MacFarlane, Peter M.

Maternal infection is a risk for preterm delivery. Preterm newborns often require supplemental oxygen to treat neonatal respiratory distress. Newborn hyperoxia exposure is associated with airway and vascular hyperreactivity, while the complications of maternal infection are variable. In a mouse model of prenatal maternal intraperitoneal lipopolysaccharide (LPS, embryonic day 18) with subsequent newborn hyperoxia (40% oxygen × 7 days) precision-cut living lung slices were used to measure intrapulmonary airway and vascular reactivity at 21 days of age. Hyperoxia increased airway reactivity to methacholine compared to room air controls. Prenatal maternal LPS did not alter airway reactivity in room air. Combined maternal LPS and hyperoxia exposures increased airway reactivity vs. controls, although maximal responses were diminished compared to hyperoxia alone. Vessel reactivity to serotonin did not significantly differ in hyperoxia or room air; however, prenatal maternal LPS appeared to attenuate vessel reactivity in room air. Following room air recovery, LPS with hyperoxia lungs displayed upregulated inflammatory and fibrosis genes compared to room air saline controls (TNFαR1, iNOS, and TGFβ). In this model, mild newborn hyperoxia increases airway but not vessel reactivity. Prenatal maternal LPS did not further increase hyperoxic airway reactivity. However, inflammatory genes remain upregulated weeks after recovery from maternal LPS and newborn hyperoxia exposures.

Supplemental O 2 is a primary mode of respiratory therapy for preterm infants. Numerous studies, however, have shown that supplemental O 2 may contribute to the pathophysiology of airway hyperreactivity (AHR) associated with wheezing disorders such as asthma later in childhood. The calcium‐sensitive receptor (CaSR) is a potent modulator of smooth muscle contractility and may be an underlying feature of childhood asthma. In the present study, we investigated whether CaSR is involved in neonatal hyperoxia induced AHR in a mouse model of BPD. We hypothesize that inhibition (CaSR antagonism), downregulation of expression (siRNA), or deletion (CaSR KO mice) of CaSR will attenuate the AHR induced by hyperoxia in neonatal mice. Newborn C57BL/6 wild‐type (background) or CaSR −/− (knockout) mice were randomized on the second day of life and assigned to room air (21% O 2 ) or hyperoxic (40% O 2 ) groups and exposed for seven days, then recovered in room air for two additional weeks. At day P21, AHR was assessed in vitro using precision‐cut living lung slice preparations in response to increasing doses of methacholine (MCh, 0.25–8 μM). Wild‐type lung slices were pre‐incubated for 48 h in a CaSR siRNA sequence (40 nM) or scrambled RNA as a negative control. In another set of experiments, slices were pre‐incubated in a CaSR antagonist – NPS 2143 (10 nM) for 1 h. Airways were imaged and airway reactivity was expressed as percent change from baseline airway lumen area (± SEM). Neonatal hyperoxia significantly increased airway contractile responses to MCh in wild‐type mice with a mean maximal effect (Emax) of 68.4±8.7% decrease in airway lumen area compared to room air controls (47.3±5.8%, p<0.001), indicating AHR. Pre‐incubation (48hrs) with siRNA targeting CaSR or pharmacologic inhibition (NPS 2143, 1hr incubation) of CaSR reversed the hyperoxia‐induced AHR and the contractile responses were normalized to control level, with hyperoxic groups Emax of 44.0±5.2% and 41.3±3.1%, respectively. AHR was not observed in CaSR knockout mice suggesting resistance to hyperoxia exposure. These data suggest that CaSR plays an important role in the long‐term effects of neonatal hyperoxia on AHR. We speculate that CaSR may be a novel therapeutic target to reverse airway hyperreactivity in former preterm infants who had received prior supplemental O 2 therapy. Support or Funding Information Funding: NIH: HL R01 138402 and NIH R01 HL 056470

