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Updated: Jul 10, 2026
Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice
Published on: October 19, 2013
Hyperoxia increases phosphodiesterase 5 expression and activity in ovine fetal pulmonary artery smooth muscle cells
Kathryn N Farrow1, Beezly S Groh, Paul T Schumacker
1Division of Neonatology, Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, Ill 60611, USA. k-farrow@northwestern.edu
Insights
High oxygen levels in newborns can increase PDE5 activity, reducing nitric oxide (NO) effectiveness in the pulmonary vasculature. Antioxidants and PDE5 inhibitors may help restore NO responsiveness in persistent pulmonary hypertension of the newborn (PPHN).
Area of Science:
- Neonatal physiology
- Pulmonary vascular research
- Pharmacology
Background:
- Persistent pulmonary hypertension of the newborn (PPHN) often requires mechanical ventilation with high oxygen concentrations.
- The impact of hyperoxia on the developing pulmonary vasculature and phosphodiesterase type 5 (PDE5) is not well understood.
- cGMP concentrations in the pulmonary vasculature are regulated by PDE5, a key enzyme in smooth muscle relaxation.
Purpose of the Study:
- To investigate the effects of hyperoxia on PDE5 expression and activity in the developing pulmonary vasculature.
- To determine the role of oxidative stress in hyperoxia-induced changes in PDE5.
- To evaluate the potential of PDE5 inhibition and antioxidant treatment to restore nitric oxide (NO) responsiveness.
Main Methods:
- Exposure of fetal pulmonary artery smooth muscle cells (FPASMCs) to hyperoxia and hydrogen peroxide (H2O2).
- Measurement of intracellular cGMP response, PDE5 mRNA and protein expression, and PDE5 hydrolytic activity.
- Treatment with sildenafil (PDE5 inhibitor) and N-acetyl-cysteine (antioxidant).
- Ventilation of neonatal sheep with 100% oxygen and analysis of pulmonary artery PDE5 expression and activity.
Main Results:
- Hyperoxia exposure in FPASMCs decreased responsiveness to NO, increased PDE5 expression and activity, and elevated oxidative stress without causing cell death.
- H2O2 mimicked hyperoxia effects, indicating reactive oxygen species mediate these changes.
- Sildenafil partially restored cGMP responsiveness, while N-acetyl-cysteine blocked hyperoxia-induced PDE5 changes and rescued NO responsiveness.
- Neonatal sheep exposed to hyperoxia showed increased PDE5 protein in pulmonary arteries and increased PDE5 activity in lung extracts.
Conclusions:
- Hyperoxia increases PDE5 expression and activity in the neonatal pulmonary vasculature, potentially through oxidative stress.
- PDE5 plays a critical role in modulating pulmonary vascular tone in response to oxygen therapy in PPHN.
- Targeting PDE5 or oxidative stress may offer therapeutic strategies for PPHN management.
Abstract:
In the pulmonary vasculature, cGMP concentrations are regulated in part by a cGMP-dependent phosphodiesterase (PDE), PDE5. Infants with persistent pulmonary hypertension of the newborn (PPHN) are often mechanically ventilated with high oxygen concentrations. The effects of hyperoxia on the developing pulmonary vasculature and PDE5 are largely unknown. Here, we demonstrate that exposure of fetal pulmonary artery smooth muscle cells (FPASMCs) to high levels of oxygen for 24 hours leads to decreased responsiveness to exogenous NO, as determined by a decreased intracellular cGMP response, increased PDE5 mRNA and protein expression, as well as increased PDE5 cGMP hydrolytic activity. We demonstrate that inhibition of PDE5 activity with sildenafil partially rescues cGMP responsiveness to exogenous NO. In FPASMCs, hyperoxia leads to increased oxidative stress without increasing cell death. Treatment of normoxic FPASMCs with H2O2 is sufficient to induce PDE5 expression and activity, suggesting that reactive oxygen species mediate the effects of hyperoxia in FPASMCs. In support of this mechanism, a chemical antioxidant, N-acetyl-cysteine, is sufficient to block the hyperoxia-mediated increase in PDE5 expression and activity and rescue cGMP responsiveness to exogenous NO. Finally, ventilation of healthy neonatal sheep with 100% O2 for 24 hours leads to increased PDE5 protein expression in the resistance pulmonary arteries and increased PDE5 activity in whole lung extracts. These data suggest that PDE5 expression and activity play a critical role in modulating neonatal pulmonary vascular tone in response to common clinical treatments for PPHN, such as oxygen and inhaled NO.
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