Decreased Liver Kinase B1 Expression and Impaired Angiogenesis in a Murine Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is characterized by impaired lung alveolar and vascular growth. We investigated the hypothesis that neonatal exposure to hyperoxia leads to persistent BPD phenotype due to decreased expression of liver kinase B1 (LKB1), a key regulator of mitochondrial function. We exposed mouse pups from postnatal day 1- day 10 (P1-P10) to 21% or 75% oxygen. Half of the pups in each group received metformin or saline intraperitoneally from P1-P10. Pups were euthanized at P4 or P10 or recovered in 21% O2 until euthanasia at P21. Lung histology/morphometry, immunofluorescence and immunoblots were done for changes in lung structure and expression of LKB1 and downstream targets, AMPK, PGC-1α, electron transport chain complexes (ETC) and Notch ligands, Jagged 1 and delta like 4 (Dll4). LKB1 signaling and in vitro angiogenesis were assessed in human pulmonary artery endothelial cells (PAEC) exposed to 21% or 95% O2 for 36h. Levels of LKB1, phosphorylated-AMPK (p-AMPK), PGC-1α, and ETC complexes were decreased in lungs at P10 and P21 in hyperoxia. Metformin increased LKB1, p-AMPK, PGC-1α, and ETC complexes at P10 and P21 in hyperoxia pups. Radial alveolar count was decreased and mean linear intercept increased in hyperoxia pups at P10 and P21; these were improved by metformin. Lung capillary density was decreased in hyperoxia at P10 and P21 and was increased by metformin. In vitro angiogenesis was decreased in HPAEC by 95% O2 and was improved by metformin. Decreased LKB1 signaling may contribute to decreased alveolar and vascular growth in a mouse model of BPD.

Cellular Senescence Contributes to the Progression of Hyperoxic Bronchopulmonary Dysplasia.

Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress sequentially occur in bronchopulmonary dysplasia (BPD), and all result in DNA damage. When DNA damage becomes irreparable, tumor suppressors increase, followed by apoptosis or senescence. Although cellular senescence contributes to wound healing, its persistence inhibits growth. Therefore, we hypothesized that cellular senescence contributes to BPD progression. Human autopsy lungs were obtained. Sprague-Dawley rat pups exposed to 95% oxygen between Postnatal Day 1 (P1) and P10 were used as the BPD phenotype. N-acetyl-lysyltyrosylcysteine-amide (KYC), tauroursodeoxycholic acid (TUDCA), and Foxo4 dri were administered intraperitoneally to mitigate myeloperoxidase oxidant generation, ER stress, and cellular senescence, respectively. Lungs were examined by histology, transcriptomics, and immunoblotting. Cellular senescence increased in rat and human BPD lungs, as evidenced by increased oxidative DNA damage, tumor suppressors, GL-13 stain, and inflammatory cytokines with decreased cell proliferation and lamin B expression. Cellular senescence-related transcripts in BPD rat lungs were enriched at P10 and P21. Single-cell RNA sequencing showed increased cellular senescence in several cell types, including type 2 alveolar cells. In addition, Foxo4-p53 binding increased in BPD rat lungs. Daily TUDCA or KYC, administered intraperitoneally, effectively decreased cellular senescence, improved alveolar complexity, and partially maintained the numbers of type 2 alveolar cells. Foxo4 dri administered at P4, P6, P8, and P10 led to outcomes similar to TUDCA and KYC. Our data suggest that cellular senescence plays an essential role in BPD after initial inducement by hyperoxia. Reducing myeloperoxidase toxic oxidant production, ER stress, and attenuating cellular senescence are potential therapeutic strategies for halting BPD progression.

Clinical course and therapeutic trial for a case of congenital secretory diarrhea due to novel GUCY2C variant.

