Redox signaling during hypoxia in mammalian cells

Hypoxia triggers a wide range of protective responses in mammalian cells, which are mediated through transcriptional and post-translational mechanisms. Redox signaling in cells by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) occurs through the reversible oxidation of cysteine thiol groups, resulting in structural modifications that can change protein function profoundly. Mitochondria are an important source of ROS generation, and studies reveal that superoxide generation by the electron transport chain increases during hypoxia. Other sources of ROS, such as the NAD(P)H oxidases, may also generate oxidant signals in hypoxia. This review considers the growing body of work indicating that increased ROS signals during hypoxia are responsible for regulating the activation of protective mechanisms in diverse cell types

Prolonged Hypoxia Increases ROS Signaling and RhoA Activation in Pulmonary Artery Smooth Muscle and Endothelial Cells

Phase I of the hypoxic pulmonary vasoconstriction (HPV) response begins upon transition to hypoxia and involves an increase in cytosolic calcium ([Ca2+]i). Phase II develops during prolonged hypoxia and involves increases in constriction without further increases in [Ca2+]i, suggesting an increase in Ca2+ sensitivity. Prolonged hypoxia activates RhoA and RhoA kinase, which may increase Ca2+ sensitivity, but the mechanism is unknown. We previously found that reactive oxygen species (ROS) trigger Phase I. We therefore asked whether ROS generation during prolonged hypoxia activates RhoA in PA smooth muscle cells (PASMCs) and endothelial cells (PAECs) during Phase II. By using a cytosolic redox sensor, RoGFP, we detected increased oxidant signaling in prolonged hypoxia in PASMCs (29.8 ± 1.3% to 39.8 ± 1.4%) and PAECs (25.9 ± 2.1% to 43.7.9 ± 3.5%), which was reversed on the return to normoxia and was attenuated with EUK-134 in both cell types. RhoA activity increased in PASMCs and PAECs during prolonged hypoxia (6.4 ± 1.2-fold and 5.8 ± 1.6-fold) and with exogenous H2O2 (4.1- and 2.3-fold, respectively). However, abrogation of the ROS signal in PASMCs or PAECs with EUK-134 or anoxia failed to attenuate the increased RhoA activity. Thus, the ROS signal is sustained during prolonged hypoxia in PASMCs and PAECs, and this is sufficient but not required for RhoA activation. Antioxid. Redox Signal. 12, 603–610

Hypoxia Increases ROS Signaling and Cytosolic Ca2+ in Pulmonary Artery Smooth Muscle Cells of Mouse Lungs Slices

Precapillary arteries constrict during alveolar hypoxia in a response known as hypoxic pulmonary vasoconstriction (HPV). The mechanism by which pulmonary arterial smooth muscle cells (PASMCs) detect a decrease in Po2 and trigger contraction is not fully understood. Previous studies in cultured PASMCs show that hypoxia induces an increase in reactive oxygen species (ROS) production, but these results may not reflect responses of PASMCs in their native tissue environment. We therefore assessed hypoxia-induced changes in cytosolic ROS in PASMCs of precision-cut mouse lung slices expressing the redox-sensitive protein, RoGFP. Superfusion of lung slices with hypoxic media (1.5% O2) resulted in a significant oxidation of RoGFP from normoxic baseline that was attenuated by overexpression of cytosolic catalase. Hypoxic superfusion also increased [Ca2+]i above normoxic baseline; this response was significantly attenuated by cytosolic catalase overexpression or by the administration of EUK134, a synthetic SOD-catalase mimetic. The hypoxia-induced increase in [Ca2+]i was abolished in the absence of extracellular Ca2+, indicating that ROS signals trigger entry of extracellular calcium. Collectively, these results indicate that an increase in cytosolic ROS signaling is required for the increase in [Ca2+]i in PASMCs in precision-cut mouse lung slices during the acute HPV response. Antioxid. Redox Signal. 12, 595—602

Aberrant cGMP signaling persists during recovery in mice with oxygen-induced pulmonary hypertension.

Bronchopulmonary dysplasia (BPD), a common complication of preterm birth, is associated with pulmonary hypertension (PH) in 25% of infants with moderate to severe BPD. Neonatal mice exposed to hyperoxia for 14d develop lung disease similar to BPD, with evidence of associated PH. The cyclic guanosine monophosphate (cGMP) signaling pathway has not been well studied in BPD-associated PH. In addition, there is little data about the natural history of hyperoxia-induced PH in mice or the utility of phosphodiesterase-5 (PDE5) inhibition in established disease. C57BL/6 mice were placed in room air or 75% O2 within 24h of birth for 14d, followed by recovery in room air for an additional 7 days (21d). Additional pups were treated with either vehicle or sildenafil for 7d during room air recovery. Mean alveolar area, pulmonary artery (PA) medial wall thickness (MWT), RVH, and vessel density were evaluated at 21d. PA protein from 21d animals was analyzed for soluble guanylate cyclase (sGC) activity, PDE5 activity, and cGMP levels. Neonatal hyperoxia exposure results in persistent alveolar simplification, RVH, decreased vessel density, increased MWT, and disrupted cGMP signaling despite a period of room air recovery. Delayed treatment with sildenafil during room air recovery is associated with improved RVH and decreased PA PDE5 activity, but does not have significant effects on alveolar simplification, PA remodeling, or vessel density. These data are consistent with clinical studies suggesting inconsistent effects of sildenafil treatment in infants with BPD-associated PH

MP PLOS One manuscript data.pdf

Data for a manuscript entitled "Aberrant cGMP Signaling Persists During Recovery in Mice with Oxygen-Induced<div>Pulmonary Hypertension".</div