*2.3. Prolonged Hyperoxia Exposure Interrupts Pulmonary Vascularization in Neonatal Mice*

We next determined pulmonary vascularization by quantifying the vWF-stained lung blood vessels. Interestingly, the changes in pulmonary vascularization followed an pattern identical to that of lung endothelial cell ERK1/2 activation in hyperoxia-exposed animals. One week of hyperoxia exposure (Figure 3A,B,E) significantly increased the number of vWF-stained lung blood vessels (7.3 ± 3.4 vs. 4.6 ± 2.7), whereas prolonged (two weeks) hyperoxia exposure (Figure 3C–E) decreased the number of blood vessels (4.9 ± 2.4 vs. 7.4 ± 2.4) in comparison with normoxia-exposed mice.

**Figure 3.** Pulmonary vascularization 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), following which the lung sections were stained with anti-von Willebrand factor (vWF) antibodies. (**A**–**D**) Representative vWF-stained lung blood vessels (red). (**E**) Quantitative analysis of vWF-stained lung blood vessels per high-power field (HPF). The values are presented as the mean ± SD. Two-way ANOVA analysis showed an effect of hyperoxia and duration of exposure and an interaction between them for the dependent variable, vWF-stained vessels, in this figure. Significant differences between normoxia- and hyperoxia-exposed mice are indicated by \* *p* < 0.01 (Two-way ANOVA). Scale bar = 100 μM.
