Endothelial Biology of PAH and HHT: A “Genotype”-Phenotype … · 2006-12-14 · Terrence Donnelly Heart Centre Terrence Donnelly Heart Centre Endothelial Biology of PAH and HHT:

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Endothelial Biology of PAH and Endothelial Biology of PAH and HHT: HHT: A A ““GenotypeGenotype””--Phenotype Phenotype

AssessmentAssessment

Duncan J. StewartDuncan J. StewartNIH Workshop NIH Workshop –– Hereditary Hemorrhagic Hereditary Hemorrhagic

TelangiectasiaTelangiectasia: Vascular Biology and : Vascular Biology and PathophysiologyPathophysiology

Hyatt Regency, Bethesda, MDHyatt Regency, Bethesda, MDJune 8June 8--9, 20069, 2006

Disclosure: DJS is the founding scientist and CSO Disclosure: DJS is the founding scientist and CSO of Northern Therapeuticsof Northern Therapeutics

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PAHPAH HHTHHT

RA

PA

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van den van den DriescheDriesche et al, 2002et al, 2002

Molecular Basis for PAH and HHTMolecular Basis for PAH and HHT

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eHow are mutations of BMPR2 related How are mutations of BMPR2 related

to the development of to the development of iPAHiPAH??Bone Morphogenetic Protein Bone Morphogenetic Protein Receptor type II Receptor type II is a member of is a member of the transforming growth factor the transforming growth factor BBreceptor family.receptor family.BMPR II is a receptor for a group BMPR II is a receptor for a group of secreted growth factors called of secreted growth factors called BMPsBMPs..In general, the BMPRIn general, the BMPR--II pathway II pathway plays a role in inhibiting cell plays a role in inhibiting cell growth (growth (SMCsSMCs))BMPsBMPs can have procan have pro-- or antior anti--apoptotic actions depending on apoptotic actions depending on cellcell--type and conditionstype and conditions

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eBMPsBMPs induce apoptosis in human induce apoptosis in human

pulmonary artery pulmonary artery SMCsSMCs

Zhang S, Zhang S, AmJAmJ PhysiolPhysiol Lung Cell Mol Lung Cell Mol PhysiolPhysiol, 2003, 2003

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Loss of inhibitory BMP signalingLoss of inhibitory BMP signaling

increased SMC increased SMC survival/proliferationsurvival/proliferation

BMPRII mutations lead to BMPRII mutations lead to dysregulateddysregulatedSMC growthSMC growth

IntimalIntimal and medial and medial hyperplasia of arterioleshyperplasia of arterioles

PAHPAH

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eBMPR2 is mainly localized to BMPR2 is mainly localized to

pulmonary vascular pulmonary vascular ECsECs

Atkinson et al. Circulation 105:1672,2002Atkinson et al. Circulation 105:1672,2002

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eEffect of BMPEffect of BMP--2 on 2 on TNFTNFαα--induced induced

HPAEC apoptosis (TUNEL)HPAEC apoptosis (TUNEL)TNF

TNF+BMP

0.0

2.0

4.0

6.0

8.0

10.0

**

TNFTNF

†

TNF+BMPTNF+BMP

TUN

EL

Pos

itive

Nuc

lei (

%)

TUN

EL

Pos

itive

Nuc

lei (

%)

Circulation Research 2005Circulation Research 2005

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bb ccControlControl

siRNAsiRNASilencingSilencing

siRNAsiRNANSNSRNAiFectRNAiFect

115kDa115kDaBMPRIIBMPRII

ββ--actinactin

ControlControlsiRNAsiRNA

SilencingSilencing

40 40 kDakDaFo

ldFo

ld-- in

crea

se in

Apo

ptos

isin

crea

se in

Apo

ptos

issiRNAsiRNA

NSNSsiRNAsiRNA

SilencingSilencing

**

00

11

22

33

44

BMPRII gene silencing by BMPRII gene silencing by siRNAsiRNA


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SMCsSMCs

upregulatedupregulated SMC SMC growthgrowth

BMPRII mutations: BMPRII mutations: ““Double JeopardyDouble Jeopardy””for PAH?for PAH?

