Overview BPD is a moving target - definitions have changed over time - PowerPoint PPT Presentation

3/8/2019 Disclosures Namasivayam Ambalavanan MD Commercial interests: Consultant, Shire Consulting Former research funding from Pfizer NIH Grant Funding: R01 HL129907 ( STOP BPD “ S ignature of T op O mic P rofiles in BPD” ) (PI) Exosomes in BPD U01 HL122626 (Alveolar DevMAP “LungMAP”) (PI) U01HL133536 (PreVENT Apnea) (PI) N. Ambalavanan MD UG1 HD034216 (Cooperative Multicenter Neonatal Research Network) (Co-Inv) Director, Division of Neonatal Research U01 HL120338 (CHRONIC HYPERTENSION AND PREGNANCY-CHAP Director, TReNDD Program CLINICAL COORDINATING CENTER) (Co-Inv) Professor of Pediatrics, UAB Relevant Patent – Airway Probiotics ambal@uab.edu What is BPD? Overview “BPD” is a moving target - definitions have changed over time • What is BPD? 1. 1980’s: Oxygen dependence for 28 days or more after birth (Tooley WH. J Pediatr 1979; 1979 BPD Workshop) • What is the pathogenesis of BPD? 2. 1990’s: Oxygen dependence at 36 wks’ corrected age (Shennan et al. Pediatrics 1988) • What are exosomes?  More correlated with abnormal pulmonary outcome at 2 years (63% PPV) vs. 28 d definition (38% PPV) • Exosomes in normal lung development 21 st century: 3. • NICHD/NHLBI/ORD Workshop definition: Mild/Moderate/Severe BPD (Jobe • Exosomes as biomarkers of BPD and Bancalari. AJRCCM 2001) • Physiologic definition of BPD (Walsh MC et al. J Perinatol 2003, Pediatrics 2004) • Exosomes as therapeutic agents for BPD Patient populations have changed over time Clinical practices have changed over time BPD in 1979 =/= BPD in 2019 1

3/8/2019 BPD is not one disease Variation in BPD Phenotype -1 • The definition of BPD is a clinical operational definition, and does not specify pathophysiology • Variation in BPD severity – Mild, Moderate, Severe BPD • Variation in BPD pathology • “Classic” BPD [airway injury, inflammation, parenchymal fibrosis] vs. “New” BPD [inhibited alveolar and microvascular development] [Jobe AH, Bancalari E. Am J Respir Crit Care Med 2001] • Infants with BPD exist along a continuum, with varying combinations of clinical and pathologic features 3 month old ex-25w 0.7 kg infant with Ureaplasma infection at birth Variation in BPD Phenotype -2 Variation in BPD Phenotype -3 3 month old ex-22w 0.4 kg infant with frequent failed extubations and 3 month old ex-24w 0.6 kg infant with supra-systemic pulmonary left sided atelectasis hypertension (RVP > 80 mm Hg; BNP >3000) 2

3/8/2019 BPD disease network: making it complex BPD pathogenesis: keeping it simple Disease –modifying genes Environmental determinants Cytokine & Lung & vascular Surfactant Immunity Ventilator Associated Hyperoxia development genes genes Fluid Lung Injury genes overload Inflammation CENTER EFFECTS Volutrauma Apoptosis Immature Impaired Immature Intermediate nutrition lung Lung with Surfactant Deficiency phenotypes Infection Hyperoxia Atelectasis Inhibition Abnormal lung Long-term of lung vascular Bronchopulmonary Dysplasia impairment of development development lung function; Pathophenotypes of BPD Lal CV, Ambalavanan N. PAH Semin Perinatol 2015 Pathology of BPD • Operational definitions (“BPD”) do not capture phenotypic and genetic heterogeneity “OLD” “NEW” BPD underlying pathogenesis BPD Severe airway Large, simplified “All normal lungs are similar; every abnormal injury alveolar lung is abnormal in its own way” structures • Endotype determined by magnitude of: Alternating Dysmorphic – Parenchymal abnormality areas of fibrosis capillary and over- configuration – Vascular abnormality inflation – Airway abnormality Pulmonary Variable hypertension interstitial cellularity and fibroproliferation Coalson et al. 2006 3

3/8/2019 Normal lung vs. BPD Baraldi & Filippone. New Engl J Med 2007 Long-term impact of BPD? Bonikos DS et al. Hum Pathol 1976 • Histology of 21 infants who died of BPD • 6 of 21 had signs of cardiac stress, including cor pulmonale Bronchiole with almost complete occlusion Baraldi & Filippone. New Engl J Med 2007 Vascular remodeling and perivascular infiltrate 4

3/8/2019 Bhat R et al. Pediatrics 2012 172 infants evaluated 21 excluded due to anomalies PMID 22311993 Pulmonary Hypertension in BPD 6 transferred to another facility 145 screened by echocardiography at 4-6 weeks of age 136 infants with no pulmonary 9 with pulmonary hypertension hypertension on initial screening on initial screening 8 received supplemental 1 infant did not receive 17 subsequently 119 not diagnosed oxygen or other therapy specific treatment (SpO 2 with pulmonary diagnosed with >90% on air) hypertension during pulmonary hospitalization hypertension 3 with resolved Pulmonary 4 with resolved 5 with persistent 10 with 3 died pulmonary hypertension pulmonary pulmonary persistent hypertension resolved, hypertension hypertension at pulmonary discharged discharge hypertension at home, no O 2 discharge or medication 2 discharged home, 1 discharged home, 2 discharged home, 2 discharged home, no O 2 /medications no O 2 /medications no O 2 /medications no O 2 /medications 1 discharged home, 2 discharged home, 1 discharged home, 1 discharged home, on O 2 / no medications on O 2 for BPD on O 2 / no medications on O 2 for BPD Bhat R et al. Pediatrics 2012 2 discharged home, 7 discharged home, 1 discharged home, on O 2 + medications on O 2 + medications on O 2 + medications PMID 22311993 Earlier Concept: Simple DNA mRNA Protein Bhat R et al. Pediatrics 2012 PMID 22311993 5

