Reiji Ito, Pediatrics/Jikei University School of Medicine, Minato-ku, Tokyo, Japan; Elizabeth Barnes, Stanford University School of Medicine, Stanford, CA, United States; Xibing Che, Stanford University School of Medicine, Palo Alto, CA, United States; Cristina M. Alvira, Stanford University School of Medicine, Stanford, CA, United States; David N. Cornfield, Stanford University, Stanford, CA, United States
Senior Research Scientist Stanford University School of Medicine Stanford, California, United States
Background: Advances in neonatal medicine have dramatically improved survival rates for preterm infants. However, many very low birth weight (VLBW) infants exposed to prolonged supplemental oxygen develop bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by an arrest of secondary septation and compromised angiogenesis. Whether and how molecular signaling in pulmonary artery smooth muscle cells (PASMC) is altered in neonatal hyperoxia-induced lung injury remains unknown.
Objective: To test the hypothesis that increased HIF protein expression in SM22α expressing cells mitigates hyperoxia-induced lung injury in neonatal mice.
Design/Methods: To stabilize HIF-1α in a cell-specific manner, SM22α-prolyl hydroxylase domain (PHD) 1/2-/- mice were generated by crossing SM22α-Cre recombinase mice with PHD1fl/fl;PHD2fl/fl mice. Neonatal mice were maintained in either normoxia (21% O2) or hyperoxia (80% O2) for 14 days, and then recovered in normoxia for 10 weeks. Tissue was obtained for analysis at both 14 days and 12 weeks.
Results: In SM22α-PHD 1/2-/- mice, protein expression of HIF-1α, but not HIF-2α, was dramatically increased in both normoxia and hyperoxia in isolated SMC. After 14 days of hyperoxia, radial alveolar count (RAC) was lower (4.6±0.2 vs. 6.8±0.2 , p< 0.001), mean linear intercept (MLI) higher (33.5±0.7µm vs. 25.8±0.9μm, p< 0.001), and microvascular density (MVD) decreased (2.7±0.3 vs. 5.9±0.8, p< 0.05) in control mice compared to SM22α-PHD1/2-/- mice. Pulmonary endothelial cells (PEC) were more proliferative and pulmonary arteries expressed more collagen IV in SM22α-PHD1/2-/- mice, compared to control mice, under both normoxic and hyperoxic conditions. Pulmonary vascular Angpt2 expression was greater in SM22α-PHD1/2-/- mice. In tube forming assays, PEC co-cultured with PASMC isolated from SM22α-PHD1/2-/- mice formed more (4.8±0.2 vs. 3.7±0.2, p< 0.05) and longer tubes (70.0±4.9µm vs. 58.9±4.5µm, p< 0.01) with increased branching (6.8±0.2 vs. 4.0±0.4, p< 0.05) compared to PEC co-cultured with PASMC isolated from SM22α-PHD1/2+/+ mice. The addition of Ang2 recombinant protein in co-cultures further amplified tube formation for both PHD1/2+/+ and PHD1/2-/- PASMC, while addition of soluble Ang2 antibody blocked tube formation.Conclusion(s): Thus, HIF-1α stabilization in SM22α expressing cells increases angiogenesis, mitigates hyperoxia-induced lung injury, and induces Ang2 and collagen IV expression. These results suggest that SM22α-specific HIF-1α stabilization may be a therapeutic target for neonatal hyperoxia-induced lung injury.
