RESUMEN

Decreased angiogenesis contributes to persistent pulmonary hypertension of the newborn (PPHN); mechanisms remain unclear. AMPK (5'AMP activated protein kinase) is a key regulator of cell metabolism. We investigated the hypothesis that a decrease in AMPK function leads to mitochondrial dysfunction and altered balance of notch ligands delta-like 4 (DLL4) and Jagged 1 (Jag1) to impair angiogenesis in PPHN. Studies were done in fetal lambs with PPHN induced by prenatal ductus arteriosus constriction and gestation-matched control lambs. PPHN lambs were treated with saline or AMPK agonist metformin. Angiogenesis was assessed in lungs with micro-computed tomography angiography and histology. AMPK function; expression of mitochondrial electron transport chain (ETC) complex proteins I-V, Dll4, and Jag1; mitochondrial number; and in vitro angiogenesis function were assessed in pulmonary artery endothelial cells (PAEC) from control and PPHN lambs. AMPK function was decreased in PPHN PAEC and lung sections. Expression of mitochondrial transcription factor, PGC-1α, ETC complex proteins I-V, and mitochondrial number were decreased in PPHN. In vitro angiogenesis of PAEC and capillary number and vessel volume fraction in the lung were decreased in PPHN. Expression of DLL4 was increased and Jag1 was decreased in PAEC from PPHN lambs. AMPK agonists A769662 and metformin increased the mitochondrial complex proteins and number, in vitro angiogenesis, and Jag1 levels and decreased DLL4 levels in PPHN PAEC. Infusion of metformin in vivo increased the vessel density in PPHN lungs. Decreased AMPK function contributes to impaired angiogenesis in PPHN by altered balance of notch ligands in PPHN.

Asunto(s)

Células Endoteliales/enzimología , Hipertensión Pulmonar/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Jagged-1/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Patológica/enzimología , Síndrome de Circulación Fetal Persistente/enzimología , Proteínas Quinasas/metabolismo , Receptores Notch/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Animales Recién Nacidos , Compuestos de Bifenilo , Conducto Arterial/embriología , Conducto Arterial/cirugía , Transporte de Electrón , Activación Enzimática , Femenino , Hipertensión Pulmonar/fisiopatología , Ligandos , Pulmón/patología , Metformina/farmacología , Metformina/uso terapéutico , Mitocondrias/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Síndrome de Circulación Fetal Persistente/tratamiento farmacológico , Síndrome de Circulación Fetal Persistente/patología , Síndrome de Circulación Fetal Persistente/fisiopatología , Fosforilación , Embarazo , Proteínas Quinasas/fisiología , Pironas/farmacología , Ovinos , Tiofenos/farmacología , Treonina/metabolismo , Transfección

RESUMEN

BACKGROUND AND PURPOSE: Dysregulation of the thromboxane A2 (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis. EXPERIMENTAL APPROACH: Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPα and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions. KEY RESULTS: The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response. CONCLUSIONS AND IMPLICATIONS: In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Síndrome de Circulación Fetal Persistente/metabolismo , Procesamiento Proteico-Postraduccional , Arteria Pulmonar/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/agonistas , Serina/metabolismo , Sustitución de Aminoácidos , Animales , Animales Recién Nacidos , Señalización del Calcio/efectos de los fármacos , Hipoxia de la Célula , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/química , Activadores de Enzimas/farmacología , Células HEK293 , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/agonistas , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Libres de Patógenos Específicos , Sus scrofa , Tromboxano A2/análogos & derivados , Tromboxano A2/metabolismo , Tromboxano A2/farmacología

