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1Departments of Pediatrics, 2Surgery, 3Pharmacology and Toxicology, and 4Cardiovascular Research Center, Medical College of Wisconsin and 5Zablocki Department of Veterans Affairs Medical Center, Milwaukee, Wisconsin
Submitted 27 April 2006 ; accepted in final form 27 November 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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–) release by an uncoupled endothelial nitric oxide synthase (eNOS) contributes to impaired pulmonary vasodilation in PPHN. We investigated the response of isolated pulmonary arteries to the NOS agonist ATP and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus and in sham-ligated controls in the presence or absence of the NOS antagonist nitro-L-arginine methyl ester (L-NAME) or the O2– scavenger 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron). ATP caused dose-dependent relaxation of pulmonary artery rings in control lambs but induced constriction of the rings in PPHN lambs. L-NAME, the NO precursor L-arginine, and Tiron restored the relaxation response of pulmonary artery rings to ATP in PPHN. Relaxation to NO was attenuated in arteries from PPHN lambs, and the response was improved by L-NAME and by Tiron. We also investigated the alteration in heat shock protein (HSP)90-eNOS interactions and release of NO and O2– in response to ATP in the pulmonary artery endothelial cells (PAEC) from these lambs. Cultured PAEC and endothelium of freshly isolated pulmonary arteries from PPHN lambs released O2– in response to ATP, and this was attenuated by the NOS antagonist L-NAME and superoxide dismutase (SOD). ATP stimulated HSP90-eNOS interactions in PAEC from control but not PPHN lambs. HSP90 immunoprecipitated from PPHN pulmonary arteries had increased nitrotyrosine signal. Oxidant stress from uncoupled eNOS contributes to impaired pulmonary vasodilation in PPHN induced by ductal ligation in fetal lambs.
ATP; superoxide dismutase; superoxide; heat shock protein 90; nitrotyrosine
–) release from NADPH oxidase in pulmonary arteries (5). Our previous studies in this model (16) demonstrated that PPHN is associated with decreased heat shock protein (HSP)90-nitric oxide synthase (NOS) interactions and impaired NO synthesis in fetal lambs. Studies in bovine aortic endothelial cells demonstrated that inhibition of HSP90-endothelial NOS (eNOS) interactions leads to uncoupling of eNOS and increased O2– release from NOS (22, 23). However, the mechanism of decreased HSP90-eNOS interactions and a potential role for NOS as a source for O2– generation in PPHN remains unknown. We investigated the hypothesis that increased generation of O2– from uncoupled eNOS and the resulting oxidative stress in the pulmonary arteries contribute to the impaired pulmonary vasodilation in this model. We also proposed that tyrosine nitration of HSP90 as a result of oxidative stress leads to decreased HSP90-eNOS interactions and impaired eNOS function in PPHN. The objectives of our studies were to investigate 1) the role of NOS-derived O2– in the impaired vasodilator responses to the physiological NOS agonist ATP and to NO, 2) the effect of ATP on NO and O2– release from pulmonary artery endothelial cells (PAEC) and pulmonary artery segments with intact endothelium in control and PPHN lambs, 3) the effect of ATP on HSP90-eNOS interactions in control PAEC and their alteration in endothelial cells from PPHN lambs, and 4) evidence of tyrosine nitration of HSP90, a marker for oxidative stress, in pulmonary arteries in PPHN. |
METHODS |
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– from eNOS. Parallel studies were done in isolated pulmonary arteries to determine the functional effects of eNOS-derived O2– in PPHN. The protocol for use of animals in this study was approved by the Institutional Animal Care and Use Committees of the Medical College of Wisconsin and Zablocki Department of Veterans Affairs Medical Center.
Isolation and study of pulmonary arteries from lungs.
