Hyperoxia suppresses DDAH1 expression and promotes proliferation in human pulmonary microvascular endothelial cells

Abstract

Objective Pulmonary hypertension (PH) is associated with mortality in patients with bronchopulmonary dysplasia (BPD), and BPD-PH is characterized by pulmonary vascular remodeling. Patients often receive chronic supplemental oxygen and nitric oxide (NO)/sildenifil therapy to dilate the pulmonary vasculature. In vitro studies of human pulmonary microvascular endothelial cells (hPMVEC) have shown that dimethylarginine dimethylaminohydrolase-1 (DDAH1) promotes NO-mediated apoptosis. In this study, we hypothesized that DDAH1 is regulated by hyperoxia, and that hyperoxia regulates hPMVEC proliferation. Methods hPMVEC were grown at 37 ̊C in a 60mm plate to ~70% confluence. hPMVEC were then exposed to 85% O2 or 21% O2 for 6, 12, or 24 hours, and RNA or protein harvested for RT-PCR or Western blot, respectively. In a separate experiment, hPMVEC were seeded at 1x105 cells in 30mm plates, exposed to 85% O2 or 21% O2 for 24 or 72 hours and viable cell number counted by trypan blue exclusion. Newborn wild-type DDAH1 mice were exposed to 85% O2 or 21% O2 for 14 days, followed by an additional 14 days of 21% O2, then protein was isolated from lung for DDAH1 protein by Western blot. Groups were compared by t-test, p<0.05 was significant. Results hPMVEC exposed to 24 hours of hyperoxia had lower DDAH1 RNA (N=5, p<0.05) and protein (N=6, p<0.05) compared to normoxic control cells, with no differences at 6 or 12 hours. The number of viable hPMVEC (x105) was greater (1.83 ± 0.07 vs. 1.38 ± 0.17; N=6, p<0.05) after 24 hours, and the number of viable hPMVEC (x105) was lower (1.26 ± 0.12 vs. 2.19 ± 0.22; N=6, p<0.01) after 72 hours of hyperoxia compared to normoxia control. DDAH1 protein expression in mouse lung homogenates was significantly lower after 85% O2 compared to 21% O2 mice (N=3, p<0.05). Conclusions These data support the concept that in hPMVEC, hyperoxia negatively regulates DDAH1. We observed that hyperoxia results in a proliferative phenotype that coincided with lower DDAH1 levels. In BPD-PH, where patients routinely receive life-saving hyperoxia treatment, the pathophysiology may include pulmonary endothelial-DDAH1 depletion, therefore, we speculate that therapies that up-regulate pulmonary endothelial-DDAH1 activity may protect endogenous NO production to prevent and/or treat PH. Future studies will determine the role of DDAH1 in hyperoxia-mediated hPMVEC proliferation, with a particular focus on the effects of hyperoxia-mediated DDAH1 suppression on NO-mediated apoptosis in hPMVEC.