Extracellular Signal-Regulated Kinase 1 Alone Is Dispensable for Hyperoxia-Mediated Alveolar and Pulmonary Vascular Simplification in Neonatal Mice

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.


Introduction
Bronchopulmonary dysplasia (BPD) remains the most common lung complication of extremely preterm neonates despite significant advances in the medical care of these infants [1]. BPD is also the most expensive neonatal disease; the costs required to provide medical care for a BPD infant are double that of a non-BPD infant in the first year of life [2]. Further, BPD has long-lasting effects on the health of preterm infants and affects the psychosocial and emotional well-being of their parents [3][4][5][6][7].
The lungs of BPD infants have fewer alveoli and blood vessels BPD [8,9]. Several studies have consistently shown the importance and necessary role of healthy lung blood vessels for normal lung development and for the ability of the lungs to recover from insults efficiently [10][11][12][13]. The well-known angiogenic molecules, vascular endothelial growth factor (VEGF) and nitric oxide (NO), are required for lung development in health and disease in neonatal animals. VEGF mitigates experimental BPD and pulmonary hypertension (PH) via the endothelial nitric oxide synthase pathway [14][15][16]. However, neither prophylactic nor rescue inhaled NO therapies decreased the BPD burden in humans [17,18]. However, Immortalized microvascular endothelial cells isolated from human neonatal lungs (HULEC-5a) were obtained from American Type Culture Collection (ATCC, Manassas, VA; CRL-3244) and grown in 95% air and 5% CO 2 at 37 • C, as described recently [33]. We used cells between Passages 6 and 12 for our studies.

Exposure of Cells to Hyperoxia
Hyperoxia experiments were conducted in a plexiglass, sealed chamber into which a mixture of 70% O 2 and 5% CO 2 was circulated continuously using a ProOx 110 Compact O 2 Controller (BioSpherix, Parish, NY, USA).

Measurement of H 2 O 2 Generation
The H 2 O 2 production was quantified by the ROS-Glo™ H 2 O 2 Assay (Promega, Madison, WI, USA; G8820) according to the manufacturer's protocol. Briefly, cells grown on 96-well plates to 60-70% confluence were transfected with 50 nM control or ERK1 siRNA for 24 h. The siRNA-transfected cells were then exposed to normoxia (21% O 2 and 5% CO 2 ) or hyperoxia (70% O 2 and 5% CO 2 ) for 24 h. Six hours prior to the completion of hyperoxia exposure experiments, the H 2 O 2 substrate was added to the wells, and the cell culture plates were returned to the incubator for the remainder of the experiment. At the end of the experiments, the cells were incubated with ROS-Glo™ detection solution for 20 min at room temperature before the relative luminescence units were measured using the Spectramax M3 luminescence microplate reader.

Statistical Analyses
We analyzed the results using the GraphPad Prism 9 software. The experiments were repeated at least more than one time. Data are expressed as mean ± SD. The differential effects of ERK1 siRNA transfection on the outcomes of interest were determined by t-test or ANOVA. A value of p < 0.05 was considered significant.

Animals
This study was approved and conducted in strict accordance with the federal guidelines for the humane care and use of laboratory animals by the Institutional Animal Care and Use Committee of Baylor College of Medicine (Protocol Number: AN-5631). ERK1 -/mice (B6.129 (Cg)-Mapk3tm1Gela/J, 019113) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Timed-pregnant mice raised in our animal facility were used for the experiments. We determined the genotype of the mice, as recommended by the Jackson Laboratory.

Analyses of Lung Alveolarization and Vascularization
On PND14, we inflated and fixed the lungs with 4% paraformaldehyde, as mentioned previously. Paraffin-embedded lung sections were obtained for the analysis of lung alveolarization and vascularization [37]. Alveolarization was determined by radial alveolar counts (RAC) and mean linear intercepts (MLI), whereas pulmonary vessel density was determined by quantifying von Willebrand factor (vWF) stained blood vessels less than 150 µm in diameter [32].

Lung Tissue Extraction for Genomic and Proteomic Studies
The lungs were snap-frozen in liquid nitrogen on PND7 and stored at −80 • C for subsequent RNA and protein studies. Total lung RNA was extracted and reverse transcribed to cDNA [38]. Whole lung protein was extracted as follows: the lung tissue was homogenized with a mortar and pestle in T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scientific; 78,510) with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific; 78,442). The homogenate was centrifuged at 10,000× g for 5 min at 4 • C, and the supernatant, protein lysate, was stored at −80 • C.