RESUMEN

Nogo-B, a reticulon-4 isoform, modulates the motility and adhesion of vascular endothelial cells after binding to its receptor, Nogo-B receptor (NgBR). Nogo-B/NgBR pathway contributes to vascular remodeling and angiogenesis, but the role of this pathway in the angiogenesis of developing lungs remains unknown. We previously reported that angiogenesis function of pulmonary artery endothelial cells (PAECs) is impaired by increased reactive oxygen species formation in a fetal lamb model of intrauterine pulmonary hypertension (IPH). Here, we report that Nogo-B/NgBR pathway is altered in IPH, and that decreased NgBR expression contributes to impaired angiogenesis in IPH. We observed a decrease in NgBR levels in lysates of whole lung or PAECs from fetal lambs with IPH compared with controls. Overexpression of NgBR in IPH PAECs rescued the in vitro angiogenesis defects and increased the phosphorylation of both Akt and endothelial nitric oxide synthase at serine(1179) as well as the levels of both manganese superoxide dismutase and GTP cyclohydrolase-1. Consistent with the phenotype of IPH PAECs, knockdown of NgBR in control PAECs decreased the levels of nitric oxide, increased the levels of reactive oxygen species, and impaired in vitro angiogenesis. Our data demonstrate that NgBR mediates PAEC angiogenesis response through the modulation of Akt/endothelial nitric oxide synthase functions, and its decreased expression is mechanistically linked to IPH-related angiogenesis defects in the developing lungs.

Asunto(s)

Células Endoteliales/enzimología , Proteínas de la Mielina/metabolismo , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Síndrome de Circulación Fetal Persistente/enzimología , Arteria Pulmonar/enzimología , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Animales , Células Cultivadas , Modelos Animales de Enfermedad , GTP Ciclohidrolasa/metabolismo , Proteínas de la Mielina/genética , Óxido Nítrico/metabolismo , Proteínas Nogo , Síndrome de Circulación Fetal Persistente/genética , Síndrome de Circulación Fetal Persistente/fisiopatología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Arteria Pulmonar/fisiopatología , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Receptores de Superficie Celular/genética , Ovinos , Superóxido Dismutasa/metabolismo , Transfección

RESUMEN

Superoxide dismutase 2 (SOD-2) is synthesized in the cytosol and imported into the mitochondrial matrix, where it is activated and functions as the primary antioxidant for cellular respiration. The specific mechanisms that target SOD-2 to the mitochondria remain unclear. We hypothesize that inducible heat shock protein 70 (iHSP70) targets SOD-2 to the mitochondria via a mechanism facilitated by ATP, and this process is impaired in persistent pulmonary hypertension of the newborn (PPHN). We observed that iHSP70 interacts with SOD-2 and targets SOD-2 to the mitochondria. Interruption of iHSP70-SOD-2 interaction with 2-phenylethylenesulfonamide-µ (PFT-µ, a specific inhibitor of substrate binding to iHSP70 COOH terminus) and siRNA-mediated knockdown of iHSP70 expression disrupted SOD-2 transport to mitochondria. Increasing intracellular ATP levels by stimulation of respiration with CaCl2 facilitated the mitochondrial import of SOD-2, increased SOD-2 activity, and decreased the mitochondrial superoxide (O2(·-)) levels in PPHN pulmonary artery endothelial cells (PAEC) by promoting iHSP70-SOD-2 dissociation at the outer mitochondrial membrane. In contrast, oligomycin, an inhibitor of mitochondrial ATPase, decreased SOD-2 expression and activity and increased O2(·-) levels in the mitochondria of control PAEC. The basal ATP levels and degree of iHSP70-SOD-2 dissociation were lower in PPHN PAEC and lead to increased SOD-2 degradation in cytosol. In normal pulmonary arteries (PA), PFT-µ impaired the relaxation response of PA rings in response to nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine. Pretreatment with Mito-Q, a mitochondrial targeted O2(·-) scavenger, restored the relaxation response in PA rings pretreated with PFT-µ. Our observations suggest that iHSP70 chaperones SOD-2 to the mitochondria. Impaired SOD-2-iHSP70 dissociation decreases SOD-2 import and contributes to mitochondrial oxidative stress in PPHN.

