AMP-activated protein kinase (AMPK) is an energy sensor activated by increases in [AMP] or by oxidant stress (reactive oxygen species [ROS]). Hypoxia increases cellular ROSEnergy-dependent cellular processes require ATP, which is derived from mitochondrial oxidative phosphorylation and/or from glycolysis. Conditions that limit the cellular oxygen supply to the point that oxidative metabolism suffers can threaten cell survival because they undermine the ability of the cell to sustain essential ATP-dependent processes. Given that ATP supply is critical for survival, multiple systems have evolved to protect cells from the consequences of oxygen supply limitation and metabolic substrate deprivation. In this regard, defense of cellular energy substrates is mediated by the AMPactivated protein kinase (AMPK) system, which has been described as a "fuel gauge of the cell" (17). Activation of AMPK leads to increased glucose uptake via translocation of the transporter GLUT4 to the plasma membrane (42) and activation of glycolytic enzymes such as 6-phosphofructo-2-kinase, leading to enhanced glycolytic capacity via increases in fructose-2,6-bisphosphate (36). These and other responses triggered by AMPK activation confer protection against hypoxic injury in tissues such as the heart by preserving energy supply, mitochondrial metabolism, and glycolytic flux (43).AMPK activation requires phosphorylation of Thr-172 in the activation loop of its ␣ subunit (21) and is regulated by increases in cellular AMP and decreases in ATP via allosteric mechanisms and by the activity of upstream kinases and phosphatases that control AMPK phosphorylation and dephosphorylation at the critical threonine residue (8,23). Perhaps the best-described AMPK kinase is the tumor suppressor LKB1, which phosphorylates the catalytic ␣ subunit of AMPK in an AMP-dependent manner (20,53). A second activation pathway occurs independently of cellular AMP levels and involves Ca 2ϩ /calmodulin-dependent protein kinase kinase (CaMKK␤) (25,52). In that pathway, activation is initiated by a rise in [Ca 2ϩ ] i which increases the activity of CaMKK␤, which then phosphorylates AMPK. In vitro studies using both endogenous and recombinant proteins show that LKB1 and CaMKK␤ phosphorylate AMPK at 20,52). Activation of AMPK can also occur in response to oxidant stress (26,47), as demonstrated by the observation that H 2 O 2 administration triggers phosphorylation of AMPK and its downstream target, acyl coenzyme A carboxylase (ACC) (7, 28). However, the mechanism underlying this reactive oxygen species (ROS)-dependent activation of AMPK is not known.During severe oxygen deprivation, limitations in mitochondrial respiration lead to increases in the AMP/ATP ratio, which then trigger AMPK activation. However, AMPK-mediated protection might be greater if the kinase was activated before the cell reached the point where the AMP/ATP ratio has increased. Some beneficial effects of AMPK signaling, including the activation of gene expression or mitochondrial biogenesis (39), are not...

Rationale: Chronic hypoxia induces pulmonary vascular remodeling, pulmonary hypertension, and right ventricular hypertrophy. At present, little is known about mechanisms driving these responses. Hypoxia-inducible factor-1a (HIF-1a) is a master regulator of transcription in hypoxic cells, up-regulating genes involved in energy metabolism, proliferation, and extracellular matrix reorganization. Systemic loss of a single HIF-1a allele has been shown to attenuate hypoxic pulmonary hypertension, but the cells contributing to this response have not been identified.Objectives: We sought to determine the contribution of HIF-1a in smooth muscle on pulmonary vascular and right heart responses to chronic hypoxia.Methods: We used mice with homozygous conditional deletion of HIF-1a combined with tamoxifen-inducible smooth muscle-specific Cre recombinase expression. Mice received either tamoxifen or vehicle followed by exposure to either normoxia or chronic hypoxia (10% O 2 ) for 30 days before measurement of cardiopulmonary responses.Measurements and Main Results: Tamoxifen-induced smooth muscle-specific deletion of HIF-1a attenuated pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, right ventricular hypertrophy was unchanged despite attenuated pulmonary pressures.Conclusions: These results indicate that HIF-1a in smooth muscle contributes to pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, loss of HIF-1 function in smooth muscle does not affect hypoxic cardiac remodeling, suggesting that the cardiac hypertrophy response is not directly coupled to the increase in pulmonary artery pressure.

