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

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Exosomes in BPD

N. Ambalavanan MD

Director, Division of Neonatal Research Director, TReNDD Program Professor of Pediatrics, UAB ambal@uab.edu

Commercial interests: Consultant, Shire Consulting Former research funding from Pfizer NIH Grant Funding: R01 HL129907 ( STOP BPD “Signature of Top Omic Profiles in BPD”) (PI) U01 HL122626 (Alveolar DevMAP “LungMAP”) (PI) U01HL133536 (PreVENT Apnea) (PI) UG1 HD034216 (Cooperative Multicenter Neonatal Research Network) (Co-Inv) U01 HL120338 (CHRONIC HYPERTENSION AND PREGNANCY-CHAP CLINICAL COORDINATING CENTER) (Co-Inv) Relevant Patent – Airway Probiotics

Disclosures

Namasivayam Ambalavanan MD

Overview

What is BPD?
What is the pathogenesis of BPD?
What are exosomes?
Exosomes in normal lung development
Exosomes as biomarkers of BPD
Exosomes as therapeutic agents for BPD

What is BPD?

“BPD” is a moving target - definitions have changed over time 1. 1980’s: Oxygen dependence for 28 days or more after birth

(Tooley WH. J Pediatr 1979; 1979 BPD Workshop)

2. 1990’s: Oxygen dependence at 36 wks’ corrected age (Shennan et al.

Pediatrics 1988)

More correlated with abnormal pulmonary outcome at 2 years (63% PPV) vs. 28 d

definition (38% PPV)

3. 21st century:

NICHD/NHLBI/ORD Workshop definition: Mild/Moderate/Severe BPD (Jobe

and Bancalari. AJRCCM 2001)

Physiologic definition of BPD (Walsh MC et al. J Perinatol 2003, Pediatrics 2004)

Patient populations have changed over time Clinical practices have changed over time BPD in 1979 =/= BPD in 2019

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BPD is not one disease

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

Variation in BPD Phenotype -1

3 month old ex-25w 0.7 kg infant with Ureaplasma infection at birth

Variation in BPD Phenotype -2

3 month old ex-24w 0.6 kg infant with supra-systemic pulmonary hypertension (RVP > 80 mm Hg; BNP >3000)

Variation in BPD Phenotype -3

3 month old ex-22w 0.4 kg infant with frequent failed extubations and left sided atelectasis

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Immature Lung with Surfactant Deficiency Bronchopulmonary Dysplasia

Ventilator Associated Lung Injury

Hyperoxia

BPD pathogenesis: keeping it simple

BPD disease network: making it complex

Intermediate phenotypes Disease –modifying genes Environmental determinants Pathophenotypes of BPD Inhibition

f lung

development Abnormal lung vascular development

Atelectasis Inflammation Apoptosis Infection Hyperoxia Volutrauma

Immature lung

Fluid

verload

Surfactant genes Lung & vascular development genes Cytokine & Immunity genes CENTER EFFECTS

Long-term impairment of lung function; PAH

Impaired nutrition

Lal CV, Ambalavanan N. Semin Perinatol 2015

Operational definitions (“BPD”) do not capture

phenotypic and genetic heterogeneity underlying pathogenesis “All normal lungs are similar; every abnormal lung is abnormal in its own way”

Endotype determined by magnitude of:

– Parenchymal abnormality – Vascular abnormality – Airway abnormality

Pathology of BPD

“OLD” BPD “NEW” BPD

Severe airway injury Large, simplified alveolar structures Alternating areas of fibrosis and over- inflation Dysmorphic capillary configuration Pulmonary hypertension Variable interstitial cellularity and fibroproliferation

Coalson et al. 2006

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Baraldi & Filippone. New Engl J Med 2007

Normal lung vs. 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 Vascular remodeling and perivascular infiltrate

Baraldi & Filippone. New Engl J Med 2007

Long-term impact of BPD?

