220 - Extracellular vesicles shed from senescent hyperoxia-exposed pulmonary vascular endothelial cells impair lung alveolar development and potentiate pulmonary vascular remodeling

Poster Number: 220
Publication Number: 220.221

Kevin G. Williams, University of Miami Leonard M. Miller School of Medicine, LAUDERHILL, FL, United States; Pingping Chen, University of Miami, Miami, FL, United States; Michael Bellio, University of Miami, Miami, FL, United States; Rahul Kumar, University of Miami, Coral Gables, FL, United States; Shu Wu, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Augusto F. Schmidt, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Merline Benny, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Karen Young, University of Miami, Miami, FL, United States

Background: Pulmonary hypertension (PH) is a leading cause of morbidity in preterm infants with bronchopulmonary dysplasia (BPD).   Oxidative stress-induced cellular senescence may contribute to BPD. Pilot studies in our laboratory show that the secretome of senescent hyperoxia-exposed pulmonary microvascular endothelial cells (PMECs) impair lung development. Senescent cells secrete cytokines and proteases and shed large numbers of extracellular vesicles (EVs). Here we test the hypothesis that EVs shed from senescent hyperoxia-exposed PMECs impair lung development and potentiate pulmonary vascular remodeling leading to the BPD-PH phenotype.

Objective: Determine whether adoptive transfer of EVs shed from senescent hyperoxia-exposed neonatal PMECs (senescent EVs) to rat pups impair lung development and potentiate vascular remodeling.

Design/Methods: PMECs were cultured in normoxia or 65% oxygen for 48 hours.  Cell senescence was verified by SA-β-gal staining and mRNA expression of senescence markers (Figure 1). EVs were isolated from the supernatant of normoxic (control EVs) and hyperoxia-exposed PMECs (senescent EVs). EVs were characterized by nanosight tracking analysis and flow cytometry. Newborn rats were randomly assigned to receive daily intraperitoneal injection from postnatal day 1-7 of one of the following: A) basal media, B) EV-free conditioned media from control PMECs, C) EV-free conditioned media from senescent PMECs, D) control EVs and E) senescent EVs.  On day 7, lung alveolarization was determined by measuring the mean linear intercept (MLI). Angiogenesis was assessed by quantifying lung vascular density. Vascular remodeling was assessed by determining the percentage of muscularized vessels per high power field. PH was determined by measuring the weight ratio of the right ventricle to left ventricle + septum (RV/LV+ S). Data are expressed as mean ± SD and analyzed by ANOVA.

Results: Adoptive transfer of senescent EVs shed from hyperoxia-exposed PMECs to neonatal rats significantly increase MLI (Figure 2), induce pulmonary vascular remodeling, reduce lung vascular density and increase RV/LV+S (Figure 3). Although EV-free conditioned media from senescent PMECs did not impair lung alveolarization or induce vascular remodeling, it significantly reduced lung angiogenesis to a similar degree as senescent EVs (Figures 2-3).Conclusion(s): Neonatal hyperoxia induces PMEC senescence and the senescent secretome of these cells contains EVs that impair lung alveolar development and potentiate vascular remodeling. These findings suggest that EVs from senescent PMECs contribute to the severe BPD-PH phenotype.
Neonatal Hyperoxia Induces Pulmonary Microvascular Endothelial Cell SenescenceA) Flow cytometric analysis demonstrates that PMECs cultured in 65% O2 exhibit increased B-gal staining. Hyperoxia exposure of neonatal PMECs increases the mRNA expression of known senescent markers B) p21, C) p16 and D) p53. Senescent PMECs also have decreased cell proliferation as evidenced by E) reduced Ki67 immunostaining (pink staining). * p < 0.05
Senescent EVs Impair Lung AlveolarizationA) Hematoxylin and eosin-stained lung sections from postnatal day 7 rat pups who received daily intraperitoneal injections of: basal media, EV-free Control Conditioned Media (CM), EV-free Senescent CM, Control EV and Senescent EV. Pups which received Senescent EVs demonstrated decreased and simplified alveoli. Scale bar is 50 um

B) Significantly increased mean linear intercept (MLI) in newborn rat pups that received the Senescent EVs. Nf6/group; * p < 0.05