Chronic diarrhea presents a significant challenge for managing nutritional and electrolyte deficiencies, especially in children, given the higher stakes of impacting growth and developmental consequence. Congenital secretory diarrhea (CSD) compounds this further, particularly in the case of the activating variants of the guanylate-cyclase 2C (GUCY2C) gene. GUCY2C encodes for the guanylate-cyclase 2C (GC-C) receptor that activates the downstream cystic fibrosis transmembrane receptor (CFTR) that primarily drives the severity of diarrhea with an unclear extent of influence on other intestinal channels. Thus far, management for CSD primarily consists of mitigating nutritional, electrolyte, and volume deficiencies with no known pathophysiology-driven treatments. For activating variants of GUCY2C, experimental compounds have shown efficacy in vitro for direct inhibition of GC-C but are not currently available for clinical use. However, Crofelemer, a CFTR inhibitory modulator with negligible systemic absorption, can theoretically help to treat this type of CSD. Herein, we describe and characterize the clinical course of a premature male infant with a de novo missense variant of GUCY2C not previously reported and highly consistent with CSD. With multi-disciplinary family-directed decision-making, a treatment for CSD was evaluated for the first time to our knowledge with Crofelemer.

Aberrant PGC-1α signaling in a lamb model of persistent pulmonary hypertension of the newborn.

BACKGROUND: Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by elevated pulmonary vascular resistance (PVR), resulting in hypoxemia. Impaired angiogenesis contributes to high PVR. Pulmonary artery endothelial cells (PAECs) in PPHN exhibit decreased mitochondrial respiration and angiogenesis. We hypothesize that Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α) downregulation leads to reduced mitochondrial function and angiogenesis in PPHN. METHODS: Studies were performed in PAECs isolated from fetal lambs with PPHN induced by ductus arteriosus constriction, with gestation-matched controls and in normal human umbilical vein endothelial cells (HUVECs). PGC-1α was knocked downed in control lamb PAECs and HUVECs and overexpressed in PPHN PAECs to investigate the effects on mitochondrial function and angiogenesis. RESULTS: PPHN PAECs had decreased PGC-1α expression compared to controls. PGC-1α knockdown in HUVECs led to reduced Nuclear Respiratory Factor-1 (NRF-1), Transcription Factor-A of Mitochondria (TFAM), and mitochondrial electron transport chain (ETC) complexes expression. PGC-1α knockdown in control PAECs led to decreased in vitro capillary tube formation, cell migration, and proliferation. PGC-1α upregulation in PPHN PAECs led to increased ETC complexes expression and improved tube formation, cell migration, and proliferation. CONCLUSION: PGC-1α downregulation contributes to reduced mitochondrial oxidative phosphorylation through control of the ETC complexes, thereby affecting angiogenesis in PPHN. IMPACT: Reveals a novel mechanism for angiogenesis dysfunction in persistent pulmonary hypertension of the newborn (PPHN). Identifies a key mitochondrial transcription factor, Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α), as contributing to the altered adaptation and impaired angiogenesis function that characterizes PPHN through its regulation of mitochondrial function and oxidative phosphorylation. May provide translational significance as this mechanism offers a new therapeutic target in PPHN, and efforts to restore PGC-1α expression may improve postnatal transition in PPHN.

OLA1 Phosphorylation Governs the Mitochondrial Bioenergetic Function of Pulmonary Vascular Cells.

Mitochondrial function and metabolic homeostasis are integral to cardiovascular function and influence how vascular cells respond to stress. However, little is known regarding how mitochondrial redox control mechanisms and metabolic regulation interact in the developing lungs. Here we show that human OLA1 (Obg-like ATPase-1) couples redox signals to the metabolic response pathway by activating metabolic gene transcription in the nucleus. OLA1 phosphorylation at Ser232/Tyr236 triggers its translocation from the cytoplasm and mitochondria into the nucleus. Subsequent phosphorylation of OLA1 at Thr325 effectively changes its biochemical function from ATPase to GTPase, promoting the expression of genes involved in the mitochondrial bioenergetic function. This process is regulated by ERK1/2 (extracellular-regulated kinases 1 and 2), which were restrained by PP1A (protein phosphatase 1A) when stress abated. Knockdown of ERK1 or OLA1 mutated to a phosphoresistant T325A mutant blocked its nuclear translocation, compromised the expression of nuclear-encoded mitochondrial genes, and consequently led to cellular energy depletion. Moreover, the lungs of OLA1 knockout mice have fewer mitochondria, lower cellular ATP concentrations, and higher lactate concentrations. The ensuing mitochondrial metabolic dysfunction resulted in abnormal behaviors of pulmonary vascular cells and significant vascular remodeling. Our findings demonstrate that OLA1 is an important component of the mitochondrial retrograde communication pathways that couple stress signals with metabolic genes in the nucleus. Thus, phosphorylation-dependent nuclear OLA1 localization that governs cellular energy metabolism is critical to cardiovascular function.

