Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased NO release and impaired pulmonary vasodilation. We investigated the hypothesis that increased superoxide (O(2)(*-)) 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 O(2)(*-) 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 O(2)(*-) 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 O(2)(*-) 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.     

Apocynin improves oxygenation and increases eNOS in persistent pulmonary hypertension of the newborn

NADPH oxidase is a major source of superoxide anions in the pulmonary arteries (PA). We previously reported that intratracheal SOD improves oxygenation and restores endothelial nitric oxide (NO) synthase (eNOS) function in lambs with persistent pulmonary hypertension of the newborn (PPHN). In this study, we determined the effects of the NADPH oxidase inhibitor apocynin on oxygenation, reactive oxygen species (ROS) levels, and NO signaling in PPHN lambs. PPHN was induced in lambs by antenatal ligation of the ductus arteriosus 9 days prior to delivery. Lambs were treated with vehicle or apocynin (3 mg/kg intratracheally) at birth and then ventilated with 100% O(2) for 24 h. A significant improvement in oxygenation was observed in apocynin-treated lambs after 24 h of ventilation. Contractility of isolated fifth-generation PA to norepinephrine was attenuated in apocynin-treated lambs. PA constrictions to NO synthase (NOS) inhibition with N-nitro-l-arginine were blunted in PPHN lambs; apocynin restored contractility to N-nitro-l-arginine, suggesting increased NOS activity. Intratracheal apocynin also enhanced PA relaxations to the eNOS activator A-23187 and to the NO donor S-nitrosyl-N-acetyl-penicillamine. Apocynin decreased the interaction between NADPH oxidase subunits p22(phox) and p47(phox) and decreased the expression of Nox2 and p22(phox) in ventilated PPHN lungs. These findings were associated with decreased superoxide and 3-nitrotyrosine levels in the PA of apocynin-treated PPHN lambs. eNOS protein expression, endothelial NO levels, and tetrahydrobiopterin-to-dihydrobiopterin ratios were significantly increased in PA from apocynin-treated lambs, although cGMP levels did not significantly increase and phosphodiesterase-5 activity did not significantly decrease. NADPH oxidase inhibition with apocynin may improve oxygenation, in part, by attenuating ROS-mediated vasoconstriction and by increasing NOS activity.     

Superoxide dismutase restores eNOS expression and function in resistance pulmonary arteries from neonatal lambs with persistent pulmonary hypertension

Endothelial nitric oxide (NO) synthase (eNOS) expression and activity are decreased in fetal lambs with persistent pulmonary hypertension (PPHN). We sought to determine the impact of mechanical ventilation with O(2) with or without inhaled NO (iNO) or recombinant human SOD (rhSOD) on eNOS in the ductal ligation model of PPHN. PPHN lambs and age-matched controls were ventilated with 100% O(2) for 24 h alone or combined with 20 ppm iNO continuously or a single dose of rhSOD (5 mg/kg) given intratracheally at delivery. In 1-day spontaneously breathing lambs, eNOS expression in resistance pulmonary arteries increased relative to fetal levels. eNOS expression increased in control lambs ventilated with 100% O(2), but not in PPHN lambs. Addition of iNO or rhSOD increased eNOS expression and decreased generation of reactive oxygen species (ROS) in PPHN lambs relative to those ventilated with 100% O(2) alone. However, only rhSOD restored eNOS function, increased tetrahydrobiopterin (BH(4)), a critical cofactor for eNOS function, and restored GTP cyclohydrolase I expression in isolated vessels and lungs from PPHN lambs. These data suggest that ventilation of PPHN lambs with 100% O(2) increases ROS production, blunts postnatal increases in eNOS expression, and decreases available BH(4) in PPHN lambs. Although the addition of iNO or rhSOD diminished ROS production and increased eNOS expression, only rhSOD improved eNOS function and levels of available BH(4). Thus therapies designed to decrease oxidative stress and restore eNOS coupling, such as rhSOD, may prove useful in the treatment of PPHN in newborn infants.     