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Pulmonary Hypertension in BPD

Bhat R et al. Pediatrics 2012 PMID 22311993

172 infants evaluated

145 screened by echocardiography at 4-6 weeks of age 9 with pulmonary hypertension

n initial screening

8 received supplemental

xygen or other therapy

1 infant did not receive specific treatment (SpO2 >90% on air) Pulmonary hypertension resolved, discharged home, no O2

r medication

3 with resolved pulmonary hypertension 5 with persistent pulmonary hypertension at discharge 136 infants with no pulmonary hypertension on initial screening

17 subsequently diagnosed with pulmonary hypertension

119 not diagnosed with pulmonary hypertension during hospitalization 10 with persistent pulmonary hypertension at discharge 3 died 4 with resolved pulmonary hypertension 21 excluded due to anomalies 6 transferred to another facility

2 discharged home, no O2 /medications 1 discharged home,

n O2 / no medications

2 discharged home,

n O2 + medications

1 discharged home, no O2 /medications 2 discharged home,

n O2 for BPD

2 discharged home, no O2 /medications 1 discharged home,

n O2 / no medications

7 discharged home,

n O2 + medications

2 discharged home, no O2 /medications 1 discharged home,

n O2 for BPD

1 discharged home,

n O2 + medications

Bhat R et al. Pediatrics 2012 PMID 22311993 Bhat R et al. Pediatrics 2012 PMID 22311993

Earlier Concept: Simple

DNA mRNA Protein

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Newer Concept: Complex DNA mRNA Protein miRNA

(and other small RNA)

lncRNA

(-) or (+) (+) or (-)/splicing (+) or (-)

DNA methylation

Metabolome

Microbiome

DNA methylation Organelle specific: Mitochondria, ER etc Cell-type specific: Endoth, Epith, Fib, MSC etc Cell-cell interactions – Exosomes, MV, mechanotransduction

How do cells communicate with each other?

Direct contact: cell-to-cell contact dependent

signaling; tunneling nanotubes (TNT)

Transfer of soluble molecules
Transfer via circulating vesicles (e.g. Exosomes)

What are exosomes? Exosomes

Discovered in 1983 (Pan & Johnstone; Cell 1983)
Defined in 1989 (Johnstone et al. Blood 1989)
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

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Mathieu M et al. Nat Cell Biol 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 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

Mathieu M et al. Nat Cell Biol 2019 ESCRT: Endosomal Sorting Complex Required for Transport

Serge P. Nana-Sinkam; Mario Acunzo; Carlo M. Croce; Kai Wang; Am J Respir Crit Care Med 1961510-1518

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

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Mechanisms of EV uptake

Mathieu M et al. Nat Cell Biol 2019

How do exosomes work?

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

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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

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Exosomes as biomarkers

Lal CV et al. JCI Insight 2018; PMID 29515035

C. Vivek Lal

BPD Susceptible BPD Resistant

(All miRs = p < 0.05 and FC > 1.5)

Discovery Cohort Validation Cohort

A B C (All miRs = p < 0.05 and FC > 1.5)

Lal CV et al. JCI Insight 2018; PMID 29515035

Exosomal miRNA from Tracheal Aspirates

miR-876-3p

AUC 0.92

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3/8/2019 11 Hyperoxia and LPS decrease miR 876-30 in vivo (also in vitro; data not shown)

Lal CV et al. JCI Insight 2018; PMID 29515035 Lal CV et al. JCI Insight 2018; PMID 29515035

miR-876-3p mimic prevents LPS+Hyperoxia induced injury and inhibition of alveolar development in newborn mice

Genschmer KR, Russell DW, Lal C….Blalock JE. Cell 2019

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

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Exosomes for therapy Bio-engineered Exosomes

Mesenchymal Stem Cell Derived-Exosomes

Intratracheal administration of clinical-grade mesenchymal stem cell-derived

extracellular vesicles reduces lung injury in a rat model of bronchopulmonary dysplasia. Porzionato A…Muraca M. Am J Physiol Lung Cell Mol Physiol. 2019 Jan 1;316(1):L6- L19.

