Identification of endothelial and mesenchymal FOXF1 enhancers involved in alveolar capillary dysplasia.

Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease.

Endothelial progenitor cells stimulate neonatal lung angiogenesis through FOXF1-mediated activation of BMP9/ACVRL1 signaling.

Pulmonary endothelial progenitor cells (EPCs) are critical for neonatal lung angiogenesis and represent a subset of general capillary cells (gCAPs). Molecular mechanisms through which EPCs stimulate lung angiogenesis are unknown. Herein, we used single-cell RNA sequencing to identify the BMP9/ACVRL1/SMAD1 pathway signature in pulmonary EPCs. BMP9 receptor, ACVRL1, and its downstream target genes were inhibited in EPCs from Foxf1WT/S52F mutant mice, a model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Expression of ACVRL1 and its targets were reduced in lungs of ACDMPV subjects. Inhibition of FOXF1 transcription factor reduced BMP9/ACVRL1 signaling and decreased angiogenesis in vitro. FOXF1 synergized with ETS transcription factor FLI1 to activate ACVRL1 promoter. Nanoparticle-mediated silencing of ACVRL1 in newborn mice decreased neonatal lung angiogenesis and alveolarization. Treatment with BMP9 restored lung angiogenesis and alveolarization in ACVRL1-deficient and Foxf1WT/S52F mice. Altogether, EPCs promote neonatal lung angiogenesis and alveolarization through FOXF1-mediated activation of BMP9/ACVRL1 signaling.

Perturbation of semaphorin and VEGF signaling in ACDMPV lungs due to FOXF1 deficiency.

BACKGROUND: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal congenital lung disorder in neonates characterized by severe progressive respiratory failure and refractory pulmonary hypertension, resulting from underdevelopment of the peripheral pulmonary tree. Causative heterozygous single nucleotide variants (SNVs) or copy-number variant (CNV) deletions involving FOXF1 or its distant lung-specific enhancer on chromosome 16q24.1 have been identified in 80-90% of ACDMPV patients. FOXF1 maps closely to and regulates the oppositely oriented FENDRR, with which it also shares regulatory elements. METHODS: To better understand the transcriptional networks downstream of FOXF1 that are relevant for lung organogenesis, using RNA-seq, we have examined lung transcriptomes in 12 histopathologically verified ACDMPV patients with or without pathogenic variants in the FOXF1 locus and analyzed gene expression profile in FENDRR-depleted fetal lung fibroblasts, IMR-90. RESULTS: RNA-seq analyses in ACDMPV neonates revealed changes in the expression of several genes, including semaphorins (SEMAs), neuropilin 1 (NRP1), and plexins (PLXNs), essential for both epithelial branching and vascular patterning. In addition, we have found deregulation of the vascular endothelial growth factor (VEGF) signaling that also controls pulmonary vasculogenesis and a lung-specific endothelial gene TMEM100 known to be essential in vascular morphogenesis. Interestingly, we have observed a substantial difference in gene expression profiles between the ACDMPV samples with different types of FOXF1 defect. Moreover, partial overlap between transcriptome profiles of ACDMPV lungs with FOXF1 SNVs and FENDRR-depleted IMR-90 cells suggests contribution of FENDRR to ACDMPV etiology. CONCLUSIONS: Our transcriptomic data imply potential crosstalk between several lung developmental pathways, including interactions between FOXF1-SHH and SEMA-NRP or VEGF/VEGFR2 signaling, and provide further insight into complexity of lung organogenesis in humans.

Lung-specific distant enhancer cis regulates expression of FOXF1 and lncRNA FENDRR.

The FOXF1 gene, causative for a neonatal lethal lung developmental disorder alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), maps 1.7 kb away from the long noncoding RNA gene FENDRR on the opposite strand, suggesting they may be coregulated. Using RNA sequencing in lung tissue from ACDMPV patients with heterozygous deletions of the FOXF1 distant enhancer located 286 kb upstream, leaving FOXF1 and FENDRR intact, we have found that the FENDRR and FOXF1 expressions were reduced by approximately 75% and 50%, respectively, and were monoallelic from the intact chromosome 16q24.1. In contrast, ACDMPV patients with FOXF1 SNVs had biallelic FENDRR expression reduced by 66%-82%. Corroboratively, depletion of FOXF1 by small interfering RNA in lung fibroblasts resulted in a 50% decrease of FENDRR expression. These data indicate that FENDRR expression in the lungs is regulated both in cis by the FOXF1 distant enhancer and in trans by FOXF1. Our findings are compatible with the involvement of FENDRR in FOXF1-related disorders, including ACDMPV.