Nitric oxide and superoxide generation from endothelial NOS: modulation by HSP90

Previously, we have shown that pulmonary arterial endothelial cells (PAECs) isolated from fetal lambs produce significant levels of nitric oxide (NO) but minimal superoxide upon stimulation, whereas PAECs isolated from 4-wk-old lambs produce significant amounts of both NO and superoxide. These data indicated that a certain degree of uncoupling of endothelial NO synthase (eNOS) occurs in PAECs during postnatal development. In this study, we sought to extend these studies by investigating the potential role of heat shock protein 90 (HSP90) in eNOS coupling. Western blot analyses revealed higher HSP90 expression in PAECs isolated from fetal compared with 4-wk-old lambs, whereas the analysis of recombinant human eNOS activation in vitro in the presence of HSP90 indicated that HSP90 significantly augmented NO production while inhibiting superoxide generation from eNOS. To further investigate whether HSP90 could be involved in uncoupling of eNOS in PAECs isolated from 4-wk-old lambs, we utilized an adenovirus to overexpress HSP90. We found that overexpression of HSP90 significantly increased the shear-stimulated association of HSP90 with eNOS and led to significant increases in NO production and reduced NOS-dependent superoxide generation. Conversely, the exposure of PAECs isolated from fetal lambs to the HSP90 inhibitor radicicol led to significant decreases in eNOS-HSP90 interactions, decreased shear-stimulated NO generation, and increased NOS-dependent superoxide production indicative of eNOS uncoupling. Finally, we examined eNOS-HSP90 interactions in our lamb model of pulmonary hypertension associated with increased pulmonary blood flow (shunt). Our data indicate that HSP90-eNOS interactions were decreased in shunt lambs and that this was associated with decreased NO generation and an increase in eNOS-dependent generation of superoxide. Together, our data support a significant role for HSP90 in promoting NO generation and inhibiting superoxide generation by eNOS and indicate that the disruption of this interaction may be involved in the endothelial dysfunction associated with pulmonary hypertension.     

rhVEGF treatment preserves pulmonary vascular reactivity and structure in an experimental model of pulmonary hypertension in fetal sheep

We have previously shown that lung VEGF expression is decreased in a fetal lamb model of PPHN and that VEGF165 inhibition causes severe pulmonary hypertension in fetal lambs. Therefore, we hypothesized that treatment with rhVEGF165 would preserve endothelium-dependent vasodilation and reduce the severity of pulmonary vascular remodeling in an experimental model of PPHN. We studied the effects of daily intrapulmonary infusions of rhVEGF after partial ligation of the ductus arteriosus (DA). We performed surgery in 24 late-gestation fetal lambs and placed catheters in the main pulmonary artery, left atrium, and aorta for pressure measurements and in the left pulmonary artery for drug infusions. A pressure transducer was placed around the LPA to measure blood flow to the left lung (Qp), and the DA was surgically constricted to induce pulmonary hypertension. rhVEGF165 or vehicle was infused for 7 or 14 days. ACh or 8-BrcGMP was infused on days 2 and 13 to assess endothelium-dependent and -independent vasodilation, respectively. ACh-induced vasodilation was reduced in PPHN lambs after 14 days (change in Qp from baseline, 106% vs. 11%). In contrast, the response to ACh was preserved in lambs treated with rhVEGF (change in Qp, 94% vs. 90%). Pulmonary vasodilation to 8-BrcGMP was not altered in PPHN lambs or enhanced by VEGF treatment. rhVEGF treatment increased expression of lung eNOS protein and decreased pulmonary artery wall thickness by 34% vs. PPHN lambs. We conclude that VEGF165 preserves endothelium-dependent vasodilation, upregulates eNOS expression, and reduces the severity of pulmonary vascular remodeling in experimental PPHN.     

Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.     

P2Y purine receptor responses and expression in the pulmonary circulation of juvenile rabbits

