Pulmonary hypertension (PH) occurs in 25 to 35% of premature infants with significant bronchopulmonary dysplasia (BPD). Neonatal mice exposed to 14 days of hyperoxia develop BPD-like lung injury and PH. To determinne the impact of hyperoxia on pulmonary artery (PA) cyclic guanosine monophosphate (cGMP) signaling in a murine model of lung injury and PH, neonatal C57BL/6 mice were placed in room air, 75% O 2 for 14 days (chronic hyperoxia [CH]) or 75% O 2 for 24 hours, followed by 13 days of room air (acute hyperoxia with recovery [AHR]) with or without sildenafil. At 14 days, mean alveolar area, PA medial wall thickness (MWT), right ventricular hypertrophy (RVH), and vessel density were assessed. PA protein was analyzed for cGMP, soluble guanylate cyclase, and PDE5 activity. CH and AHR mice had RVH, but only CH mice had increased alveolar area and MWT and decreased vessel density. In CH and AHR PAs, soluble guanylate cyclase activity was decreased, and PDE5 activity was increased. In CH mice, sildenafil attenuated MWT and RVH but did not improve mean alveolar area or vessel density. In CH and AHR PAs, sildenafil decreased PDE5 activity and increased cGMP. Our results indicate that prolonged hyperoxia leads to lung injury, PH, RVH, and disrupted PA cGMP signaling.Furthermore, 24 hours of hyperoxia causes RVH and disrupted PA cGMP signaling that persists for 13 days. Sildenafil reduced RVH and restored vascular cGMP signaling but did not attenuate lung injury. Thus, hyperoxia can rapidly disrupt PA cGMP signaling in vivo with sustained effects, and concurrent sildenafil therapy can be protective.Keywords: bronchopulmonary dysplasia; phosphodiesterases; soluble guanylate cyclase; right ventricular hypertrophy Clinical RelevanceUsing a murine model of hyperoxia-induced lung injury and pulmonary hypertension, we demonstrate that 24 hours of exposure to 75% O 2 causes disrupted cyclic guanosine monophosphate (cGMP) signaling in the small pulmonary arteries of mice and right ventricular hypertrophy that persists long after exposure. In addition, treatment with low doses of sildenafil prevents hyperoxia-induced pulmonary hypertension and restores cGMP signaling in the small pulmonary arteries.Bronchopulmonary dysplasia (BPD) is a well-described and common complication of prematurity. It has been recognized that 25 to 35% of infants with moderate to severe BPD develop pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) (1-3). Infants with BPD have simplified alveolarization and stunted vascularization. Although there has been mixed success in the prevention of BPD using inhaled nitric oxide, vitamin A, and caffeine, there is no definitive treatment to prevent BPD (4-7).Moreover, the underlying pathophysiology involving BPD-associated PH is poorly understood, and there are no proven therapeutic options.In this study, we used a previously described murine model to approximate

In the pulmonary vasculature, phosphodiesterase-5 (PDE5) degrades cGMP and inhibits nitric oxide-mediated, cGMP-dependent vasorelaxation. We previously reported that ventilation with 100% O 2 increased PDE5 activity in pulmonary arteries (PA) of pulmonary hypertension lambs (PPHN) more than in control lambs. In the present study, PA smooth muscle cells (PASMC) from PPHN lambs had increased basal PDE5 activity, decreased cGMP-responsiveness to NO, and increased mitochondrial matrix oxidant stress compared to control PASMC. Hyperoxia (24h) increased PDE5 activity and mitochondrial matrix oxidant stress above baseline to a similar degree in PPHN and control PASMC. Mitochondrially-targeted catalase decreased PDE5 activity at baseline and after hyperoxia in PPHN PASMC. Similarly, catalase treatment of PPHN lambs ventilated with 100% O 2 decreased PDE5 activity and increased cGMP in PA. We conclude that baseline PDE5 activity and oxidative stress is increased in PPHN PASMC, and scavenging H 2 O 2 is sufficient to block oxidant-mediated increases in PDE5 activity in PPHN.

