Aberrant cGMP signaling persists during recovery in mice with oxygen-induced pulmonary hypertension.

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.

Dose-dependent effects of glucocorticoids on pulmonary vascular development in a murine model of hyperoxic lung injury.

BACKGROUND: Exposure of neonatal mice to hyperoxia results in pulmonary vascular remodeling and aberrant phosphodiesterase type 5 (PDE5) signaling. Although glucocorticoids are frequently utilized in the NICU, little is known about their effects on the developing pulmonary vasculature and on PDE5. We sought to determine the effects of hydrocortisone (HC) on pulmonary vascular development and on PDE5 in a neonatal mouse model of hyperoxic lung injury. METHODS: C57BL/6 mice were placed in 21% O2 or 75% O2 within 24 h of birth and received HC (1, 5, or 10 mg/kg subcutaneously every other day) or vehicle. At 14 d, right ventricular hypertrophy (RVH), medial wall thickness (MWT), lung morphometry, and pulmonary artery (PA) PDE5 activity were assessed. PDE5 activity was measured in isolated pulmonary artery smooth muscle cells exposed to 21 or 95% O2 ± 100 nmol/l HC for 24 h. RESULTS: Hyperoxia resulted in alveolar simplification, RVH, increased MWT, and increased PA PDE5 activity. HC decreased hyperoxia-induced RVH and attenuated MWT. HC had dose-dependent effects on alveolar simplification. HC decreased hyperoxia-induced PDE5 activity both in vivo and in vitro. CONCLUSIONS: HC decreases hyperoxia-induced pulmonary vascular remodeling and attenuates PDE5 activity. These findings suggest that HC may protect against hyperoxic injury in the developing pulmonary vasculature.

Right ventricular cyclic nucleotide signaling is decreased in hyperoxia-induced pulmonary hypertension in neonatal mice.

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.

SOD2 activity is not impacted by hyperoxia in murine neonatal pulmonary artery smooth muscle cells and mice.

Pulmonary hypertension (PH) complicates bronchopulmonary dysplasia (BPD) in 25% of infants. Superoxide dismutase 2 (SOD2) is an endogenous mitochondrial antioxidant, and overexpression protects against acute lung injury in adult mice. Little is known about SOD2 in neonatal lung disease and PH. C57Bl/6 mice and isogenic SOD2+/+ and SOD2-/+ mice were placed in room air (control) or 75% O2 (chronic hyperoxia, CH) for 14 days. Right ventricular hypertrophy (RVH) was assessed by Fulton's index. Medial wall thickness (MWT) and alveolar area were assessed on formalin fixed lung sections. Pulmonary artery smooth muscle cells (PASMC) were placed in 21% or 95% O2 for 24 h. Lung and PASMC protein were analyzed for SOD2 expression and activity. Oxidative stress was measured with a mitochondrially-targeted sensor, mitoRoGFP. CH lungs have increased SOD2 expression, but unchanged activity. SOD2-/+ PASMC have decreased expression and activity at baseline, but increased SOD2 expression in hyperoxia. Hyperoxia increased mitochondrial ROS in SOD2+/+ and SOD2-/+ PASMC. SOD2+/+ and SOD2-/+ CH pups induced SOD2 expression, but not activity, and developed equivalent increases in RVH, MWT, and alveolar area. Since SOD2-/+ mice develop equivalent disease, this suggests other antioxidant systems may compensate for partial SOD2 expression and activity in the neonatal period during hyperoxia-induced oxidative stress.

Disrupted pulmonary artery cyclic guanosine monophosphate signaling in mice with hyperoxia-induced pulmonary hypertension.

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% O2 for 14 days (chronic hyperoxia [CH]) or 75% O2 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.

Isolation of pulmonary artery smooth muscle cells from neonatal mice.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al. that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 µM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

Cadherin point mutations alter cell sorting and modulate GTPase signaling.

This study investigated the impact of cadherin binding differences on both cell sorting and GTPase activation. The use of N-terminal domain point mutants of Xenopus C-cadherin enabled us to quantify binding differences and determine their effects on cadherin-dependent functions without any potential complications arising as a result of differences in cytodomain interactions. Dynamic cell-cell binding measurements carried out with the micropipette manipulation technique quantified the impact of these mutations on the two-dimensional binding affinities and dissociation rates of cadherins in the native context of the cell membrane. Pairwise binding affinities were compared with in vitro cell-sorting specificity and ligation-dependent GTPase signaling. Two-dimensional affinity differences greater than five-fold correlated with cadherin-dependent in vitro cell segregation, but smaller differences failed to induce cell sorting. Comparison of the binding affinities with GTPase signaling amplitudes further demonstrated that differential binding also proportionally modulates intracellular signaling. These results show that differential cadherin affinities have broader functional consequences than merely controlling cell-cell cohesion.

Brief hyperoxia increases mitochondrial oxidation and increases phosphodiesterase 5 activity in fetal pulmonary artery smooth muscle cells.

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 cGMP-dependent protein kinase-dependent manner in pulmonary vascular smooth muscle cells.

Mitochondrial oxidant stress increases PDE5 activity in persistent pulmonary hypertension of the newborn.

In the pulmonary vasculature, phosphodiesterase-5 (PDE5) degrades cGMP and inhibits nitric oxide-mediated, cGMP-dependent vasorelaxation. We previously reported that ventilation with 100% O2 increased PDE5 activity in pulmonary arteries (PAs) of pulmonary hypertension lambs (PPHN) more than in control lambs. In the present study, PA smooth muscle cells (PASMCs) from PPHN lambs had increased basal PDE5 activity, decreased cGMP-responsiveness to NO, and increased mitochondrial matrix oxidant stress compared to control PASMC. Hyperoxia (24 h) 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% O2 decreased PDE5 activity and increased cGMP in PA. We conclude that baseline PDE5 activity and oxidative stress is increased in PPHN PASMC, and scavenging H2O2 is sufficient to block oxidant-mediated increases in PDE5 activity in PPHN.