Details of methods for isolation and study of pulmonary arteries from fetal lambs in tissue bath were reported previously (16). Third- to fifth-generation intrapulmonary arteries with an internal diameter of 300–500 µm were dissected and isolated from the lung. The arteries were cut into rings 1-mm in length, suspended with stainless steel hooks in water-jacketed chambers, and connected to force displacement transducers (FTO3, Grass Instruments). The artery rings were bathed in 2 ml of physiological salt solution (PSS) kept at 37°C and aerated to maintain normal acid-base status and oxygenation of tissue. They were allowed to equilibrate for 45 min, stretched to a passive tension of 0.8 g, and preconstricted with 10–6–10–7 M norepinephrine. The tension reached with norepinephrine constriction for each ring was normalized to 100%, and the percent change from this tension with each dose of ATP or S-nitroso-N-acetylpenicillamine [SNAP (NO donor); Sigma, St. Louis, MO] was expressed as mean (SD). Relaxation responses to 10–8–10–3 M doses of ATP and SNAP were determined, and the responses were compared with rings treated with PSS alone. Separate rings were pretreated with 10–4 M nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, L-arginine, NOS substrate, sepiapterin, a tetrahydrobiopterin (BH4) analog, and 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron), a O2– scavenger (all from Sigma). Relaxation responses to ATP and NO were then studied after 15 min of incubation with these agents.
Isolation of PAEC.
PAEC were isolated and characterized with techniques described previously (16). Briefly, the right and left pulmonary arteries were dissected up to the third-generation branches in the lung. The branches were tied and cut distally, and endothelial cells were isolated with 0.1% collagenase. PAEC were grown in six-well plates for measurement of NO, 100-mm flasks for immunoprecipitation studies, and eight-well chamber glass slides (Lab-Tek II, Nalgene, Naperville, IL) for assessment of O2– release with dihydroethidium (DHE) fluorescence. We previously reported (16) that PAEC from PPHN lambs continue to show a decrease in HSP90-eNOS interactions and impaired NO release with NOS agonists compared with control cells up to the fourth passage. We therefore studied PAEC from control and PPHN lambs up to passage 4 to investigate altered eNOS biology in PPHN.
Measurement of NO release. PAEC in six-well plates were placed in serum-free medium overnight and washed with Hanks’ balanced salt solution (HBSS). NOS activity was assessed by accumulation of cGMP in the cells, detected with an enzyme immunoassay (EIA) method as described previously (34). Cells in selected wells were treated with the NOS inhibitor L-NAME (10–4 M) for 30 min. PAEC were then exposed to HBSS with or without 10–6–10–5 M ATP for 15 min. The reaction was stopped by addition of cell lysis buffer, and cGMP content of the lysate was measured with an EIA method (Amersham). The protein concentration in each well was estimated to calculate the amount of cGMP per milligram of protein. The cGMP in control, unstimulated cells was normalized to 100%, and the percent change from the baseline was compared between different treatments.
Measurement of O2– release from PAEC.
PAEC from control and PPHN lambs were grown to confluence in eight-well chamber glass slides. PAEC in selected wells were pretreated with 10–4 M L-NAME (NOS antagonist) or superoxide dismutase (SOD, 1,000 U/ml; Sigma) for 30 min. The PAEC were then treated with HBSS alone or with 10–5 M ATP and DHE (10–5 M) to detect increases in intracellular O2– generation (23). After 15-min incubation with ATP or HBSS, supernatant was aspirated and cells were rinsed with ice-cold HBSS. Slides were coverslipped, visualized, and imaged with a fluorescent microscope (NIKON Eclipse 600) equipped with a SPOT RT Slide camera and software (Diagnostic Instruments). DHE fluorescence from PAEC and pulmonary artery segments was quantified with MetaVue software (Universal Imaging, Downingtown, PA).
DHE fluorescence in pulmonary artery segments with intact endothelium. The second-generation branches of pulmonary arteries were dissected fresh from the lungs of control and PPHN lambs. A 1-mm length of the artery was cut open to expose endothelium and placed in six-well plates with 1 ml of HBSS. The pulmonary artery segments were incubated in HBSS alone or with 10–4 M L-NAME or 1,000 U/ml SOD for 30 min. DHE (10–5 M) and HBSS with or without 10–5 M ATP were then added (23). After 15 min, pulmonary artery segments were washed, placed in microwells of chamber glass slides, and coverslipped. DHE fluorescence of endothelium was visualized and quantified as described above.