The purine nucleotide ATP mediates pulmonary vasodilation at birth by stimulation of P2Y purine receptors in the pulmonary circulation. The specific P2Y receptors in the pulmonary circulation and the segmental distribution of their responses remain unknown. We investigated the effects of purine nucleotides, ATP, ADP, and AMP, and pyrimidine nucleotides, UTP, UDP, and UMP, in juvenile rabbit pulmonary arteries for functional characterization of P2Y receptors. We also studied the expression of P2Y receptor subtypes in pulmonary arteries and the role of nitric oxide (NO), prostaglandins, and cytochrome P-450 metabolites in the response to ATP. In conduit size arteries, ATP, ADP, and AMP caused greater relaxation responses than UTP, UDP, and UMP. In resistance vessels, ATP and UTP caused comparable vasodilation. The response to ATP was attenuated by the P2Y antagonist cibacron blue, the NO synthase antagonist N(omega)-nitro-l-arginine methyl ester (l-NAME), and the cytochrome P-450 inhibitor 17-octadecynoic acid but not by the P2X antagonist alpha,beta-methylene ATP or the cyclooxygenase inhibitor indomethacin in conduit arteries. In the resistance vessels, l-NAME caused a more complete inhibition of the responses to ATP and UTP. Responses to AMP and UMP were NO and endothelium dependent, whereas responses to ADP and UDP were NO and endothelium independent in the conduit arteries. RT-PCR showed expression of P2Y(1), P2Y(2), and P2Y(4) receptors, but not P2Y(6) receptors, in lung parenchyma, pulmonary arteries, and pulmonary artery endothelial cells. These data suggest that distinct P2Y receptors mediate the vasodilator responses to purine and pyrimidine nucleotides in the juvenile rabbit pulmonary circulation. ATP appears to cause NO-mediated vasodilation predominantly through P2Y2 receptors on endothelium.     

Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress

Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 microg/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO(x)), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P < 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P < 0.05). Conversely, NO(x) levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P < 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P < 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P < 0.05) and were greater in shunted lambs than controls at all ages (P < 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P < 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO.     

Pulmonary vascular effects of nitric oxide-cGMP augmentation in a model of chronic pulmonary hypertension in fetal and neonatal sheep

Persistent pulmonary hypertension of the newborn (PPHN) is partly due to impaired nitric oxide (NO)-cGMP signaling. BAY 41-2272 is a novel direct activator of soluble guanylate cyclase, but whether this drug may be an effective therapy for PPHN is unknown. We hypothesized that BAY 41-2272 would cause pulmonary vasodilation in a model of severe PPHN. To test this hypothesis, we compared the hemodynamic response of BAY 41-2272 to acetylcholine, an endothelium-dependent vasodilator, and sildenafil, a selective inhibitor of PDE5 in chronically instrumented fetal lambs at 1 and 5 days after partial ligation of the ductus arteriosus. After 9 days, we delivered the animals by cesarean section to measure their hemodynamic responses to inhaled NO (iNO), sildenafil, and BAY 41-2272 alone or combined with iNO. BAY 41-2272 caused marked pulmonary vasodilation, as characterized by a twofold increase in blood flow and a nearly 60% fall in PVR at day 1. Effectiveness of BAY 41-2272-induced pulmonary vasodilation increased during the development of pulmonary hypertension. Despite a similar effect at day 1, the pulmonary vasodilator response to BAY 41-2272 was greater than sildenafil at day 5. At birth, BAY 41-2272 dramatically reduced PVR and augmented the pulmonary vasodilation induced by iNO. We concluded that BAY 41-2272 causes potent pulmonary vasodilation in fetal and neonatal sheep with severe pulmonary hypertension. We speculate that BAY 41-2272 may provide a novel treatment for severe PPHN, especially in newborns with partial response to iNO therapy.     

Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased cGMP-mediated vasodilation in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are increased in pulmonary arterial smooth muscle cells (PASMC) exposed to ET-1. Thus the initial objective of this study was to determine whether the development of pulmonary hypertension in utero is associated with elevated production of the ROS hydrogen peroxide (H(2)O(2)) and if this is associated with alterations in antioxidant capacity. Second we wished to determine whether chronic exposure of PASMC isolated from fetal lambs to H(2)O(2) would mimic the decrease in soluble guanylate cyclase expression observed in the ovine model of PPHN. Our results indicate that H(2)O(2) levels are significantly elevated in pulmonary arteries isolated from 136-day-old fetal PPHN lambs (P 0.05). In addition, we determined that catalase and glutathione peroxidase expression and activities remain unchanged. Also, we found that the overnight exposure of fetal PASMC to a H(2)O(2)-generating system resulted in significant decreases in soluble guanylate cyclase expression and nitric oxide (NO)-dependent cGMP generation (P 0.05). Finally, we demonstrated that the addition of the ROS scavenger catalase to isolated pulmonary arteries normalized the vasodilator responses to exogenous NO. As these scavengers had no effect on the vasodilator responses in pulmonary arteries isolated from age-matched control lambs this enhancement appears to be unique to PPHN. Overall our data suggest a role for H(2)O(2) in the abnormal vasodilation associated with the pulmonary arteries of PPHN lambs.     

Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and decreased vascular growth. The role of Rho kinase activity in modulating endothelial function and regulating angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases Rho kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho kinase activity was measured by RhoA, Rho GTP, and phosphorylated MYPT-1 protein content. The effects of Rho kinase activity on angiogenesis, endothelial nitric oxide (NO) synthase (eNOS) protein expression, and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632 (a Rho kinase inhibitor) and calpeptin (a Rho kinase activator) in the presence/absence of N-nitro-l-arginine (l-NA, an NOS inhibitor). RhoA, Rho GTP, and phosphorylated MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced 29% in PPHN PAECs (P < 0.001) and increased with Y-27632 treatment in normal and PPHN PAECs, with PPHN PAECs achieving levels similar to those of normal PAECs. l-NA inhibited the Y-27632-induced increase in tube formation in normal, but not PPHN, PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression was reduced 42% in PPHN PAECs (P < 0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs but reduced NO production in normal and PPHN PAECs. We conclude that Rho kinase activity is increased in PPHN PAECs and impairs angiogenesis and downregulates eNOS protein and NO production in vitro.     

Increased pulmonary vascular resistance and defective pulmonary artery filling in caveolin-1-/- mice

Caveolin-1, the structural and signaling protein of caveolae, is an important negative regulator of endothelial nitric oxide synthase (eNOS). We observed that mice lacking caveolin-1 (Cav1(-/-)) had twofold increased plasma NO levels but developed pulmonary hypertension. We measured pulmonary vascular resistance (PVR) and assessed alterations in small pulmonary arteries to determine the basis of the hypertension. PVR was 46% greater in Cav1(-/-) mice than wild-type (WT), and increased PVR in Cav1(-/-) mice was attributed to precapillary sites. Treatment with NG-nitro-l-arginine methyl ester (l-NAME) to inhibit NOS activity raised PVR by 42% in WT but 82% in Cav1(-/-) mice, indicating greater NO-mediated pulmonary vasodilation in Cav1(-/-) mice compared with WT. Pulmonary vasculature of Cav1(-/-) mice was also less reactive to the vasoconstrictor thromboxane A2 mimetic (U-46619) compared with WT. We observed redistribution of type I collagen and expression of smooth muscle alpha-actin in lung parenchyma of Cav1(-/-) mice compared with WT suggestive of vascular remodeling. Fluorescent agarose casting also showed markedly decreased density of pulmonary arteries and artery filling defects in Cav1(-/-) mice. Scanning electron microscopy showed severely distorted and tortuous pulmonary precapillary vessels. Thus caveolin-1 null mice have elevated PVR that is attributed to remodeling of pulmonary precapillary vessels. The elevated basal plasma NO level in Cav1(-/-) mice compensates partly for the vascular structural abnormalities by promoting pulmonary vasodilation.     

Endothelial nitric oxide synthase mediates cutaneous vasodilation during local heating and is attenuated in middle-aged human skin

Local skin heating is used to assess microvascular function in clinical populations because NO is required for full expression of the response; however, controversy exists as to the precise NO synthase (NOS) isoform producing NO. Human aging is associated with attenuated cutaneous vasodilation but little is known about the middle aged, an age cohort used for comparison with clinical populations. We hypothesized that endothelial NOS (eNOS) is the primary isoform mediating NO production during local heating, and eNOS-dependent vasodilation would be reduced in middle-aged skin. Vasodilation was induced by local heating (42°C) and during acetylcholine dose-response (ACh-DR: 0.01, 0.1, 1.0, 5.0, 10.0, 50.0, 100.0 mmol/l) protocols. Four microdialysis fibers were placed in the skin of 24 men and women; age cohorts were 12 middle-aged (53 ± 1 yr) and 12 young (23 ± 1 yr). Sites served as control, nonselective NOS inhibited [N(G)-nitro-l-arginine methyl ester (l-NAME)], inducible NOS (iNOS) inhibited (1400W), and neuronal NOS (nNOS) inhibited (N(ω)-propyl-l-arginine). After full expression of the local heating response, l-NAME was perfused at all sites. Cutaneous vascular conductance was measured and normalized to maximum (%CVC(max): Nitropress). l-NAME reduced %CVCmax at baseline, all phases of the local heating response, and at all ACh concentrations compared with all other sites. iNOS inhibition reduced the initial peak (53 ± 2 vs. 60 ± 2%CVC(max); P < 0.001); however, there were no other differences between control, nNOS-, and iNOS-inhibited sites during the phases of local heating or ACh-DR. When age cohorts were compared, NO-dependent vasodilation during local heating (52 ± 6 vs. 68 ± 4%CVC(max); P = 0.013) and ACh perfusion (50 mmol/l: 83 ± 3 vs. 93 ± 2%CVC(max); 100 mmol/l: 83 ± 4 vs. 92 ± 3%CVC(max); both P = 0.03) were reduced in middle-aged skin. There were no differences in NOS isoform expression obtained from skin biopsy samples between groups (all P > 0.05). These data suggest that eNOS mediates the production of NO during local heating and that cutaneous vasodilation is attenuated in middle-aged skin.     