In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term = 150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.

Rationale: The role of reactive oxygen species (ROS) signaling in the O 2 sensing mechanism underlying acute hypoxic pulmonary vasoconstriction (HPV) has been controversial. Although mitochondria are important sources of ROS, studies using chemical inhibitors have yielded conflicting results, whereas cellular models using genetic suppression have precluded in vivo confirmation. Hence, genetic animal models are required to test mechanistic hypotheses. Objectives: We tested whether mitochondrial Complex III is required for the ROS signaling and vasoconstriction responses to acute hypoxia in pulmonary arteries (PA). Methods: A mouse permitting Cre-mediated conditional deletion of the Rieske iron-sulfur protein (RISP) of Complex III was generated. Adenoviral Cre recombinase was used to delete RISP from isolated PA vessels or smooth muscle cells (PASMC). Measurements and Main Results: In PASMC, RISP depletion abolished hypoxia-induced increases in ROS signaling in the mitochondrial intermembrane space and cytosol, and it abrogated hypoxia-induced increases in [Ca 21 ] i . In isolated PA vessels, RISP depletion abolished hypoxia-induced ROS signaling in the cytosol. Breeding the RISP mice with transgenic mice expressing tamoxifen-activated Cre in smooth muscle permitted the depletion of RISP in PASMC in vivo. Precision-cut lung slices from those mice revealed that RISP depletion abolished hypoxia-induced increases in [Ca 21 ] i of the PA. In vivo RISP depletion in smooth muscle attenuated the acute hypoxia-induced increase in right ventricular systolic pressure in anesthetized mice. Conclusions: Acute hypoxia induces superoxide release from Complex III of smooth muscle cells. These oxidant signals diffuse into the cytosol and trigger increases in [Ca 21 ] i that cause acute hypoxic pulmonary vasoconstriction.Keywords: oxygen sensing; Rieske iron-sulfur protein; reactive oxygen species; roGFP; hypoxic pulmonary vasoconstrictionIn the lung, alveolar hypoxia triggers acute constriction of small pulmonary arteries (PA), a response termed hypoxic pulmonary vasoconstriction (HPV). This response is recapitulated in cultured PA smooth muscle cells (PASMC), indicating that the oxygen-sensing mechanism underlying HPV is intrinsic to the PASMC (1-12). Our previous work has implicated increases in reactive oxygen species (ROS) signaling during hypoxia (9,10, 12). Previous studies using mitochondrial inhibitors and mitochondria-deficient (r 0 ) cells suggested that the electron transport chain (ETC) is required for hypoxia-induced ROS signaling in the pulmonary circulation (9, 11-18). We subsequently assessed ROS signaling in hypoxic PASMC using roGFP, a thiol-containing, redox-sensitive reporter (19-23) targeted to compartments within mitochondria or the cytosol (10). Unlike other methods (24-26), this targeted approach permitted the differentiation of hypoxia-induced ROS changes between mitochondrial subcompartments. During hypoxia, increased oxidation was detected in the mitochondrial intermembrane space (IMS) and the cytoso...