Immunoprecipitation studies. Confluent PAEC in 100-mm flasks were treated with either HBSS or 10–5 M ATP in HBSS for 10 min. The supernatant was then aspirated, and cells were lysed in modified radioimmunoprecipitation assay (RIPA) buffer (8). eNOS was immunoprecipitated from cell lysate with an eNOS antibody (H32, BioMol). The immunoprecipitate was immunoblotted for eNOS and HSP90 (16) with appropriate antibodies [9D10 (Zymed) and H38220 [GenBank] (Transduction Laboratories), respectively]. Bands were visualized with the appropriate anti-immunoglobulin horseradish peroxidase conjugate (Sigma), ECL reagents (Amersham Pharmacia), and Kodak X-OMAT film. An aliquot of protein (1 mg) from control and PPHN PAEC was used to perform Western blots for eNOS and soluble guanylate cyclase (sGC) to assess for potential changes in the expression of these proteins in PPHN. Autoradiograms were imaged with Adobe PhotoShop version 5.5 software, and relative band densities were quantified with NIH Image 1.62.
Third- to fifth-generation pulmonary arteries were dissected clear of surrounding parenchyma, flash frozen in liquid nitrogen, pulverized, and placed in modified RIPA buffer (16). The mixture was homogenized and sonicated to break the cells, and cell debris was removed by centrifugation. An aliquot (1 mg) of the protein was removed for immunoprecipitation with HSP90 antibody (Transduction Laboratories). The immunoprecipitate was immunoblotted for HSP90, and the HSP90 blot was probed with antibody for nitrotyrosine (Upstate) as described above. In some studies, nitrotyrosine was immunoprecipitated from protein lysate with appropriate antibody (Upstate) and the immunoprecipitate was immunoblotted for HSP90 as described above. Expression of HSP90 was assessed by Western blotting for this protein in samples from control and PPHN pulmonary arteries, and the ratio of nitrated HSP90 to HSP90 was calculated for the samples from control and PPHN lambs.
Statistical analysis. Data are shown as means (SD). Changes in vascular ring tension with incremental doses of ATP and with different blockers were analyzed by two-way ANOVA (37). Comparison of cGMP and DHE fluorescence values with different treatments was also done by two-way ANOVA. When a significant difference (P < 0.05) was found, a Duncan's multiple-range test was done to determine which means were different. Comparison of densitometric data for eNOS, HSP90, and nitrated HSP90 from control and pulmonary hypertension groups was done by unpaired t-tests.
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RESULTS |
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– scavenger Tiron (Fig. 2B) in PPHN. Sepiapterin had no significant effect on the relaxation response to SNAP in either control or PPHN arteries (data not shown). These data indicate that NOS-derived oxidant stress impairs the response of the guanylate cyclase-cGMP system in vascular smooth muscle to NO.
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– release.
There was no increase in DHE fluorescence with ATP in control cells (Fig. 4), and no changes were noted with L-NAME or with SOD. In contrast, PAEC from ductal ligation lambs had increased DHE fluorescence at baseline and in response to stimulation with ATP (Fig. 4). The increase in DHE fluorescence observed in hypertensive PAEC with ATP was attenuated by the NOS antagonist L-NAME and by SOD (Fig. 4). Pulmonary artery segments harvested from control lambs had no changes in DHE fluorescence with ATP or with L-NAME and SOD (Fig. 5). Pulmonary arteries from PPHN lambs showed increased DHE fluorescence in the endothelium in response to ATP exposure (Fig. 5). The increase in DHE fluorescence with ATP was attenuated by L-NAME and by SOD, suggesting an increase in NOS-derived O2– release in response to ATP in PPHN.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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–/decreased NO release in response to ATP, a physiological agonist. The increase in O2– appears to impair the vasodilator response to both endothelium-dependent vasodilator ATP and -independent vasodilator NO. These data suggest that ROS contribute to vascular adaptation in both endothelium and vascular smooth muscle in PPHN.