IntimalIntimal and medial and medial hyperplasia of arterioleshyperplasia of arterioles

ECsECs

EC apoptosisEC apoptosis

Regression and loss of Regression and loss of precapillaryprecapillary pulmonary pulmonary

arteriolesarterioles

PAHPAH

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FMA SMA

Pulmonary Microvasculature in the Rat Pulmonary Microvasculature in the Rat MonocrotalineMonocrotaline model of PAH: model of PAH: 21 Days post MCT21 Days post MCT

NormalNormal MCT (3 wks)MCT (3 wks)

Caspase 3 IHC

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eThe PreThe Pre--Capillary Arteriole: the Achilles Capillary Arteriole: the Achilles Heel of Pulmonary Microvasculature?Heel of Pulmonary Microvasculature?

Precapillary arteriolar discontinuity

Central Role of EC Apoptosis in PAH?

EC injuryEC injury

EC apoptosisEC apoptosis

Normal lungNormal lungGenetic susceptibilityGenetic susceptibility

-- BMPR2 mutationBMPR2 mutation-- Tie2Tie2-- otherother

Environmental triggerEnvironmental trigger--ToxinToxin-- AnorexoginsAnorexogins-- HIVHIV-- Shear stressShear stress

IncreasedPVR

IncreasedPVR

Microvasculardropout

Microvasculardropout

PAHPAH

SMC hyperplasiaSMC hyperplasia

VasoconstrictionVasoconstriction

Endothelialdysfunction

Endothelialdysfunction

Emergence of apoptosis-resistant hyperproliferative

EC clones

Emergence of apoptosis-resistant hyperproliferative

EC clones

Plexiform arteriopathyPlexiform arteriopathy

?

EC proliferationEC proliferation

Vascular repairVascular repair

EPCsEPCs

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Role of EPCs in pulmonary vascular regeneration?Role of EPCs in pulmonary vascular regeneration?

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Isolation and characterization of EPCsIsolation and characterization of EPCs

Bone marrow was harvested Bone marrow was harvested from tibia and femur of from tibia and femur of syngeneic Fisher 344 ratssyngeneic Fisher 344 ratsMononuclear cells were Mononuclear cells were isolated by Ficoll gradient isolated by Ficoll gradient centrifugationcentrifugationDifferential culture in EBMDifferential culture in EBM--2 2 medium supplemented with medium supplemented with endothelial growth factors for 7endothelial growth factors for 7--10 days10 days

DiIDiI acLDLacLDL

vWFvWF

LectinLectin (UEA(UEA--1)1)

Flk1Flk1

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eHeterogenousHeterogenous response to BMPresponse to BMP--2 in 2 in EPCsEPCs

from patients with from patients with iPAHiPAH

NormalNormal PAHPAH-100

0

100

200

Apo

ptos

is (%

cha

nge)

Apo

ptos

is (%

cha

nge)

0 25 50 75 100-50

0

50

100

150r2=0.53p<0.05

mPAPmPAP (mm Hg)(mm Hg)

Apo

ptos

is (%

cha

nge)

Apo

ptos

is (%

cha

nge)


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1 week post MCT1 week post MCT

PrePre--capillarycapillaryarteriolearteriole

PulmonaryPulmonaryarteriolearteriole

Engraftment of EPCs into lung microcirculation Engraftment of EPCs into lung microcirculation and reand re--endothelializationendothelialization of distal arteriolesof distal arterioles

15 minutes15 minutes

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ControlControl0

10

20

30

40

50

60

Right Ventricular Systolic Pressure Right Ventricular Systolic Pressure (RVSP)(RVSP)

RV

SP

(mm

Hg)

RV

SP

(mm

Hg)

= p < 0.001 vs. Control= p < 0.001 vs. Control= p < 0.05 vs. MCT/ = p < 0.05 vs. MCT/

MCT+FB MCT+FB **††

***

FBFBFB

††

EPCEPC

**

MCT MCT

Zhao et al. Circ Res. 2005; 96(4):442Zhao et al. Circ Res. 2005; 96(4):442--5050

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eEffect of EPC transplant on lung Effect of EPC transplant on lung

microvascularmicrovascular structure: structure: 21 Days post MCT21 Days post MCT

ControlControl MCTMCT--FBFB MCTMCT--EPCEPC

100 x100 x 100 x100 x 100 x100 x

FMA SMA


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eRight Ventricular Systolic Pressure Right Ventricular Systolic Pressure

(RVSP)(RVSP)R

VSP

(mm

Hg)

RVS

P (m

mH

g)

MCT MCT EPCsEPCs EPCsEPCs//eNOSeNOS

00

2020

4040

6060

8080

ControlControl

Day 21

Day 35

**

* **** p<0.001 vs. d21 MCTp<0.001 vs. d21 MCT** p<0.01 vs. d21 MCTp<0.01 vs. d21 MCT


