Hyperoxia-induced airflow restriction and Renin-Angiotensin System expression in a bronchopulmonary dysplasia mouse model.

Mechanisms underlying hyperoxia-induced airflow restriction in the pediatric lung disease Bronchopulmonary dysplasia (BPD) are unclear. We hypothesized a role for Renin-Angiotensin System (RAS) activity in BPD. RAS is comprised of a pro-developmental pathway consisting of angiotensin converting enzyme-2 (ACE2) and angiotensin II receptor type 2 (AT2), and a pro-fibrotic pathway mediated by angiotensin II receptor type 1 (AT1). We investigated associations between neonatal hyperoxia, airflow restriction, and RAS activity in a BPD mouse model. C57 mouse pups were randomized to normoxic (FiO2 = 0.21) or hyperoxic (FiO2 = 0.75) conditions for 15 days (P1-P15). At P15, P20, and P30, we measured airflow restriction using plethysmography and ACE2, AT1, and AT2 mRNA and protein expression via polymerase chain reaction and Western Blot. Hyperoxia increased airflow restriction P15 and P20, decreased ACE2 and AT2 mRNA, decreased AT2 protein, and increased AT1 protein expression. ACE2 mRNA and protein remained suppressed at P20. By P30, airflow restriction and RAS expression did not differ between groups. Hyperoxia caused high airflow restriction, increased pulmonary expression of the pro-fibrotic RAS pathway, and decreased expression of the pro-developmental in our BPD mouse model. These associated findings may point to a causal role for RAS in hyperoxia-induced airflow restriction.

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.

Endothelial Adenosine Monophosphate-Activated Protein Kinase-Alpha1 Deficiency Potentiates Hyperoxia-Induced Experimental Bronchopulmonary Dysplasia and Pulmonary Hypertension.

Bronchopulmonary dysplasia and pulmonary hypertension, or BPD-PH, are serious chronic lung disorders of prematurity, without curative therapies. Hyperoxia, a known causative factor of BPD-PH, activates adenosine monophosphate-activated protein kinase (AMPK) α1 in neonatal murine lungs; however, whether this phenomenon potentiates or mitigates lung injury is unclear. Thus, we hypothesized that (1) endothelial AMPKα1 is necessary to protect neonatal mice against hyperoxia-induced BPD-PH, and (2) AMPKα1 knockdown decreases angiogenesis in hyperoxia-exposed neonatal human pulmonary microvascular endothelial cells (HPMECs). We performed lung morphometric and echocardiographic studies on postnatal day (P) 28 on endothelial AMPKα1-sufficient and -deficient mice exposed to 21% O2 (normoxia) or 70% O2 (hyperoxia) from P1-P14. We also performed tubule formation assays on control- or AMPKα1-siRNA transfected HPMECs, exposed to 21% O2 or 70% O2 for 48 h. Hyperoxia-mediated alveolar and pulmonary vascular simplification, pulmonary vascular remodeling, and PH were significantly amplified in endothelial AMPKα1-deficient mice. AMPKα1 siRNA knocked down AMPKα1 expression in HPMECs, and decreased their ability to form tubules in normoxia and hyperoxia. Furthermore, AMPKα1 knockdown decreased proliferating cell nuclear antigen expression in hyperoxic conditions. Our results indicate that AMPKα1 is required to reduce hyperoxia-induced BPD-PH burden in neonatal mice, and promotes angiogenesis in HPMECs to limit lung injury.

ProLung™-budesonide Inhibits SARS-CoV-2 Replication and Reduces Lung Inflammation.

BACKGROUND: Inhaled budesonide benefits patients with COVID-19. ProLung™-budesonide enables the sustained, low dose administration of budesonide within a delivery vehicle similar to lung surfactant. ProLung™-budesonide may offer anti-inflammatory and protective effects to the lung in COVID-19, yet it's effect on SARS-CoV-2 replication is unknown. OBJECTIVE: To determine the efficacy of ProLung™-budesonide against SARS-CoV-2-infection in vitro, evaluate its ability to decrease inflammation, and airway hyperresponsiveness in an animal model of lung inflammation. METHODS: SARS-CoV-2-infected Vero 76 cells were treated with ProLung™-budesonide ([0.03-100 µg/ml]) for 3 days, and virus yield in the supernatant was measured. Ovalbumin-sensitized C57BL/6 mice received aerosolized (a) ProLung™-budesonide weekly, (b) only budesonide, either daily or weekly, or (c) weekly empty ProLung™ carrier (without budesonide). All treatment groups were compared to sensitized untreated, or normal mice using histopathologic examination, electron microscopy (EM), airway hyperresponsiveness (AHR) to Methacholine (Mch) challenge, and eosinophil peroxidase activity (EPO) measurements in bronchioalveolar lavage (BAL). RESULTS: ProLung™-budesonide showed significant inhibition of viral replication of SARS-CoV-2-infected cells with the selectivity index (SI) value >24. Weekly ProLung™-budesonide and daily budesonide therapy significantly decreased lung inflammation and EPO in BAL. ProLung™-budesonide localized in type II pneumocytes, and was the only group to significantly decrease AHR, and EPO in BAL with Mch challenge. CONCLUSIONS: ProLung™-budesonide significantly inhibited viral replication in SARS-CoV-2-infected cells. It localized into type II pneumocytes, decreased lung inflammation, AHR and EPO activity with Mch challenge. This novel drug formulation may offer a potential inhalational treatment for COVID-19.

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.

Pediatric Pulmonary Hypertension: Definitions, Mechanisms, Diagnosis, and Treatment.

Pediatric pulmonary hypertension (PPH) is a multifactorial disease with diverse etiologies and presenting features. Pulmonary hypertension (PH), defined as elevated pulmonary artery pressure, is the presenting feature for several pulmonary vascular diseases. It is often a hidden component of other lung diseases, such as cystic fibrosis and bronchopulmonary dysplasia. Alterations in lung development and genetic conditions are an important contributor to pediatric pulmonary hypertensive disease, which is a distinct entity from adult PH. Many of the causes of pediatric PH have prenatal onset with altered lung development due to maternal and fetal conditions. Since lung growth is altered in several conditions that lead to PPH, therapy for PPH includes both pulmonary vasodilators and strategies to restore lung growth. These strategies include optimal alveolar recruitment, maintaining physiologic blood gas tension, nutritional support, and addressing contributing factors, such as airway disease and gastroesophageal reflux. The outcome for infants and children with PH is highly variable and largely dependent on the underlying cause. The best outcomes are for neonates with persistent pulmonary hypertension (PPHN) and reversible lung diseases, while some genetic conditions such as alveolar capillary dysplasia are lethal. © 2021 American Physiological Society. Compr Physiol 11:2135-2190, 2021.

Perinatal Hypoxemia and Oxygen Sensing.

The development of the control of breathing begins in utero and continues postnatally. Fetal breathing movements are needed for establishing connectivity between the lungs and central mechanisms controlling breathing. Maturation of the control of breathing, including the increase of hypoxia chemosensitivity, continues postnatally. Insufficient oxygenation, or hypoxia, is a major stressor that can manifest for different reasons in the fetus and neonate. Though the fetus and neonate have different hypoxia sensing mechanisms and respond differently to acute hypoxia, both responses prevent deviations to respiratory and other developmental processes. Intermittent and chronic hypoxia pose much greater threats to the normal developmental respiratory processes. Gestational intermittent hypoxia, due to maternal sleep-disordered breathing and sleep apnea, increases eupneic breathing and decreases the hypoxic ventilatory response associated with impaired gasping and autoresuscitation postnatally. Chronic fetal hypoxia, due to biologic or environmental (i.e. high-altitude) factors, is implicated in fetal growth restriction and preterm birth causing a decrease in the postnatal hypoxic ventilatory responses with increases in irregular eupneic breathing. Mechanisms driving these changes include delayed chemoreceptor development, catecholaminergic activity, abnormal myelination, increased astrocyte proliferation in the dorsal respiratory group, among others. Long-term high-altitude residents demonstrate favorable adaptations to chronic hypoxia as do their offspring. Neonatal intermittent hypoxia is common among preterm infants due to immature respiratory systems and thus, display a reduced drive to breathe and apneas due to insufficient hypoxic sensitivity. However, ongoing intermittent hypoxia can enhance hypoxic sensitivity causing ventilatory overshoots followed by apnea; the number of apneas is positively correlated with degree of hypoxic sensitivity in preterm infants. Chronic neonatal hypoxia may arise from fetal complications like maternal smoking or from postnatal cardiovascular problems, causing blunting of the hypoxic ventilatory responses throughout at least adolescence due to attenuation of carotid body fibers responses to hypoxia with potential roles of brainstem serotonin, microglia, and inflammation, though these effects depend on the age in which chronic hypoxia initiates. Fetal and neonatal intermittent and chronic hypoxia are implicated in preterm birth and complicate the respiratory system through their direct effects on hypoxia sensing mechanisms and interruptions to the normal developmental processes. Thus, precise regulation of oxygen homeostasis is crucial for normal development of the respiratory control network. © 2021 American Physiological Society. Compr Physiol 11:1653-1677, 2021.

N-acetyl-lysyltyrosylcysteine amide, a novel systems pharmacology agent, reduces bronchopulmonary dysplasia in hyperoxic neonatal rat pups.

Bronchopulmonary dysplasia (BPD) is caused primarily by oxidative stress and inflammation. To induce BPD, neonatal rat pups were raised in hyperoxic (>90% O2) environments from day one (P1) until day ten (P10) and treated with N-acetyl-lysyltyrosylcysteine amide (KYC). In vivo studies showed that KYC improved lung complexity, reduced myeloperoxidase (MPO) positive (+) myeloid cell counts, MPO protein, chlorotyrosine formation, increased endothelial cell CD31 expression, decreased 8-OH-dG and Cox-1/Cox-2, HMGB1, RAGE, TLR4, increased weight gain and improved survival in hyperoxic pups. EPR studies confirmed that MPO reaction mixtures oxidized KYC to a KYC thiyl radical. Adding recombinant HMGB1 to the MPO reaction mixture containing KYC resulted in KYC thiylation of HMGB1. In rat lung microvascular endothelial cell (RLMVEC) cultures, KYC thiylation of RLMVEC proteins was increased the most in RLMVEC cultures treated with MPO + H2O2, followed by H2O2, and then KYC alone. KYC treatment of hyperoxic pups decreased total HMGB1 in lung lysates, increased KYC thiylation of HMGB1, terminal HMGB1 thiol oxidation, decreased HMGB1 association with TLR4 and RAGE, and shifted HMGB1 in lung lysates from a non-acetylated to a lysyl-acetylated isoform, suggesting that KYC reduced lung cell death and that recruited immune cells had become the primary source of HMGB1 released into the hyperoxic lungs. MPO-dependent and independent KYC-thiylation of Keap1 were both increased in RLMVEC cultures. Treating hyperoxic pups with KYC increased KYC thiylation and S-glutathionylation of Keap1, and Nrf2 activation. These data suggest that KYC is a novel system pharmacological agent that exploits MPO to inhibit toxic oxidant production and is oxidized into a thiyl radical that inactivates HMGB1, activates Nrf2, and increases antioxidant enzyme expression to improve lung complexity and reduce BPD in hyperoxic rat pups.

Surf early to higher tides: surfactant therapy to optimize tidal volume, lung recruitment, and iNO response.

Inhaled nitric oxide is approved by FDA for the management of hypoxemic respiratory failure in term and near-term infants. However, approximately a third of patients treated with inhaled nitric oxide fail to have a sustained improvement in oxygenation. Recruitment of the lung with surfactant enables optimal delivery of nitric oxide to the alveolar space leading to effective pulmonary vasodilation.

Decreased AMP-activated protein kinase (AMPK) function and protective effect of metformin in neonatal rat pups exposed to hyperoxia lung injury.

We investigated the hypothesis that exposure of lungs at the saccular stage of development to hyperoxia leads to persistent growth arrest and dysfunction of 5'AMP-activated protein kinase (AMPK), a key energy sensor in the cell. We exposed neonatal rat pups from postnatal day 1- day 10 (P1-P10) to ≥90% oxygen or control normoxia. Pups were euthanized at P4 or P10 or recovered in normoxia until euthanasia at P21. Half of the pups in each group received AMPK activator, metformin, or saline intraperitoneally from P1 to P10. Lung histology, morphometric analysis, immunofluorescence, and immunoblots were done for changes in lung structure at P10 and P21 and AMPK function at P4, P10, and P21. Phosphorylation of AMPK (p-AMPK) was decreased in lungs at P10 and P21 in hyperoxia-exposed pups. Metformin increased the levels of p-AMPK and PGC-1α, a downstream AMPK target which regulates mitochondrial biogenesis, at P4, P10, and P21 in hyperoxia pups. Lung ATP levels decreased during hyperoxia and were increased by metformin at P10 and P21. Radial alveolar count and alveolar septal tips were decreased and mean linear intercept increased in hyperoxia-exposed pups at P10 and the changes persisted at P21; these were improved by metformin. Lung capillary number was decreased in hyperoxia-exposed pups at P10 and P21 and was restored by metformin. Hyperoxia leads to impaired AMPK function, energy balance and alveolar simplification. The AMPK activator, metformin improves AMPK function and alveolar and vascular growth in this rat pup model of hyperoxia-induced lung injury.

Development of in-house genetic screening for pediatric hearing loss.

OBJECTIVES: To evaluate the efficiency of in-house genetic testing for mutations causing the most common types of inherited, nonsyndromic, sensorineural hearing loss (SNHL). METHODS: Retrospective cohort study of 200 patients at a single, pediatric medical center with suspected or confirmed hearing loss who underwent either send out vs in-house genetic testing for mutations in GJB2/GJB6, SLC26A4, and MTRNR1. Primary outcome measure was the difference in mean turnaround time for send-out vs in-house genetic testing. Additional outcomes included associations between audiometric findings and genetic test results. RESULTS: One hundred four send-out tests were performed between October 2010 and June 2014, and 100 in-house tests were performed between November 2014 and November 2016. The mean turnaround time for send-out testing was 53.7 days. The mean turnaround time for in-house testing was 18.9 days. This difference was statistically significant (P < .001). The largest component of turnaround time was the amount of time elapsed between receipt of specimen in the lab and final test result. These intervals were 47.0 and 18.3 days for send-out and in-house tests, respectively. Notably, the longest turnaround time for in-house testing (43 days) was less than the average turnaround time for send-out testing. In addition, we identified two simple audiometric parameters (ie, bilateral newborn hearing screen referral and audiometry showing symmetric SNHL) that may increase diagnostic yield of genetic testing. CONCLUSIONS: The development of in-house genetic testing programs for inherited SNHL can significantly reduce testing turnaround times. Newborn hearing screening and audiometry results can help clinicians identify patients most likely to benefit from genetic testing. LEVEL OF EVIDENCE: IV.

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.

Decreased OLA1 (Obg-Like ATPase-1) Expression Drives Ubiquitin-Proteasome Pathways to Downregulate Mitochondrial SOD2 (Superoxide Dismutase) in Persistent Pulmonary Hypertension of the Newborn.

Persistent pulmonary hypertension of the newborn (PPHN) is a failure of pulmonary vascular resistance to decline at birth rapidly. One principal mechanism implicated in PPHN development is mitochondrial oxidative stress. Expression and activity of mitochondrial SOD2 (superoxide dismutase) are decreased in PPHN; however, the mechanism remains unknown. Recently, OLA1 (Obg-like ATPase-1) was shown to act as a critical regulator of proteins controlling cell response to stress including Hsp70, an obligate chaperone for SOD2. Here, we investigated whether OLA1 is causally linked to PPHN. Compared with controls, SOD2 expression is reduced in distal-pulmonary arteries (PAs) from patients with PPHN and fetal-lamb models. Disruptions of the SOD2 gene reproduced PPHN phenotypes, manifested by elevated right ventricular systolic pressure, PA-endothelial cells apoptosis, and PA-smooth muscle cells proliferation. Analyses of SOD2 protein dynamics revealed higher ubiquitinated-SOD2 protein levels in PPHN-lambs, suggesting dysregulated protein ubiquitination. OLA1 controls multiple proteostatic mechanisms and is overexpressed in response to stress. We demonstrated that OLA1 acts as a molecular chaperone, and its activity is induced by stress. Strikingly, OLA1 expression is decreased in distal-PAs from PPHN-patients and fetal-lambs. OLA1 deficiency enhanced CHIP affinity for Hsp70-SOD2 complexes, facilitating SOD2 degradation. Consequently, mitochondrial H2O2 formation is impaired, leading to XIAP (X-linked inhibitor of apoptosis) overexpression that suppresses caspase activity in PA-smooth muscle cells, allowing them to survive and proliferate, contributing to PA remodeling. In-vivo, ola1-/- downregulated SOD2 expression, induced distal-PA remodeling, and right ventricular hypertrophy. We conclude that decreased OLA1 expression accounts for SOD2 downregulation and, therefore, a therapeutic target in PPHN treatments.

Adverse early-life environment impairs postnatal lung development in mice.

BACKGROUND: Fetal growth restriction (FGR) is a major risk factor for bronchopulmonary dysplasia (BPD). Maternal stress and poor diet are linked to FGR. Effect of perinatal stress on lung development remains unknown. OBJECTIVE: Using a murine model of adverse early life environment (AELE), we hypothesized that maternal exposure to perinatal environmental stress and high-fat diet (Western diet) lead to impaired lung development in the offspring. METHODS: Female mice were placed on either control diet or Western diet before conception. Those exposed to Western diet were also exposed to perinatal environmental stress, the combination referred to as AELE. Pups were either euthanized at postnatal day 21 (P21) or weaned to control diet and environment until adulthood (8-14 wk old). Lungs were harvested for histology, gene expression by quantitative RT-PCR, microRNA profiling, and immunoblotting. RESULTS: AELE increased the mean linear intercept and decreased the radial alveolar count and secondary septation in P21 and adult mice. Capillary count was also decreased in P21 and adult mice. AELE lungs had decreased vascular endothelial growth factor A (VEGFA), VEGF receptor 2, endothelial nitric oxide synthase, and hypoxia inducible factor-1α protein levels and increased expression of genes that regulate DNA methylation and upregulation of microRNAs that target genes involved in lung development at P21. CONCLUSION: AELE leads to impaired lung alveolar and vascular growth, which persists into adult age despite normalizing the diet and environment at P21. AELE also alters the expression of genes involved in lung remodeling.

Altered hypoxia-inducible factor-1α (HIF-1α) signaling contributes to impaired angiogenesis in fetal lambs with persistent pulmonary hypertension of the newborn (PPHN).

Previous studies in adult pulmonary hypertension reported that increased hypoxia-inducible factor-1α (HIF-1α) signaling contributes to pulmonary vascular remodeling. However, alterations in endothelial HIF-1α signaling and its contribution to impaired angiogenesis in persistent pulmonary hypertension of the newborn (PPHN) remain unclear. We investigated the hypothesis that HIF-1α levels are increased in lung endothelial cells in PPHN and contribute to impaired angiogenesis function. We examined HIF-1α expression and promoter activity in the isolated pulmonary artery endothelial cells (PAEC) from fetal lambs with or without PPHN induced by prenatal ductus arteriosus constriction. We measured the levels of HIF-1α downstream targets, vascular endothelial growth factor (VEGF) and glycolytic protein, hexokinase 2 (Hek-2) in PAEC from PPHN, and control lambs. We examined the effect of small interfering-RNA (siRNA) mediated knockdown of native HIF-1α on VEGF expression and in vitro angiogenesis function of PPHN-PAEC. HIF-1α protein levels were higher in the isolated PAEC from PPHN-lambs compared to controls. HIF-1α promoter activity and Hek-2 protein levels were higher in PPHN. VEGF protein levels and in vitro angiogenesis function were decreased in PAEC from PPHN lambs. HIF-1α silencing significantly increased the expression of VEGF and improved the angiogenesis function of PPHN PAEC. Aberrant HIF-1α signaling contributes to endothelial dysfunction and decreased angiogenesis in PPHN.

Dynamic Phosphorylation of the C Terminus of Hsp70 Regulates the Mitochondrial Import of SOD2 and Redox Balance.

