Correction: Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

[This corrects the article DOI: 10.1371/journal.pbio.2005924.].

Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

The heart exhibits the highest basal oxygen (O2) consumption per tissue mass of any organ in the body and is uniquely dependent on aerobic metabolism to sustain contractile function. During acute hypoxic states, the body responds with a compensatory increase in cardiac output that further increases myocardial O2 demand, predisposing the heart to ischemic stress and myocardial dysfunction. Here, we test the utility of a novel engineered protein derived from the heme-based nitric oxide (NO)/oxygen (H-NOX) family of bacterial proteins as an O2 delivery biotherapeutic (Omniox-cardiovascular [OMX-CV]) for the hypoxic myocardium. Because of their unique binding characteristics, H-NOX-based variants effectively deliver O2 to hypoxic tissues, but not those at physiologic O2 tension. Additionally, H-NOX-based variants exhibit tunable binding that is specific for O2 with subphysiologic reactivity towards NO, circumventing a significant toxicity exhibited by hemoglobin (Hb)-based O2 carriers (HBOCs). Juvenile lambs were sedated, mechanically ventilated, and instrumented to measure cardiovascular parameters. Biventricular admittance catheters were inserted to perform pressure-volume (PV) analyses. Systemic hypoxia was induced by ventilation with 10% O2. Following 15 minutes of hypoxia, the lambs were treated with OMX-CV (200 mg/kg IV) or vehicle. Acute hypoxia induced significant increases in heart rate (HR), pulmonary blood flow (PBF), and pulmonary vascular resistance (PVR) (p < 0.05). At 1 hour, vehicle-treated lambs exhibited severe hypoxia and a significant decrease in biventricular contractile function. However, in OMX-CV-treated animals, myocardial oxygenation was improved without negatively impacting systemic or PVR, and both right ventricle (RV) and left ventricle (LV) contractile function were maintained at pre-hypoxic baseline levels. These data suggest that OMX-CV is a promising and safe O2 delivery biotherapeutic for the preservation of myocardial contractility in the setting of acute hypoxia.

Disrupted NOS signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow.

Associated abnormalities of the lymphatic circulation are well described in congenital heart disease. However, their mechanisms remain poorly elucidated. Using a clinically relevant ovine model of a congenital cardiac defect with chronically increased pulmonary blood flow (shunt), we previously demonstrated that exposure to chronically elevated pulmonary lymph flow is associated with: 1) decreased bioavailable nitric oxide (NO) in pulmonary lymph; and 2) attenuated endothelium-dependent relaxation of thoracic duct rings, suggesting disrupted lymphatic endothelial NO signaling in shunt lambs. To further elucidate the mechanisms responsible for this altered NO signaling, primary lymphatic endothelial cells (LECs) were isolated from the efferent lymphatic of the caudal mediastinal node in 4-wk-old control and shunt lambs. We found that shunt LECs (n = 3) had decreased bioavailable NO and decreased endothelial nitric oxide synthase (eNOS) mRNA and protein expression compared with control LECs (n = 3). eNOS activity was also low in shunt LECs, but, interestingly, inducible nitric oxide synthase (iNOS) expression and activity were increased in shunt LECs, as were total cellular nitration, including eNOS-specific nitration, and accumulation of reactive oxygen species (ROS). Pharmacological inhibition of iNOS reduced ROS in shunt LECs to levels measured in control LECs. These data support the conclusion that NOS signaling is disrupted in the lymphatic endothelium of lambs exposed to chronically increased pulmonary blood and lymph flow and may contribute to decreased pulmonary lymphatic bioavailable NO.

Right ventricular nitric oxide signaling in an ovine model of congenital heart disease: a preserved fetal phenotype.

We recently reported superior right ventricle (RV) performance in response to acute afterload challenge in lambs with a model of congenital heart disease with chronic left-to-right cardiac shunts. Compared with control animals, shunt lambs demonstrated increased contractility because of an enhanced Anrep effect (the slow increase in contractility following myocyte stretch). This advantageous physiological response may reflect preservation of a fetal phenotype, since the RV of shunt lambs remains exposed to increased pressure postnatally. Nitric oxide (NO) production by NO synthase (NOS) is activated by myocyte stretch and is a necessary intermediary of the Anrep response. The purpose of this study was to test the hypothesis that NO signaling is increased in the RV of fetal lambs compared with controls and shunt lambs have persistence of this fetal pattern. An 8-mm graft was placed between the pulmonary artery and aorta in fetal lambs (shunt). NOS isoform expression, activity, and association with activating cofactors were determined in fetal tissue obtained during late-gestation and in 4-wk-old juvenile shunt and control lambs. We demonstrated increased RNA and protein expression of NOS isoforms and increased total NOS activity in the RV of both shunt and fetal lambs compared with control. We also found increased NOS activation and association with cofactors in shunt and fetal RV compared with control. These data demonstrate preserved fetal NOS phenotype and NO signaling in shunt RV, which may partially explain the mechanism underlying the adaptive response to increased afterload seen in the RV of shunt lambs.