Aims: Oxygen is a pulmonary vasodilator, but data suggest high O 2 concentrations impede that response. We previously reported 24 h of 100% O 2 increased phosphodiesterase 5 (PDE5) activity in fetal pulmonary artery smooth muscle cells (FPASMC) and in ventilated neonatal lambs. PDE5 degrades cyclic GMP (cGMP) and inhibits nitric oxide (NO)-mediated cGMP-dependent vasorelaxation. We sought to determine the mechanism by which hyperoxia initiates reactive oxygen species (ROS) production and regulates PDE5. Results: Thirty minutes of hyperoxia increased mitochondrial ROS versus normoxia (30.3 -1.7% vs. 21.1 -2.8%), but had no effect on cytosolic ROS, measured by roGFP, a ratiometric protein thiol redox sensor. Hyperoxia increased PDE5 activity (220 -39%) and decreased cGMP responsiveness to NO (37 -17%). Mitochondrial catalase overexpression attenuated hyperoxia-induced mitochondrial roGFP oxidation, compared to FPASMC infected with empty adenoviral vector (50 -3% of control) or mitochondrial superoxide dismutase. MitoTEMPO, mitochondrial catalase, and DT-3, a cGMP-dependent protein kinase I alpha inhibitor, decreased PDE5 activity (32 -13%, 26 -21%, and 63 -10% of control, respectively), and restored cGMP responsiveness to NO (147 -16%,172 -29%, and 189 -43% of control, respectively). C57Bl6 mice exposed to 90%-100% O 2 for 45 min -mechanical ventilation had increased PA PDE5 activity (206 -39% and 235 -75%, respectively). Innovation: This is the first description that hyperoxia induces ROS in the mitochondrial matrix prior to the cytosol. Our results indicate that short hyperoxia exposures can produce significant changes in critical cellular signaling pathways. Conclusions: These results indicate that mitochondrial matrix oxidant signals generated during hyperoxia, specifically H 2 O 2 , activate PDE5 in a cGMPdependent protein kinase-dependent manner in pulmonary vascular smooth muscle cells. Antioxid. Redox Signal. 17, 460-470.

Pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) affect 25-35% of premature infants with significant bronchopulmonary dysplasia (BPD), increasing morbidity and mortality. We sought to determine the role of phosphodiesterase 5 (PDE5) in the right ventricle (RV) and left ventricle (LV) in a hyperoxia-induced neonatal mouse model of PH and RVH. After birth, C57BL/6 mice were placed in room air (RA) or 75% O2 (CH) for 14 days to induce PH and RVH. Mice were euthanized at 14 days or recovered in RA for 14 days or 42 days prior to euthanasia at 28 or 56 days of age. Some pups received sildenafil or vehicle (3 mg·kg(-1)·dose(-1) sc) every other day from P0. RVH was assessed by Fulton's index [RV wt/(LV + septum) wt]. PDE5 protein expression was analyzed via Western blot, PDE5 activity was measured by commercially available assay, and cGMP was measured by enzyme-linked immunoassay. Hyperoxia induced RVH in mice after 14 days, and RVH did not resolve until 56 days of age. Hyperoxia increased PDE5 expression and activity in RV, but not LV + S, after 14 days. PDE5 expression normalized by 28 days of age, but PDE5 activity did not normalize until 56 days of age. Sildenafil given during hyperoxia prevented RVH, decreased RV PDE5 activity, and increased RV cGMP levels. Mice with cardiac-specific overexpression of PDE5 had increased RVH in RA. These findings suggest normal RV PDE5 function is disrupted by hyperoxia, and elevated PDE5 contributes to RVH and remodeling. Therefore, in addition to impacting the pulmonary vasculature, sildenafil also targets PDE5 in the neonatal mouse RV and decreases RVH.

Bronchopulmonary dysplasia (BPD), a common complication of preterm birth, is associated with pulmonary hypertension (PH) in 25% of infants with moderate to severe BPD. Neonatal mice exposed to hyperoxia for 14d develop lung disease similar to BPD, with evidence of associated PH. The cyclic guanosine monophosphate (cGMP) signaling pathway has not been well studied in BPD-associated PH. In addition, there is little data about the natural history of hyperoxia-induced PH in mice or the utility of phosphodiesterase-5 (PDE5) inhibition in established disease. C57BL/6 mice were placed in room air or 75% O2 within 24h of birth for 14d, followed by recovery in room air for an additional 7 days (21d). Additional pups were treated with either vehicle or sildenafil for 7d during room air recovery. Mean alveolar area, pulmonary artery (PA) medial wall thickness (MWT), RVH, and vessel density were evaluated at 21d. PA protein from 21d animals was analyzed for soluble guanylate cyclase (sGC) activity, PDE5 activity, and cGMP levels. Neonatal hyperoxia exposure results in persistent alveolar simplification, RVH, decreased vessel density, increased MWT, and disrupted cGMP signaling despite a period of room air recovery. Delayed treatment with sildenafil during room air recovery is associated with improved RVH and decreased PA PDE5 activity, but does not have significant effects on alveolar simplification, PA remodeling, or vessel density. These data are consistent with clinical studies suggesting inconsistent effects of sildenafil treatment in infants with BPD-associated PH.