Asunto(s)

Células Endoteliales/enzimología , Proteínas HSP70 de Choque Térmico/fisiología , Mitocondrias/enzimología , Estrés Oxidativo , Síndrome de Circulación Fetal Persistente/enzimología , Adenosina Trifosfato/biosíntesis , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Humanos , Peróxido de Hidrógeno/metabolismo , Recién Nacido , Pulmón/patología , Fosforilación Oxidativa , Síndrome de Circulación Fetal Persistente/patología , Transporte de Proteínas , Proteolisis , Arteria Pulmonar/patología , Ovinos

RESUMEN

Exposure to chronic hypoxia during gestation predisposes infants to neonatal pulmonary hypertension, but the underlying mechanisms remain unclear. Here, we test the hypothesis that moderate continuous hypoxia during gestation causes changes in the rho-kinase pathway that persist in the newborn period, altering vessel tone and responsiveness. Lambs kept at 3,801 m above sea level during gestation and the first 2 wk of life were compared with those with gestation at low altitude. In vitro studies of isolated pulmonary arterial rings found a more forceful contraction in response to KCl and 5-HT in high-altitude compared with low-altitude lambs. There was no difference between the effects of blockers of various pathways of extracellular Ca(2+) entry in low- and high-altitude arteries. In contrast, inhibition of rho-kinase resulted in significantly greater attenuation of 5-HT constriction in high-altitude compared with low-altitude arteries. High-altitude lambs had higher baseline pulmonary artery pressures and greater elevations in pulmonary artery pressure during 15 min of acute hypoxia compared with low-altitude lambs. Despite evidence for an increased role for rho-kinase in high-altitude arteries, in vivo studies found no significant difference between the effects of rho-kinase inhibition on hypoxic pulmonary vasoconstriction in intact high-altitude and low-altitude lambs. We conclude that chronic hypoxia in utero results in increased vasopressor response to both acute hypoxia and serotonin, but that rho-kinase is involved only in the increased response to serotonin.

Asunto(s)

Hipoxia Fetal/complicaciones , Síndrome de Circulación Fetal Persistente/etiología , Arteria Pulmonar/enzimología , Vasoconstricción , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Altitud , Animales , Animales Recién Nacidos , Presión Arterial , Bloqueadores de los Canales de Calcio/farmacología , Enfermedad Crónica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hipoxia Fetal/enzimología , Hipoxia Fetal/fisiopatología , Edad Gestacional , Humanos , Recién Nacido , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/fisiopatología , Inhibidores de Proteínas Quinasas/farmacología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiopatología , Serotonina/farmacología , Ovinos , Factores de Tiempo , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores

RESUMEN

Alveolar capillary dysplasia (ACD) is a congenital, lethal disorder of the pulmonary vasculature. Phosphatase and tensin homologue deleted from chromosome 10 (Pten) encodes a lipid phosphatase controlling key cellular functions, including stem/progenitor cell proliferation and differentiation; however, the role of PTEN in mesodermal lung cell lineage formation remains unexamined. To determine the role of mesodermal PTEN in the ontogeny of various mesenchymal cell lineages during lung development, we specifically deleted Pten in early embryonic lung mesenchyme in mice. Pups lacking Pten died at birth, with evidence of failure in blood oxygenation. Analysis at the cellular level showed defects in angioblast differentiation to endothelial cells and an accompanying accumulation of the angioblast cell population that was associated with disorganized capillary beds. We also found decreased expression of Forkhead box protein F1 (Foxf1), a gene associated with the ACD human phenotype. Analysis of human samples for ACD revealed a significant decrease in PTEN and increased activated protein kinase B (AKT). These studies demonstrate that mesodermal PTEN has a key role in controlling the amplification of angioblasts as well as their differentiation into endothelial cells, thereby directing the establishment of a functional gas exchange interface. Additionally, these mice could serve as a murine model of ACD.

