ABSTRACT Moderate (<60%) O2 (hyperoxia) in premature infants promotes bronchial airway hyperresponsiveness (AHR) via effects on airway smooth muscle (ASM), a cell type that also contributes to impaired bronchodilation, and remodeling (proliferation, altered extracellular matrix (ECM)). Thus understanding mechanisms by which O2 affects bronchial airways is critical for therapeutic strategies in a vulnerable population. We focus on a novel, targetable mechanism in ASM: cellular senescence (Sen). Sen cells are long-living, and secrete factors (senescence-associated secretory phenotype; SASP) that promote inflammation and fibrosis via paracrine effects on naïve cells. Appeal lies in novel drugs that kill Sen cells (senolytics) such as dasatanib+quercetin (D+Q) and fisetin. Little is known regarding Sen cells in perinatal airways but our data indicate moderate O2 enhances detrimental Sen in human fetal ASM (fASM) with increased inflammatory, pro- fibrotic SASP that promotes proliferation and ECM of naïve ASM: effects inhibited by D+Q. We find that ROS and ER stress promote fASM Sen, and in newborn mice exposed to O2 (which results in AHR and fibrosis) D+Q alleviates O2 effects. Thus, we hypothesize perinatal O2 induces detrimental Sen cell burden that, via SASP, initiates and promotes AHR and remodeling: effects alleviated by senolytics. We propose 3 Aims using human fetal lung and in vivo neonatal mouse models of O2: Aim 1: Determine mechanisms by which hyperoxia induces cellular Sen in developing human ASM; Aim 2: Determine the role of cellular Sen in hyperoxia effects on developing human ASM; Aim 3: Determine effects of detrimental Sen on contractility and remodeling in mouse model of neonatal hyperoxia. In Aims 1 and 2, we will use 18-22 wk gestation human fASM and lung slices to examine mechanisms of Sen induction, focusing on ROS, mitochondria and ER stress (Aim 1) and downstream effects of Sen/SASP in the context of contractility and remodeling (Aim 2) following 40% O2. Alleviation by senolytics D+Q or fisetin (Aim 1, 2) are explored. In vitro studies are integrated in the newborn mouse model (Aim 3) where extent of Sen is assessed, and alleviation of airway hyperreactivity and remodeling by senolytics are tested. Clinical significance lies in establishing detrimental Sen in O2 effects on developing airway towards future therapeutic targeting for neonatal asthma.