3/8/2019 Newer Concept: Complex How do cells communicate with each other? DNA methylation Microbiome DNA • Direct contact: cell-to-cell contact dependent DNA methylation signaling; tunneling nanotubes (TNT) (+) or (-) • Transfer of soluble molecules miRNA lncRNA mRNA • Transfer via circulating vesicles (e.g. Exosomes) (and other small RNA) (-) or (+) (+) or (-)/splicing Protein Metabolome Organelle specific: Mitochondria, ER etc Cell-type specific: Endoth, Epith, Fib, MSC etc Cell-cell interactions – Exosomes , MV, mechanotransduction Exosomes • Discovered in 1983 (Pan & Johnstone; Cell 1983) • Defined in 1989 (Johnstone et al. Blood 1989) • Exosomes: What are exosomes? – Membrane-bound vesicles 40-100 nm in diameter – Present in almost all biological fluids – Released from most cell types – Requires enzymes, ATP, and sorting by parent cells – Contain lipids, proteins, mRNA, miRNA and other ncRNA 6

3/8/2019 Extracellular vesicle biogenesis. Extracellular vesicles are divided into three main types: exosomes , microvesicles, and apoptotic bodies. Exosomes are formed in multivesicular endosomes and contain molecules including nucleic acids, proteins, lipids, and metabolites. The exosomal content can be transferred to other cells through different processes, including endocytosis, phagocytosis, macropinocytosis, or direct membrane fusion. Am J Respir Crit Care Med, 2017 https://www.atsjournals.org/doi/abs/10.1164/rccm.201612-2457PP Mathieu M et al. Nat Cell Biol 2019 Published in: Serge P. Nana-Sinkam; Mario Acunzo; Carlo M. Croce; Kai Wang; Am J Respir Crit Care Med 1961510-1518. Mechanisms of EV secretion ESCRT : E ndosomal S orting C omplex R equired for Serge P. Nana-Sinkam; Mario Acunzo; Carlo M. Croce; Kai Wang; Am J Respir Crit Care Med 1961510-1518 T ransport Exocarta (http://exocarta.org/exosome_markers): Top 25 proteins HSPA8, CD9, GAPDH, ACTB, CD63 , CD81 , ANXA2, ENO1, HSP90AA1, EEF1A1, PKM2, YWHAE, SDCBP, PDCD6IP, ALB, YWHAZ, EEF2, ACTG1, LDHA, HSP90AB1, ALDOA, MSN, ANXA5, PGK1, and CFL1 Mathieu M et al. Nat Cell Biol 2019 7

3/8/2019 Mechanisms of EV uptake How do exosomes work? Mathieu M et al. Nat Cell Biol 2019 How do exosomes work? – it’s controversial a) They don’t work – MVs do the work • Both exosomes and MVs have reporter proteins and mRNA but only MVs transferred reporter function to recipient cells (Kanada M et al. PNAS 2015) b) miRNA • miRNA  mRNA in producer cells  miRNA in exosomes (e.g. Macrophages to Endothelial cells) (Squadrito ML et al. Cell Rep 2014) c) Proteins • MSC exosomes probably work through protein rather than mRNA (Toh WS et al. Biochem Soc Trans 2018) • MSC exosomes have mostly miRNA and ncRNA, not mRNA 8

3/8/2019 Exosomes in normal lung development Olave N et al. AJP LCMP 2016; PMID 26719145 LNA-miR-489 attenuates hyperoxia-induced inhibition of neonatal mouse lung development, while miR-489 overexpression inhibits normal lung development Olave N et al. AJP LCMP 2016; PMID 26719145 Olave N et al. AJP LCMP 2016; PMID 26719145 9

3/8/2019 C. Vivek Lal Exosomes as biomarkers Lal CV et al. JCI Insight 2018; PMID 29515035 Exosomal miRNA from Tracheal Aspirates miR-876-3p Discovery Cohort B A Validation Cohort (All miRs = p < 0.05 and FC > 1.5) (All miRs = p < 0.05 and FC > 1.5) C BPD Resistant BPD Susceptible AUC 0.92 Lal CV et al. JCI Insight 2018; PMID 29515035 10

3/8/2019 miR-876-3p mimic prevents LPS+Hyperoxia induced Hyperoxia and LPS decrease miR 876-30 in vivo injury and inhibition of alveolar development in (also in vitro ; data not shown) newborn mice Lal CV et al. JCI Insight 2018; PMID 29515035 Lal CV et al. JCI Insight 2018; PMID 29515035 Tracheal Aspirate Exosomes from BPD Subjects Confer a BPD-like Phenotype to Exposed Neonatal Mice Genschmer KR, Russell DW, Lal C ….Blalock JE. Cell 2019 Genschmer KR, Russell DW, Lal C….Blalock JE. Cell 2019 11