*2.10. ERK1/2 Inhibition Alters the Level of Proteins That Regulate Cell Cycle Progression in HPAECs*

We finally investigated the mechanisms by which ERK1/2 inhibition disrupts angiogenesis in vitro. Because cell cycle regulation is the major biological pathway affected by ERK1/2 signaling, we determined the effects of ERK1/2 inhibition on the expression of proteins that modulate the crucial G1/S phase of cell cycle. Cyclins A and D and Cdk4 promote G1/S phase transition, while the cyclin-dependent kinase inhibitor p27 prevents this transition. Exposure of cells to the ERK1/2 inhibitor for 24 h decreased the protein levels of cyclin A and Cdk4 and increased the protein levels of p27. Cyclin D expression was similar between the vehicle- and PD-treated cells (Figure 10). These

findings indicate that repression of cell cycle progression is one of the mechanisms by which ERK1/2 inhibition interrupts angiogenesis.

**Figure 9.** Suppression of ERK1/2 activity decreases HPAEC tubule and mesh formation. HPAECs were pre-treated with dimethylsulfoxide (DMSO) or 30 μM PD98059 (PD 30) for 30 min before being loaded on growth factor-reduced Matrigel (BD Bioscience) in 96-well plates. Following an incubation period of 18 h, tubule formation was quantified. (**A**,**B**) Representative photographs showing tubule formation in growth factor-reduced Matrigel. (**C**,**D**) Quantitative analysis of tubule (**C**) and mesh (**D**) formation. The values are presented as mean ± SD (*n* = 9/group). Significant differences between DMSO- and PD-treated cells are indicated by \* *p* < 0.001 (*t*-test). Scale bar = 100 μM.

**Figure 10.** ERK1/2 inhibition affects the expression of cell cycle regulatory proteins. HPAECs were treated with dimethylsulfoxide (DMSO) or 30 μM PD98059 (PD 30) for 24 h, after which whole-cell protein were extracted, and immunoblotting was performed using antibodies against the following proteins: cyclin A, cyclin D, Cdk4, p27, and β-actin. Representative immunoblots showing the expression of the above proteins (**A**). Densitometric analyses wherein cyclin A (**B**), cyclin D (**C**), Cdk4 (**D**), and p27 Kip 1 (**E**) band intensities were quantified and normalized to those of total β-actin. The values are presented as mean ± SD (*n* = 6/group). Significant differences between DMSO- and PD-treated cells are indicated by \* *p* < 0.05 (*t*-test).

#### **3. Discussion**

In this study, we investigated the interaction between hyperoxia and ERK1/2 activation in mouse lungs and fetal HPAECs. Previously, we demonstrated that moderate hyperoxia exposure leads to increased lung oxidative stress and inflammation and causes alveolar and pulmonary vascular simplification, pulmonary vascular remodeling, and PH [25]. Further, we showed in the same model that exposure of mice to neonatal hyperoxia causes lung developmental abnormalities that persist into adolescence [30]. These findings indicate that the phenotype of our mouse model closely aligns with that of preterm infants with BPD and PH. Therefore, we chose the same model for this study and found that hyperoxia exposure transiently increases ERK1/2 activation before decreasing it to below-baseline levels. Further, we show that hyperoxia exposure affects mouse lung vascularization in an identical pattern. Finally, using fetal HPAECs, we demonstrate that inhibition of ERK1/2 pathway disrupts angiogenesis by repressing cell cycle progression.

Lung angiogenesis actively contributes to alveologenesis during development, and healthy lung blood vessels are necessary to maintain the structural and functional integrity of alveolar structures later in life. Numerous in vitro studies have demonstrated that ERK1/2 promote angiogenesis. For example, Mavria et al. [31] demonstrated that these kinases promote endothelial cell survival and sprouting by repressing Rho kinase signaling. Similarly, Murphy et al. [32] showed that sorafenib exerts tumoricidal anti-angiogenic effects by inhibiting of ERK1/2 signaling. Further, an elegant in vivo study using mice lacking endothelial ERK1/2 genes demonstrated that ERK1/2 genes are necessary for embryonic angiogenesis [33]. Therefore, we initially determined the effects of hyperoxia on ERK1/2 activation in the lungs. Hyperoxia exposure was shown to increase ERK1/2 activation in whole lung homogenates and lung epithelial cells by several investigators [34–39]; however, the effects of hyperoxia on lung endothelial cell ERK1/2 activation are poorly understood. Our study demonstrates that hyperoxia exposure increases lung endothelial cell ERK1/2 activation at postnatal day (PND) 7, but decreases their activation at PND14. Importantly, PND14 is still a critical time period for lung development in mice. Lung development occurs at an accelerated rate between PND5 and PND14, and the maximal alveolar number is reached by PND39 [40]. These findings indicate that there may be a mechanistic link between endothelial ERK1/2 signaling and hyperoxia-induced developmental lung injury. To this end, we determined the time-dependent effects of hyperoxia on pulmonary vascularization in our mouse model.

