Serotonin-Deficient Neonatal Murine Models Not Protected Against Hypoxia-Induced Bronchopulmonary Dysplasia or Pulmonary Hypertension

Background: development of pulmonary hypotension (PH) associated with bronchopulmonary dysplasia (BPD) in neonates

Bronchopulmonary dysplasia (BPD) is a common, chronic lung disease that affects preterm infants and may result in long-term respiratory impairment. There is no cure for BPD and the incidence is rising. 

Most therapies for pulmonary hypertension (PH) associated with BPD target NO—cGMP signaling, with the goal of decreasing pulmonary pressures, and includes inhaled nitric oxide.

Alterations in serotonin (5-hydroxytryptamine or 5-HT) signaling may lead to PH associated with BPD:

• Serotonin is increased in the lungs of an experimental fetal model of PH and is proposed to contribute to the development of PH by several mechanisms.
• Serotonin is synthesized from L-tryptophan through tryptophan hydroxylase.

The enzyme tryptophan hydroxylase is the rate limiting step in serotonin synthesis. It exists as two isoforms:

• TPH1
  • Highest expression in enterochromaffin cells of the small intestine
  • Expressed in pulmonary artery endothelial cells
• TPH2
  • Limited to brain, enteric nervous system
  • TPH1 knock-out murine models still able to synthesize peripheral serotonin via TPH2, which is expressed in the enteric nervous system

• Serotonin immunoreactive cells present at 8 weeks gestation in human fetal lungs.
• Pulmonary serotonin is increased 34-fold increase in infants who died from severe BPD compared to controls.
• Maternal use of selective serotonin reuptake inhibitors in third trimester increases the risk of persistent PH of newborn.

Previous studies by a team of neonatal and pediatric critical care researchers at Children’s Hospital Colorado and the University of Colorado School of Medicine found:

• Serotonin is a powerful pulmonary vasoconstrictor in the ovine fetus.
• Serotonin contributes to fetal pulmonary vascular resistance.
• Increased pulmonary TPH1 expression and pulmonary artery endothelial cell synthesis of serotonin in experimental fetal models with PH.
• Increased lung TPH1 expression and plasma serotonin in neonatal murine models with bleomycin-induced PH associated with BPD.
• Increased platelet (which store the majority of serotonin) accumulation in murine lungs with bleomycin-induced neonatal PH.

In neonatal murine models of bleomycin-induced BPD and PH, studies -- including two from Children’s Colorado and CU School of Medicine –– found pharmacological blockade of serotonin via inhibition of the 2A receptor:

• Decreases fetal pulmonary vascular resistance in the ovine fetus with PH
• Protects against development of PH and pulmonary vascular remodeling in a murine bleomycin model of PH and BPD

In this study, researchers Cassidy Delaney, MD, Danielle Roberts, MD, Laura Sherlock, MD, Jamie Archambault, MD, Eva Nozik, MD, and Janelle Posey hypothesized:

• Circulating (platelet and plasma) and pulmonary serotonin would be increased in neonatal wild-type (WT) hypoxic murine models
• TPH1 knock-out (KO) neonatal murine models would be protected against:
  • Hypoxia-induced alveolar simplification
  • Impaired pulmonary vessel development
  • PH

Methods: measurement and evaluation of hypoxic, normoxic murine models

Newborn WT and TPH1 KO murine models were exposed to normoxia or hypoxia for 2 weeks.

• This model produces similar major pathologic findings to infants with BPD and PH, these include
  • Impaired alveolar development
  • Vascular injury
  • PH and right ventricular hypertrophy

Results: TPH1 inhibition did not improve experimental PH associated with BPD

Circulating and lung serotonin is decreased in TPH-1 KO murine model

• Pulmonary expression of TPH1 gene not detected in TPH1 KO murine model
• KO averages:
  • ~6 ng/ml plasma serotonin
  • ~30 ng/ml platelet serotonin
• WT averages:
  • ~30 ng/ml plasma serotonin
  • ~285 ng/ml platelet serotonin

Similarities between WT and TPH1 normoxic murine models

Both model genotypes showed similar baseline:

• Alveolar development
• Pulmonary vessel density
• Pulmonary pressures
• Right heart size

Similarities between hypoxic WT and TPH1 models

TPH1 KO models displayed attenuated hypoxia-induced

PH compared to WT hypoxic models

• Hypoxia-induced increase in right ventricular systolic pressure was attenuated in TPH1 KO models (26% in KO, 36% in WT)
• Hypoxia-induced right ventricular hypertrophy increase was same in both WT and KO models (33% in each genotype)

Hypothesis disproved: circulating and pulmonary serotonin did not increase in hypoxia exposed murine models; TPH1 inhibition did not prevent hypoxia-induced PH associated with BPD

• To the surprise of study authors, serotonin decreased in platelets and platelet-poor plasma in murine models two weeks of hypoxia exposure
• Lung serotonin decreased 23% in hypoxia-exposed KO models when compared to normoxic KO models

Discussion and conclusion: tph1 inhibition not beneficial in neonates with PH associated with BPD

Major findings from this study include:

• Alveolar development, pulmonary vascular density, right ventricular systolic pressures and right heart size are similar between WT and TPH1 KO models
• Platelet and plasma serotonin are decreased in neonatal murine models with hypoxia-induced PH
• Findings in hypoxic 2-week-old murine model differ from the authors’ prior findings in bleomycin-treated neonatal murine mode where plasma serotonin increased, emphasizing importance of evaluating disease processes at various time points and in multiple models
• Hypoxia-induced increase in right ventricular systolic pressures was attenuated in TPH1 KO murine models compared with WT murine models, but they were not protected against hypoxia-induced right ventricular hypertrophy

Study authors concluded that TPH1 KO murine models are not protected against hypoxia-induced alveolar simplification, reduction in pulmonary vessel density, or right ventricular hypertrophy. While genetic and pharmacologic inhibition of TPH1 has protective effects in adult models of PH, the results of this study suggest TPH1 inhibition may decrease pulmonary vasoconstriction but may not benefit neonates with PH associated with BPD.