Decreased Cyclic Guanosine Monophosphate-Protein Kinase G Signaling Impairs Angiogenesis in a Lamb Model of Persistent Pulmonary Hypertension of the Newborn.

Impaired angiogenesis function in pulmonary artery endothelial cells (PAEC) contributes to persistent pulmonary hypertension of the newborn (PPHN). Decreased nitric oxide (NO) amounts in PPHN lead to impaired mitochondrial biogenesis and angiogenesis in the lung; the mechanisms remain unclear. We hypothesized that decreased cyclic guanosine monophosphate (cGMP)-PKG (protein kinase G) signaling downstream of NO leads to decreased mitochondrial biogenesis and angiogenesis in PPHN. PPHN was induced by ductus arteriosus constriction from 128-136 days' gestation in fetal lambs. Control animals were gestation-matched lambs that did not undergo ductal constriction. PAEC isolated from PPHN lambs were treated with the sGC (soluble guanylate cyclase) activator cinaciguat, the PKG activator 8-bromo-cGMP, or the PDE-V (PDE type V) inhibitor sildenafil. Lysates were immunoblotted for mitochondrial transcription factors and electron transport chain C-I (complex I), C-II, C-III, C-IV, and C-V proteins. The in vitro angiogenesis of PAEC was evaluated by using tube-formation and scratch-recovery assays. cGMP concentrations were measured by using an enzyme immunoassay. Fetal lambs with ductal constriction were given sildenafil or control saline through continuous infusion in utero, and the lung histology, capillary counts, vessel density, and right ventricular pressure were assessed at birth. PPHN PAEC showed decreased mitochondrial transcription factor levels, electron transport chain protein levels, and in vitro tube formation and cell migration; these were restored by cinaciguat, 8-bromo-cGMP, and sildenafil. Cinaciguat and sildenafil increased cGMP concentrations in PPHN PAEC. Radial alveolar and capillary counts and vessel density were lower in PPHN lungs, and the right ventricular pressure and Fulton Index were higher in PPHN lungs; these were improved by in utero sildenafil infusion. cGMP-PKG signaling is a potential therapeutic target to restore decreased mitochondrial biogenesis and angiogenesis in PPHN.

AMP-Kinase Dysfunction Alters Notch Ligands to Impair Angiogenesis in Neonatal Pulmonary Hypertension.

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.

Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function.

Stress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at 445GERAMT450 Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity in vitro and in vivo To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu446 and Arg447 cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.

Attenuation of endoplasmic reticulum stress by caffeine ameliorates hyperoxia-induced lung injury.

Rodent pups exposed to hyperoxia develop lung changes similar to bronchopulmonary dysplasia (BPD) in extremely premature infants. Oxidative stress from hyperoxia can injure developing lungs through endoplasmic reticulum (ER) stress. Early caffeine treatment decreases the rate of BPD, but the mechanisms remain unclear. We hypothesized that caffeine attenuates hyperoxia-induced lung injury through its chemical chaperone property. Sprague-Dawley rat pups were raised either in 90 (hyperoxia) or 21% (normoxia) oxygen from postnatal day 1 (P1) to postnatal day 10 (P10) and then recovered in 21% oxygen until P21. Caffeine (20 mg/kg) or normal saline (control) was administered intraperitoneally daily starting from P2. Lungs were inflation-fixed for histology or snap-frozen for immunoblots. Blood caffeine levels were measured in treated pups at euthanasia and were found to be 18.4 ± 4.9 µg/ml. Hyperoxia impaired alveolar formation and increased ER stress markers and downstream effectors; caffeine treatment attenuated these changes at P10. Caffeine also attenuated the hyperoxia-induced activation of cyclooxygenase-2 and markers of apoptosis. In conclusion, hyperoxia-induced alveolar growth impairment is mediated, in part, by ER stress. Early caffeine treatment protects developing lungs from hyperoxia-induced injury by attenuating ER stress.