Brief exposures to a high inspired O2 concentration (>95% O2) or prolonged exposures to a moderate oxygen concentration was shown to inhibit lung angiogenesis. Here, we show that exposure to a moderate oxygen concentration (70% O2) initially increased and later induced a significant decline in angiogenesis. Interestingly, hyperoxia-induced changes in lung vascularization paralleled those of ERK1/2 activation. Further, the decreased ERK1/2 activation correlated with increased apoptosis in lung endothelial cells. It is possible that upon exposure to moderate hyperoxia, several angiogenic molecules, such as ERK1/2, are activated to promote or maintain angiogenesis and facilitate lung repair. However, with prolonged hyperoxia exposure, these changes are not sustained to promote healing and prevent further damage from hyperoxic injury. Therefore, we hypothesized that the early ERK1/2 activation upon hyperoxia exposure is an adaptive response to mitigate rather than to potentiate hyperoxic lung injury. To test this hypothesis and to examine the clinical significance of our animal studies, we investigated the effects of hyperoxia exposure on ERK1/2 activation and the effects of ERK1/2 inhibition on angiogenesis using fetal HPAECs. HPAECs were selected because: (1) their proliferation and maturation are crucial for alveolarization and lung growth; (2) their dysfunction contributes to BPD pathogenesis; (3) arterial endothelial cells are enriched in ERK1/2 proteins. Similar to our findings in mouse lungs, hyperoxia exposure transiently increased ERK1/2 activation before decreasing it, when compared with normoxia exposure. Activation of ERK1/2 promotes cell proliferation and differentiation in health [41] and protects against cell death in pathological states [42–44]. However, it is unclear if ERK1/2 signaling attenuates or potentiates hyperoxia-mediated cytotoxicity. Several investigators have demonstrated that ERK1/2

activation protects lung epithelial cells against hyperoxia-induced cell death [28,29,45,46]. Similarly, Ahmad et al. [47] showed that ERK1/2 activation protects adult human lung pulmonary microvascular endothelial cells against hyperoxia-induced cell death. On the other hand, Zhang et al. [36] have demonstrated that inhibition of ERK1/2 signaling decreases cytochrome c release, caspase-9 and -3 activation, and poly (ADP-ribosyl) polymerase cleavage and attenuates lung epithelial cell death in hyperoxic conditions. Similarly, Carnesecchi et al. [46] demonstrated that NADPH oxidase 1 inhibition decreases oxidative stress-mediated ERK1/2 activation and attenuates acute hyperoxic lung injury in adult mice. Further, it was also shown that ERK1/2 activation potentiates hyperoxia-induced developmental lung injury, primarily by regulating the proliferation and differentiation of fibroblasts [35]. The fate of alveolar interstitial fibroblasts influences lung epithelial proliferation and differentiation, i.e., lung development. Differentiation of alveolar interstitial fibroblasts into lipofibroblast promotes lung epithelial proliferation and differentiation [48,49], whereas their differentiation into myofibroblasts interrupts lung development [49]. Under hyperoxic conditions, ERK1/2 activation was shown to be associated with increased myofibroblast differentiation [35]. It is possible that the biological response of ERK1/2 activation may be dependent upon several factors, including the cell and tissue types, magnitude and duration of ERK1/2 activation, and the interactions between ERK1/2 and other activated pathways. Further studies using ERK1/2 transgenic mice are needed to address these knowledge gaps.

