Altered endothelium-dependent relaxations in lambs with high pulmonary blood flow and pulmonary hypertension

Congenital heart disease associated with increased pulmonary blood flow produces pulmonary hypertension. To characterize vascular alterations in the nitric oxide (NO)-cGMP cascade induced by increased pulmonary blood flow and pulmonary hypertension, 10 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). When the lambs were 4-6 wk of age, we assessed responses of pulmonary arteries (PAs) and pulmonary veins (PVs) isolated from lungs of control and shunted lambs. PVs from control and shunted lambs relaxed similarly to exogenous NO (S-nitrosyl-acetyl-penicillamine), to NO produced endogenously (zaprinast and A-23187), and to cGMP (atrial natriuretic peptide). In contrast, relaxations to A-23187 and zaprinast were blunted in PAs isolated from shunted lambs relative to controls. Inhibitors of NO synthase (NOS) and soluble guanylate cyclase constricted control but not shunt PAs, indicating reduced basal NOS activity in shunt PAs. Pretreatment of shunt PAs with the substrates L-arginine and sepiapterin, a precursor for tetrahydrobiopterin synthesis, did not augment A-23187 relaxations. However, pretreatment with superoxide dismutase and catalase significantly enhanced A-23187 relaxations in shunt PAs. We conclude that increased pulmonary blood flow induces an impairment of endothelium-dependent relaxation that is selective to PAs. The impaired relaxation may be mediated in part by excess superoxide production.     

Chronic hypoxia attenuates cGMP-dependent pulmonary vasodilation

Chronic hypoxia (CH) augments endothelium-derived nitric oxide (NO)-dependent pulmonary vasodilation; however, responses to exogenous NO are reduced following CH in female rats. We hypothesized that CH-induced attenuation of NO-dependent pulmonary vasodilation is mediated by downregulation of vascular smooth muscle (VSM) soluble guanylyl cyclase (sGC) expression and/or activity, increased cGMP degradation by phosphodiesterase type 5 (PDE5), or decreased VSM sensitivity to cGMP. Experiments demonstrated attenuated vasodilatory responsiveness to the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate and to arterial boluses of dissolved NO solutions in isolated, saline-perfused lungs from CH vs. normoxic female rats. In additional experiments, the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked vasodilation to NO donors in lungs from each group. However, CH was not associated with decreased pulmonary sGC expression or activity as assessed by Western blotting and cGMP radioimmunoassay, respectively. Consistent with our hypothesis, the selective PDE5 inhibitors dipyridamole and T-1032 augmented NO-dependent reactivity in lungs from CH rats, while having little effect in lungs from normoxic rats. However, the attenuated vasodilatory response to NO in CH lungs persisted after PDE5 inhibition. Furthermore, CH similarly inhibited vasodilatory responses to 8-bromoguanosine 3'5'-cyclic monophosphate. We conclude that attenuated NO-dependent pulmonary vasodilation after CH is not likely mediated by decreased sGC expression, but rather by increased cGMP degradation by PDE5 and decreased pulmonary VSM reactivity to cGMP.     

Expression of VEGF and its receptors Flt-1 and Flk-1/KDR is altered in lambs with increased pulmonary blood flow and pulmonary hypertension

Utilizing in utero aortopulmonary vascular graft placement, we developed a lamb model of congenital heart disease and increased pulmonary blood flow. We showed previously that these lambs have increased pulmonary vessel number at 4 wk of age. To determine whether this was associated with alterations in VEGF signaling, we investigated vascular changes in expression of VEGF and its receptors, Flt-1 and KDR/Flk-1, in the lungs of shunted and age-matched control lambs during the first 8 wk of life. Western blot analysis demonstrated that VEGF, Flt-1, and KDR/Flk-1 expression was higher in shunted lambs. VEGF and Flt-1 expression was increased at 4 and 8 wk of age (P <0.05). However, KDR/Flk-1 expression was higher in shunted lambs only at 1 and 4 wk of age (P <0.05). Immunohistochemical analysis demonstrated that, in control and shunted lambs, VEGF localized to the smooth muscle layer of vessels and airways and to the pulmonary epithelium while increased VEGF expression was localized to the smooth muscle layer of thickened media in remodeled vessels in shunted lambs. VEGF receptors were localized exclusively in the endothelium of pulmonary vessels. Flt-1 was increased in the endothelium of small pulmonary arteries in shunted animals at 4 and 8 wk of age, whereas KDR/Flk-1 was increased in small pulmonary arteries at 1 and 4 wk of age. Our data suggest that increased pulmonary blood flow upregulates expression of VEGF and its receptors, and this may be important in development of the vascular remodeling in shunted lambs.     