Adaptive right ventricular performance in response to acutely increased afterload in a lamb model of congenital heart disease: evidence for enhanced Anrep effect.

Patients with pulmonary hypertension associated with congenital heart disease survive longer with preserved right ventricular (RV) function compared with those with primary pulmonary hypertension. The purpose of this study was to test the hypothesis that superior RV performance can be demonstrated, at baseline and when challenged with increased RV afterload, in lambs with chronic left-to-right cardiac shunts compared with control lambs. A shunt was placed between the pulmonary artery and the aorta in fetal lambs (shunt). RV pressure-volume loops were obtained 4 wk after delivery in shunt and control lambs, before and after increased afterload was applied using pulmonary artery banding (PAB). Baseline stroke volume (8.7 ± 1.8 vs. 15.8 ± 2.7 ml, P = 0.04) and cardiac index (73.0 ± 4.0 vs. 159.2 ± 25.1 ml·min(-1)·kg(-1), P = 0.02) were greater in shunts. After PAB, there was no difference in the change in cardiac index (relative to baseline) between groups; however, heart rate (HR) was greater in controls (168 ± 7.3 vs. 138 ± 6.6 beats/min, P = 0.01), and end-systolic elastance (Ees) was greater in shunts (2.63 vs. 1.31 × baseline, P = 0.02). Control lambs showed decreased mechanical efficiency (71% baseline) compared with shunts. With acute afterload challenge, both controls and shunts maintained cardiac output; however, this was via maladaptive responses in controls, while shunts maintained mechanical efficiency and increased contractility via a proposed enhanced Anrep effect-the second, slow inotropic response in the biphasic ventricular response to increased afterload, a novel finding in the RV. The mechanisms related to these physiological differences may have important therapeutic implications.

Altered reactivity and nitric oxide signaling in the isolated thoracic duct from an ovine model of congenital heart disease with increased pulmonary blood flow.

We have previously shown decreased pulmonary lymph flow in our lamb model of chronically increased pulmonary blood flow, created by the in utero placement of an 8-mm aortopulmonary shunt. The purpose of this study was to test the hypothesis that abnormal lymphatic function in shunt lambs is due to impaired lymphatic endothelial nitric oxide (NO)-cGMP signaling resulting in increased lymphatic vascular constriction and/or impaired relaxation. Thoracic duct rings were isolated from 4-wk-old shunt (n = 7) and normal (n = 7) lambs to determine length-tension properties, vascular reactivity, and endothelial NO synthase protein. At baseline, shunt thoracic duct rings had 2.6-fold higher peak to peak tension and a 2-fold increase in the strength of contractions compared with normal rings (P < 0.05). In response to norepinephrine, shunt thoracic duct rings had a 2.4-fold increase in vascular tone compared with normal rings (P < 0.05) and impaired relaxation in response to the endothelium-dependent dilator acetylcholine (63% vs. 13%, P < 0.05). In vivo, inhaled NO (40 ppm) increased pulmonary lymph flow (normalized for resistance) ∼1.5-fold in both normal and shunt lambs (P < 0.05). Inhaled NO exposure increased bioavailable NO [nitrite/nitrate (NOx); ∼2.5-fold in normal lambs and ∼3.4-fold in shunt lambs] and cGMP (∼2.5-fold in both) in the pulmonary lymph effluent (P < 0.05). Chronic exposure to increased pulmonary blood flow is associated with pulmonary lymphatic endothelial injury that disrupts NO-cGMP signaling, leading to increased resting vasoconstriction, increased maximal strength of contraction, and impaired endothelium-dependent relaxation. Inhaled NO increases pulmonary lymph NOx and cGMP levels and pulmonary lymph flow in normal and shunt lambs. Therapies that augment NO-cGMP signaling within the lymphatic system may provide benefits, warranting further study.