Statistical Analyses
GraphPad Prism 9 software was used to analyze the results. The experiments were repeated at least more than one time. Data are expressed as mean ± SD. The individual and interactive effects of ERK1 gene expression and hyperoxia exposure on lung inflammation, antioxidant enzyme expression, alveolarization, and pulmonary vascularization were assessed using analysis of variance (ANOVA). A p value of < 0.05 was considered significant.

Results
Expression and activation of ERK1 and ERK2 in HULEC-5a at baseline and following hyperoxia exposure: We initially sought to determine the relative expression and activation of ERK1 and ERK2 protein at baseline and following hyperoxia exposure in transformed human lung microvascular endothelial cells derived from a neonate (HULEC-5a) because hyperoxia and pulmonary microvascular endothelial cells play crucial roles in BPD pathogenesis. Our immunoblotting analyses suggest that the expression and activation of ERK2 protein ( Figure 1A,C,E) are 1.3-fold to 2.5-fold greater than that of ERK1 ( Figure 1A,B,D) at baseline in human pulmonary microvascular endothelial cells. Following hyperoxia exposure, the activation of ERK1 and ERK2 decrease initially at 6 h, followed by an increase at 48 h ( Figure 1A-E). Consistent with the findings at baseline, the expression and activation of ERK2 ( Figure 1A,C,E) were 1.2-fold to 2.7-fold greater than that of ERK1 ( Figure 1A,B,D) following hyperoxia exposure.
ERK1 knockdown increases ERK2 activation in HULEC-5a: We next used HULEC-5a to investigate the effects of ERK1 deficiency in hyperoxic lung injury. We used ERK1 siRNA to knockdown ERK1 protein. ERK1 siRNA reduced ERK1 protein expression and activation by greater than 2-fold to 7-fold ( Figure 2A,B,D). ERK1 knockdown increased ERK2 activation at basal conditions, as evidenced by increased phosphorylated ERK2 protein levels in ERK1-deficient cells (Figure 2A,E).
ing hyperoxia exposure, the activation of ERK1 and ERK2 decrease initially at 6 h, followed by an increase at 48 h ( Figure 1A-E). Consistent with the findings at baseline, the expression and activation of ERK2 ( Figure 1A,C,E) were 1.2-fold to 2.7-fold greater than that of ERK1 ( Figure 1A,B,D) following hyperoxia exposure. Figure 1. ERK1 and ERK2 expression and activation in HULEC-5a at baseline and following hyperoxia exposure. Cells grown on six-well plates to 60-70% confluence were exposed to normoxia (21% O2 and 5% CO2) or hyperoxia (70% O2 and 5% CO2) for up to 48 h. The cells were then harvested for protein expression studies at 6 h, 24 h, and 48 h of exposure. (A) Determination of total and phosphorylated ERK1 and ERK2 protein levels by immunoblotting. (B-E) Quantification and normalization of total (t)-ERK1 (B) and t-ERK2 (C) band intensities and phospho (p)-ERK1 (D) and p-ERK2 (E) band intensities to those of vinculin. Values represent the mean ± SD (n = 4/group). Significant differences between exposures at different time points are indicated by *, p < 0.05 and **, p < 0.01 (ANOVA). ns = not significant. ERK1 knockdown increases ERK2 activation in HULEC-5a: We next used HULEC-5a to investigate the effects of ERK1 deficiency in hyperoxic lung injury. We used ERK1 siRNA to knockdown ERK1 protein. ERK1 siRNA reduced ERK1 protein expression and activation by greater than 2-fold to 7-fold ( Figure 2A,B,D). ERK1 knockdown increased ERK2 activation at basal conditions, as evidenced by increased phosphorylated ERK2 protein levels in ERK1-deficient cells (Figure 2A,E). . ERK1 and ERK2 expression and activation in HULEC-5a at baseline and following hyperoxia exposure. Cells grown on six-well plates to 60-70% confluence were exposed to normoxia (21% O 2 and 5% CO 2 ) or hyperoxia (70% O 2 and 5% CO 2 ) for up to 48 h. The cells were then harvested for protein expression studies at 6 h, 24 h, and 48 h of exposure. (A) Determination of total and phosphorylated ERK1 and ERK2 protein levels by immunoblotting. (B-E) Quantification and normalization of total (t)-ERK1 (B) and t-ERK2 (C) band intensities and phospho (p)-ERK1 (D) and p-ERK2 (E) band intensities to those of vinculin. Values represent the mean ± SD (n = 4/group). Significant differences between exposures at different time points are indicated by *, p < 0.05 and **, p < 0.01 (ANOVA). ns = not significant. Figure 1. ERK1 and ERK2 expression and activation in HULEC-5a at baseline and following hyperoxia exposure. Cells grown on six-well plates to 60-70% confluence were exposed to normoxia (21% O2 and 5% CO2) or hyperoxia (70% O2 and 5% CO2) for up to 48 h. The cells were then harvested for protein expression studies at 6 h, 24 h, and 48 h of exposure. (A) Determination of total and phosphorylated ERK1 and ERK2 protein levels by immunoblotting. (B-E) Quantification and normalization of total (t)-ERK1 (B) and t-ERK2 (C) band intensities and phospho (p)-ERK1 (D) and p-ERK2 (E) band intensities to those of vinculin. Values represent the mean ± SD (n = 4/group). Significant differences between exposures at different time points are indicated by *, p < 0.05 and **, p < 0.01 (ANOVA). ns = not significant. ERK1 knockdown increases ERK2 activation in HULEC-5a: We next used HULEC-5a to investigate the effects of ERK1 deficiency in hyperoxic lung injury. We used ERK1 siRNA to knockdown ERK1 protein. ERK1 siRNA reduced ERK1 protein expression and activation by greater than 2-fold to 7-fold ( Figure 2A,B,D). ERK1 knockdown increased ERK2 activation at basal conditions, as evidenced by increased phosphorylated ERK2 protein levels in ERK1-deficient cells (Figure 2A,E).

Figure 2.
Effects of ERK1 knockdown on ERK1/2 expression and activation in HULEC-5a. Cells grown on six-well plates to 60% confluence were transfected with 50 nM control or ERK1 siRNA and exposed to normoxia (21% O 2 and 5% CO 2 ) for 48 h. The transfected cells were then harvested for protein expression studies. (A) Determination of total and phosphorylated ERK1 and ERK2 protein levels by immunoblotting. (B-E) Quantification and normalization of total (t)-ERK1 (B) and t-ERK2 (C) band intensities and phospho (p)-ERK1 (D) and p-ERK2 (E) band intensities to those of vinculin. Values represent the mean ± SD (n = 3/group). Significant differences between transfected cells are indicated by *, p < 0.05 and ***, p < 0.001 (t-test). ns = not significant. ERK1 is not required for HULEC-5a tubule formation: Our previous studies demonstrated that pharmacological inhibition of ERK1/2 signaling [27] and genetic inhibition of ERK2 signaling [28] decrease human pulmonary microvascular endothelial cell tubule formation. So, we investigated if ERK1 signaling is necessary for human neonatal lung angiogenesis by performing the tubule formation assay using normoxia (21% O 2 and 5% CO 2 ) and hyperoxia (70% O 2 and 5% CO 2 ) exposed ERK1-sufficient and -deficient HULEC-5a cells. Unlike genetic inhibition of ERK2 signaling, knockdown of ERK1 did not decrease HULEC-5a tubule formation either in normoxic or hyperoxic conditions ( Figure 3). ERK1 is not required for HULEC-5a tubule formation: Our previous studies demonstrated that pharmacological inhibition of ERK1/2 signaling [27] and genetic inhibition of ERK2 signaling [28] decrease human pulmonary microvascular endothelial cell tubule formation. So, we investigated if ERK1 signaling is necessary for human neonatal lung angiogenesis by performing the tubule formation assay using normoxia (21% O2 and 5% CO2) and hyperoxia (70% O2 and 5% CO2) exposed ERK1-sufficient and -deficient HULEC-5a cells. Unlike genetic inhibition of ERK2 signaling, knockdown of ERK1 did not decrease HULEC-5a tubule formation either in normoxic or hyperoxic conditions ( Figure 3). Figure 3. Effects of ERK1 knockdown on HULEC-5a tubule formation. Cells grown on six-well plates to 60% confluence were transfected with control or ERK1 siRNA and exposed to normoxia (21% O2 and 5% CO2) or hyperoxia (70% O2 and 5% CO2) for 48 h. The transfected cells were then harvested for tubule formation assay. (A-D) Representative photographs showing tubule formation of cells transfected with control (A,C) and ERK1 (B,D) siRNA and exposed to normoxia (A,B) or hyperoxia (C,D). Scale bar = 100 µm. (E) Quantification of tubule formation. Values are presented as mean ± SD (n = 7-8/group). Significant differences between exposures are indicated by ***, p < 0.001 and ****, p < 0.0001 (ANOVA). ns = not significant.
Knockdown of ERK1 does not potentiate hyperoxia-induced H2O2 generation in HULEC-5a: Reactive oxygen species (ROS) such as H2O2 are widely implicated in BPD pathogenesis. So, we investigated the effects of decreased ERK1 signaling on H2O2 generation in our in vitro model of hyperoxic lung injury. In normoxic conditions, H2O2 levels were comparable between ERK1-sufficient and -deficient cells (Figure 4), indicating that the knockdown of ERK1 does not affect H2O2 generation at baseline. Hyperoxic exposure increased H2O2 generation; however, the extent of this hyperoxia effect was similar in ERK1-sufficient and -deficient cells (Figure 4), suggesting that ERK1 is not a major regulator of H2O2 generation even in hyperoxic conditions in HULEC-5a cells. Figure 3. Effects of ERK1 knockdown on HULEC-5a tubule formation. Cells grown on six-well plates to 60% confluence were transfected with control or ERK1 siRNA and exposed to normoxia (21% O 2 and 5% CO 2 ) or hyperoxia (70% O 2 and 5% CO 2 ) for 48 h. The transfected cells were then harvested for tubule formation assay. (A-D) Representative photographs showing tubule formation of cells transfected with control (A,C) and ERK1 (B,D) siRNA and exposed to normoxia (A,B) or hyperoxia (C,D). Scale bar = 100 µm. (E) Quantification of tubule formation. Values are presented as mean ± SD (n = 7-8/group). Significant differences between exposures are indicated by ***, p < 0.001 and ****, p < 0.0001 (ANOVA). ns = not significant.
Knockdown of ERK1 does not potentiate hyperoxia-induced H 2 O 2 generation in HULEC-5a: Reactive oxygen species (ROS) such as H 2 O 2 are widely implicated in BPD pathogenesis. So, we investigated the effects of decreased ERK1 signaling on H 2 O 2 generation in our in vitro model of hyperoxic lung injury. In normoxic conditions, H 2 O 2 levels were comparable between ERK1-sufficient and -deficient cells (Figure 4), indicating that the knockdown of ERK1 does not affect H 2 O 2 generation at baseline. Hyperoxic exposure increased H 2 O 2 generation; however, the extent of this hyperoxia effect was similar in ERK1-sufficient and -deficient cells (Figure 4), suggesting that ERK1 is not a major regulator of H 2 O 2 generation even in hyperoxic conditions in HULEC-5a cells.
Global ERK1 deficiency does not potentiate hyperoxia-induced pulmonary vascular simplification in neonatal mice: In addition to alveolar simplification, fewer and dysmorphic lung blood vessels, i.e., pulmonary vascular simplification, is a hallmark of experimental BPD. Neonatal mice exposed to hyperoxia displayed fewer von Willebrand factor (vWF)-stained lung blood vessels ( Figure 7A,C,E). However, the extent of hyperoxiainduced pulmonary vascular simplification was comparable between ERK1 -/and ERK1 +/+ mice ( Figure 7C Figure 8E) without affecting the expression of the pro-inflammatory cytokines, IL-1β ( Figure 8D) and TNF-α ( Figure 8F) in our model. However, the effects of hyperoxia on the mRNA levels of these cytokines were comparable between ERK1 -/and ERK1 +/+ mice ( Figure 8A-F), indicating that isolated ERK1 deficiency does not potentiate or attenuate hyperoxia-induced lung inflammation in neonatal mice.  Figure  8C), and decreased the mRNA level of the anti-inflammatory cytokine, IL-10 ( Figure 8E) without affecting the expression of the pro-inflammatory cytokines, IL-1β ( Figure 8D) and TNF-α ( Figure 8F) in our model. However, the effects of hyperoxia on the mRNA levels of these cytokines were comparable between ERK1 -/-and ERK1 +/+ mice ( Figure 8A-F), indicating that isolated ERK1 deficiency does not potentiate or attenuate hyperoxia-induced lung inflammation in neonatal mice.