Hydrocortisone normalizes oxygenation and cGMP regulation in lambs with persistent pulmonary hypertension of the newborn

In the pulmonary vasculature, cGMP levels are regulated by soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). We previously reported that lambs with persistent pulmonary hypertension of the newborn (PPHN) demonstrate increased reactive oxygen species (ROS) and altered sGC and PDE5 activity, with resultant decreased cGMP. The objective of this study was to evaluate the effects of hydrocortisone on pulmonary vascular function, ROS, and cGMP in the ovine ductal ligation model of PPHN. PPHN lambs were ventilated with 100% O(2) for 24 h. Six lambs received 5 mg/kg hydrocortisone every 8 h times three doses (PPHN-hiHC), five lambs received 3 mg/kg hydrocortisone followed by 1 mg·kg(-1)·dose(-1) times two doses (PPHN-loHC), and six lambs were ventilated with O(2) alone (PPHN). All groups were compared with healthy 1-day spontaneously breathing lambs (1DSB). O(2) ventilation of PPHN lambs decreased sGC activity, increased PDE5 activity, and increased ROS vs. 1DSB lambs. Both hydrocortisone doses significantly improved arterial-to-alveolar ratios relative to PPHN lambs, decreased PDE5 activity, and increased cGMP relative to PPHN lambs. High-dose hydrocortisone also increased sGC activity, decreased PDE5 expression, decreased ROS, and increased total vascular SOD activity vs. PPHN lambs. These data suggest that hydrocortisone treatment in clinically relevant doses improves oxygenation and decreases hyperoxia-induced changes in sGC and PDE5 activity, increasing cGMP levels. Hydrocortisone reduces ROS levels in part by increasing SOD activity in PPHN lambs ventilated with 100% O(2.) We speculate that hydrocortisone increases cGMP by direct effects on sGC and PDE5 expression and by attenuating abnormalities induced by oxidant stress.     

Alterations in TGF-beta1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension

The mechanisms responsible for pulmonary vascular remodeling in congenital heart disease with increased pulmonary blood flow remain unclear. We developed a lamb model of congenital heart disease and increased pulmonary blood flow utilizing an in utero placed aortopulmonary vascular graft (shunted lambs). Morphometric analysis of barium-injected pulmonary arteries indicated that by 4 wk of age, shunts had twice the pulmonary arterial density of controls (P < 0.05), and their pulmonary vessels showed increased muscularization and medial thickness at both 4 and 8 wk of age (P < 0.05). To determine the potential role of TGF-beta1 in this vascular remodeling, we investigated vascular changes in expression and localization of TGF-beta1 and its receptors TbetaRI, ALK-1, and TbetaRII in lungs of shunted and control lambs at 1 day and 1, 4, and 8 wk of life. Western blots demonstrated that TGF-beta1 and ALK-1 expression was elevated in shunts compared with control at 1 and 4 wk of age (P < 0.05). In contrast, the antiangiogenic signaling receptor TbetaRI was decreased at 4 wk of age (P < 0.05). Immunohistochemistry demonstrated shunts had increased TGF-beta1 and TbetaRI expression in smooth muscle layer and increased TGF-beta1 and ALK-1 in endothelium of small pulmonary arteries at 1 and 4 wk of age. Moreover, TbetaRI expression was significantly reduced in endothelium of pulmonary arteries in the shunt at 1 and 4 wk. Our data suggest that increased pulmonary blood flow dysregulates TGF-beta1 signaling, producing imbalance between pro- and antiangiogenic signaling that may be important in vascular remodeling in shunted lambs.     