Intraperitoneal injection of MSC-derived exosomes prevent experimental

bronchopulmonary dysplasia. Braun RK…Eldridge MW. Biochem Biophys Res Commun. 2018 Sep 18;503(4):2653- 2658.

Early gestational mesenchymal stem cell secretome attenuates experimental

bronchopulmonary dysplasia in part via exosome-associated factor TSG-6. Chaubey S….Bhandari V. Stem Cell Res Ther. 2018 Jun 26;9(1):173.

Mesenchymal Stromal Cell Exosomes Ameliorate Experimental

Bronchopulmonary Dysplasia and Restore Lung Function through Macrophage Immunomodulation. Willis GR…Kourembanas S. Am J Respir Crit Care Med. 2018 Jan 1;197(1):104-116.

MSC EV in rat BPD.

Porzionato et al. AJP LCMP 2019

Newborn rats: Air x 2 w vs. 60% O2 x 2w
Rats in 60% O2 received saline or MSC or MSC-

EV i.t on P3, P7, and P10

(Total dose 0.64 x 1010 EV or 6 x 106 MSC; MSC from human umbilical cords)

MSC increased only alveolar number
MSC-EV increased alveolar number, alveolar

volume, and prevented increase in medial thickness of small pulmonary vessels

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MSC-EV in rat BPD

Braun RK et al. BBRC 2018

Rats exposed from P1-P14 to air vs. 85% O2
Rats given MSC-EV or saline every day until P15

(3.4 x 109 exosomes; 15 µg daily; MSC from rat bone marrow)

MSC-EV improved lung development, lung

blood vessel density, and RV hypertrophy

In vitro, HUVEC tube formation was induced by

MSC-EV, and MSC-EV contained VEGF

Preterm infant MSC-EV in mouse BPD

Chaubey S et al. Stem Cell Res Ther 2018

Conditioned media (CM) and exosome (Exo) were

isolated from hUC-MSC (25 and 30w GA)

Newborn mice exposed to 95% O2 from P1-P4

were given MSC-CM or MSC-CM Exo at P2 and P4

Additional experiments used rhTSG-6, TSG-6

neutralizing Ab, TSG-6 siRNA-MSC-CM Exo

CM and Exo improved lung development,

pulmonary hypertension, and RV hypertrophy

TSG-6 also improved BPD pathology, and TSG-6

knockdown abrogated therapeutic effects of Exo

MSC-Exo in mouse BPD

Willis GR et al. AJRCCM 2018

MSC-exo isolated from hUC-MSC
Mice exposed to 75% O2 from P1-7, given

MSC-exo i.v on P4

MSC –exo (similar to bone marrow MSC-exo):

– improved lung architecture – reduced pulmonary hypertension, improved microvasculature and vascular remodeling – Reduced lung inflammation (M1 MacsM2 Macs)

Willis GR et al AJRCCM 2018

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Willis GR et al AJRCCM 2018

Conclusion

BPD is a complex multifactorial disorder with

pathophysiologic and phenotypic heterogeneity

Unbiased evaluation of biomarkers, such as exosomes in

biofluids (blood, tracheal aspirates) may help early diagnosis, prognosis, following response to therapy, and may occasionally yield insight into underlying disease mechanisms

A better understanding of optimal exosome source,

concentration, delivery, and effects may lead to therapeutic strategies for BPD (and other lung disorders)

Recent reviews

Nanotherapies for micropreemies: Stem cells and the secretome

in bronchopulmonary dysplasia. Lesage F, Thébaud B. Semin

Perinatol. 2018 Nov;42(7):453-458.
Exosome-based Therapy for Bronchopulmonary Dysplasia.

Matthay MA, Abman SH. Am J Respir Crit Care Med. 2018 Jan 1;197(1):10-12.

Therapeutic Applications of Extracellular Vesicles: Perspectives