Cinaciguat, a soluble guanylate cyclase activator, augments cGMP after oxidative stress and causes pulmonary vasodilation in neonatal pulmonary hypertension

Although inhaled NO (iNO) therapy is often effective in treating infants with persistent pulmonary hypertension of the newborn (PPHN), up to 40% of patients fail to respond, which may be partly due to abnormal expression and function of soluble guanylate cyclase (sGC). To determine whether altered sGC expression or activity due to oxidized sGC contributes to high pulmonary vascular resistance (PVR) and poor NO responsiveness, we studied the effects of cinaciguat (BAY 58-2667), an sGC activator, on pulmonary artery smooth muscle cells (PASMC) from normal fetal sheep and sheep exposed to chronic intrauterine pulmonary hypertension (i.e., PPHN). We found increased sGC α(1)- and β(1)-subunit protein expression but lower basal cGMP levels in PPHN PASMC compared with normal PASMC. To determine the effects of cinaciguat and NO after sGC oxidation in vitro, we measured cGMP production by normal and PPHN PASMC treated with cinaciguat and the NO donor, sodium nitroprusside (SNP), before and after exposure to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an sGC oxidizer), hyperoxia (fraction of inspired oxygen 0.50), or hydrogen peroxide (H(2)O(2)). After treatment with ODQ, SNP-induced cGMP generation was markedly reduced but the effects of cinaciguat were increased by 14- and 64-fold in PPHN fetal PASMC, respectively (P < 0.01 vs. controls). Hyperoxia or H(2)O(2) enhanced cGMP production by cinaciguat but not SNP in PASMC. To determine the hemodynamic effects of cinaciguat in vivo, we compared serial responses to cinaciguat and ACh in fetal lambs after ductus arteriosus ligation. In contrast with the impaired vasodilator response to ACh, cinaciguat-induced pulmonary vasodilation was significantly increased. After birth, cinaciguat caused a significantly greater fall in PVR than either 100% oxygen, iNO, or ACh. We conclude that cinaciguat causes more potent pulmonary vasodilation than iNO in experimental PPHN. We speculate that increased NO-insensitive sGC may contribute to the pathogenesis of PPHN, and cinaciguat may provide a novel treatment of severe pulmonary hypertension.     

Stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to globular adiponectin-induced NO production: vasorelaxation in response to globular adiponectin

The present study examined potential interactions between endothelial NO synthase (eNOS), heat shock protein (HSP)90, and Akt in vascular endothelial cells stimulated with globular adiponectin to produce nitric oxide (NO). Globular adiponectin-induced eNOS phosphorylation was accompanied by eNOS-HSP90-Akt complex formation, resulting in a dose-dependent increase in NO release. Globular adiponectin stimulated binding of HSP90 to eNOS, and inhibition of HSP90 significantly suppressed globular adiponectin-stimulated NO release. Globular adiponectin also caused Akt phosphorylation, and inhibition of PI3 kinase significantly suppressed globular adiponectin-stimulated NO release. This study also examined whether globular adiponectin really induces endothelial-dependent vasodilation using rings from rat thoracic aorta. It was observed that globular adiponectin caused dose-dependent vasorelaxation in the aorta. These results indicate that stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to globular adiponectin-induced eNOS phosphorylation and NO production, and to endothelium-dependent vasorelaxation.     