Altered lymphatics in an ovine model of congenital heart disease with increased pulmonary blood flow.

Abnormalities of the lymphatic circulation are well recognized in patients with congenital heart defects. However, it is not known how the associated abnormal blood flow patterns, such as increased pulmonary blood flow (PBF), might affect pulmonary lymphatic function and structure. Using well-established ovine models of acute and chronic increases in PBF, we cannulated the efferent lymphatic duct of the caudal mediastinal node and collected and analyzed lymph effluent from the lungs of lambs with acutely increased PBF (n = 6), chronically increased PBF (n = 6), and age-matched normal lambs (n = 8). When normalized to PBF, we found that lymph flow was unchanged following acute increases in PBF but decreased following chronic increases in PBF. The lymph:plasma protein ratio decreased with both acute and chronic increases in PBF. Lymph bioavailable nitric oxide increased following acute increases in PBF but decreased following chronic increases in PBF. In addition, we found perturbations in the transit kinetics of contrast material through the pleural lymphatics of lambs with chronic increases in PBF. Finally, there were structural changes in the pulmonary lymphatic system in lambs with chronic increases in PBF: lymphatics from these lambs were larger and more dilated, and there were alterations in the expression of vascular endothelial growth factor-C, lymphatic vessel endothelial hyaluronan receptor-1, and Angiopoietin-2, proteins known to be important for lymphatic growth, development, and remodeling. Taken together these data suggest that chronic increases in PBF lead to both functional and structural aberrations of lung lymphatics. These findings have important therapeutic implications that warrant further study.

Nitric oxide alterations following acute ductal constriction in the fetal lamb: a role for superoxide.

Acute partial compression of the fetal ductus arteriosus (DA) results in an initial abrupt increase in pulmonary blood flow (PBF), which is followed by a significant reduction in PBF to baseline values over the ensuing 2-4 h. We have previously demonstrated that this potent vasoconstricting response is due, in part, to an endothelin-1 (ET-1)-mediated decrease in nitric oxide synthase (NOS) activity. In addition, in vitro data demonstrate that ET-1 increases superoxide levels in pulmonary arterial smooth muscle cells and that oxidative stress alters NOS activity. Therefore, the objectives of this study were to determine the potential role of superoxide in the alterations of hemodynamics and NOS activity following acute ductal constriction in the late-gestation fetal lamb. Eighteen anesthetized near-term fetal lambs were instrumented, and a lung biopsy was performed. After a 48-h recovery, acute constriction of the DA was performed by inflating a vascular occluder. Polyethylene glycol-superoxide dismutase (PEG-SOD; 1,000-1,500 units/kg, n = 7) or PEG-alone (vehicle control group, n = 5) was injected into the pulmonary artery before ductal constriction. Six animals had a sham operation. In PEG-alone-treated lambs, acute ductal constriction rapidly decreased pulmonary vascular resistance (PVR) by 88%. However, by 4 h, PVR returned to preconstriction baseline. This vasoconstriction was associated with an increase in lung superoxide levels (82%), a decrease in total NOS activity (50%), and an increase in P-eNOS-Thr495 (52%) (P < 0.05). PEG-SOD prevented the increase of superoxide after ductal constriction, attenuated the vasoconstriction, preserved NOS activity, and increased P-eNOS Ser1177 (307%, P < 0.05). Sham procedure induced no changes. These data suggest that an acute decrease in NOS activity that is mediated, in part, by increased superoxide levels, and alterations in the phosphorylation status of the endothelial NOS isoform, underlie the pulmonary vascular response to acute ductal constriction.

Alterations in cGMP, soluble guanylate cyclase, phosphodiesterase 5, and B-type natriuretic peptide induced by chronic increased pulmonary blood flow in lambs.

OBJECTIVE: The objective of the study was to determine alterations in cGMP, soluble guanylate cyclase (sGC), phosphodiesterase type 5 (PDE5), and B-type natriuretic peptide (BNP), in an animal model of a congenital cardiac defect with increased pulmonary blood flow. DESIGN: Prospective, comparative, experimental study. SUBJECTS: Lambs, from birth until 8 weeks of age. METHODOLOGY: Late gestation fetal lambs underwent in utero placement of an 8 mm aortopulmonary vascular graft (shunt). In shunted and normal age-matched control lambs, at 2, 4, and 8 weeks of age, cGMP and BNP levels were measured, and sGC subunit and PDE5 protein expression were determined by Western blot analysis and immunohistochemistry. RESULTS: In shunted lambs, tissue and plasma cGMP levels were greater than normal throughout the 8-week study period (P < 0.05). sGCalpha protein was greater at 2 and 4 weeks (P < 0.05), and sGCbeta and PDE5 protein were greater at 4 weeks in shunted lambs (P < 0.05). Plasma BNP levels did not change in normal lambs but increased in shunted lambs by 8 weeks of age (P < 0.05). BNP levels were greater in shunted lambs than normal at 4 and 8 weeks (P < 0.05). CONCLUSIONS: Alterations in sGC subunit protein expression during the first post-natal month, and increased BNP levels during the second post-natal month contribute to elevations in plasma and lung tissue cGMP in lambs with increased pulmonary blood flow.

