PulmonaryDevelopmentandDiseaseinDownSyndrome:aScien7ficJourneyfromthe

BedtoBenchSite

Csaba Galambos MD, PhD Department of Pathology and Laboratory Medicine & Pediatric Heart Lung Center,

Children’s Hospital Colorado & University of Colorado School of Medicine

Diagnos7cPediatricPathology(Lung)

BenchResearchonLungDevelopment&

Disease

PediatricPulmonaryVascularBiology&Pathology

-4montholdmalewithtrisomy21-bornat364/7weeksgesta7onalage-birthweightof3080grams-deliveredbyCesareansec7ontoa34yearoldG2P1mother-mul7plerespiratoryproblemssincebirthincludingpulmonaryhypertension,chronicrespiratoryfailure,andtracheobronchomalaciarequiringassistedven7la7on-Nocardiacorotherorgananomalies-AVeralonghospitalcoursewithoutimprovementinhiscardiorespiratorystatus,hisfamilydecidedtowithdrawlifesupportandanautopsypermitwasgranted

Casestudy

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PAPA

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BrDEFECTIVEVASCULARREMODELING

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Control Downsyndrome

DIMINISHEDALVEOLARIZATION

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IMPAIREDMICROVASCULARGROWTH

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Histopathology

Respiratory:severechroniclungdisease,pulmonaryhypoplasiaandpulmonaryhypertensionrelatedtoDownsyndrome

CauseofDeath

Babies with DS succumb to respiratory disease without any co-morbid condition

v DSassociatedwithincreasedriskfordevelopingrespiratorydisordersincludingseverepulmonaryhypertension(PHT)andpersistentpulmonaryhypertensionofthenewborn(PPHN)

v PrevalencestudyatSieCenterforDownSyndrome(ongoing):-1,252childrenwithDS,27.6%(n=346)withPHT-PPHNincidence:9.9%(vs0.1ingeneralpopulaJon)

v ChildrenwithDSandco-morbidi7es,suchascongenitalheartdiseaseorobstruc7vesleepapnea,aremoresuscep7blefordevelopingacceleratedPHTthanchildrenwithoutDS

v OneoftheleadingcausesofdeathinpeoplewithDSisrespiratoryrelated

Thegene7candmolecularmechanismsresponsibleforpulmonaryhypoplasiaandPHTinDSareunknown

PulmonarydiseaseandDownsyndrome

Downsyndromeassociatedlungdisordersareunderstudied

ChallengesofDownsyndromeresearch

v manygenes(300+):onegenedysfunc5on-onepathway-onediseasephenotypeparadigmdoesnotapply

v genesareoverexpressed

v ~80clinicalphenotypesinvariouscombina7ons

GENETICPATHWAYSANDCONFOUNDINGFACTORSLEADTOVARIABLEDSPHENOTYPES

80clinicalphenotypesofDS

1.5foldoverexpressionoftrisomicgenes

(“dosagesensi5vegenes”)

celltype&7meofdevelopmentstage

1

upand/ordownregula7onofdisomicgenes(“modifiablegenes”viatranscrip5onalregula5on)

2

confoundingfactors(hypoxemia)

3

3

3

BronchopulmonaryDysplasia

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20x

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An7-angiogenicDisorder Control

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Downsyndrome

AreDS-relatedlunghypoplasiaandPHTan5-angiogenicdisorders?

JudahFolkman,MDSurgeon-Scien7st

Founderofangiogenesisresearch

v Pa7entswithDShaveadecreasedincidenceofpro-angiogenesisrelateddiseasesincludingsolidtumors,atherosclerosis,diabe7cre7nopathy,andvascularanomalies

v Genesforpotentan7-angiogenicfactorsarepresentonthetriplicatedchromosome21andoverexpressedinpa7entswithDSandinDSanimalmodels

Endosta7n(ES),B-amyloidpep7de(BAP)andCalcineurinRegulator-1(RCAN-1/DSCR-1)

v AllprominentendogenousanJ-angiogenicfactors

v AlllocatedonChromosome21andallindividualswithDShaveanextracopy

v PaJentswithDShaveincreasedserumand/orJssuelevels

v TheyspecificallyinhibittheproliferaJonandmigraJonofvascularendothelialcells

v TheysuppressVEGF-VEGFR2inducedsignalingcausingmarkedangiogenesisinhibiJon

ItisunknownwhetherES/BAP/RCAN1-relatedan7angiogenicmechanismscontribute

toabnormallungdevelopmentandPHTinDS

OVERRIDINGHYPOTHESIS

Overexpressionofchromosome21-relatedan7-angiogenicfactorsplayacri7calroleinthe

developmentoflunghypoplasiaandpulmonaryhypertensionininfantsand

childrenwithDownsyndrome

Specificstudyques7ons

1.   DolungsinpeoplewithDSshowimpairedalveolarandvasculargrowth/remodeling?

2.   DolungsofafetuswithDownsyndromeoverexpressES,BAP,RCAN-1andotheran7-angiogenicgenes(disomic)andshowvasculargrowthimpairment?

3.   Areangiogenicfunc7onsofendothelialcellsandprogenitorsisolatedfromindividualswithDSimpaired?

Studyques7on-1

DolungsinpeoplewithDSshowimpairedalveolarandvasculargrowth/remodeling?