BackgroundPercutaneous closure of patent ductus arteriosus (PDA) in term neonates is established, but data regarding outcomes in infants born very preterm (<32 weeks of gestation) are minimal, and no published criteria exist establishing a minimal weight of 4 kg as a suitable cutoff. We sought to analyze outcomes of percutaneous PDA occlusion in infants born very preterm and referred for PDA closure at weights <4 kg.Methods and ResultsRetrospective analysis (January 2005–January 2014) was done at a single pediatric center. Procedural successes and adverse events were recorded. Markers of respiratory status (need for mechanical ventilation) were determined, with comparisons made before and after catheterization. A total of 52 very preterm infants with a median procedural weight of 2.9 kg (range 1.2–3.9 kg) underwent attempted PDA closure. Twenty‐five percent (13/52) of infants were <2.5 kg. Successful device placement was achieved in 46/52 (88%) of infants. An adverse event occurred in 33% of cases, with an acute arterial injury the most common complication. We observed no association between weight at time of procedure and the risk of an adverse event. No deaths were attributable to the PDA closure. Compared to precatheterization trends, percutaneous PDA closure resulted in improved respiratory status, including less exposure to mechanical ventilation (mixed effects logistic model, P<0.01).ConclusionsAmong infants born very preterm, percutaneous PDA closure at weights <4 kg is generally safe and may improve respiratory health, but risk of arterial injury is noteworthy. Randomized clinical trials are needed to assess clinically relevant differences in outcomes following percutaneous PDA closure versus alternative (surgical ligation) management strategies.

Intratracheal lipopolysaccharide (LPS) causes acute inflammation and injurious mechanical ventilation results in pulmonary and systemic inflammation. We aimed to determine in preterm lungs if continuous positive airway pressure (CPAP) protects against pulmonary and systemic inflammation, compared with conventional mechanical ventilation (CMV) after intratracheal LPS. Preterm fetuses were exposed to maternal betamethasone and Epostane 36 h before delivery at 133 d gestational age (term ϭ 150 d). Lambs were intubated and randomized to receive gentle CMV (tidal volume 8 mL/kg) or CPAP with 8 cm H 2 O pressure. Surfactant (10 mg/kg) mixed with 1 mg LPS or saline was instilled into the trachea at 15 min. Blood gas status, ventilation variables, and arterial pressures were recorded for 3 h. Static pressure-volume curves and lung and systemic inflammation were assessed postmortem. CPAP lambs had elevated Paco 2 and minute ventilation compared with the CMV lambs. Cytokine mRNA was increased in the lungs and liver of CPAP and CMV lambs relative to unventilated controls. Intratracheal LPS amplified the cytokine mRNA responses of IL-1␤, IL-6, and IL-8 in the lung and liver. Blood neutrophils decreased similarly after LPS in CPAP and CMV groups. Cytokine markers of lung injury or the systemic response to intratracheal LPS were not decreased by CPAP relative to CMV, in preterm lambs. . Similarly, ventilation with lung volumes that approach or exceed total lung capacity will stretch the lungs and cause injury (3,4). The preterm fetal lung is particularly vulnerable to lung injury during the transition from a fluid-filled lung to airway breathing because surfactant deficiency increases the pressures required to open the lung, the lung aerates nonuniformly, and the delicate tissues of the preterm lung are easily stretched (5,6). The use of continuous positive airway pressure (CPAP) is being advocated for transitioning the preterm fetal lung to air breathing as a way to decrease lung injury (7,8). Although CPAP can cause lung injury in sepsis models (9), CPAP can decrease indicators of lung injury in preterm lambs (10) compared with conventional mechanical ventilation (CMV) and its use has been associated with decreased bronchopulmonary dysplasia (11).Prior exposure of the adult lung to pro-inflammatory mediators such as endotoxin amplifies lung injury caused by ventilation from low lung volumes or elevated lung volumes (9,12,13), and the mediators can enter the systemic circulation and cause systemic inflammatory responses (14,15). Termventilated lambs do not leak endotoxin from the airspaces into the systemic circulation unless the lungs are over-stretched (16). In contrast, preterm lungs leak endotoxin or IL-1 with conventional ventilation (16,17). Chorioamnionitis is associated with many preterm deliveries before 32 wk gestational age, and inflammatory mediators are present in the fetal lung (18,19). We hypothesized that spontaneous breathing with CPAP would protect the preterm lung and minimize a systemic inflamma...

Among infants <6 kg, transcatheter PDA occlusion is technically feasible, but risks of MAE are noteworthy. These findings may help inform patient selection and procedural approach for transcatheter PDA occlusion and direct targeted research efforts to support the practice of evidence-based medicine.

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