**1. Introduction**

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants that is characterized by interrupted lung development [1]. The incidence of BPD has remained unchanged over the past few decades, and BPD is still the most common long-term morbidity of preterm infants [2]. Importantly, there are no specific therapies for BPD. In addition, BPD is the second most expensive childhood disease after asthma. Therefore, there is a need for improved therapies to prevent and treat BPD.

Decreased alveolarization or alveolar simplification and dysmorphic lung vascularization are histopathological hallmarks of BPD [3,4]. Lung blood vessels are crucial for healthy lungs. Abnormal lung angiogenesis is a characteristic feature of BPD [5]. Lung angiogenesis facilitates alveolarization (lung development), and disrupted angiogenesis can interrupt alveolarization in the developing lungs [6]. Therefore, understanding the mechanisms that promote the development and function of the lung blood vessels is vital to prevent and treat this human disease. Toward this end, vascular endothelial growth factor (VEGF) and nitric oxide (NO) signaling pathways have been extensively investigated and have been shown to be necessary for lung development in health and disease in neonatal animals [7–11]. VEGF restores the alveolar and pulmonary vascular structure and function via the endothelial nitric oxide synthase pathway in experimental BPD and pulmonary hypertension (PH) [12–14]. However, these results were not replicated in clinical studies [15,16]. Recent evidence suggests that inhaled NO combined with vitamin A can decrease the incidence of BPD better than NO therapy alone [17]. Thus, there is a need to identify additional druggable molecular targets that can complement the inhaled NO therapy to promote the development and function of the lung vascular system.

Lung development is orchestrated by a complex process involving signaling by growth factors [18], which mediate their effects mostly via the activation of mitogen-activated protein (MAP) and phosphatidylinositol 3-OH kinases. Among the four major families of MAP kinases, the extracellular signal-regulated kinases (ERK)1/2 were shown to primarily mediate proliferation and differentiation of many cell types, whereas c-Jun NH2-terminal kinases and p38 kinase mainly induce cell apoptosis [19]. In fact, ERK1/2 are activated during development in many organisms [20,21] and regulate morphogenesis in several organs including the lungs [22–24]. Thus, it seems logical that disruption of these signaling pathways may mechanistically contribute to a developmental lung disease such as BPD.

Supplemental oxygen is frequently used as a life-saving therapy in preterm infants with respiratory failure; however, excessive oxygen exposure or hyperoxia contributes to BPD pathogenesis. We [25] and others [26,27] have demonstrated that hyperoxia-induced lung parenchymal and vascular injury in newborn mice leads to a phenotype that is similar to that of human BPD. So, we used this model to investigate the effects of hyperoxia on the expression and activation of ERK1/2 proteins in the developing lungs. ERK1/2 activation is shown to protect alveolar epithelial cells against hyperoxic injury [28,29]. However, there are several knowledge gaps, including: (1) the effects of hyperoxia on the expression and activation of ERK1/2 in neonatal mouse and fetal human lung endothelial cells; (2) the effects of ERK1/2 signaling on lung angiogenesis in preterm infants. Therefore, using neonatal C57BL/6J wild type (WT) and fetal human lung cells, we tested the following hypotheses: (1) hyperoxia exposure will increase endothelial ERK1/2 signaling in neonatal C57BL/6J (WT) mouse lungs and fetal human lung endothelial cells; (2) inhibition of ERK1/2 signaling will disrupt angiogenesis in vitro by repressing cell cycle progression.

#### **2. Results**

#### *2.1. Hyperoxia Exposure Transiently Activates ERK1/2 in Neonatal Mouse Lungs*

The level of protein phosphorylation correlates strongly with the activity of a protein. Therefore, we quantified phosphorylated (p) ERK1/2 protein levels in whole lung homogenates to determine if hyperoxia activates ERK1/2. Western blot analyses (Figure 1) showed that hyperoxia exposure for one week increased ERK1/2 phosphorylation (0.6 ± 0.07 vs. 0.38 ± 0.02). However, prolonged hyperoxia exposure (14 d) decreased ERK1/2 activation compared to age-matched controls (0.31 ± 0.06 vs. 0.43 ± 0.08), suggesting that hyperoxia transiently activates ERK1/2 in the developing lungs.

#### *2.2. Hyperoxia Exposure Transiently Activates ERK1/2 in Neonatal Mouse Lung Endothelial Cells*

To investigate if hyperoxia activates ERK1/2 in mouse lung endothelial cells, we performed immunofluorescence colocalization experiments using lung sections from neonatal mice exposed to normoxia or hyperoxia for one or two weeks. We localized pERK1/2 protein expression in endothelial cells by immunofluorescence labelling using anti-pERK1/2 and anti-von Willebrand factor (vWF) antibodies. Figure 2B shows a clear overlap between the green (pERK1/2) and red (vWF) signals, indicating that pERK1/2 is expressed in lung endothelial cells. Similar to our results of immunoblotting experiments with whole lung homogenates, hyperoxia increased ERK1/2 activation in lung endothelial cells compared with the normoxia group at one week of life (Figure 2B). However, analyses of

the time-dependent effects of hyperoxia revealed that ERK1/2 activation significantly decreased in the hyperoxia group at two weeks of life (Figure 2D), a time point at which lung development is still occurring.

**Figure 1.** Lung phosphorylated extracellular signal-regulated kinases (ERK)1/2 protein levels in neonatal wild-type (WT) mice exposed to hyperoxia. Lung proteins obtained from neonatal WT mice exposed to 21% O2 (normoxia) or 70% O2 (hyperoxia) for up to two weeks (*n* = 6/exposure) were subjected to immunoblotting using antibodies against total ERK1/2, phosphorylated ERK1/2, or β-actin. Representative immunoblot showing total ERK1/2 and phosphorylated ERK1/2 protein expression (**A**). Densitometric analyses wherein the phosphorylated ERK1/2 band intensities were quantified and normalized to those of total ERK1/2 (**B**). The values are presented as mean ± SD. Significant differences between the normoxia and hyperoxia groups are indicated by \* *p* < 0.05. Significant differences between the hyperoxia groups are indicated by † *p* < 0.001 (Two-way ANOVA).

**Figure 2.** Phosphorylated ERK1/2 protein levels in lung endothelial cells of neonatal WT mice exposed to hyperoxia. One-day-old WT mice were exposed to either 21% O2 (normoxia) or 70% O2 (hyperoxia) for one or two weeks (*n* = 6/exposure/time-point), after which lung sections were processed for colocalization studies. (**A**–**D**) Representative merged images of lung sections stained with anti-pERK1/2 (green) and anti-vWF (red) antibodies, and DAPI (blue). Scale bar = 50 μM.
