Pabelick, Christina B.

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