Continuous positive airway pressure (CPAP) is a common mode of respiratory support for preterm infants. Recent studies from our lab, however, have shown that CPAP may contribute to the pathophysiology of airway hyperreactivity (AHR) associated with wheezing disorders in former preterm infants. The long‐term effects of CPAP on lung‐development, however, still remain largely unknown. Both hyaluronan synthase 3 (HAS3) and the calcium‐sensitive receptor (CaSR) have been associated with various respiratory morbidities. In the present study, we used a neonatal mouse model of daily CPAP treatment to investigate whether the long‐term effects on AHR are associated with changes in airway HAS3 and CaSR expression. Neonatal mice were exposed to CPAP (6cm H 2 O) for 3 hours per day for the first 7 postnatal days. Control animals experienced the same conditions as experimental animals only with no CPAP. On postnatal day 21, the lungs from these animals were harvested for either real time rt‐PCR or immunohistochemistry to assess changes in HAS3 and CaSR expression. We observed increased expression of multiple genes involved in hylauraonan synthesis, most notably has3 , as well as increased CaSR expression, in the lungs of mice two weeks after CPAP ended when compared to control mice. The increased gene expression of has3 and CaSR was also reflected in increased protein expression as confirmed by immunohistochemistry. HAS3 was expressed predominantly in airway epithelium and the increased CaSR expression correlated with increased αSM actin. To further investigate the role of CaSR in CPAP‐induced AHR, CaSR KO mice ( CaSR (−/−) ) were exposed to CPAP and airway reactivity to methacholine challenge was assessed two weeks later (P21 days). Compared to control mice, AHR was not expressed in CaSR (−/−) mice suggesting resistance to CPAP treatment. These data suggest increased airway HAS3 and CaSR may be important modulators of CPAP‐induced AHR and remodeling. Support or Funding Information HLR01138402 HLR01056470

Abstract Airway smooth muscle (ASM) regulation of airway structure and contractility is critical in fetal/neonatal physiology in health and disease. Fetal lungs experience higher Ca 2+ environment that may impact extracellular Ca 2+ ([Ca 2+ ] o ) sensing receptor (CaSR). Well‐known in the parathyroid gland, CaSR is also expressed in late embryonic lung mesenchyme. Using cells from 18‐22 week human fetal lungs, we tested the hypothesis that CaSR regulates intracellular Ca 2+ ([Ca 2+ ] i ) in fetal ASM (fASM). Compared with adult ASM, CaSR expression was higher in fASM, while fluorescence Ca 2+ imaging showed that [Ca 2+ ] i was more sensitive to altered [Ca 2+ ] o . The fASM [Ca 2+ ] i responses to histamine were also more sensitive to [Ca 2+ ] o (0–2 mM) compared with an adult, enhanced by calcimimetic R568 but blunted by calcilytic NPS2143. [Ca 2+ ] i was enhanced by endogenous CaSR agonist spermine (again higher sensitivity compared with adult). Inhibition of phospholipase C (U73122; siRNA) or inositol 1,4,5‐triphosphate receptor (Xestospongin C) blunted [Ca 2+ ] o sensitivity and R568 effects. NPS2143 potentiated U73122 effects. Store‐operated Ca 2+ entry was potentiated by R568. Traction force microscopy showed responsiveness of fASM cellular contractility to [Ca 2+ ] o and NPS2143. Separately, fASM proliferation showed sensitivity to [Ca 2+ ] o and NPS2143. These results demonstrate functional CaSR in developing ASM that modulates airway contractility and proliferation.

Lung diseases, such as bronchopulmonary dysplasia (BPD), wheezing, and asthma, remain significant causes of morbidity and mortality in the pediatric population, particularly in the setting of premature birth. Pulmonary outcomes in these infants are highly influenced by perinatal exposures including prenatal inflammation, postnatal intensive care unit interventions, and environmental agents. Here, there is strong evidence that perinatal supplemental oxygen administration has significant effects on pulmonary development and health. This is of particular importance in the preterm lung, where premature exposure to room air represents a hyperoxic insult that may cause harm to a lung primed to develop in a hypoxic environment. Preterm infants are also subject to increased episodes of hypoxia, which may also result in pulmonary damage and disease. Here, we summarize the current understanding of the effects of oxygen on the developing lung and how low vs. high oxygen may predispose to pulmonary disease that may extend even into adulthood. Better understanding of the underlying mechanisms will help lead to improved care and outcomes in this vulnerable population.

Wheezing is a major long-term respiratory morbidity in preterm infants with and without bronchopulmonary dysplasia. We hypothesized that mild vs. severe hyperoxic exposure in neonatal mice differentially affects airway smooth muscle hypertrophy and resultant airway reactivity. Newborn mice were exposed to 7 days of mild (40% oxygen) or severe (70% oxygen) hyperoxia vs. room air controls. Respiratory system resistance (Rrs), compliance (Crs), and airway reactivity were measured 14 days after oxygen exposure ended under ketamine/xylazine anesthesia. Baseline Rrs increased and Crs decreased in both treatment groups. Methacholine challenge dose dependently increased Rrs and decreased Crs in 40% oxygen-exposed mice, whereas Rrs and Crs responses were similar between 70% oxygen-exposed and normoxic controls. Airway smooth muscle thickness was increased in 40%- but not 70%-exposed mice, whereas collagen increased and both alveolar number and radial alveolar counts decreased after 40% and 70% oxygen. These data indicate that severity of hyperoxia may differentially affect structural and functional changes in the developing mouse airway that contribute to longer-term hyperreactivity. These findings may be important to our understanding of the complex role of neonatal supplemental oxygen therapy in postnatal development of airway responsiveness.