Manlove, Logan

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.

Airway remodeling in asthma driven by inflammation involves proliferation of epithelial cells and airway smooth muscle (ASM), as well as enhanced extracellular matrix (ECM) generation and deposition, i.e., fibrosis. Accordingly, understanding profibrotic mechanisms is important for developing novel therapeutic strategies in asthma. Recent studies, including our own, have suggested a role for locally produced growth factors such as brain-derived neurotrophic factor (BDNF) in mediating and modulating inflammation effects. In this study, we explored the profibrotic influence of BDNF in the context of asthma by examining expression, activity, and deposition of ECM proteins in primary ASM cells isolated from asthmatic vs. nonasthmatic patients. Basal BDNF expression and secretion, and levels of the high-affinity BDNF receptor TrkB, were higher in asthmatic ASM. Exogenous BDNF significantly increased ECM production and deposition, especially of collagen-1 and collagen-3 (less so fibronectin) and the activity of matrix metalloproteinases (MMP-2, MMP-9). Exposure to the proinflammatory cytokine TNFα significantly increased BDNF secretion, particularly in asthmatic ASM, whereas no significant changes were observed with IL-13. Chelation of BDNF using TrkB-Fc reversed TNFα-induced increase in ECM deposition. Conditioned media from asthmatic ASM enhanced ECM generation in nonasthmatic ASM, which was blunted by BDNF chelation. Inflammation-induced changes in MMP-2, MMP-9, and tissue inhibitor metalloproteinases (TIMP-1, TIMP-2) were reversed in the presence of TrkB-Fc. These novel data suggest ASM as an inflammation-sensitive source of BDNF within human airways, with autocrine effects on fibrosis relevant to asthma.

Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases.