*2.4. Hyperoxia Exposure Increases Pulmonary Endothelial Cell Apoptosis in Neonatal Mice*

To identify the mechanisms through which hyperoxia interrupts lung angiogenesis, we performed immunofluorescence colocalization experiments using lung sections from neonatal mice exposed to normoxia or hyperoxia for one or two weeks. We determined apoptosis in lung endothelial cells by immunofluorescence labelling using an indirect TUNEL assay and anti-vWF antibodies. Figure 4 shows an increased intensity of apoptotic stain (green) in vWF-stained endothelial cells (red) in animals exposed to hyperoxia (Figure 4B,D), indicating that hyperoxia causes lung endothelial cell apoptosis.

**Figure 4.** Lung endothelial cell apoptosis in 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 the lung sections were processed for colocalization studies. (**A**–**D**) Representative merged images of lung sections stained with TUNEL (green), anti-vWF (red) antibody, and DAPI (blue). The frames in the original magnification figures represent the zoomed regions. Scale bar = 50 μM.

#### *2.5. Hyperoxia Exposure Activates ERK1/2 in HPAECs*

To examine the clinical significance of our findings, we used fetal HPAECs to determine if hyperoxia similarly activates ERK1/2 in the developing lungs of preterm infants. Similar to our findings in neonatal mouse lungs, time-dependent studies showed that hyperoxia increased ERK1/2 phosphorylation (0.35 ± 0.01 vs. 0.22 ± 0.03), and the extent of phosphorylation declined (0.28 ± 0.01 vs. 0.21 ± 0.02) as the duration of hyperoxia was prolonged (Figure 5).

**Figure 5.** Phosphorylated ERK1/2 protein levels in human pulmonary artery endothelial cells (HPAECs) exposed to hyperoxia. HPAECs were exposed to normoxia or hyperoxia for 24 or 48 h, following which whole-cell proteins were extracted, and immunoblotting was performed 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 (*n* = 6/group). Two-way ANOVA analysis showed an effect of hyperoxia and duration of exposure and an interaction between them for the dependent variable, p-ERK1/2, in this figure. Significant differences between normoxia- and hyperoxia-exposed cells are indicated by \* *p* < 0.001. Significant differences between hyperoxia-exposed cells are indicated by † *p* < 0.01 (Two-way ANOVA).

#### *2.6. PD98059 Efficiently Inhibits ERK1/2 Activation in HPAECs*

To investigate if ERK1/2 plays a direct role in angiogenesis in the developing lungs, we performed in vitro angiogenesis assays after inhibiting ERK1/2 activity by PD98059 in HPAECs. To this end, we first validated ERK1/2 inhibition by determining the expression of phosphorylated ERK1/2 protein levels in PD98059-treated cells. As expected, PD98059 decreased phosphorylated ERK1/2 protein expression in a dose-dependent manner (Figure 6), indicating that PD98059 efficiently inhibits ERK1/2 activation also in these cells.

**Figure 6.** Decreased phosphorylated ERK1/2 protein levels in HPAECs treated with PD98059. HPAECs were treated with dimethylsulfoxide (DMSO) or PD98059 at concentrations of 10 (PD 10) or 30 (PD 30) μM for 30 min, after which whole-cell proteins were extracted, and immunoblotting was performed 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 (*n* = 6/group). Significant differences between DMSO- and PD-treated cells are indicated by \* *p* < 0.001 (One-way ANOVA).

#### *2.7. ERK1/2 Inhibition Decreases HPAEC Migration*

Endothelial cell migration, proliferation, and tubule formation are essential steps in angiogenesis. To determine the effects of ERK1/2 inhibition on these steps, we performed scratch, proliferation, and tubule formation assays in cells treated with varying concentrations of PD98059. We initially performed the scratch assay and quantified cell migration by assessing the extent of wound closure in mitomycin-treated cell monolayers exposed to the vehicle or PD98059. The extent of wound closure was significantly decreased by PD98059 in a dose-dependent manner (Figure 7), indicating that ERK1/2 inhibition decreases cell migration. When compared with vehicle-treated cells, 10, 20, and 30 μM of PD98059 inhibited cell migration by 29.6, 55.5, and 91.8%, respectively (Figure 7I).

### *2.8. ERK1/2 Inhibition Decreases HPAEC Proliferation*

The MTT activity reflects the cell number, and, thus, the measured absorbance positively correlates with cell proliferation. As expected, there was a time-dependent effect on cell proliferation in vehicle-treated cells. Their proliferation rate increased by 38% in 24 h (Figure 8). However, PD98059 decreased cell proliferation in a dose-dependent manner (Figure 8). When compared with vehicle-treated cells, 10, 20, and 30 μM of PD98059 inhibited cell proliferation by 6.7, 26.4, and 29.1%, respectively, at 24 h and by 25, 29, and 30.8%, respectively, at 48 h.

### *2.9. ERK1/2 Inhibition Decreases HPAEC Tubule and Mesh Formation*

A Matrigel assay was performed to determine the extent of tubule and mesh formation in cells treated with the vehicle or with 30 μM PD98059. Consistent with its effects on cell proliferation and migration, PD98059 decreased HPAEC tubule (1.9 ± 1.3 vs. 5.1 ± 2.3) (Figure 9A–C) and mesh (0.4 ± 0.6 vs. 1.7 ± 0.9) (Figure 9A,B,D) formation, in comparison with vehicle-treated cells.

**Figure 7.** Suppression of ERK1/2 activity decreases HPAEC migration. HPAECs grown as monolayers in six-well plates were treated with 10 μg/mL of mitomycin for 2 h and scratched with a 200 μL pipette tip. The cells were then treated with dimethylsulfoxide (DMSO) or PD98059 at concentrations of 10 (PD 10), 20 (PD 20), or 30 (PD 30) μM. The wound closure area was analyzed using Image J software after 16 h of treatment. (**A**–**H**) Representative photographs showing cell migration. (**I**) Quantitative analysis of cell migration. The values are presented as mean ± SD (*n* = 6/group). Significant differences between DMSO- and PD-treated cells are indicated by \*, (DMSO vs. PD 10 [*p* < 0.05]; DMSO vs. PD 20 and PD 30 [*p* < 0.001]) (One-way ANOVA). Scale bar = 100 μM.

**Figure 8.** Suppression of ERK1/2 activity decreases HPAEC proliferation. HPAECs were treated with dimethylsulfoxide (DMSO) or PD98059 at concentrations of 10 (PD 10), 20 (PD 20), or 30 (PD 30) μM for 24 or 48 h, following which cell proliferation was assessed by the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay. The values are presented as mean ± SD (*n* = 10/group). Two-way ANOVA analysis showed an effect of PD and duration of exposure and an interaction between them for the dependent variable, absorbance at 570 nm, in this figure. Significant differences between DMSO- and PD-treated cells are indicated by \* *p* < 0.001 (Two-way ANOVA).