Asunto(s)

Diferenciación Celular , Células Endoteliales/enzimología , Pulmón/embriología , Mesodermo/embriología , Fosfohidrolasa PTEN/metabolismo , Síndrome de Circulación Fetal Persistente/embriología , Animales , Linaje de la Célula , Células Endoteliales/patología , Activación Enzimática , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Pulmón/enzimología , Pulmón/patología , Mesodermo/enzimología , Mesodermo/patología , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/genética , Síndrome de Circulación Fetal Persistente/patología , Fenotipo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Alveolos Pulmonares/anomalías , Alveolos Pulmonares/embriología , Alveolos Pulmonares/enzimología , Alveolos Pulmonares/patología

RESUMEN

A rapid increase in the synthesis and release of nitric oxide (NO) facilitates the pulmonary vasodilation that occurs during birth-related transition. Alteration of this transition in persistent pulmonary hypertension of the newborn (PPHN) is associated with impaired function of endothelial nitric oxide synthase (eNOS) and an increase in oxidative stress. We investigated the hypothesis that a decrease in expression and activity of mitochondrial localized manganese superoxide dismutase (MnSOD) in pulmonary artery endothelial cells (PAEC) increases oxidative stress and impairs eNOS function in PPHN. We isolated PAEC and pulmonary arteries from fetal lambs with PPHN induced by prenatal ductus arteriosus ligation or sham ligation (control). We investigated MnSOD expression and activity, tyrosine nitration of MnSOD, and mitochondrial O(2)(-) levels in PAEC from control and PPHN lambs. We introduced exogenous MnSOD via an adenoviral vector (ad-MnSOD) transduction into PAEC and pulmonary arteries of PPHN lambs. The effect of ad-MnSOD was investigated on: mitochondrial O(2)(-) levels, MnSOD and eNOS expression and activity, intracellular hydrogen peroxide (H(2)O(2)) levels, and catalase expression in PAEC. MnSOD mRNA and protein levels and activity were decreased and MnSOD tyrosine nitration was increased in PPHN-PAEC. ad-MnSOD transduction of PPHN-PAEC increased its activity two- to threefold, decreased mitochondrial O(2)(-) levels, and increased H(2)O(2) levels and catalase expression. ad-MnSOD transduction improved eNOS expression and function and the relaxation response of PPHN pulmonary arteries. Our observations suggest that decreased MnSOD expression and activity contribute to the endothelial dysfunction observed in PPHN.

Asunto(s)

Células Endoteliales/enzimología , Regulación Enzimológica de la Expresión Génica , Estrés Oxidativo , Síndrome de Circulación Fetal Persistente/enzimología , Arteria Pulmonar/enzimología , Superóxido Dismutasa/biosíntesis , Adenoviridae , Animales , Modelos Animales de Enfermedad , Células Endoteliales/patología , Vectores Genéticos , Humanos , Peróxido de Hidrógeno/metabolismo , Recién Nacido , Mitocondrias/enzimología , Mitocondrias/genética , Mitocondrias/patología , Óxido Nítrico/biosíntesis , Óxido Nítrico/genética , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Óxido Nítrico Sintasa de Tipo III/genética , Síndrome de Circulación Fetal Persistente/genética , Síndrome de Circulación Fetal Persistente/patología , Síndrome de Circulación Fetal Persistente/fisiopatología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ovinos , Superóxido Dismutasa/genética , Superóxidos/metabolismo , Transducción Genética , Vasodilatación/genética

RESUMEN

Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.

Asunto(s)

Células Endoteliales/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Arteria Pulmonar/patología , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Animales , Calcimicina/farmacología , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/patología , Expresión Génica , Histamina/fisiología , Humanos , Recién Nacido , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/genética , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/metabolismo , Síndrome de Circulación Fetal Persistente/patología , Unión Proteica , Mapeo de Interacción de Proteínas , Ovinos , Canal Aniónico 1 Dependiente del Voltaje/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/genética

RESUMEN

AIM: To evaluate the association of angiotensin-converting enzyme (ACE) gene polymorphism with risk/severity of persistent pulmonary hypertension of the newborn (PPHN) among at risk infants. METHODS: Infants ≥ 34 weeks with respiratory distress at birth were recruited. PPHN was diagnosed clinically and by cardiac echocardiogram. Control group consisted of infants with respiratory distress who did not develop PPHN. ACE genotyping (DD, II, DI genotypes) and serum ACE levels were determined. RESULTS: A total of 120 infants were included (PPHN = 44; control = 76). Frequency of ACE DD genotype was not different between the two groups of infants (25% versus 33%). Among PPHN infants, severity of illness did not differ between genotypes. Mean (SD) serum ACE levels [15 (9) versus 24 (13) versus 29 (14) U/L] were positively associated with the number of D alleles and inversely associated with infants' gestational age (GA) and level of cardiovascular support. CONCLUSION: Angiotensin-converting enzyme gene polymorphism did not impact the risk or severity of PPHN among infants ≥ 34 weeks GA.