Nogo-B receptor deficiency increases liver X receptor alpha nuclear translocation and hepatic lipogenesis through an adenosine monophosphate-activated protein kinase alpha-dependent pathway.

Nogo-B receptor (NgBR) was identified as a specific receptor for binding Nogo-B and is essential for the stability of Niemann-Pick type C2 protein (NPC2) and NPC2-dependent cholesterol trafficking. Here, we report that NgBR expression levels decrease in the fatty liver and that NgBR plays previously unrecognized roles in regulating hepatic lipogenesis through NPC2-independent pathways. To further elucidate the pathophysiological role of NgBR in mammals, we generated NgBR liver-specific knockout mice and investigated the roles of NgBR in hepatic lipid homeostasis. The results showed that NgBR knockout in mouse liver did not decrease NPC2 levels or increase NPC2-dependent intracellular cholesterol levels. However, NgBR deficiency still resulted in remarkable cellular lipid accumulation that was associated with increased free fatty acids and triglycerides in hepatocytes in vitro and in mouse livers in vivo. Mechanistically, NgBR deficiency specifically promotes the nuclear translocation of the liver X receptor alpha (LXRα) and increases the expression of LXRα-targeted lipogenic genes. LXRα knockout attenuates the accumulation of free fatty acids and triglycerides caused by NgBR deficiency. In addition, we elucidated the mechanisms by which NgBR bridges the adenosine monophosphate-activated protein kinase alpha signaling pathway with LXRα nuclear translocation and LXRα-mediated lipogenesis. CONCLUSION: NgBR is a specific negative regulator for LXRα-dependent hepatic lipogenesis. Loss of NgBR may be a potential trigger for inducing hepatic steatosis. (Hepatology 2016;64:1559-1576).

Caffeine ameliorates hyperoxia-induced lung injury by protecting GCH1 function in neonatal rat pups.

BackgroundBronchopulmonary dysplasia (BPD) is a major morbidity in premature infants, and impaired angiogenesis is considered a major contributor to BPD. Early caffeine treatment decreases the incidence of BPD; the mechanism remains incompletely understood.MethodsSprague-Dawley rat pups exposed to normoxia or hyperoxia since birth were treated daily with either 20 mg/kg caffeine or normal saline by an intraperitoneal injection from day 2 of life. The lungs were obtained for studies at days 10 and 21.ResultsHyperoxia impaired somatic growth and lung growth in the rat pups. The impaired lung growth during hyperoxia was associated with decreased levels of cyclic AMP (cAMP) and tetrahydrobiopterin (BH4) in the lungs. Early caffeine treatment increased cAMP levels in the lungs of hyperoxia-exposed pups. Caffeine also increased the levels of phosphorylated endothelial nitric oxide synthase (eNOS) at serine1177, total and serine51 phosphorylated GTP cyclohydrolase 1 (GCH1), and BH4 levels, with improved alveolar structure and angiogenesis in hyperoxia-exposed lungs. Reduced GCH1 levels in hyperoxia were due, in part, to increased degradation by the ubiquitin-proteasome system.ConclusionOur data support the notion that early caffeine treatment can protect immature lungs from hyperoxia-induced damage by improving eNOS activity through increased BH4 bioavailability.

Nogo-B Receptor Modulates Pulmonary Artery Smooth Muscle Cell Function in Developing Lungs.

Nogo-B and its receptor (NgBR) are involved in blood vessel growth in developing lungs, but their role in pulmonary artery smooth muscle cell (PASMC) growth is unknown. We hypothesized that NgBR regulates growth of PASMCs by modulating the function of endoplasmic reticulum (ER) and formation of reactive oxygen species (ROS). In utero constriction of the ductus arteriosus created pulmonary hypertension in fetal lambs (hypertensive fetal lamb [HTFL]). PASMCs isolated 8 days after surgery were assessed for the alteration of protein levels by immunoblots and ROS formation by dihydroethidium and Cell ROX deep red fluorescence. NgBR small interfering RNA and plasmid DNA were used to manipulate NgBR levels. Proliferation and wound healing were assessed by cell counts and scratch recovery assay, respectively. Acute ER stress was induced by tunicamycin. Differences of mitogen-activated protein kinase and Akt pathway activation in HTFL versus control PASMCs were evaluated. Results showed that HTFL PASMCs had decreased NgBR levels and increased proliferation, wound healing, ER stress, and ROS formation compared with controls. Knockdown of NgBR in control PASMCs generated a phenotype similar to HTFL, and overexpression in HTFL restored the defective phenotype to control. Decreased NgBR levels were associated with increased ROS formation in HTFL PASMCs. Subsequently, scavenging ROS decreased proliferation and wound healing. Mechanistically, ROS formation decreases NgBR expression, which induces ER stress. This leads to extracellular signal-regulated kinase pathway activation and PASMC phenotype alteration. Our data suggest that decreased NgBR expression in pulmonary hypertension of the newborn contributes to increased PASMC proliferation and oxidative stress, which lead to the pathogenesis of lung injury.