Study1-Design

Retrospective Autopsy Review Children 0-8 years

With DS or typical with CHD Excluded disorders of lung development

Study Population Patients with DS (n=13) Typical patients age and CHD matched controls

(n=4) Clinical data obtained from autopsy reports Routine H&E lung histology reviewed Serial sectioning and 3D image reconstruction to

clarify microvascular anatomy

Study1-Results

Study1-Summary

Infants and Children with DS who died of severe cardiopulmonary disease have: Histologic evidence of abnormal lung development

Alveolar simplification Histologic evidence of abnormal pulmonary vascular development

Prominent arterial hypertensive remodeling Prominent bronchial vessels Persistence of double capillary network Prominent intrapulmonary anastomotic vessels

Findings present in those with DS with and without CHD are more prevalent than typical patients who were age and CHD matched controls Study 1 supports angiogenic hypothesis

DBushetal.JPeds2017

Studyques7on-2

DolungsofafetuswithDSoverexpressES,BAP,RCAN-1andotheran7-angiogenicgenes

(disomic)andshowvasculargrowthimpairment?

Study2-DesignHuman fetal lung tissue* from

Down syndrome (n=4) Typical controls (n=4)

Individual qPCR (mRNA) and Western Blot Analysis (Protein)

Endostatin B-Amyloid Protein (BAP) Regulator of Calcineurin-1 (DSCR1)

Angiogenesis-associated mRNA microarray (84 angiogenic

genes) IHC evaluation for impaired angiogenesis:

Vessel Density (CD31) Vessel Thickness (SMA)

*U.Maryland,Bal:more/NICHDBrain&TissueBankforDevelopmentalDisorders

Study2-ResultsmRNA mRNA

Study2-Results

(EndostaJn)

(TumstaJn)

(TissueMetalloproteinaseinhibitor3)

Study 2 - Results

Study2-SummaryAnti-angiogenic factors are over-expressed in the lungs of fetuses with DS Chromosome 21-related Genes (trisomic) and Proteins

ENDOSTATIN BAP RCAN1/DSCR1

Non Chromosome 21-related Genes (disomic) TIMP3 COL4A3

Early abnormal vascular growth in the lungs of fetuses with DS Reduced vessel density Increased vessel wall thickness

Study 2 supports angiogenic hypothesis

CGalambosetal.PLOS-12016

Areangiogenicfunc7onsofendothelialcellsandprogenitorsisolatedfromindividualswithDS

impaired?

Studyques7on-3

Study3-Results(ongoing)Endothelialcells(HUVEC)andprecursors(endothelialcolonyformingcells)

isolatedfromindividualswithDSshowimpairedangiogenicfunc7ons

Dr.CBaker,PHLC

DiseasespecificDSmousemodels•  tofurthertestourangiogenichypothesis•  studyspecificmolecularmechanisms•  exploreavarietyofinterven7ons/treatmentop7ons

www.down-syndrome.org/research-practice

MousemodelsofDownsyndromeNocharacteriza7on/researchoflungdisordershasbeendone

inanyofDSmousemodels• Mouse orthologues of human Chr21 on 3 separate mouse chromosomes

• Mmu 10 – ENDOSTATIN (37 genes) • Mmu 16 – DSCR1/RCAN1 & BAP* (102 genes) • Mmu 17 – NO ANTIANGIOGENIC GENE (17 genes)

Guptaetal,MammGenome,2016

Summary•  Lungdisease(PHT/PPHN)isasignificant

contributortomorbidityandmortalityofinfantsandchildrenwithDS

•  DSLunghistologyischaracterizedbyvascularandalveolargrowthabnormali7es

•  LungdiseaseinDSisunderstudied•  LungpathologyofDSisdrivenbyan7-

angiogenicmechanisms•  Earlyinterven7onwithangiogenic

s7mulatorsorblockersofan7-angiogenicpathwaysmaypreventlungdiseaseinthosewithDS

•  AngiogenicbiomarkerswillhelppredictlungdiseaseinthosewithDS

Summary

MembersofDept.ofPathologyDrs.AnnThorandMarkLovell

MembersofPediatricHeartLungCenterAngelaMinic,DougBush,GregorySeedorf,BlairDodson,ChrisBakerDr.SteveAbman—Scien7ficDirector

FundsFounda7onJérômeLejeuneforDownSyndrome Children’sHospitalColoradoResearchIns7tuteTheLindaCrnicIns7tuteforDownSyndromeChallengeGrantJaydendeLucaFounda7on(Dr.DunbarIvy)forPulmonaryHypertensionDean’sBridgeFund,UniversityofColoradoDenver

Collabora7onDr.CharlesHoefferLabatUniversityofColoradoBoulderTheLindaCrnicIns7tuteforDownSyndrome-Drs.FranHickey,JoaquinEspinosaUniv.Maryland,Bal7more/NICHDBrain&TissueBankforDevelopmentalDisorders

“DownSyndromeTakesTheBreathAway”“PeoplewithDSaregiA.Bystudyingtheirbiologycanhelpthemandtherestofmankind.”

----DrTomBlumenthal,FormerExecuJveDirectorofLCIforDS

Self-Advocates,Families,&ResearchercelebrateWorldDownSyndromeDayintheCOCapitoltogetherwithGovernorJohnW.Hickenlooper(3/22/17)