Role of oxidative phosphorylation and ATP release in mediating birth-related pulmonary vasodilation in fetal lambs

We investigated the hypothesis that birth-related pulmonary vasodilation is mediated in part by an increase in oxidative phosphorylation and ATP release in response to oxygen exposure at birth. Studies were done in fetal lambs to evaluate the independent effects of oxygen, lung distension alone, or lung distension accompanied by oxygenation and shear stress on fetal pulmonary blood flow and resistance and plasma ATP levels in the pulmonary artery. The effect of each intervention was evaluated in lambs assigned to one of three groups: control or pretreatment with 2,4-dinitrophenol or antimycin-A, inhibitors of oxidative phosphorylation. Exposure to oxygen alone or with lung distension was associated with increases in plasma ATP levels and pulmonary blood flow and a decrease in pulmonary vascular resistance. Plasma ATP levels did not change during lung distension alone. 2,4-Dinitrophenol and antimycin-A attenuated the pulmonary vasodilator response to oxygen but did not attenuate the response to lung distension alone. An increase in oxidative phosphorylation and ATP release during oxygen exposure may contribute to birth-related pulmonary vasodilation in fetal lambs.     

Estrogen stimulates heat shock protein 90 binding to endothelial nitric oxide synthase in human vascular endothelial cells. Effects on calcium sensitivity and NO release

Estradiol (E(2)) causes endothelium-dependent vasodilation, mediated, in part, by enhanced nitric oxide (NO) release. We have previously shown that E(2)-induced activation of endothelial nitric oxide synthase (eNOS) reduces its calcium dependence. This pathway of eNOS activation is unique to a limited number of stimuli, including shear stress, the response to which is herbimycin-inhibitable. Consistent with this, herbimycin and geldanamycin pretreatment of human umbilical vein endothelial cells (HUVEC) abrogated E(2)-stimulated NO release and cGMP production, respectively. These benzoquinone ansamycins are potent inhibitors of Hsp90 function, which has recently been shown to play a role in stimulus-dependent eNOS activation. As in response to shear, E(2) induced an Hsp90-eNOS association, peaking at 30 min and completely inhibited by the conventional estrogen receptor antagonist ICI 182,780. These findings suggest that Hsp90 plays an important role in the rapid, estrogen receptor-mediated modulation of eNOS activation by estrogen.     

Effect of long-term high-altitude hypoxia on fetal pulmonary vascular contractility.

Hypoxia in the fetus and/or newborn is associated with an increased risk of pulmonary hypertension. The present study tested the hypothesis that long-term high-altitude hypoxemia differentially regulates contractility of fetal pulmonary arteries (PA) and veins (PV) mediated by differences in endothelial NO synthase (eNOS). PA and PV were isolated from near-term fetuses of pregnant ewes maintained at sea level (300 m) or high altitude of 3,801 m for 110 days (arterial Po(2) of 60 Torr). Hypoxia had no effect on the medial wall thickness of pulmonary vessels and did not alter KCl-induced contractions. In PA, hypoxia significantly increased norepinephrine (NE)-induced contractions, which were not affected by eNOS inhibitor N(G)-nitro-l-arginine (l-NNA). In PV, hypoxia had no effect on NE-induced contractions in the absence of l-NNA. l-NNA significantly increased NE-induced contractions in both control and hypoxic PV. In the presence of l-NNA, NE-induced contractions of PV were significantly decreased in hypoxic lambs compared with normoxic animals. Acetylcholine caused relaxations of PV but not PA, and hypoxia significantly decreased both pD(2) and the maximal response of acetylcholine-induced relaxation in PV. Additionally, hypoxia significantly decreased the maximal response of sodium nitroprusside-induced relaxations of both PA and PV. eNOS was detected in the endothelium of both PA and PV, and eNOS protein levels were significantly higher in PV than in PA in normoxic lambs. Hypoxia had no significant effect on eNOS levels in either PA or PV. The results demonstrate heterogeneity of fetal pulmonary arteries and veins in response to long-term high-altitude hypoxia and suggest a likely common mechanism downstream of NO in fetal pulmonary vessel response to chronic hypoxia in utero.     

Asymmetric dimethylarginine inhibits HSP90 activity in pulmonary arterial endothelial cells: role of mitochondrial dysfunction

Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states.