Inhibition of phosphodiesterase 1 augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs with acute pulmonary hypertension

Phosphodiesterase 1 (PDE1) modulates vascular tone and the development of tolerance to nitric oxide (NO)-releasing drugs in the systemic circulation. Any role of PDE1 in the pulmonary circulation remains largely uncertain. We measured the expression of genes encoding PDE1 isozymes in the pulmonary vasculature and examined whether or not selective inhibition of PDE1 by vinpocetine attenuates pulmonary hypertension and augments the pulmonary vasodilator response to inhaled NO in lambs. Using RT-PCR, we detected PDE1A, PDE1B, and PDE1C mRNAs in pulmonary arteries and veins isolated from healthy lambs. In 13 lambs, the thromboxane A(2) analog U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mmHg. Four animals received an intravenous infusion of vinpocetine at incremental doses of 0.3, 1, and 3 mg.kg(-1).h(-1). In nine lambs, inhaled NO was administered in a random order at 2, 5, 10, and 20 ppm before and after an intravenous infusion of 1 mg.kg(-1).h(-1) vinpocetine. Administration of vinpocetine did not alter pulmonary and systemic hemodynamics or transpulmonary cGMP or cAMP release. Inhaled NO selectively reduced mean pulmonary arterial pressure, pulmonary capillary pressure, and pulmonary vascular resistance index, while increasing transpulmonary cGMP release. The addition of vinpocetine enhanced pulmonary vasodilation and transpulmonary cGMP release induced by NO breathing without causing systemic vasodilation but did not prolong the duration of pulmonary vasodilation after NO inhalation was discontinued. Our findings demonstrate that selective inhibition of PDE1 augments the therapeutic efficacy of inhaled NO in an ovine model of acute chemically induced pulmonary hypertension.     

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.     

Alterations in ET-1, not nitric oxide,in 1-week-old lambs with increased pulmonary blood flow

Altered pulmonary vascular reactivity is a source of morbidity and mortality for children with congenital heart disease and increased pulmonary blood flow. Nitric oxide (NO) and endothelin (ET)-1 are important mediators of pulmonary vascular reactivity. We hypothesize that early alterations in endothelial function contribute to the altered vascular reactivity associated with congenital heart disease. The objective of this study was to characterize endothelial function in our lamb model of increased pulmonary blood flow at 1 wk of life. Eleven fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 7 days after delivery. The pulmonary vasodilator response to both intravenous ACh (endothelium dependent) and inhaled NO (endothelium independent) was similar in shunted and control lambs. In addition, tissue NO(x), NO synthase (NOS) activity, and endothelial NOS protein levels were similar. Conversely, the vasodilator response to both ET-1 and 4Ala-ET-1 (an ET(B) receptor agonist) were attenuated in shunted lambs, and tissue ET-1 concentrations were increased (P < 0.05). Associated with these changes were an increase in ET-converting enzyme-1 protein and a decrease in ET(B) receptor protein levels (P < 0.05). These data demonstrate that increased pulmonary blood flow induces alterations in ET-1 signaling before NO signaling and suggest an early role for ET-1 in the altered vascular reactivity associated with increased pulmonary blood flow.     

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.     

Increased pulmonary blood flow does not alter surfactant protein gene expression in lambs within the first week of life

Neonates and infants with congenital heart disease with increased pulmonary blood flow suffer morbidity from poor oxygenation and decreased lung compliance. In a previous experiment involving 4-wk-old lambs with pulmonary hypertension secondary to increased pulmonary blood flow following an in utero placement of an aortopulmonary vascular graft, we found a decrease in surfactant protein (SP)-A gene expression as well as a decrease in SP-A and SP-B protein contents. To determine the timing of these changes, the objective of the present study was to characterize the effect of increased pulmonary blood flow and pulmonary hypertension on SP-A, -B, and -C gene expressions and protein contents within the first week of life. Of eight fetal lambs that underwent the in utero placement of the shunt, there was no difference in the expression of SP-A, -B, and -C mRNA levels or SP-A and -B protein contents compared with age-matched controls. The results showed that, in this model of congenital heart disease with pulmonary hypertension and increased pulmonary blood flow, the effect of the shunt on SP gene expression and protein content was not apparent within the first week of life.     

In vivo attenuation of endothelium-dependent pulmonary vasodilation by methylene blue.

