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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 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.

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., (Copyright © 2016 the American Physiological Society.)