PPHN affects 0.2% of full-term infants during their transition to postnatal life (35). Prenatal ductal constriction from in utero exposure to nonsteroidal anti-inflammatory drugs is associated with PPHN (3). Prenatal ductal constriction in fetal lambs reproduces the hemodynamic and structural features of PPHN (2, 18). Although decreases in eNOS expression and NO release occur in this model, they do not fully account for the impaired pulmonary vasodilation in PPHN. Lambs with PPHN show impaired pulmonary vasodilator response to inhaled NO (28) and enhancement of this response with exogenous SOD (28). These data suggest that oxidant stress impairs pulmonary vasodilation in PPHN. The source of ROS in PPHN was not clear from previous studies. NADPH oxidase activity and expression are increased in pulmonary arteries in lambs with PPHN (5). However, a potential role for eNOS also as a source of O2– was suggested by our previous observation of decreased HSP90-eNOS interactions in the pulmonary arteries in this model (16). Altered HSP90-eNOS interactions are associated with uncoupling of eNOS activity and increased O2– release (22, 23) in cultured endothelial cells. Our present studies provide in vivo evidence that eNOS uncoupling from decreased HSP90-eNOS interaction impairs pulmonary vascular responses to vasodilator stimuli. We observed that both cultured PAEC and endothelium in intact pulmonary artery segments demonstrate increased NOS-derived O2– in PPHN. The mechanism of eNOS uncoupling in PPHN remains unclear. Previous studies demonstrated that ductal constriction leads to increased pulmonary artery pressure without a change in flow in fetal lambs (2, 16). Murata et al. (19) reported that a decrease in HSP90-eNOS interactions occurs in hypoxia-induced pulmonary hypertension in rats. Increased pressure load on endothelial cells in pulmonary hypertension may therefore result in decreased HSP90 interaction with eNOS and uncoupling of NOS activity. The mechanism of decreased HSP90-eNOS interaction with the pressure load on endothelium remains unclear. HSP90 associates with cytoskeleton proteins actin and tubulin when the cells are stressed and experience a decrease in intracellular ATP levels (10). We observed that the PPHN cells stimulated with ATP showed a trend for decreased HSP90-eNOS interactions, associated with a significant increase in oxidative stress. The potential targeting of HSP90 to cytoskeleton to stabilize the endothelial cell in the presence of oxidative stress in pulmonary hypertension requires further investigation.
Previous studies have suggested that the association of HSP90 with eNOS is facilitated by tyrosine phosphorylation of HSP90 (21, 31). We observed that ATP promotes association of HSP90 with eNOS in control PAEC. In contrast, hypertensive PAEC did not show this response to ATP. We observed increased tyrosine nitration of HSP90 in the pulmonary arteries from PPHN lambs. Since nitration of tyrosine causes steric hindrance for phosphorylation, this may serve as a potential mechanism for decreased HSP90-eNOS interactions in PPHN lambs. Peroxynitrite (ONOO–), generated by reaction of O2– with NO, was shown previously to increase tyrosine nitration in blood vessels (24). Increased activity of NADPH oxidase has been demonstrated as early as 48 h after ductal constriction in fetal lambs (5). Release of O2– early in the course of PPHN and its reaction with NO from eNOS to form ONOO– can potentially lead to tyrosine nitration of HSP90 and decrease its interaction with eNOS in PPHN. The potential role of ONOO– in causing tyrosine nitration of HSP90 in the endothelial cell and the specific tyrosine residues nitrated by ONOO– require further investigation.