Emergence of smooth muscle cell endothelin B-mediated vasoconstriction in lambs with experimental congenital heart disease and increased pulmonary blood flow.

BACKGROUND: Endothelin-1 (ET-1) has been implicated in the pathophysiology of pulmonary hypertension. In 1-month-old lambs with increased pulmonary blood flow, we have demonstrated early alterations in the ET-1 cascade. The objective of this study was to investigate the role of potential later alterations of the ET cascade in the pathophysiology of pulmonary hypertension secondary to increased pulmonary blood flow. METHODS AND RESULTS: Eighteen fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 8 weeks after spontaneous delivery. Compared with age-matched control lambs, lung tissue ET-1 levels were increased in shunt lambs (317.2+/-113.8 versus 209.8+/-61.8 pg/g, P<0.05). In shunt lambs (n=9), exogenous ET-1 induced potent pulmonary vasoconstriction, which was blocked by the ETA receptor antagonist PD 156707 (n=3). This pulmonary vasoconstriction was mimicked by exogenous Ala1,3,11,15 ET-1 (4 Ala ET-1), the ETB receptor agonist, and was blocked by the ETB receptor antagonist BQ 788 (n=3). However, in control lambs (n=7), ET-1 and 4 Ala ET-1 did not change pulmonary vascular tone. In contrast to 4-week-old shunt lambs, immunohistochemistry revealed the emergence of ETB receptors on smooth muscle cells in the vasculature of 8-week-old shunt lambs. CONCLUSIONS: Over time, increased pulmonary blood flow and/or pressure results in the emergence of ETB-mediated vasoconstriction, which coincides with the emergence of ETB receptors on smooth muscle cells. These data suggest an important role for ETB receptors in the pathophysiology of pulmonary hypertension in this animal model of increased pulmonary blood flow.

Inhaled nitric oxide decreases pulmonary soluble guanylate cyclase protein levels in 1-month-old lambs.

BACKGROUND: Inhaled nitric oxide produces potent pulmonary vasodilation by activating soluble guanylate cyclase and increasing smooth muscle cell concentrations of cyclic guanosine monophosphate. However, responses are often nonsustained, and clinically significant increases in pulmonary vascular resistance have been noted on its acute withdrawal. In vitro and in vivo data suggest that inhaled nitric oxide decreases endogenous nitric oxide synthase activity. The effects of inhaled nitric oxide on the downstream mediators of the nitric oxide/cyclic guanosine monophosphate cascade, soluble guanylate cyclase and phosphodiesterase 5, have not been investigated. We sought to determine the effects of inhaled nitric oxide on endogenous cyclic guanosine monophosphate levels, soluble guanylate cyclase, and phosphodiesterase 5 protein levels in the intact lamb. METHODS: Eleven 1-month-old lambs were mechanically ventilated. In 7 lambs, inhaled nitric oxide (40 ppm) was administered for 24 hours and then acutely withdrawn. Intermittent lung biopsy samples were obtained for cyclic guanosine monophosphate concentrations and soluble guanylate cyclase and phosphodiesterase 5 protein levels (Western blot analysis). RESULTS: Initiation of nitric oxide decreased left pulmonary vascular resistance by 26.2%, and withdrawal rapidly increased pulmonary vascular resistance by 77.8% (P <.05). Tissue cyclic guanosine monophosphate concentrations initially increased during nitric oxide therapy but were not maintained during the 24-hour exposure. In addition, cyclic guanosine monophosphate concentrations rapidly decreased after nitric oxide withdrawal (P <.05). The alpha soluble guanylate cyclase (-45.7%) and beta soluble guanylate cyclase (-48.4%) protein levels decreased during nitric oxide therapy (P <.05), whereas phosphodiesterase 5 proteins levels were unchanged. CONCLUSIONS: These data suggest a role for decreased soluble guanylate cyclase and its resulting decrease in cyclic guanosine monophosphate concentrations in the nonsustained response to nitric oxide and the rebound pulmonary hypertension noted on its acute withdrawal. Phosphodiesterase 5 inhibitors may be a useful adjunct therapy during inhaled nitric oxide to preserve cyclic guanosine monophosphate levels and thereby preserve nitric oxide responsiveness and prevent rebound pulmonary hypertension.