Discussion
In this study, we investigated the effects of ERK1 deficiency on human neonatal lung microvascular endothelial cell tubule formation and redox homeostasis in vitro and those of global ERK1 deficiency on hyperoxic lung injury in neonatal mice in vivo. Our in vitro experiments suggest that ERK1 deficiency alone is not necessary for both lung angiogenesis and maintaining pulmonary redox homeostasis. Further, our in vivo experiments indicate that global ERK1 deficiency alone does not augment alveolar and pulmonary vascular simplification, inflammation, and oxidative stress in hyperoxia-exposed neonatal murine lungs.

Discussion
In this study, we investigated the effects of ERK1 deficiency on human neonatal lung microvascular endothelial cell tubule formation and redox homeostasis in vitro and those of global ERK1 deficiency on hyperoxic lung injury in neonatal mice in vivo. Our in vitro experiments suggest that ERK1 deficiency alone is not necessary for both lung angiogenesis and maintaining pulmonary redox homeostasis. Further, our in vivo experiments indicate that global ERK1 deficiency alone does not augment alveolar and pulmonary vascular simplification, inflammation, and oxidative stress in hyperoxia-exposed neonatal murine lungs.
Lung endothelial cell health plays a vital role in maintaining lung health across the life span in both humans and rodents. In BPD infants, lung angiogenesis is disrupted [8][9][10]12,39]. Similarly, in rodents with experimental BPD, the structurally and functionally abnormal lung vasculature promotes alveolar simplification and lung dysfunction and delays the resolution of neonatal lung injury [1,11,[13][14][15][16]40]. These findings emphasize that lung endothelial cells are important in BPD pathogenesis and, thereby, are important therapeutic targets to improve the mortality and morbidities of BPD infants. However, the mechanisms through which these cells contribute to BPD pathogenesis remain unclear and are the focus of many preclinical and translational studies. Our previous studies demonstrate that pharmacological inhibition of ERK1/2 signaling [27] and genetic inhibition of ERK2 signaling [28] decrease HPMEC angiogenesis in vitro. We also demonstrated that deficient ERK2 signaling in the endothelial cells potentiates experimental BPD and PH [28]. However, whether ERK1 and ERK2 have distinct or redundant functions in developing lungs remains unclear. Therefore, we sought to investigate the mechanistic role of ERK1 signaling in BPD. Initially, we investigated the relative expression of ERK1 and ERK2 protein levels in the human lung microvascular endothelial cells. Consistent with the existent literature [41,42], we observed that levels of ERK2 protein are greater than those of ERK1 in these cells at baseline. We also observed a similar finding when these cells were exposed to hyperoxia. Further, ERK1 inhibition did not exert an antiangiogenic effect in vitro. Additionally, we observed a similar finding in vivo. Global ERK1 deficiency in neonatal mice did not decrease lung angiogenesis at baseline and following hyperoxia exposure. Several studies [43,44], including ours [27], have demonstrated that inhibition of both ERK1 and ERK2 decreases angiogenesis. We have also shown a similar antiangiogenic effect when ERK2 signaling is inhibited [28]. However, this study is one of the few studies to investigate the effect of ERK1 signaling on lung endothelial cell heath. Our in vitro and in vivo findings suggest that isolated ERK1 deficiency does not have a vascular phenotype, which is consistent with the findings by Pages and colleagues [45]. In a recent study by Ricard et al. [46], isolated endothelial ERK1 deficiency did not affect arteriogenesis, whereas global ERK1 deficiency or combined endothelial and macrophage ERK1 deficiency resulted in increased but dysfunctional arteriogenesis in a mouse model of acute hindlimb ischemia. They observed increased macrophage infiltration at the arteriogenesis site after inducing ischemia.