In vitro evidence suggests that resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation are mediated by changes in vascular smooth muscle concentrations of guanosine 3',5'-cyclic monophosphate (cGMP). We investigated this hypothesis in vivo in 19 mechanically ventilated intact lambs by determining the hemodynamic effects of methylene blue (a guanylate cyclase inhibitor) and then by comparing the hemodynamic response to five vasodilators during pulmonary hypertension induced by the infusion of U-46619 (a thromboxane A2 mimic) or methylene blue. Methylene blue caused a significant time-dependent increase in pulmonary arterial pressure. During U-46619 infusions, acetylcholine, ATP-MgCl2, sodium nitroprusside, isoproterenol, and 8-bromo-cGMP decreased pulmonary arterial pressure. During methylene blue infusions, the decreases in pulmonary arterial pressure caused by acetylcholine and ATP-MgCl2 (endothelium-dependent vasodilators) and sodium nitroprusside (an endothelium-independent guanylate cyclase-dependent vasodilator) were attenuated by greater than 50%. The decreases in pulmonary arterial pressure caused by isoproterenol and 8-bromo-cGMP (endothelium-independent vasodilators) were unchanged. This study in intact lambs supports the in vitro evidence that changes in vascular smooth muscle cell concentrations of cGMP in part mediate resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation.     

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.     

Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension

Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P < 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P < 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow.     

Chronic endothelin A receptor blockade in lambs with increased pulmonary blood flow and pressure

Endothelin receptor blockade is an emerging therapy for pulmonary hypertension. However, hemodynamic and structural effects and potential changes in endogenous nitric oxide (NO)-cGMP and endothelin-1 signaling of chronic endothelin A receptor blockade in pulmonary hypertension secondary to congenital heart disease are unknown. Therefore, the objectives of this study were to determine hemodynamic and structural effects and potential changes in endogenous NO-cGMP and endothelin-1 signaling of chronic endothelin A receptor blockade in a lamb model of increased pulmonary blood flow following in utero placement of an aortopulmonary shunt. Immediately after spontaneous birth, shunt lambs were treated lifelong with either an endothelin A receptor antagonist (PD-156707) or placebo. At 4 wk of age, PD-156707-treated shunt lambs (n = 6) had lower pulmonary vascular resistance and right atrial pressure than placebo-treated shunt lambs (n = 8, P < 0.05). Smooth muscle thickness or arterial number per unit area was not different between the two groups. However, the number of alveolar profiles per unit area was increased in the PD-156707-treated shunt lambs (190.7 +/- 5.6 vs. 132.9 +/- 10.0, P < 0.05). Plasma endothelin-1 and cGMP levels and lung NOS activity, cGMP, eNOS, preproendothelin-1, endothelin-converting enzyme-1, endothelin A, and endothelin B receptor protein levels were similar in both groups. We conclude that chronic endothelin A receptor blockade attenuates the progression of pulmonary hypertension and augments alveolar growth in lambs with increased pulmonary blood flow.     

Decreased surfactant proteins in lambs with pulmonary hypertension secondary to increased blood flow

Infants with increased pulmonary blood flow secondary to congenital heart disease suffer from tachypnea, dyspnea, and recurrent pulmonary infections. We have recently established a model of pulmonary hypertension secondary to increased pulmonary blood flow in lambs after in utero placement of an aortopulmonary vascular graft. The purpose of the present study was to utilize our animal model to determine the effects on the expression of surfactant proteins A (SP-A), B (SP-B), and C (SP-C). At age 4 wk, SP-A mRNA content in lambs decreased to 61.4 +/- 8% of age-matched control value (n = 5; P < 0.05). In addition, SP-A protein content was decreased to 50 +/- 12% of control value (n = 6; P < 0.0001). Although we did not observe statistically significant changes in SP-B mRNA content, SP-B protein was decreased to 74 +/- 25% of control value (n = 4; P < 0.02). There was no difference in SP-C mRNA. These data show that in a model of congenital heart disease with pulmonary hypertension secondary to increased pulmonary blood flow, there is a decrease in SP-A gene expression as well as a decrease in SP-A and SP-B protein contents.     

Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice

High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V(T). Ventilation with 20 ml/kg V(T) for 80 min increased the filtration coefficient (K(f)), an index of vascular permeability. The increased cGMP and K(f) caused by HV(T) were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 muM ODQ) also blocked cGMP generation and inhibited the increase in K(f), suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wet-to-dry weight ratio in an anesthetized mouse model of HV(T) injury. Additional activation of sGC (1.5 muM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K(f) in lungs ventilated with 15 ml/kg V(T). HV(T) endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 muM IBMX) as well as an inhibitor (10 muM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V(T)-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV(T)-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.     

Endothelial alterations during inhaled NO in lambs with pulmonary hypertension: implications for rebound hypertension

Clinically significant increases in pulmonary vascular resistance (PVR) have been noted upon acute withdrawal of inhaled nitric oxide (iNO). Previous studies in the normal pulmonary circulation demonstrate that iNO increases endothelin-1 (ET-1) levels and decreases endogenous nitric oxide synthase (NOS) activity, implicating an endothelial etiology for the increase in resistance upon iNO withdrawal. However, the effect of iNO on endogenous endothelial function in the clinically relevant pulmonary hypertensive circulation is unknown. The objective of this study was to determine the effects of iNO on endogenous NO-cGMP and ET-1 signaling in lambs with preexisting pulmonary hypertension secondary to increased pulmonary blood flow. Eight fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt lambs). After delivery (4 wk), the shunt lambs were mechanically ventilated with iNO (40 ppm) for 24 h. After 24 h of inhaled NO, plasma ET-1 levels increased by 34.8% independently of changes in protein levels (P < 0.05). Contrary to findings in normal lambs, total NOS activity did not decrease during iNO. In fact, Western blot analysis demonstrated that tissue endothelial NOS protein levels decreased by 43% such that NOS activity relative to protein levels actually increased during iNO (P < 0.05). In addition, the beta-subunit of soluble guanylate cyclase decreased by 70%, whereas phosphodiesterase 5 levels were unchanged (P < 0.05). Withdrawal of iNO was associated with an acute increase in PVR, which exceeded baseline PVR by 45%, and a decrease in cGMP concentrations to levels that were below baseline. These data suggest that the endothelial response to iNO and the potential mechanisms of rebound pulmonary hypertension are dependent upon the underlying pulmonary vasculature.     

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.     

The role of nitric oxide synthase-derived reactive oxygen species in the altered relaxation of pulmonary arteries from lambs with increased pulmonary blood flow

Congenital cardiac defects associated with increased pulmonary blood flow (Q(p)) produce pulmonary hypertension. We have previously reported attenuated endothelium-dependent relaxations in pulmonary arteries (PA) isolated from lambs with increased Q(p) and pulmonary hypertension. To better characterize the vascular alterations in the nitric oxide-superoxide system, 12 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). Twin lambs served as controls. PA were isolated from these lambs at 4-6 wk of age. Electron paramagnetic resonance spectroscopy on fourth-generation PA showed significantly increased superoxide anion generation in shunt PA that were decreased to control levels following inhibition of nitric oxide synthase (NOS) with 2-ethyl-2-thiopseudourea. Preconstricted fifth-generation PA rings were relaxed with a NOS agonist (A-23187), a nitric oxide donor [S-nitrosyl amino penicillamine (SNAP)], polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), or H(2)O(2). A-23187-, PEG-SOD-, and H(2)O(2)-mediated relaxations were impaired in shunt PA compared with controls. Pretreatment with PEG-SOD significantly enhanced the relaxation response to A-23187 and SNAP in shunt but not control PA. Inhibition of NOS with nitro-L-arginine or scavenging superoxide anions with tiron enhanced relaxation to SNAP and inhibited relaxation to PEG-SOD in shunt PA. Pretreatment with catalase inhibited relaxation of shunt PA to A-23187, SOD, and H(2)O(2). We conclude that NOS catalyzes the production of superoxide anions in shunt PA. PEG-SOD relaxes shunt PA by converting these anions to H(2)O(2), a pulmonary vasodilator. The redox environment, influenced by the balance between production and scavenging of ROS, may have important consequences on pulmonary vascular reactivity in the setting of increased Q(p).     

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.