We observed that scavenging O2– is more effective than NOS inhibition in decreasing DHE fluorescence in PAEC and improving the relaxation response to ATP in PPHN. This may be due to increased bioavailability of endogenous NO or contribution of O2– from sources other than eNOS in PPHN (5). The enhancement of pulmonary vasodilator response to ATP by L-NAME in PPHN may appear incongruous since ATP stimulates NO release in control PAEC. However, ATP causes vasodilation in part by NO-independent mechanisms. These include stimulation of prostacyclin release (9) and direct activation of purinergic receptors and K+ channels on the vascular smooth muscle (12, 25). Inhibition of O2– release from eNOS by L-NAME may facilitate the initiation of vasodilator response to ATP by these NO-independent mechanisms. Similarly, L-NAME also enhanced the response to the NO donor SNAP, suggesting that NOS-derived O2– may impair sensitivity of sGC on smooth muscle to NO (17). The mechanism of decreased response of the guanylyl cyclase-cGMP system in PPHN remains unclear. ONOO– has been shown to decrease the sensitivity of guanylyl cyclase to NO in vitro (17). Expression of sGC is decreased in the pulmonary arteries of PPHN lambs (4, 30). Hydrogen peroxide, a product of oxidant stress, has been shown to decrease the guanylate cyclase expression and NO-generated cGMP levels in cultured fetal pulmonary artery smooth muscle cells (36). However, it is unclear whether endothelium-derived O2– or ONOO– is transported to the vascular smooth muscle. The SOD mimetic Tiron enhanced the relaxation response to ATP in the control pulmonary arteries, suggesting that O2– modulates the response of pulmonary artery rings to ATP. Although we did not observe an increase in O2– release in control pulmonary artery segments or endothelial cells exposed to ATP, some O2– release may have occurred in the pulmonary arteries under the tissue bath conditions in vitro.
We observed that the eNOS protein levels in the endothelial cells are similar in control and PPHN lambs. Previous studies reported a decrease in eNOS protein levels in pulmonary arteries in PPHN. However, these studies were done in protein lysate from homogenized isolated pulmonary arteries and lung tissue (16, 27, 32) in PPHN. The apparent increase in eNOS expression in the immunoblots shown for PAEC from PPHN lambs is probably related to differences in the proliferative state of the cells. We attempted to minimize the variation in immunoprecipitation technique by performing the assays on control and PPHN cells on the same day. Previous studies reported higher eNOS expression in proliferating endothelial cells compared with confluent cells (22).
Studies in mice with hyperphenylalanine phenotype (hph-1 mice) have reported a role for BH4 depletion in eNOS uncoupling and pulmonary hypertension (11, 20). We did not measure BH4 levels in the lungs or PAEC in our model. However, the BH4 analog sepiapterin had minimal effect on the relaxation response to ATP in our studies. We previously reported (16) that alteration of HSP90-NOS interactions by geldanamycin impairs the vasodilator response to ATP in fetal pulmonary arteries. These data suggest that in fetal pulmonary hypertension HSP90-NOS interactions play an important role in the modulation of pulmonary vascular tone. Whether oxidized BH4 plays a role in the altered HSP90-eNOS interactions or eNOS uncoupling in the ductal ligation model of PPHN remains unknown.
In conclusion, our studies provide evidence that eNOS is uncoupled and serves as a source of oxidative stress in pulmonary hypertension induced by ductal ligation in fetal lambs. NOS-derived oxidant stress contributes to impaired pulmonary vasodilation during transition of the fetus to postnatal life in PPHN. The mechanisms of eNOS uncoupling in PPHN require further investigation.
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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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from ATP alone. The relaxation response to ATP in control rings was attenuated by the nitric oxide synthase (NOS) antagonist nitro-L-arginine methyl ester (L-NAME; A) and enhanced by the superoxide (O2
from ATP. OD, optical density; IOD, integrated optical density. NO release was estimated by cGMP accumulation in PAEC, and the values for untreated cells at baseline were adjusted to 100%. ATP stimulated NO release in a dose-dependent manner from control, but not from PPHN cells. C: representative Western blot (WB) for soluble guanylate cyclase (sGC) protein from control and PPHN PAEC. D: summarized data from Western blots performed on PAEC from 6 different control and PPHN lambs each. sGC protein levels are similar in PAEC from control and PPHN lambs. These data suggest that the decrease in cGMP levels in PPHN cells is not due to decreased expression of sGC.
from untreated PPHN cells, 