Nitrotyrosine impairs mitochondrial function in fetal lamb pulmonary artery endothelial cells.

Nitration of both protein-bound and free tyrosine by reactive nitrogen species results in the formation of nitrotyrosine (NT). We previously reported that free NT impairs microtubule polymerization and uncouples endothelial nitric oxide synthase (eNOS) function in pulmonary artery endothelial cells (PAEC). Because microtubules modulate mitochondrial function, we hypothesized that increased NT levels during inflammation and oxidative stress will lead to mitochondrial dysfunction in PAEC. PAEC isolated from fetal lambs were exposed to varying concentrations of free NT. At low concentrations (1-10 µM), NT increased nitration of mitochondrial electron transport chain (ETC) protein subunit complexes I-V and state III oxygen consumption. Higher concentrations of NT (50 µM) caused decreased microtubule acetylation, impaired eNOS interactions with mitochondria, and decreased ETC protein levels. We also observed increases in heat shock protein-90 nitration, mitochondrial superoxide formation, and fragmentation of mitochondria in PAEC. Our data suggest that free NT accumulation may impair microtubule polymerization and exacerbate reactive oxygen species-induced cell damage by causing mitochondrial dysfunction.

Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension.

Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2 , partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen.

Interaction of endothelial nitric oxide synthase with mitochondria regulates oxidative stress and function in fetal pulmonary artery endothelial cells.

An increase in oxygen tension at birth is one of the key signals that initiate pulmonary vasodilation in the fetal lung. We investigated the hypothesis that targeting endothelial nitric oxide synthase (eNOS) to the mitochondrial outer membrane regulates reactive oxygen species (ROS) formation in the fetal pulmonary artery endothelial cells (PAEC) during this transition. We isolated PAEC and pulmonary arteries from 137-day gestation fetal lambs (term = 144 days). We exposed PAEC to a simulated transition from fetal to (3% O2) to normoxic (21%) or hyperoxic (95% O2) postnatal Po2 or to the nitric oxide synthase (NOS) agonist ATP. We assessed the effect of O2 and ATP on eNOS interactions with the mitochondrial outer membrane protein porin and with the chaperone hsp90. We also investigated the effect of decoy peptides that blocked eNOS interactions with porin or hsp90 on PAEC angiogenesis and vasodilator function of pulmonary arteries. Transition of fetal PAEC from 3 to 21% O2 but not to 95% O2 or exposure to ATP increased eNOS association with hsp90 and porin. Decoy peptides that blocked eNOS interactions decreased NO release, increased O2 consumption and mitochondrial ROS levels, and impaired PAEC angiogenesis. Decoy peptides also inhibited the relaxation responses of pulmonary artery rings and dilation of resistance size pulmonary arteries to ATP. The mitochondrial-antioxidant mito-ubiquinone restored the response to ATP in decoy peptide-treated pulmonary arteries. These data indicate that targeting eNOS to mitochondria decreases endothelial oxidative stress and facilitates vasodilation in fetal pulmonary circulation at birth.

Altered prostanoid metabolism contributes to impaired angiogenesis in persistent pulmonary hypertension in a fetal lamb model.

