Thursday, December 26, 2024
12/26/2024
ABSTRACT Premature birth necessitates interventions such as supplemental O2 (hyperoxia; typically <60% O2) with/without respiratory support primarily using non-invasive nasal CPAP. Studies from the previous cycle of this longstanding R01 have shown neonatal O2 is associated with development of airway hyperreactivity (AHR) and remodeling (cell proliferation, fibrosis): effects that contribute to a wheezing phenotype and predispose to asthma. While CPAP is initially beneficial, long-term effects on highly compliant bronchial airways are less known. We previously found static stretch of CPAP induces sustained AHR and airway thickening even after CPAP is stopped and involves airway smooth muscle (ASM). What we do not fully understand are upstream mechanosensitive transducers of stretch, and downstream pathways by which O2 and stretch interact to produce these effects, Based on preliminary data, we hypothesize that mechanosensitive Piezo (PZ) channels in developing ASM contribute to CPAP-induced AHR and remodeling. There are currently limited data on PZ channels in lung (which does express them) and none in airways. Preliminary data show PZ1 and PZ2 are expressed in human fetal ASM (fASM) and in neonatal mouse, and that PZs are functional in enhancing Ca2+ responses, proliferation and ECM formation. 50% O2 and stretch each increase fASM PZ: effects also noted in newborn mice where 7 days CPAP results in sustained AHR and remodeling at 3 wks, and inhibition of PZ blunts CPAP effects. Downstream of PZ, we find that canonical Wnt/β-catenin signaling is important, and can be increased by stretch/CPAP. Our overall hypothesis/model is that in developing airways, ASM PZ channels and downstream Wnt/β-catenin pathways are linked in the context of O2 and/or stretch effects, leading to AHR and remodeling. Our 3 Specific Aims to explore our model are: Aim 1: Determine the role of PZ channels in O2- and stretch-induced responses of human fASM; Aim 2: Determine mechanisms that mediate PZ effects in O2 and stretch-induced human fASM responses; Aim 3: In a neonatal mouse model of O2 and CPAP, determine role of PZ in AHR and remodeling. In Aims 1 and 2, 18-22 wk human fASM cells are exposed to normoxia vs. moderate hyperoxia with/without static stretch on a background of cyclic strain, mimicking clinical application of O2 +/- CPAP in spontaneously breathing premies. In Aim 1, complementary imaging and biochemical techniques are used with PZ modulators to assess roles of PZ in fASM [Ca2+]i (imaging), contractility (traction force microscopy), proliferation and fibrosis. In Aim 2, complementary biochemical and molecular tools are used to examine mechanisms that are involved in PZ effects on remodeling in particular with a focus on Wnt/β-catenin. In vitro results are integrated in Aim 3 using clinically- relevant neonatal mouse models of moderate hyperoxia (50% O2) with/without intermittent CPAP applied for the first 1 wk followed by 2 wks recovery in WT and inducible smooth muscle PZ1 KO (sm-PZ1 KO) mice. Effects of inhibiting PZ1 vs. PZ2 are tested towards establishing clinical significance of targeting these novel pathways to alleviate adverse long-term effects of hyperoxia and CPAP.
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