Generation of Pulmonary Endothelial Progenitor Cells for Cell-based Therapy Using Interspecies Mouse-Rat Chimeras.

Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.Methods: Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with S52F Foxf1 mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.Measurements and Main Results: We identified a unique population of EPCs, FOXF1+cKIT+ EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1+cKIT+ gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1+cKIT+ gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1+cKIT+ gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1+cKIT+ endothelial progenitors may be considered for treatment of human ACDMPV.

Optimal Oxygen Targets in Term Lambs with Meconium Aspiration Syndrome and Pulmonary Hypertension.

Optimal oxygen saturation as measured by pulse oximetry (SpO2) in neonatal lung injury, such as meconium aspiration syndrome (MAS) and persistent pulmonary hypertension of newborn (PPHN), is not known. Our goal was to determine the SpO2 range in lambs with MAS and PPHN that results in the highest brain oxygen delivery (bDO2) and pulmonary blood flow (Qp) and the lowest pulmonary vascular resistance and oxidative stress. Meconium was instilled into endotracheal tubes in 25 near-term gestation lambs, and the umbilical cord was occluded to induce asphyxia and gasping, causing MAS and PPHN. Lambs were randomized into four groups and ventilated for 6 hours with fixed fraction of inspired oxygen (FiO2) = 1.0 irrespective of SpO2, and three groups had FiO2 titrated to keep preductal SpO2 between 85% and 89%, 90% and 94%, and 95% and 99%, respectively. Tissues were collected to measure nitric oxide synthase activity, 3-nitrotyrosine, and 8-isoprostanes. Throughout the 6-hour exposure period, lambs in the 95-99% SpO2 target group had the highest Qp, lowest pulmonary vascular resistance, and highest bDO2 but were exposed to higher FiO2 (0.5 ± 0.21 vs. 0.29 ± 0.17) with higher lung 3-nitrotyrosine (0.67 [interquartile range (IQR), 0.43-0.73] ng/mcg protein vs. 0.1 [IQR, 0.09-0.2] ng/mcg protein) and lower lung nitric oxide synthase activity (196 [IQR, 192-201] mMol nitrite/mg protein vs. 270 [IQR, 227-280] mMol nitrite/mg protein) compared with the 90-94% target group. Brain 3-nitrotyrosine was lower in the 85-89% target group, and brain/lung 8-isoprostane levels were not significantly different. In term lambs with MAS and PPHN, Qp and bDO2 through the first 6 hours are higher with target SpO2 in the 95-99% range. However, the 90-94% target range is associated with significantly lower FiO2 and lung oxidative stress. Clinical trials comparing the 90-94% versus the 95-99% SpO2 target range in term infants with PPHN are warranted.

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.

Optimal oxygenation and role of free radicals in PPHN.

Effective ventilation of the lungs is essential in mediating pulmonary vasodilation at birth to allow effective gas exchange and an increase in systemic oxygenation. Unsuccessful transition prevents the increase in pulmonary blood flow after birth resulting in hypoxemia and persistent pulmonary hypertension of the newborn (PPHN). Management of neonates with PPHN includes ventilation of the lungs with supplemental oxygen to correct hypoxemia. Optimal oxygenation should meet oxygen demand to the tissues and avoid hypoxic pulmonary vasoconstriction (HPV) while preventing oxidative stress. The optimal target for oxygenation in PPHN is not known. Animal models have demonstrated that PaO2<45 mmHg exacerbates HPV. However, there are no practical methods of assessing oxygen levels associated with oxidant stress. Oxidant stress can be due to free radical generation from underlying lung disease or from free radicals generated by supplemental oxygen. Free radicals act on the nitric oxide pathway reducing cGMP and promoting pulmonary vasoconstriction. Antioxidant therapy improves systemic oxygenation in an animal model of PPHN but there are no clinical trials to support such therapy. Targeting preductal SpO2 between 90 and 97% and PaO2 at 50-80 mmHg appears prudent in PPHN but clinical trials to support this practice are lacking. Preterm infants with PPHN present unique challenges due to lack of antioxidant defenses and functional and structural immaturity of the lungs. This review highlights the need for additional studies to mitigate the impact of oxidative stress in the lung and pulmonary vasculature in PPHN.

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.

A novel compound heterozygous mutation in VARS2 in a newborn with mitochondrial cardiomyopathy: a case report of a Chinese family.

BACKGROUND: Genetic defects in the mitochondrial aminoacyl-tRNA synthetase are important causes of mitochondrial disorders. VARS2 is one of the genes encoding aminoacyl-tRNA synthetases. Recently, an increasing number of pathogenic variants of VARS2 have been reported. CASE PRESENTATION: We report the novel compound heterozygous pathogenic VARS2 mutations c.643 C > T (p. His215Tyr) and c.1354 A > G (p. Met452Val) in a female infant who presented with poor sucking at birth, poor activity, hyporeflexia, hypertonia, persistent pulmonary hypertension of newborn (PPHN), metabolic acidosis, severe lactic acidosis, expansion and hypertrophic cardiomyopathy. These heterozygous mutations were carried individually by the proband's parents and elder sister; the two mutations segregated in the family and were the cause of the disease in the proband.The c.643 C > T (p. His215Tyr) mutation was not described in the ExaC, GNomAD and 1000 Genomes Project databases, and the frequency of c.1354 A > G (p. Met452Val) was < 0.001 in these gene databases.The two mutated amino acids were located in a highly conserved region of the VARS2 protein that is important for its interaction with the cognate tRNA. The two missense mutations were predicted by online tools to be damaging and deleterious. CONCLUSIONS: Our report expands the spectrum of known pathogenicVARS2 variants associated with mitochondrial disorders in humans.VARS2 deficiency may cause a severe neonatal presentation with structural cardiac abnormalities.

Hypoxia inhibits adenylyl cyclase catalytic activity in a porcine model of persistent pulmonary hypertension of the newborn.

Persistent pulmonary hypertension of the newborn (PPHN) features hypoxemia, pulmonary vasoconstriction, and impaired cardiac inotropy. We previously reported low basal and stimulated cAMP in hypoxic pulmonary artery smooth muscle cells (PASMCs). We now examine pulmonary arterial adenylyl cyclase (AC) activity and regulation in hypoxic PPHN. PPHN was induced in newborn swine by normobaric hypoxia (fraction of inspired oxygen 0.10) for 72 h and compared with age-matched normoxic controls. We studied relaxation of pulmonary arterial (PA) rings to AC activator forskolin and cGMP activator sodium nitroprusside (SNP) by isometric myography, ATP content, phosphodiesterase activity, AC content, isoform expression, and catalytic activity in presence or absence of Gαs-coupled receptor agonists, forskolin, or transnitrosylating agents in human and neonatal porcine PASMCs and HEK293T stably expressing AC isoform 6, after 72 h hypoxia (10% O2) or normoxia (21% O2). Relaxation to forskolin and SNP were equally impaired in PPHN PA. AC-specific activity decreased in hypoxia. PASMC from PPHN swine had reduced AC activity despite exposure to normoxia in culture; transient hypoxia in vitro further decreased AC activity. Prostacyclin receptor ligand affinity decreased, but its association with Gαs increased in hypoxia. Total AC content was unchanged by hypoxia, but AC6 increased in hypoxic cells and PPHN pulmonary arteries. Impairment of AC6 activity in hypoxia was associated with nitrosylation. PPHN PA relaxation is impaired because of loss of AC activity. Hypoxic AC is inhibited because of S-nitrosylation; inhibition persists after removal from hypoxia. Downregulation of AC-mediated relaxation in hypoxic PA has implications for utility of Gαs-coupled receptor agonists in PPHN treatment.

Localization and pneumococcal alteration of junction proteins in the human alveolar-capillary compartment.

Loss of alveolar barrier function with subsequent respiratory failure is a hallmark of severe pneumonia. Although junctions between endo- and epithelial cells regulate paracellular fluid flux, little is known about their composition and regulation in the human alveolar compartment. High autofluorescence of human lung tissue in particular complicates the determination of subcellular protein localization. By comparing conventional channel mode confocal imaging with spectral imaging and linear unmixing, we demonstrate that background fluorescent spectra and fluorophore signals could be rigorously separated resulting in complete recovery of the specific signal at a high signal-to-noise ratio. Using this technique and Western blotting, we show the expression patterns of tight junction proteins occludin, ZO-1 as well as claudin-3, -4, -5 and -18 and adherence junction protein VE-cadherin in naive or Streptococcus pneumoniae-infected human lung tissue. In uninfected tissues, occludin and ZO-1 formed band-like structures in alveolar epithelial cells type I (AEC I), alveolar epithelial cells type II (AEC II) and lung capillaries, whereas claudin-3, -4 and -18 were visualised in AEC II. Claudin-5 was detected in the endothelium only. Claudin-3, -5, -18 displayed continuous band-like structures, while claudin-4 showed a dot-like expression. Pneumococcal infection reduced alveolar occludin, ZO-1, claudin-5 and VE-cadherin but did not change the presence of claudin-3, -4 and -18. Spectral confocal microscopy allows for the subcellular structural analysis of proteins in highly autofluorescent human lung tissue. The thereby observed deterioration of lung alveolar junctional organisation gives a structural explanation for alveolar barrier disruption in severe pneumococcal pneumonia.

The Role of Serotonin Transporter in Human Lung Development and in Neonatal Lung Disorders.

Introduction. Failure of the vascular pulmonary remodeling at birth often manifests as pulmonary hypertension (PHT) and is associated with a variety of neonatal lung disorders including a uniformly fatal developmental disorder known as alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV). Serum serotonin regulation has been linked to pulmonary vascular function and disease, and serotonin transporter (SERT) is thought to be one of the key regulators in these processes. We sought to find evidence of a role that SERT plays in the neonatal respiratory adaptation process and in the pathomechanism of ACD/MPV. Methods. We used histology and immunohistochemistry to determine the timetable of SERT protein expression in normal human fetal and postnatal lungs and in cases of newborn and childhood PHT of varied etiology. In addition, we tested for a SERT gene promoter defect in ACD/MPV patients. Results. We found that SERT protein expression begins at 30 weeks of gestation, increases to term, and stays high postnatally. ACD/MPV patients had diminished SERT expression without SERT promoter alteration. Conclusion. We concluded that SERT/serotonin pathway is crucial in the process of pulmonary vascular remodeling/adaptation at birth and plays a key role in the pathobiology of ACD/MPV.

Milrinone Pharmacokinetics and Pharmacodynamics in Neonates with Persistent Pulmonary Hypertension of the Newborn.

Objective To describe the pharmacokinetics and pharmacodynamics of milrinone in infants with persistent pulmonary hypertension of the newborn (PPHN) and to explore the impact of age on milrinone disposition. Design Randomized, open label pilot study. Setting Multicenter; level 3 and level 4 neonatal intensive care units. Patients Six infants ≥34 weeks' gestational age and <10 days of life with persistent hypoxemia receiving inhaled nitric oxide. Intervention Intravenous milrinone lactate in one of two dosing regimens: (1) low dose, 20 mcg/kg bolus followed by 0.2 mcg/kg/minute, and (2) standard dose, 50 mcg/kg bolus followed by 0.5 mcg/kg/minute. Measurements and Main Results The final structural model was a two-compartment disposition model with interindividual variability estimated on clearance (CL). The estimated value of CL is 7.65 mL/minute/3.4 kg (3.05 mL/minute/kg). The addition of age improved the precision of the CL estimate, and CL increased with chronological age in days. The oxygenation index was highly variable within each participant and improved with time. There were no observed safety concerns in either dosing group. Conclusion The CL of milrinone in newborns with PPHN is reduced and increases with age. In this pilot study, we did not see significant pharmacodynamic or safety effects associated with drug exposure.

Hypoxia and hyperoxia potentiate PAF receptor-mediated effects in newborn ovine pulmonary arterial smooth muscle cells: significance in oxygen therapy of PPHN.

Platelet-activating factor (PAF) acting via its receptor (PAFR) is implicated in the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Effects of long-term oxygen therapy on newborn lung are not well understood; therefore, we studied the effect of oxygen tension on ovine newborn pulmonary artery smooth muscle cells (NBPASMC). Our global hypothesis is that PPHN results from failure of newborn lamb pulmonary system to downregulate PAFR activity or to upregulate vasodilatory cyclic nucleotides (Cnucs) activity. NBPASMC from newborns 6-12 days old were studied in vitro at three different oxygen tensions (pO2, [Torr]: hypoxia, <40; normoxia, 80-100; and hyperoxia, >100 Torr often clinically imposed upon newborns with PPHN) PAFR- and Cnucs mediated effects were determined. PAFR and PKA Cα mRNA expression as well as prostacyclin, thromboxane, cAMP production, and DNA synthesis was studied to assess PAFR-mediated hypertrophy and/or hyperplasia. Hypoxia and hyperoxia increased specific PAFR binding. PAF treatment during hyperoxia increased PAFR gene, but decreased PKA-Cα gene expression. Hypoxia and hyperoxia increased NBPASMC proliferation via PAFR signaling. Baseline prostacyclin level was ninefold greater than in fetal PASMC, whereas baseline thromboxane was sevenfold less suggesting greater postnatal cyclooxygenase activity in NBPASMC PAF decreased, while forskolin and 8-Br-cAMP increased cAMP production. Decrease of PAFR effects by Cnucs indicates that normal newborn PA physiology favors vasodilator pathways to minimize PAF-induced hypertrophy or hyperplasia. We speculate that failure of newborn lung to anchor downregulation of vasoconstrictors with upregulation of vasodilators leads to PPHN.

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.

Cyclic stretch stimulates mitochondrial reactive oxygen species and Nox4 signaling in pulmonary artery smooth muscle cells.

This study was designed to determine whether cyclic stretch induces a persistent pulmonary hypertension of the newborn (PPHN) phenotype of increased NADPH oxidase (Nox) 4 signaling in control pulmonary artery smooth muscle cells (PASMC), and to identify the signal transduction molecules involved. To achieve this, PPHN was induced in lambs by antenatal ligation of the ductus arteriosus at 128 days gestation. After 9 days, lungs and PASMC were isolated from control (twin) and PPHN lambs. Control PASMC were exposed to cyclic stretch at 1 Hz and 15% elongation for 24 h. Stretch-induced Nox4 expression was attenuated by inhibition of mitochondrial complex III and NF-κB, and stretch-induced protein thiol oxidation was attenuated by Nox4 small interfering RNA and complex III inhibition. NF-κB activity was increased by stretch in a complex III-dependent fashion, and stretch-induced cyclin D1 expression was attenuated by complex III inhibition and Nox4 small interfering RNA. This is the first study to show that cyclic stretch increases Nox4 expression via mitochondrial complex III-induced activation of NF-κB in fetal PASMC, resulting in ROS signaling and increased cyclin D1 expression. Targeting these signaling molecules may attenuate pulmonary vascular remodeling associated with PPHN.

Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling.

In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms regulating thromboxane-mediated actin polymerization are potential targets for future PPHN pharmacotherapy.

Comparative analyses of lung transcriptomes in patients with alveolar capillary dysplasia with misalignment of pulmonary veins and in foxf1 heterozygous knockout mice.

Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a developmental disorder of the lungs, primarily affecting their vasculature. FOXF1 haploinsufficiency due to heterozygous genomic deletions and point mutations have been reported in most patients with ACDMPV. The majority of mice with heterozygous loss-of-function of Foxf1 exhibit neonatal lethality with evidence of pulmonary hemorrhage in some of them. By comparing transcriptomes of human ACDMPV lungs with control lungs using expression arrays, we found that several genes and pathways involved in lung development, angiogenesis, and in pulmonary hypertension development, were deregulated. Similar transcriptional changes were found in lungs of the postnatal day 0.5 Foxf1+/- mice when compared to their wildtype littermate controls; 14 genes, COL15A1, COL18A1, COL6A2, ESM1, FSCN1, GRINA, IGFBP3, IL1B, MALL, NOS3, RASL11B, MATN2, PRKCDBP, and SIRPA, were found common to both ACDMPV and Foxf1 heterozygous lungs. Our results advance knowledge toward understanding of the molecular mechanism of ACDMPV, lung development, and its vasculature pathology. These data may also be useful for understanding etiologies of other lung disorders, e.g. pulmonary hypertension, bronchopulmonary dysplasia, or cancer.

Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324.

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.