Asunto(s)

Peptidil-Dipeptidasa A/genética , Síndrome de Circulación Fetal Persistente/genética , Polimorfismo Genético , Estudios de Casos y Controles , Femenino , Genotipo , Humanos , Recién Nacido , Modelos Logísticos , Masculino , Peptidil-Dipeptidasa A/sangre , Síndrome de Circulación Fetal Persistente/sangre , Síndrome de Circulación Fetal Persistente/enzimología , Estudios Prospectivos , Riesgo , Índice de Severidad de la Enfermedad

RESUMEN

BACKGROUND: Neonatal hypoxemic respiratory failure (NHRF) is usually associated with reversible persistent pulmonary hypertension (PPHN). Congenital diaphragmatic hernia (CDH), a cause of refractory NHRF, is associated with irreversible pulmonary hypertension. Nitric oxide (NO) generated in the pulmonary vascular endothelium by endothelial nitric oxide synthase (eNOS) plays a pivotal role in perinatal circulatory adaptation. OBJECTIVE: To compare the expression of eNOS using IHC in postmortem lung tissue from newborns diagnosed clinically with PPHN and CDH. DESIGN/METHODS: Formalin-fixed lung tissue from infants who died following treatment for PPHN (n=12) or CDH (n=8) and age and gender matched controls who died from non-respiratory causes (Control, n=14) was evaluated for expression and staining intensity (1-4 scale) of eNOS using IHC. RESULTS: Mean gestational and postnatal age was comparable across groups. Histological evidence of chronic lung disease, pulmonary hypoplasia and pulmonary hypertension were seen more frequently in CDH compared to PPHN and control infants. eNOS expression was increased in arteriolar media of PPHN infants compared to Controls (p=0.027). CDH infants had increased intensity of staining for eNOS in the arteriolar endothelium (p=0.022) compared to control and PPHN infants and in the alveolar lining (p=0.002) compared to Controls. CONCLUSIONS: Upregulation of eNOS was seen both in infants with CDH and PPHN but was more marked in infants with CDH. These findings may have implications for understanding disease pathophysiology in cases with fatal outcome and development of novel therapies for neonatal pulmonary hypertension.

Asunto(s)

Hipertensión Pulmonar/enzimología , Pulmón/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estudios de Casos y Controles , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Femenino , Hernia Diafragmática/complicaciones , Hernia Diafragmática/enzimología , Hernia Diafragmática/patología , Hernias Diafragmáticas Congénitas , Humanos , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/patología , Inmunohistoquímica , Recién Nacido , Pulmón/irrigación sanguínea , Pulmón/patología , Masculino , Óxido Nítrico/metabolismo , Síndrome de Circulación Fetal Persistente/complicaciones , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/patología , Estudios Retrospectivos , Regulación hacia Arriba

RESUMEN

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.

Asunto(s)

Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Pulmón/enzimología , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Síndrome de Circulación Fetal Persistente/tratamiento farmacológico , Arteria Pulmonar/enzimología , Superóxido Dismutasa/farmacología , Animales , Animales Recién Nacidos , Biopterinas/análogos & derivados , Biopterinas/biosíntesis , GTP Ciclohidrolasa/biosíntesis , Humanos , Recién Nacido , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Síndrome de Circulación Fetal Persistente/enzimología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/farmacología , Respiración Artificial , Ovinos

