the fetal lamb (27, 38). The decrease in eNOS activity occurs before a decrease in eNOS expression (27). Villamor et al. (41) demonstrated that immunoreactive eNOS persists in the vascular endothelium, 10 days after ductal constriction. Thus an impairment of NOS activity appears to contribute significantly to the impaired NO release after ductal ligation. However, the mechanism of the impaired NOS activity after constriction of ductus arteriosus remains unknown.
Our previous studies demonstrated that release of ATP contributes to
perinatal pulmonary vasodilation in fetal lambs. The plasma levels of ATP
increased during exposure of fetal lambs to oxygen, alone or accompanied by
lung distension (15,
18). Infusion of ATP and its
metabolites into fetal pulmonary artery reproduced the pulmonary vasodilation
that occurs at birth in fetal lambs
(19). Inhibition of ATP
synthesis (16) or receptors
for ATP and its metabolite adenosine
(15,
18) attenuated the
birth-related pulmonary vasodilation in fetal lambs. ATP causes vasodilation
in fetal lambs, partly by NO-dependent mechanism and partly by its direct
effect on vascular smooth muscle
(17). The potential alteration
in the vasodilator response to ATP in fetal pulmonary hypertension was
previously unknown. Our data suggest that in the intrapulmonary resistance
vessels, ATP causes vasodilation primarily by NO-dependent pathway. The
attenuation of the vasodilator response to ATP in pulmonary arteries from
lambs with pulmonary hypertension was greater than what we observed with
L-NAME pretreatment in control vessels. These data suggest that in
pulmonary hypertension, lack of vasodilation was due to both loss of NO
release and alteration in other mechanisms that contribute to vasodilation in
response to ATP. In addition, both L-NAME and Tiron have improved
the vasodilator response to ATP in PPHN vessels. These data suggest that
inhibition of NOS-dependent
· allows these vessels
to relax in response to ATP by NO-independent mechanisms. Our previous studies
(33) in isolated rabbit
pulmonary arteries suggested that ATP causes NO-independent vasodilation by
activation of voltage-dependent K+ channels (Kv) on
vascular smooth muscle.
· has been shown to
inhibit smooth muscle Kv channels in coronary arteries
(20). These studies suggest
that a decrease in ·
may allow ATP to cause vasodilation by stimulation of smooth muscle
Kv channels in PPHN.
Regulation of NOS activity in the endothelial cell is a complex and dynamic
process. NOS has both reductase and oxygenase domains and occurs as a dimer
under physiological conditions
(25). Activation of NOS is
followed by its phosphorylation at Ser1177 site, which increases the electron
flow through the enzyme (23).
The balance of NO/·
released by activated NOS depends on its conformation, which is regulated by
cofactors such as HSP90 (30).
HSP90 association with eNOS facilitates NO release in response to a number of
physiological stimuli, such as shear stress, vascular endothelial growth
factor, and estrogen (7,
12,
32). The absence of HSP90 in
the NOS complex (10,
32) or inhibition of the HSP90
conformational change (30)
impairs NO release and increases
· release from NOS
(10,
30) in response to agonist
stimulation. Our studies demonstrated that ATP, a mediator of NO release in
response to shear stress (14)
and oxygen (15,
16), promoted the association
of HSP90 with eNOS in control endothelial cells. Although ATP increased
phosphorylation of eNOS in hypertensive cells, it failed to increase HSP90
association with eNOS and NO release. The increase in electron flow, which
occurs with phosphorylation of eNOS, increases the release of NO or
· from NOS
(23). Our data suggest that
activation of NOS is uncoupled from NO synthesis in pulmonary hypertension.
Steinhorn et al. (37) reported
an increase in · in
pulmonary arteries from lambs with ductal ligation and observed an improvement
in NO-mediated vasodilation when these vessels are treated with superoxide
dismutase and catalase. We observed an improvement in vasodilator response to
ATP by L-NAME and
· scavenger Tiron in
these pulmonary arteries. These observations suggest that
· release from
uncoupled NOS in pulmonary hypertension contributes to impaired
vasodilation.
Pulmonary arteries harvested from lambs with ductal ligation showed a
decrease in basal association of HSP90 with eNOS, suggesting an altered
regulation of NOS function in these arteries. Although the total amount of NOS
protein in ductal ligation lambs showed a decrease by Western blot, our
immunoprecipitation procedure ensured equal loading of eNOS in samples from
different arteries. Because immunoprecipitation is done with a limited amount
of eNOS antibody relative to eNOS, it is not quantitative with respect to
total amount of eNOS protein in the sample. However, the technique is useful
to evaluate the relative amounts of associated proteins that coprecipitate
with eNOS. The striking decrease in HSP90 coprecipitated with eNOS therefore
indicates an alteration in the HSP90/NOS interaction. Because this ratio is an
important determinant of the
NO/· balance coming
from NOS, these data support our concept of uncoupled NOS contributing to
impaired pulmonary vasodilation in PPHN.
The limitation of our study is that we did not address the alterations in other mechanisms that contribute to pulmonary vasodilation during the perinatal period. Previous studies (36) have demonstrated that ductal ligation is associated with a decreased sensitivity of vascular smooth muscle to NO with potential alterations in mechanisms downstream from release of NO. In addition, NOS function is regulated by several factors other than its association with HSP90 (25). Potentially, an alteration in its function caused by these factors may contribute to impaired NO release and pulmonary vasodilation in PPHN. We also observed a small but significant decrease in PaO2 by day 7 of ductal ligation. Whether the decrease in HSP90-NOS interaction is secondary to the effects of decreased PaO2 (39) or increased pulmonary artery pressure remains unknown.
In conclusion, our study provides evidence that decreased association of HSP90 with eNOS occurs in pulmonary hypertension induced by prenatal ductal ligation in fetal lambs. A decrease in this interaction is associated with significant impairment of NO release and vasodilator response to ATP, an important mediator of perinatal pulmonary vasodilation. The mechanisms involved in the decreased HSP90-eNOS interactions in pulmonary hypertension require further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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