Disrupted Pulmonary Artery Cyclic Guanosine Monophosphate Signaling in Mice with Hyperoxia-Induced Pulmonary Hypertension

Abstract

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

Selective depletion of vascular EC-SOD augments chronic hypoxic pulmonary hypertension

“…For studies of sGC and PDE5 expression and activity, frozen lung was pulverized and homogenized in 1ϫ Mg 2ϩ lysis buffer (EMD Millipore) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and a phosphatase inhibitor cocktail (EMD Biosciences). Lung protein extracts were sonicated, and protein concentration was determined using the Bradford assay (13,20). Pulmonary artery protein was isolated from pulmonary arteries as previously described (13).…”

“…For normalization, bands were quantified by densitometry using ImageJ software from the National Institutes of Health (NIH) or Image Lab Software (Bio-Rad). Data are shown as fold Ϯ SE relative to EC-SOD control mice (13,20).…”

“…cGMP levels were measured as previously described (13,20). Briefly, lung tissue was homogenized in 10 volumes of 5% TCA (Sigma-Aldrich).…”

“…Total lung protein was freshly prepared for sGC activity assays, as previously described (10,20). Each sample was dried, resuspended in cGMP EIA buffer, and acetylated according to the manufacturer's protocol.…”

Pulmonary Hypertension in Premature Infants. Sharpening the Tools of Detection

“…After birth, pulmonary arterial pressure decreases more slowly in preterm relative to term infants, especially in the presence of respiratory failure (11). Alterations of NO-cyclic guanosine monophosphate signaling disrupt lung and pulmonary vascular development in animal models of prematurity and lung injury (12)(13)(14), but these findings have not been successfully translated into therapeutic advances. Vascular agents such as iNO and sildenafil may acutely improve pulmonary artery pressure, but their long-term efficacy remains uncertain and poorly studied (15)(16)(17).…”