RESUMEN

BACKGROUND/PURPOSE: Heme oxygenase (HO-1), an inducible isoform of HO is a regulator of vascular tone and cell proliferation through the production of endogenous carbon monoxide (CO). Endothelium-derived nitric oxide (NO) occurs in the endothelial layers of blood vessels and mediates vasorelaxation. Both CO and NO have similar properties and are potent vasodilators. The aim of this study was to examine the expression of HO-1 and endothelial nitric oxide synthase (eNOS) in the Congenital diaphragmatic hernia (CDI) lung. METHODS: RNA was extracted from archival formalin-fixed paraffin-embedded lung tissue from 11 patients with CDH complicated by persistent pulmonary hypertension (PPH). Five age-matched newborns served as controls. Reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers for human HO-1 and eNOS. Immunohistochemistry using HO-1 and eNOS antibodies was performed and examined using laser scanning microscope. RESULTS: HO-1 and eNOS mRNA expression was significantly decreased in CDH lung compared with controls (P <.05). HO-1 and eNOS immunoreactivity was reduced markedly reduced in the endothelium and arterial wall in the CDH samples compared with normal lung. CONCLUSIONS: Decreased expression of HO-1 and eNOS in the CDH lung suggests deficiency of endogenous NO and CO, which may contribute to altered vascular tone causing PPH.

Asunto(s)

Hemo Oxigenasa (Desciclizante)/metabolismo , Hernias Diafragmáticas Congénitas , Pulmón/enzimología , Óxido Nítrico Sintasa/metabolismo , Síndrome de Circulación Fetal Persistente/enzimología , Análisis de Varianza , Estudios de Casos y Controles , Hemo-Oxigenasa 1 , Hernia Diafragmática/complicaciones , Hernia Diafragmática/enzimología , Humanos , Recién Nacido , Proteínas de la Membrana , Microscopía Confocal , Óxido Nítrico Sintasa de Tipo III , Síndrome de Circulación Fetal Persistente/complicaciones , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Ligation of the ductus arteriosus in utero produces pulmonary hypertension and vascular remodeling in fetal and newborn lambs. However, the mechanisms producing these vascular changes are not well defined. Because reactive oxygen species (ROS) have been implicated as mediators of smooth muscle cell proliferation, we hypothesized that increased formation of ROS may be involved in the pathophysiology of pulmonary hypertension after in utero ductal ligation. Using ethidium fluorescence, we demonstrated an increase in superoxide levels after 9 days of ductal ligation compared with control lungs (P<0.05) that was localized to the adventitia and smooth muscle cells of hypertensive vessels. SOD-1 and SOD-2 protein levels and activities in lung, vein, and artery of hypertensive lambs were unchanged relative to controls after 2 days of ductal ligation. However, after 9 days, superoxide dismutase (SOD) activity was significantly decreased in arteries from ligated lambs without associated changes in SOD protein expression (P<0.05). Examination of NADPH oxidase expression as a potential source of the superoxide production indicated that the levels of p67phox, a subunit of the NADPH oxidase complex, were significantly increased in the pulmonary arteries, but not veins, from the ligated lung as early as 2 days (P<0.05). Functional analyses demonstrated that reducing superoxide levels significantly increased the NO-mediated relaxation of pulmonary arteries isolated after 9 days, but not 2 days, of ductal ligation (P<0.05). These results suggest that increased NADPH oxidase expression may increase levels of superoxide in persistent pulmonary hypertension of the newborn lung tissue, and that increased superoxide blunts vascular relaxations to exogenous NO while stimulating smooth muscle cell growth.

Asunto(s)

NADPH Oxidasas/fisiología , Penicilamina/análogos & derivados , Síndrome de Circulación Fetal Persistente/etiología , Superóxidos/metabolismo , Animales , Técnicas de Cultivo , Feto/metabolismo , Humanos , Recién Nacido , Chaperonas Moleculares/análisis , Donantes de Óxido Nítrico/farmacología , Penicilamina/farmacología , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/metabolismo , Síndrome de Circulación Fetal Persistente/fisiopatología , Fosfoproteínas/análisis , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiopatología , Ovinos , Superóxido Dismutasa/análisis , Vasodilatación