Their findings indicate that ERK1 may interact with other cells and affect vascular health in pathologic states. However, we did not observe increased lung inflammation or lung angiogenesis after neonatal exposure in global ERK1 deficient mice. Some of the reasons for these discrepant findings may be due to the differences in the nature of the insult, organs studied, and the developmental stage of mice. Our recent and current findings suggest that ERK2 may play a major role than ERK1 in maintaining lung endothelial cell health, both at baseline and under pathological conditions. Hyperoxia causes experimental and human BPD by affecting pathways necessary for lung development and repair [47,48]. Further, the lung phenotype of our hyperoxia mouse model resembles human BPD [31,32]. So, we investigated if ERK1 signaling regulates hyperoxic lung injury using global ERK1 deficient mice. Kim et al. [49] showed that the formyl peptide receptor 2 agonist, WKYMVm hexapeptide-mediated ERK1/2 activation mitigates hyperoxia-induced neonatal lung injury in mice. We also recently showed that endothelial ERK2 deficiency potentiates hyperoxic lung injury in neonatal mice [28]. However, Zhang et al. [50] and Carnesecchi et al. [51] noted an opposite effect of ERK1/2 activation on hyperoxia-induced lung injury in adult rodents. A similar finding of potentiation of hyperoxic lung injury due to ERK1/2 activation was noted in neonatal rodents, mainly mediated through fibroblasts [52,53]. Differentiation of alveolar interstitial fibroblast into lipofibroblast is necessary for lung development [54,55], whereas their differentiation into myofibroblast inhibits alveolarization [55]. These discrepant findings may be due to differences in the cell type and organ studied, activation of ERK1 and ERK2 isoforms, and the interactions between ERK1/2 and other pathways. Therefore, we investigated the effects of the ERK1 isoform and found that isolated deficiency of this isoform does not potentiate hyperoxia-induced alveolar simplification in neonatal mice. Additionally, ERK1 deficiency did not potentiate hyperoxia-induced lung inflammation in neonatal mice. Finally, we determined the effect of isolated ERK1 deficiency on hyperoxia-induced oxidative stress because the latter is one of the major BPD risk factors [56,57]. We determined oxidative stress in vivo indirectly by quantifying the expression of antioxidant enzymes since it is difficult to quantify the highly unstable reactive oxygen species in real time.
Our findings indicate that, unlike endothelial ERK2 deficiency, isolated deficiency of ERK1 does not influence the hyperoxia-induced expression of antioxidant enzyme expression in neonatal mice. We also show that ERK1 knockdown does not affect hyperoxia-induced H 2 O 2 generation in HPMECs. Collectively, the findings from our recent [28] and current studies highlight that ERK2 rather than ERK1 plays a mechanistic role in the pathogenesis of hyperoxia-induced neonatal lung injury. It is possible that increased ERK2 activation in global ERK1 knockout mice may have protected them from worsening lung injury when exposed to hyperoxia because the outcomes may be ultimately dependent on the total ERK1/2 activity, as demonstrated recently. In an elegant study, Fremin et al. [41] demonstrated that the severe developmental defects seen in ERK2-knockout mice can be rescued by increasing the total ERK1/2 activity from the transgenic expression of ERK1. These findings indicate that the phenotype positively correlates with the total ERK1/2 activity, highlighting the functional redundancy of ERK1 and ERK2. In our future studies, we will use genetic approaches to increase ERK1 expression in ERK2-deficient endothelial cells to further differentiate the redundant and distinct functions of ERK1 and ERK2 in neonatal hyperoxic lung injury. It is also possible that ERK1 may regulate neonatal lung injury in a sex-specific manner. Additionally, ERK1 may preferentially affect lung function rather than lung development. Finally, ERK1 may have a pathogenic role in developmental lung injury mediated by other insults, including lipopolysaccharide. We plan to address these gaps in our future studies. Nevertheless, we used robust genetic approaches using both human pulmonary microvascular endothelial cells and neonatal mice and performed rigorous studies to determine the mechanistic role of ERK1 signaling in hyperoxia-induced neonatal lung injury.
In summary, we demonstrate that isolated ERK1 signaling is not required for HPMEC tubule formation and, thereby, probably for human lung angiogenesis. We also show that ERK1 is not a major regulator of hyperoxia-induced oxidative stress in these cells. Further, our in vivo studies demonstrate that global ERK1 deficiency is dispensable for hyperoxiainduced neonatal murine lung injury. To the best of our knowledge, this is the first study to investigate the role of isolated ERK1 signaling in hyperoxia-induced experimental BPD. Our findings reinforce that targeting ERK2 rather than ERK1 may improve BPD outcomes.

Conclusions
ERK1 is dispensable for hyperoxia-induced experimental BPD due to compensatory ERK2 overactivation.