BACKGROUND: Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased lung angiogenesis and impaired pulmonary vasodilatation at birth. Prostanoids are important modulators of vascular tone and angiogenesis. We hypothesized that altered levels of prostacyclin (PGI2), a potent vasodilator, and thromboxane A2 (TXA2), a vasoconstrictor, contribute to impaired angiogenesis of pulmonary artery endothelial cells (PAEC) in PPHN. METHODS: PAEC were isolated from fetal lambs with PPHN induced by prenatal ductus arteriosus constriction or from sham operated controls. Expression and activity of PGI2 synthase (PGIS) and TXA2 synthase (TXAS), expression of cyclooxygenases 1 and 2 (COX-1 and COX-2), and the role of PGIS/TXAS alterations in angiogenesis were investigated in PAEC from PPHN and control lambs. RESULTS: PGIS protein and activity were decreased and PGIS protein tyrosine nitration was increased in PPHN PAEC. In contrast, TXAS protein and its stimulated activity were increased in PPHN PAEC. COX-1 and COX-2 proteins were decreased in PPHN PAEC. Addition of PGI2 improved in vitro tube formation by PPHN PAEC, whereas indomethacin decreased tube formation by control PAEC. PGIS knockdown decreased the in vitro angiogenesis in control PAEC, whereas TXAS knockdown increased the in vitro angiogenesis in PPHN PAEC. CONCLUSION: Reciprocal alterations in PGI2 and TXA2 may contribute to impaired angiogenesis in PPHN.

Cord blood level of insulin-like growth factor-1 and IGF binding protein-3 in monochorionic twins.

BACKGROUND/PURPOSE: Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are known to modulate fetal growth but their role in intrauterine growth of monochorionic twins (MCT) has not been studied. METHODS: Cord venous blood was collected directly after birth. IGF-1 and IGFBP-3 in the cord venous blood were quantified by radioimmunoassay. Birth weights (BWs) were obtained electronically. Placentas were examined for chorionicity. RESULTS: Cord blood was collected in 37 pairs of MCT (15 pairs were males). BWs ranged from 564 to 3240 g, and gestational ages (GAs) were between 24 weeks and 39 weeks. There was a correlation between BW and cord venous blood IGFBP-3 concentration (r = 0.28, p = 0.015), but not between BW and cord venous blood IGF-1 level. There was no difference in IGF-1 between the heavier twins (30.8 ± 61.8 ng/mL) and lighter twins (33.2 ± 63.7 ng/mL), but a trend (p = 0.096) of higher IGFBP-3 level was demonstrated in heavier twins (3.14 ± 1.23 µg/mL) than in lighter twins (2.71 ± 1.19 µg/mL). The IGFBP-3 levels were higher (p = 0.042) in female twins (3.20 ± 1.33 µg/mL) than in male twins (2.64 ± 1.04 µg/mL). The IGF-1 level of the heavier twins correlated significantly to their lighter co-twin (r = 0.73, p < 0.001). CONCLUSION: Our data showed that cord venous blood IGF-1 level might be controlled mainly by genetic factors. IGFBP-3 might play an important role in fetal growth.

Efficacy of recombinant human granulocyte colony stimulating factor in very-low-birth-weight infants with early neutropenia.

BACKGROUND/PURPOSE: Neutropenia is a risk factor for nosocomial infections (NI) in very-low-birth-weight (VLBW) infants. Although recombinant human granulocyte colony stimulating factor (rhG-CSF) increases the neutrophil counts in neutropenic VLBW infants, its long-term efficacy for early neutropenia (EN) remains unknown. METHODS: In this case-controlled study, charts of VLBW recipients of rhG-CSF for EN (total neutrophil count <1.5 × 10(9)/L during first 7 days) were reviewed and compared to gestational age, total neutrophil count, and birth weight matched infants unexposed to rhG-CSF. RESULTS: Twenty-seven infants were identified in each group. Mortality and morbidity did not differ between the two groups. Rate of NI (16/27 vs. 4/27, p = 0.002, odds ratio = 8.36) as well as the total number of episodes of NI (22 vs. 4, p = 0.007) were higher in rhG-CSF (+) group than in the rhG-CSF (-) group. CONCLUSION: Our experience does not show benefit in empirical use of rhG-CSF in preventing NI in VLBW infants with EN.

Nogo-B receptor modulates angiogenesis response of pulmonary artery endothelial cells through eNOS coupling.

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.

Inducible HSP70 regulates superoxide dismutase-2 and mitochondrial oxidative stress in the endothelial cells from developing lungs.

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.