RESUMEN

Superoxide plays an important role in pulmonary arterial smooth muscle cell (SMC) proliferation and survival. The rapid reaction between superoxide and nitric oxide (NO) to form peroxynitrite suggests that endothelium-derived NO may influence adjacent SMC growth. To investigate this possibility, we determined the dose-dependent effects of NO on the proliferation and viability of pulmonary arterial SMC isolated from fetal lambs (FPASMC). Using fluorescence microscopy we found a dose-dependent decrease in superoxide levels in FPASMC treated with the NO donor spermine NONOate. This was associated with an increase in peroxynitrite-mediated protein nitration. At doses between 50 and 250 microM, spermine NONOate attenuated serum-induced FPASMC proliferation resulting in a G(0)/G(1) cell cycle arrest. This process involved a decrease in levels of cyclin A and an increase in the nuclear localization of p21 and p27. Furthermore, 500 microM spermine NONOate decreased viable cell number by inducing programmed cell death: FPASMC treated with 500 microM spermine NONOate displayed a loss of mitochondrial membrane potential, followed by caspase activation and DNA fragmentation. These data suggest that NO inhibits superoxide-induced proliferation of FPASMC and at higher doses induces apoptosis. NO donors may therefore prove to be useful therapeutic tools to treat diseases resulting from excessive proliferation of vascular smooth muscle.

Asunto(s)

Apoptosis/efectos de los fármacos , Músculo Liso Vascular/citología , Óxido Nítrico/farmacología , Arteria Pulmonar/citología , Espermina/análogos & derivados , Animales , Apoptosis/fisiología , Caspasas/fisiología , Ciclo Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Feto , Humanos , Recién Nacido , Potenciales de la Membrana/fisiología , Microscopía Fluorescente , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiología , Donantes de Óxido Nítrico/farmacología , Óxidos de Nitrógeno , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/metabolismo , Síndrome de Circulación Fetal Persistente/patología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/enzimología , Arteria Pulmonar/metabolismo , Ovinos , Espermina/farmacología , Superóxidos/metabolismo

RESUMEN

Previous studies in adults have shown that chronic pulmonary hypertension is associated with decreased endothelial nitric oxide synthase (eNOS) expression in pulmonary arteries. However, the role of decreased eNOS expression in persistent pulmonary hypertension of the newborn (PPHN) is unknown. We investigated the hypothesis that umbilical vein endothelial cells cultured from infants with PPHN will have decreased eNOS expression. Umbilical cords were collected from meconium-stained infants at birth, and endothelial cells were isolated if the infants developed PPHN. Endothelial cells were grown in primary culture, and total RNA was isolated. cDNA was reverse transcribed from mRNA and amplified by PCR. An expected product of approximately 550 bp was found in all control infants but only in two of the six infants with PPHN. Identity of the PCR product was confirmed by Southern hybridization to a separate internal eNOS-specific probe. Amplification of beta-actin cDNA, an internal control, was detected in all controls and in all infants with PPHN, including the four infants without the eNOS band. There was no difference in the course and outcome of patients with presence or absence of the eNOS band. However, there was an acidotic arterial blood pH (7.19-7.29) and intrapartum fetal heart rate decelerations in all four infants without eNOS expression. In conclusion, eNOS mRNA was detected in all normal term infants but was notably absent in the majority of infants with PPHN in this pilot study. The development of PPHN is multifactorial, and a decrease in eNOS gene expression may occur in some infants. Whether the decreased eNOS transcript is a cause of PPHN or a result of intrapartum stress remains to be determined.