Tezosentan, a combined parenteral endothelin receptor antagonist, produces pulmonary vasodilation in lambs with acute and chronic pulmonary hypertension.

OBJECTIVE: To investigate the hemodynamic effects of tezosentan in the intact lamb both at rest and during acute and chronic pulmonary hypertension. DESIGN: Prospective, randomized experimental study. SETTING: University-based research laboratory. SUBJECTS: Lambs with and without pulmonary hypertension. INTERVENTIONS: Six newborn lambs were instrumented to measure vascular pressures and left pulmonary blood flow. The hemodynamic effects of tezosentan (0.5, 1.0, 5.0 mg/kg, intravenously) were studied at rest and during U46619-induced pulmonary hypertension. Following in utero placement of an aortopulmonary vascular graft, nine additional lambs with increased pulmonary blood flow and chronic pulmonary hypertension (shunt) were also studied at 1 wk (n = 5) and 8 wks (n = 4) of age. MEASUREMENTS AND MAIN RESULTS: At rest, tezosentan had no significant effect on any of the variables. During acute U46619-induced pulmonary hypertension, tezosentan caused a dose-dependent decrease in pulmonary arterial pressure (from 5.9% +/- 4.7 to 16.0% +/- 10.7; p < .05) and pulmonary vascular resistance (from 6.2% +/- 8.0 to 21% +/- 8.8; p < .05). Mean systemic arterial pressure was unchanged. In 1- and 8-wk-old shunt lambs with increased pulmonary blood flow, tezosentan (1 mg/kg) produced potent nonselective pulmonary vasodilation. CONCLUSIONS: Tezosentan, a combined endothelin receptor antagonist optimized for parenteral use, induces potent selective pulmonary vasodilation during acute U46619-induced pulmonary hypertension and potent nonselective vasodilation in chronic pulmonary hypertension secondary to increased pulmonary blood flow. In general, the hemodynamic effects of bolus doses of tezosentan occurred within 60 secs of administration and lasted approximately 5-10 mins. The hemodynamic profile of intravenous tezosentan may make it a useful adjunct therapy for acute pulmonary hypertensive disorders and warrants further study.

Phosphodiesterase-3 inhibition prevents the increase in pulmonary vascular resistance following inhaled nitric oxide withdrawal in lambs.

OBJECTIVES: To determine the effects of inhaled nitric oxide on endogenous cyclic adenosine monophosphate in the intact lamb, and to determine the potential role of cyclic adenosine monophosphate in the rebound pulmonary hypertension associated with nitric oxide withdrawal. DESIGN: Prospective, placebo-controlled experimental study. SETTING: University-based basic science research laboratory. SUBJECTS: One-month-old lambs. INTERVENTIONS: Six 1-month-old control lambs, and 6 milrinone- (phosphodiesterase-3 inhibitor) treated lambs, were mechanically ventilated. Inhaled nitric oxide (40 ppm) was administered for 24 hrs and then acutely withdrawn. Sequential peripheral lung biopsies were obtained before, during, and 2 hrs after withdrawing inhaled nitric oxide therapy. MEASUREMENTS AND MAIN RESULTS: In control lambs, initiation of nitric oxide decreased left pulmonary vascular resistance by 29.6%, and withdrawal rapidly increased pulmonary vascular resistance by 77.1% (p <.05). Lung tissue cyclic adenosine monophosphate concentrations decreased by 25.3% during nitric oxide therapy (p <.05). In milrinone-treated lambs, nitric oxide decreased pulmonary vascular resistance by 26.6% (p <.05), but pulmonary vascular resistance was unchanged after acute withdrawal. Lung tissue cyclic adenosine monophosphate concentrations were preserved during nitric oxide therapy. CONCLUSIONS: Inhaled nitric oxide produces potent pulmonary vasodilation by activating soluble guanylate cyclase and increasing smooth muscle cell concentrations of guanosine-3',5'-cyclic monophosphate. However, alterations in endogenous nitric oxide/guanosine-3',5'-cyclic monophosphate during inhaled nitric oxide have been implicated in the clinically significant increases in pulmonary vascular resistance noted upon its acute withdrawal. Previous in vitro data suggest that exogenous nitric oxide/guanosine-3',5'-cyclic monophosphate can also alter cyclic adenosine monophosphate concentrations via their effect on cyclic adenosine monophosphate production and metabolism. The current in vivo study demonstrates that lung tissue cyclic adenosine monophosphate concentrations are decreased during inhaled nitric oxide and suggests a role for decreased cyclic adenosine monophosphate in the rebound pulmonary hypertension noted upon inhaled nitric oxide withdrawal. Milrinone may be a useful adjunct therapy during inhaled nitric oxide to preserve cyclic adenosine monophosphate concentrations and prevent rebound pulmonary hypertension.

Antenatal maternally-administered phosphodiesterase type 5 inhibitors normalize eNOS expression in the fetal lamb model of congenital diaphragmatic hernia.

PURPOSE: Pulmonary hypertension (pHTN), a main determinant of survival in congenital diaphragmatic hernia (CDH), results from in utero vascular remodeling. Phosphodiesterase type 5 (PDE5) inhibitors have never been used antenatally to treat pHTN. The purpose of this study is to determine if antenatal PDE5 inhibitors can prevent pHTN in the fetal lamb model of CDH. METHODS: CDH was created in pregnant ewes. Postoperatively, pregnant ewes received oral placebo or tadalafil, a PDE5 inhibitor, until delivery. Near term gestation, lambs underwent resuscitations, and lung tissue was snap frozen for protein analysis. RESULTS: Mean cGMP levels were 0.53±0.11 in placebo-treated fetal lambs and 1.73±0.21 in tadalafil-treated fetal lambs (p=0.002). Normalized expression of eNOS was 82%±12% in Normal-Placebo, 61%±5% in CDH-Placebo, 116%±6% in Normal-Tadalafil, and 86%±8% in CDH-Tadalafil lambs. Normalized expression of ß-sGC was 105%±15% in Normal-Placebo, 82%±3% in CDH-Placebo, 158%±16% in Normal-Tadalafil, and 86%±8% in CDH-Tadalafil lambs. Endothelial NOS and ß-sGC were significantly decreased in CDH (p=0.0007 and 0.01 for eNOS and ß-sGC, respectively), and tadalafil significantly increased eNOS expression (p=0.0002). CONCLUSIONS: PDE5 inhibitors can cross the placental barrier. ß-sGC and eNOS are downregulated in fetal lambs with CDH. Antenatal PDE5 inhibitors normalize eNOS and may prevent in utero vascular remodeling in CDH.

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.

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.

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.

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.

Nitric oxide-endothelin-1 interactions after acute ductal constriction in fetal lambs.

Acute partial compression of the fetal ductus arteriosus (DA) results in an initial increase in pulmonary blood flow (PBF) that is followed by acute vasoconstriction. The objective of the present study was to determine the role of nitric oxide (NO)-endothelin-1 (ET-1) interactions in the acute changes in pulmonary vascular tone after in utero partial constriction of the DA. Twelve late-gestation fetal lambs (132-140 days) were instrumented to measure vascular pressures and left PBF. After a 24-h recovery period, acute constriction of the DA was performed by partially inflating a vascular occluder, and the hemodynamic variables were observed for 4 h. In control lambs (n = 7), acute ductal constriction initially increased PBF by 627% (P < 0.05). However, this was followed by active vasoconstriction, such that PBF was restored to preconstriction values by 4 h. This was associated with a 43% decrease in total NO synthase (NOS) activity (P < 0.05) and a 106% increase in plasma ET-1 levels (P < 0.05). Western blot analysis demonstrated no changes in lung tissue endothelial NOS, preproET-1, endothelin-converting enzyme-1, or ET(B) receptor protein levels. The infusion of PD-156707 (an ET(A) receptor antagonist, n = 5) completely blocked the vasoconstriction and preserved NOS activity. These data suggest that the fetal pulmonary vasoconstriction after acute constriction of the DA is mediated by NO-ET-1 interactions. These include an increase in ET(A) receptor-mediated vasoconstriction and an ET(A) receptor-mediated decrease in NOS activity. The mechanisms of these NO-ET-1 interactions, and their role in mediating acute changes in PBF, warrant further studies.