The import of superoxide dismutase-2 (SOD2) into mitochondria is vital for the survival of eukaryotic cells. SOD2 is encoded within the nuclear genome and translocated into mitochondria for activation after translation in the cytosol. The molecular chaperone Hsp70 modulates SOD2 activity by promoting import of SOD2 into mitochondria. In turn, the activity of Hsp70 is controlled by co-chaperones, particularly CHIP, which directs Hsp70-bound proteins for degradation in the proteasomes. We investigated the mechanisms controlling the activity of SOD2 to signal activation and maintain mitochondrial redox balance. We demonstrate that Akt1 binds to and phosphorylates the C terminus of Hsp70 on Serine631, which inhibits CHIP-mediated SOD2 degradation thereby stabilizing and promoting SOD2 import. Conversely, increased mitochondrial-H2O2 formation disrupts Akt1-mediated phosphorylation of Hsp70, and non-phosphorylatable Hsp70 mutants decrease SOD2 import, resulting in mitochondrial oxidative stress. Our findings identify Hsp70 phosphorylation as a physiological mechanism essential for regulation of mitochondrial redox balance.

Persistent pulmonary hypertension alters the epigenetic characteristics of endothelial nitric oxide synthase gene in pulmonary artery endothelial cells in a fetal lamb model.

Decreased expression of endothelial nitric oxide synthase (eNOS), a key mediator of perinatal transition, characterizes persistent pulmonary hypertension of the newborn (PPHN) in neonates and a fetal lamb model; the mechanisms are unclear. We investigated whether increased DNA CpG methylation at the eNOS promoter in estrogen response elements (EREs) and altered histone code together contribute to decreased eNOS expression in PPHN. We isolated pulmonary artery endothelial cells (PAEC) from fetal lambs with PPHN induced by prenatal ductus arteriosus constriction from 128 to 136 days gestation or gestation-matched twin controls. We measured right ventricular systolic pressure (RVSP) and Fulton index and determined eNOS expression in PAEC in control and PPHN lambs. We determined DNA CpG methylation by pyrosequencing and activity of ten eleven translocase demethylases (TET) by colorimetric assay. We quantified the occupancy of transcription factors, specificity protein 1 (Sp1), and estrogen receptors and density of four histone marks around Sp1 binding sites by chromatin immunoprecipitation (ChIP) assays. Fetal lambs with PPHN developed increased RVSP and Fulton index. Levels of eNOS mRNA and protein were decreased in PAEC from PPHN lambs. PPHN significantly increased the DNA CpG methylation in eNOS promoter and decreased TET activity in PAEC. PPHN decreased Sp1 occupancy and density of the active mark, lysine 12 acetylation of histone 4, and increased density of the repression mark, lysine 9 trimethylation of histone 3 around Sp1 binding sites in eNOS promoter. These results suggest that epigenetic modifications are primed to decrease Sp1 binding at the eNOS gene promoter in PPHN.

Effects of neonatal hyperoxia on the critical period of postnatal development of neurochemical expressions in brain stem respiratory-related nuclei in the rat.

We have identified a critical period of respiratory development in rats at postnatal days P12-13, when inhibitory influence dominates and when the response to hypoxia is at its weakest. This critical period has significant implications for Sudden Infant Death Syndrome (SIDS), the cause of which remains elusive. One of the known risk factors for SIDS is prematurity. A common intervention used in premature infants is hyperoxic therapy, which, if prolonged, can alter the ventilatory response to hypoxia and induce sustained inhibition of lung alveolar growth and pulmonary remodeling. The goal of this study was to test our hypothesis that neonatal hyperoxia from postnatal day (P) 0 to P10 in rat pups perturbs the critical period by altering the normal progression of neurochemical development in brain stem respiratory-related nuclei. An in-depth, semiquantitative immunohistochemical study was undertaken at P10 (immediately after hyperoxia and before the critical period), P12 (during the critical period), P14 (immediately after the critical period), and P17 (a week after the cessation of hyperoxia). In agreement with our previous findings, levels of cytochrome oxidase, brain-derived neurotrophic factor (BDNF), TrkB (BDNF receptor), and several serotonergic proteins (5-HT1A and 2A receptors, 5-HT synthesizing enzyme tryptophan hydroxylase [TPH], and serotonin transporter [SERT]) all fell in several brain stem respiratory-related nuclei during the critical period (P12) in control animals. However, in hyperoxic animals, these neurochemicals exhibited a significant fall at P14 instead. Thus, neonatal hyperoxia delayed but did not eliminate the critical period of postnatal development in multiple brain stem respiratory-related nuclei, with little effect on the nonrespiratory cuneate nucleus.