Hyperoxia contributes to the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of infants that is characterized by interrupted alveologenesis. Disrupted angiogenesis inhibits alveologenesis, but the mechanisms of disrupted angiogenesis in the developing lungs are poorly understood. In pre-clinical BPD models, hyperoxia increases the expression of extracellular signal-regulated kinases (ERK) 1/2; however, its effects on the lung endothelial ERK1/2 signaling are unclear. Further, whether ERK1/2 activation promotes lung angiogenesis in infants is unknown. Hence, we tested the following hypotheses: (1) hyperoxia exposure will increase lung endothelial ERK1/2 signaling in neonatal C57BL/6J (WT) mice and in fetal human pulmonary artery endothelial cells (HPAECs); (2) ERK1/2 inhibition will disrupt angiogenesis in vitro by repressing cell cycle progression. In mice, hyperoxia exposure transiently increased lung endothelial ERK1/2 activation at one week of life, before inhibiting it at two weeks of life. Interestingly, hyperoxia-mediated decrease in ERK1/2 activation in mice was associated with decreased angiogenesis and increased endothelial cell apoptosis. Hyperoxia also transiently activated ERK1/2 in HPAECs. ERK1/2 inhibition disrupted angiogenesis in vitro, and these effects were associated with altered levels of proteins that modulate cell cycle progression. Collectively, these findings support our hypotheses, emphasizing that the ERK1/2 pathway is a potential therapeutic target for BPD infants with decreased lung vascularization.

Bronchopulmonary dysplasia and pulmonary hypertension, or BPD-PH, are serious chronic lung disorders of prematurity, without curative therapies. Hyperoxia, a known causative factor of BPD-PH, activates adenosine monophosphate-activated protein kinase (AMPK) α1 in neonatal murine lungs; however, whether this phenomenon potentiates or mitigates lung injury is unclear. Thus, we hypothesized that (1) endothelial AMPKα1 is necessary to protect neonatal mice against hyperoxia-induced BPD-PH, and (2) AMPKα1 knockdown decreases angiogenesis in hyperoxia-exposed neonatal human pulmonary microvascular endothelial cells (HPMECs). We performed lung morphometric and echocardiographic studies on postnatal day (P) 28 on endothelial AMPKα1-sufficient and -deficient mice exposed to 21% O₂ (normoxia) or 70% O₂ (hyperoxia) from P1–P14. We also performed tubule formation assays on control- or AMPKα1-siRNA transfected HPMECs, exposed to 21% O₂ or 70% O₂ for 48 h. Hyperoxia-mediated alveolar and pulmonary vascular simplification, pulmonary vascular remodeling, and PH were significantly amplified in endothelial AMPKα1-deficient mice. AMPKα1 siRNA knocked down AMPKα1 expression in HPMECs, and decreased their ability to form tubules in normoxia and hyperoxia. Furthermore, AMPKα1 knockdown decreased proliferating cell nuclear antigen expression in hyperoxic conditions. Our results indicate that AMPKα1 is required to reduce hyperoxia-induced BPD-PH burden in neonatal mice, and promotes angiogenesis in HPMECs to limit lung injury.

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.

Bronchopulmonary dysplasia (BPD)-associated pulmonary hypertension (PH) is a significant lung morbidity of infants, and disrupted lung angiogenesis is a hallmark of this disease. We observed that extracellular signal-regulated kinases (ERK) 1/2 support angiogenesis in vitro, and hyperoxia activates ERK1/2 in fetal human pulmonary microvascular endothelial cells (HPMECs) and in neonatal murine lungs; however, their role in experimental BPD and PH is unknown. Therefore, we hypothesized that Tie2 Cre-mediated deficiency of ERK2 in the endothelial cells of neonatal murine lungs would potentiate hyperoxia-induced BPD and PH. We initially determined the role of ERK2 in in vitro angiogenesis using fetal HPMECs. To disrupt endothelial ERK2 signaling in the lungs, we decreased ERK2 expression by breeding ERK2^flox/flox mice with Tie-Cre mice. One-day-old endothelial ERK2-sufficient (eERK2^+/+) or –deficient (eERK2^+/−) mice were exposed to normoxia or hyperoxia (FiO₂ 70%) for 14 d. We then performed lung morphometry, gene and protein expression studies, and echocardiography to determine the extent of inflammation, oxidative stress, and development of lungs and PH. The knockdown of ERK2 in HPMECs decreased in vitro angiogenesis. Hyperoxia increased lung inflammation and oxidative stress, decreased lung angiogenesis and alveolarization, and induced PH in neonatal mice; however, these effects were augmented in the presence of Tie2-Cre mediated endothelial ERK2 deficiency. Therefore, we conclude that endothelial ERK2 signaling is necessary to mitigate hyperoxia-induced experimental BPD and PH in neonatal mice. Our results indicate that endothelial ERK2 is a potential therapeutic target for the management of BPD and PH in infants.

We describe a 39-year-old man referred for surgical aortic valve replacement for severe symptomatic aortic stenosis. Intraoperative inspection was unexpectedly consistent with marantic endocarditis. Pathology confirmed nonbacterial thrombotic endocarditis. We present high-resolution intraoperative, diagnostic, and pathology images of nonbacterial thrombotic endocarditis in a patient with antiphospholipid syndrome with atypical presentation.