Asunto(s)

Endotelio Vascular/enzimología , Regulación Enzimológica de la Expresión Génica , Óxido Nítrico Sintasa/genética , Síndrome de Circulación Fetal Persistente/genética , Células Cultivadas , Endotelio Vascular/patología , Humanos , Recién Nacido , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo III , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/patología , Reacción en Cadena de la Polimerasa , Cordón Umbilical

RESUMEN

Endothelial (e) nitric oxide synthase (NOS) activity modulates pulmonary vascular tone in the normal fetus and decreases pulmonary vascular resistance (PVR) at birth. Mechanisms contributing to sustained elevations of PVR and the failure of postnatal adaptation at birth are uncertain but may include decreased eNOS activity. To test this hypothesis, we studied the effects of chronic intrauterine pulmonary hypertension on lung eNOS content and NOS activity in an ovine model of perinatal pulmonary hypertension and in normal lambs. We measured eNOS mRNA and protein content by Northern and Western blot analyses, respectively. Calcium-dependent and total NOS activities were determined by assaying the conversion of L-[14C]arginine to L-[14C]citrulline from lung homogenates. To determine the effects of intrauterine hypertension on lung eNOS content, fetal lung tissue was harvested 8-12 days after intrauterine closure of the ductus arteriosus (DA) performed at 125-128 days of gestation (term = 147 days). Although positive immunostaining for eNOS persisted in lung vascular endothelium, eNOS protein content was reduced by 48%, as measured by Western analysis (P < 0.001). Chronic hypertension reduced lung eNOS mRNA content by 30% (P < 0.05). Compared with age-matched controls, Ca(2+)-dependent NOS activity was decreased after DA ligation by 75% (P < 0.01). We conclude that chronic intrauterine pulmonary hypertension decreases eNOS in the fetal lung. We speculate that decreased NO production contributes to failure of postnatal adaptation in this experimental model of persistent pulmonary hypertension of the newborn.

Asunto(s)

Endotelio Vascular/embriología , Enfermedades Fetales/enzimología , Feto/enzimología , Hipertensión Pulmonar/enzimología , Óxido Nítrico Sintasa/metabolismo , Animales , Northern Blotting , Enfermedad Crónica , Endotelio Vascular/enzimología , Humanos , Recién Nacido , Óxido Nítrico Sintasa/genética , Síndrome de Circulación Fetal Persistente/enzimología , ARN Mensajero/metabolismo , Ovinos/embriología

RESUMEN

Acetylhydrolase is an acid-labile, 43 kd protein that catalyzes the degradation of platelet activating factor (PAF), a potent phospholipid inflammatory mediator, to its biologically inactive metabolite lysoPAF. PAF has a short half-life, thus acetylhydrolase plays an important role in its regulation. Since previous work suggests that PAF may be involved in certain neonatal diseases such as necrotizing enterocolitis, we studied the effect of age on acetylhydrolase activity. Serum acetylhydrolase activity was quantified using radio-labelled PAF and measuring reaction products. Serum samples were obtained prospectively from 70 subjects ranging in age from 4 hr to 48 yr. Acetylhydrolase activity was lower for newborns (less than 3 wk) than all other age ranges (8.2 +/- 1.4 nmole/ml/min vs 30.0 +/- 1.6 nmole/ml/min, p less than .01). Furthermore, enzyme activity increased linearly with respect to the natural logarithm of age from 0 days to 6 weeks (r = 0.65, p less than .001). By 6 weeks of life acetylhydrolase activity approached values of older children and adults. Newborn acetylhydrolase activity was similar between term and preterm infants (8.6 +/- 1.9 nmole/ml/min vs 7.2 +/- 2.4 nmole/ml/min, p = NS). We conclude that acetylhydrolase activity is low in human neonates and increases during the first 6 weeks of life. These results suggest that newborn infants may be at increased risk for pathophysiologic processes mediated by PAF.

Asunto(s)

Envejecimiento/sangre , Fosfolipasas A/sangre , Fosfolipasas/sangre , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Adulto , Niño , Cardiopatías Congénitas/sangre , Cardiopatías Congénitas/enzimología , Humanos , Hiperbilirrubinemia Hereditaria/sangre , Hiperbilirrubinemia Hereditaria/enzimología , Lactante , Recién Nacido , Recien Nacido Prematuro , Síndrome de Circulación Fetal Persistente/sangre , Síndrome de Circulación Fetal Persistente/enzimología , Valores de Referencia , Análisis de Regresión , Síndrome de Dificultad Respiratoria del Recién Nacido/sangre , Síndrome de Dificultad Respiratoria del Recién Nacido/enzimología