Bronchopulmonary dysplasia (BPD) is a morbid lung disease distinguished by lung alveolar and vascular simplification. Hyperoxia, an important BPD causative factor, increases extracellular signal-regulated kinases (ERK)-1/2 expression, whereas decreased lung endothelial cell ERK2 expression reduces angiogenesis and potentiates hyperoxia-mediated BPD in mice. However, ERK1′s role in experimental BPD is unclear. Thus, we hypothesized that hyperoxia-induced experimental BPD would be more severe in global ERK1-knockout (ERK1^-/-) mice than their wild-type (ERK1^+/+ mice) littermates. We determined the extent of lung development, ERK1/2 expression, inflammation, and oxidative stress in ERK1^-/- and ERK1^+/+ mice exposed to normoxia (FiO₂ 21%) or hyperoxia (FiO₂ 70%). We also quantified the extent of angiogenesis and hydrogen peroxide (H₂O₂) production in hyperoxia-exposed neonatal human pulmonary microvascular endothelial cells (HPMECs) with normal and decreased ERK1 signaling. Compared with ERK1^+/+ mice, ERK1^-/- mice displayed increased pulmonary ERK2 activation upon hyperoxia exposure. However, the extent of hyperoxia-induced inflammation, oxidative stress, and interrupted lung development was similar in ERK1^-/- and ERK1^+/+ mice. ERK1 knockdown in HPMECs increased ERK2 activation at baseline, but did not affect in vitro angiogenesis and hyperoxia-induced H₂O₂ production. Thus, we conclude ERK1 is dispensable for hyperoxia-induced experimental BPD due to compensatory ERK2 activation.

Inflammation causes bronchopulmonary dysplasia (BPD), a common lung disease of preterm infants. One reason this disease lacks specific therapies is the paucity of information on the mechanisms regulating inflammation in developing lungs. We address this gap by characterizing the lymphatic phenotype in an experimental BPD model because lymphatics are major regulators of immune homeostasis. We hypothesized that hyperoxia (HO), a major risk factor for experimental and human BPD, disrupts lymphatic endothelial homeostasis using neonatal mice and human dermal lymphatic endothelial cells (HDLECs). Exposure to 70% O₂ for 24–72 h decreased the expression of prospero homeobox 1 (Prox1) and vascular endothelial growth factor c (Vegf-c) and increased the expression of heme oxygenase 1 and NAD(P)H dehydrogenase [quinone]1 in HDLECs, and reduced their tubule formation ability. Next, we determined Prox1 and Vegf-c mRNA levels on postnatal days (P) 7 and 14 in neonatal murine lungs. The mRNA levels of these genes increased from P7 to P14, and 70% O₂ exposure for 14 d (HO) attenuated this physiological increase in pro-lymphatic factors. Further, HO exposure decreased VEGFR3⁺ and podoplanin⁺ lymphatic vessel density and lymphatic function in neonatal murine lungs. Collectively, our results validate the hypothesis that HO disrupts lymphatic endothelial homeostasis.

Interrupted lung angiogenesis is a hallmark of bronchopulmonary dysplasia (BPD); however, druggable targets that can rescue this phenotype remain elusive. Thus, our investigation focused on amphiregulin (Areg), a growth factor that mediates cellular proliferation, differentiation, migration, survival, and repair. While Areg promotes lung branching morphogenesis, its effect on endothelial cell (EC) homeostasis in developing lungs is understudied. Therefore, we hypothesized that Areg promotes the proangiogenic ability of the ECs in developing murine lungs exposed to hyperoxia. Lung tissues were harvested from neonatal mice exposed to normoxia or hyperoxia to determine Areg expression. Next, we performed genetic loss-of-function and pharmacological gain-of-function studies in normoxia- and hyperoxia-exposed fetal murine lung ECs. Hyperoxia increased Areg mRNA levels and Areg+ cells in whole lungs. While Areg expression was increased in lung ECs exposed to hyperoxia, the expression of its signaling receptor, epidermal growth factor receptor, was decreased, indicating that hyperoxia reduces Areg signaling in lung ECs. Areg deficiency potentiated hyperoxia-mediated anti-angiogenic effects. In contrast, Areg treatment increased extracellular signal-regulated kinase activation and exerted proangiogenic effects. In conclusion, Areg promotes EC tubule formation in developing murine lungs exposed to hyperoxia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease commonly affecting premature infants, with limited therapeutic options and increased long-term consequences. Adrenomedullin (Adm), a proangiogenic peptide hormone, has been found to protect rodents against experimental BPD. This study aims to elucidate the molecular and cellular mechanisms through which Adm influences BPD pathogenesis using a lipopolysaccharide (LPS)-induced model of experimental BPD in mice. Bulk RNA sequencing of Adm-sufficient (wild-type or Adm^+/+) and Adm-haplodeficient (Adm^+/−) mice lungs, integrated with single-cell RNA sequencing data, revealed distinct gene expression patterns and cell type alterations associated with Adm deficiency and LPS exposure. Notably, computational integration with cell atlas data revealed that Adm-haplodeficient mouse lungs exhibited gene expression signatures characteristic of increased inflammation, natural killer (NK) cell frequency, and decreased endothelial cell and type II pneumocyte frequency. Furthermore, in silico human BPD patient data analysis supported our cell type frequency finding, highlighting elevated NK cells in BPD infants. These results underscore the protective role of Adm in experimental BPD and emphasize that it is a potential therapeutic target for BPD infants with an inflammatory phenotype.