The proangiogenic effects of ERK1/2 signaling is well established in the field of cancer biology. However, it is important to note that endothelial cells display substantial organ- and tissue-specific diversity [50,51]. Further, whether ERK1/2 signaling regulates angiogenesis in the developing lungs of humans needs to be determined. Consequently, we inhibited ERK1/2 activation by PD98035 and determined the resulting effects on in vitro angiogenesis using fetal HPAECs. Angiogenesis is a highly coordinated multistep process that includes cell migration, proliferation, and tubule formation [52]. Inhibition of any of these processes disrupts angiogenesis [53]. We observed that ERK1/2 inhibition decreased fetal lung endothelial cell migration, proliferation, and tubule and mesh formation, indicating that ERK1/2 signaling regulates angiogenesis in the developing lungs. Others have shown that ERK1/2 activation is associated with pulmonary vascular development [54]; however, to the best of our knowledge, ours is the first study to indicate that ERK1/2 signaling is necessary for pulmonary vascular development. The angiogenic molecule VEGF promotes lung angiogenesis via ERK1/2 activation [54], but the downstream effectors of ERK1/2 activation are poorly characterized. Hence, we finally investigated the mechanisms by which ERK1/2 signaling regulates lung angiogenesis. A large body of evidence indicate that cell cycle regulation is the predominant biological pathway regulated by ERK1/2 signaling. G1/S phase transition is critical for cell cycle progression. Cyclins A and D and Cdk4 promote this transition, whereas cyclin-dependent kinase inhibitor p27 prevents this transition [55]. Importantly, cyclin A regulates cell cycle progression at multiple levels and therefore can be considered a master regulator of the cell cycle [56]. The Cdks determine the biological activity of cyclin A. For example, cyclin A-mediated activation of Cdk2 promotes G1/S transition, whereas activation of Cdk1 promotes G2/M phase transition. We demonstrate that ERK1/2 inhibition prevented cell cycle progression by decreasing the expression of cyclin A and Cdk4 and increasing that of p27, a conclusion supported by other investigators [33,57,58].

The major limitation of this study is that the interactions between ERK1/2 signaling and lung angiogenesis were determined by in vitro studies. However, our studies in a clinically relevant model of hyperoxia-induced developmental lung injury suggest that there may be similar interactions in vivo. Our future studies will address this limitation by performing in vivo studies using ERK1/2 transgenic mice.

In summary, we demonstrate that exposure of neonatal mice to hyperoxia causes parallel changes in lung endothelial cell ERK1/2 activation and lung vascularization, wherein it initially increases ERK1/2 activation and lung vascularization and later induces a significant decline of these biological processes. Further, our in vitro studies using primary fetal human lung endothelial cells show that ERK1/2 signaling may be necessary for pulmonary vascular development. Our findings signify that targeting ERK1/2 signaling may be beneficial for BPD infants who have decreased lung vascularization.

#### **4. Materials and Methods**

#### *4.1. In Vivo Experiments*

#### 4.1.1. 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 (AN-5631, 12/12/2016). C57BL/6J wild-type (WT) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Timed-pregnant mice raised in our animal facility were used for the experiments. The dams were fed standard mice food and water ad libitum, and all the experimental animals were maintained in 12 h day–night cycles.

#### 4.1.2. Hyperoxia Experiments

Within 24 h of birth, WT dams and their male and female pups were exposed to 21% O2 (normoxia, *n* = 18) or 70% O2 (hyperoxia, *n* = 18) for up to two weeks. The dams were rotated between normoxiaand hyperoxia-exposed litters every 24 h during the experiment to prevent oxygen toxicity in the dams and to control for the maternal effects between the groups. Oxygen exposures were conducted in plexiglass chambers, and the animals were monitored as described previously [59].

#### 4.1.3. Lung Tissue Harvest and Protein Extraction

The lungs from a subset of study animals (*n* = 6/exposure) were snap-frozen in liquid nitrogen and stored at −80 ◦C for the subsequent isolation of total proteins. A mortar and pestle were used to homogenize the lung tissue in a buffer containing 50 mM Tris-HCL (pH 7.5), 0.5 M KCL, 1 M MgCL, and 0.5 M EDTA. The homogenates were centrifuged at 2400× *g* for 5 min at 4 ◦C. The supernatants (protein lysate) were stored at −80 ◦C.

#### 4.1.4. Western Blot Assays

The protein lysates from the experimental animals were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight at 4 ◦C with the following primary antibodies: anti-β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-47778, dilution 1:1000), anti-total ERK1/2 (Cell Signaling, Danvers, MA, USA; 4695, dilution 1:1000), and anti-phospho-ERK1/2 (Cell Signaling, Danvers, MA, USA; 9106, dilution 1:1000). The primary antibodies were detected by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies. The immunoreactive bands were detected by chemiluminescence methods, and the band densities were quantified by Image lab 5.2.1 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
