Calcium-Sensing Receptor Contributes to Hyperoxia Effects on Human Fetal Airway Smooth Muscle.

Supplemental O2 (hyperoxia), necessary for maintenance of oxygenation in premature infants, contributes to neonatal and pediatric airway diseases including asthma. Airway smooth muscle (ASM) is a key resident cell type, responding to hyperoxia with increased contractility and remodeling [proliferation, extracellular matrix (ECM) production], making the mechanisms underlying hyperoxia effects on ASM significant. Recognizing that fetal lungs experience a higher extracellular Ca2+ ([Ca2+]o) environment, we previously reported that the calcium sensing receptor (CaSR) is expressed and functional in human fetal ASM (fASM). In this study, using fASM cells from 18 to 22 week human fetal lungs, we tested the hypothesis that CaSR contributes to hyperoxia effects on developing ASM. Moderate hyperoxia (50% O2) increased fASM CaSR expression. Fluorescence [Ca2+]i imaging showed hyperoxia increased [Ca2+]i responses to histamine that was more sensitive to altered [Ca2+]o, and promoted IP3 induced intracellular Ca2+ release and store-operated Ca2+ entry: effects blunted by the calcilytic NPS2143. Hyperoxia did not significantly increase mitochondrial calcium which was regulated by CaSR irrespective of oxygen levels. Separately, fASM cell proliferation and ECM deposition (collagens but not fibronectin) showed sensitivity to [Ca2+]o that was enhanced by hyperoxia, but blunted by NPS2143. Effects of hyperoxia involved p42/44 ERK via CaSR and HIF1α. These results demonstrate functional CaSR in developing ASM that contributes to hyperoxia-induced contractility and remodeling that may be relevant to perinatal airway disease.

Cellular clocks in hyperoxia effects on [Ca2+]i regulation in developing human airway smooth muscle.

Supplemental O2 (hyperoxia) is necessary for preterm infant survival but is associated with development of bronchial airway hyperreactivity and childhood asthma. Understanding early mechanisms that link hyperoxia to altered airway structure and function are key to developing advanced therapies. We previously showed that even moderate hyperoxia (50% O2) enhances intracellular calcium ([Ca2+]i) and proliferation of human fetal airway smooth muscle (fASM), thereby facilitating bronchoconstriction and remodeling. Here, we introduce cellular clock biology as a novel mechanism linking early oxygen exposure to airway biology. Peripheral, intracellular clocks are a network of transcription-translation feedback loops that produce circadian oscillations with downstream targets highly relevant to airway function and asthma. Premature infants suffer circadian disruption whereas entrainment strategies improve outcomes, highlighting the need to understand relationships between clocks and developing airways. We hypothesized that hyperoxia impacts clock function in fASM and that the clock can be leveraged to attenuate deleterious effects of O2 on the developing airway. We report that human fASM express core clock machinery (PER1, PER2, CRY1, ARNTL/BMAL1, CLOCK) that is responsive to dexamethasone (Dex) and altered by O2. Disruption of the clock via siRNA-mediated PER1 or ARNTL knockdown alters store-operated calcium entry (SOCE) and [Ca2+]i response to histamine in hyperoxia. Effects of O2 on [Ca2+]i are rescued by driving expression of clock proteins, via effects on the Ca2+ channels IP3R and Orai1. These data reveal a functional fASM clock that modulates [Ca2+]i regulation, particularly in hyperoxia. Harnessing clock biology may be a novel therapeutic consideration for neonatal airway diseases following prematurity.

Hydrogen sulfide, oxygen, and calcium regulation in developing human airway smooth muscle.

Preterm infants can develop airway hyperreactivity and impaired bronchodilation following supplemental O2 (hyperoxia) in early life, making it important to understand mechanisms of hyperoxia effects. Endogenous hydrogen sulfide (H2 S) has anti-inflammatory and vasodilatory effects with oxidative stress. There is little understanding of H2 S signaling in developing airways. We hypothesized that the endogenous H2 S system is detrimentally influenced by O2 and conversely H2 S signaling pathways can be leveraged to attenuate deleterious effects of O2 . Using human fetal airway smooth muscle (fASM) cells, we investigated baseline expression of endogenous H2 S machinery, and effects of exogenous H2 S donors NaHS and GYY4137 in the context of moderate hyperoxia, with intracellular calcium regulation as a readout of contractility. Biochemical pathways for endogenous H2 S generation and catabolism are present in fASM, and are differentially sensitive to O2 toward overall reduction in H2 S levels. H2 S donors have downstream effects of reducing [Ca2+ ]i responses to bronchoconstrictor agonist via blunted plasma membrane Ca2+ influx: effects blocked by O2 . However, such detrimental O2 effects are targetable by exogenous H2 S donors such as NaHS and GYY4137. These data provide novel information regarding the potential for H2 S to act as a bronchodilator in developing airways in the context of oxygen exposure.

Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium.

Consistent daylight oscillations and abundant oxygen availability are fundamental to human health. Here, we investigate the intersection between light-sensing (Period 2 [PER2]) and oxygen-sensing (hypoxia-inducible factor [HIF1A]) pathways in cellular adaptation to myocardial ischemia. We demonstrate that intense light is cardioprotective via circadian PER2 amplitude enhancement, mimicking hypoxia-elicited adenosine- and HIF1A-metabolic adaptation to myocardial ischemia under normoxic conditions. Whole-genome array from intense light-exposed wild-type or Per2-/- mice and myocardial ischemia in endothelial-specific PER2-deficient mice uncover a critical role for intense light in maintaining endothelial barrier function via light-enhanced HIF1A transcription. A proteomics screen in human endothelia reveals a dominant role for PER2 in metabolic reprogramming to hypoxia via mitochondrial translocation, tricarboxylic acid (TCA) cycle enzyme activity regulation, and HIF1A transcriptional adaption to hypoxia. Translational investigation of intense light in human subjects identifies similar PER2 mechanisms, implicating the use of intense light for the treatment of cardiovascular disease.