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eSurvival analysis of Survival analysis of eNOSeNOS EPC EPC

treatment in reversal PAH modeltreatment in reversal PAH model

353533333131292927272525

1.01.0

0.90.9

0.80.8

0.70.7

0.60.6

0.50.5

0.40.4

0.30.3

Days post MCTDays post MCT

Cum

ulat

ive

Sur

viva

lC

umul

ativ

e S

urvi

val

MCTMCT

MCTMCT--EPCEPC

MCTMCT--EPC/EPC/eNOSeNOS

P<0.05P<0.02

N = 63N = 63


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Safety study (18 patients)Safety study (18 patients)–– IIoo EP: tolerability of cell transplantation in patients with EP: tolerability of cell transplantation in patients with

PAH PAH refractory to all standard therapiesrefractory to all standard therapiesCell deliveryCell delivery–– eNOSeNOS transfectedtransfected autologousautologous EPCsEPCs–– Delivery via SG catheterDelivery via SG catheter

Pacing port (i.e. RV delivery) Pacing port (i.e. RV delivery) –– allows continuous monitoring of PA pressureallows continuous monitoring of PA pressure–– exclude intraexclude intra--cardiac shunting (echo bubble study)cardiac shunting (echo bubble study)

–– Cell dose: extrapolation from rat and porcine modelsCell dose: extrapolation from rat and porcine modelsDose ranging up to 150 x 10Dose ranging up to 150 x 1066 eNOSeNOS transfectedtransfected cells given over 3 cells given over 3 days in divided dosesdays in divided doses

PPulmonary ulmonary HHypertension ypertension AAnd nd CeCell ll TTherapy (herapy (PHACeTPHACeT) Trial) Trial

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e HypothesisHypothesis

Do mutations in the TGFDo mutations in the TGF--ββ receptor superreceptor super--family effect progenitor cell number and family effect progenitor cell number and function? function? Do the specific mutations associated with Do the specific mutations associated with PAH and HHT produce distinct diseasePAH and HHT produce distinct disease--specific abnormalities in EPC phenotype?specific abnormalities in EPC phenotype?

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e Circulating Circulating ““EPCsEPCs””Flow Cytometry

0.0

0.1

0.2

0.3

0.4

0.5

0.6

*P<0.05 v. Control

*

*

% o

f MN

Cs

Posi

tive

for M

arke

r

CD34+

CD133++ +-- + +

+ +-- + +

+ +-- + +

Controls (n=10)Controls (n=10) IPAH (n=14)IPAH (n=14) HHT (n=14)HHT (n=14)

Liana Liana ZuccoZucco, PhD Candidate, PhD Candidate

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Day 5Day 5 Day 7Day 7

Day 14Day 14 Day 7Day 7

100um

EPC Isolation and CultureEPC Isolation and Culture

Endoglin: n=23ALK-1: n= 8Smad4: n = 144.5 ± 14.449.4 ± 22.2239HHT (32)

None74.047.033CTEPH (6)

546.3N/A41FPAH (5)

N/A (Possibly 2 Familial)

None

Mutation

45.2 ± 12.348.5 ± 5.58255IPAH (30)

40.7 ± 8.335.8 ± 6.5219Control (30)

Average Age (F)Average Age (M)FemalesMales

Liana Liana ZuccoZucco, PhD Candidate, PhD Candidate

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Effect on ApoptosisEffect on Apoptosis

Basal Basal Basal0

10

20

30

ControlsControls IPAHIPAH HHTHHT

n = 24 n = 27 n = 16SF

n = 17

*

SFn = 8

*

≤≠ ≤*P 0.05 v. BasalP 0.05 v. Control

SFn = 16

*≠

≠

% o

f Cel

ls U

nder

goin

g A

popt

osis

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e Effect on Gene ExpressionEffect on Gene Expression

VEGF mRNA

Controls IPAH HHT0.0

0.1

0.2

0.3

0.4

* P=0.1 v. Control

**

n=6 n=6 n=6ALK-1 ENG

n=3 n=3

Expr

essi

on re

lativ

e to

B-a

ctin

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Endoglin mRNA


0.5

1.0

1.5

P< 0.05 v. Control

n=12 n=12 n=12ALK-1 ENG

n=3 n= 9

Expr

essi

on re

lativ

e to

B-a

ctin

ALK-1 mRNA


0.25

0.50

0.75

1.00

P≤0.05 vs. Control

n=6 n=6 n=6ALK-1 ENG

n=3 n=3

Expr

essi

on re

lativ

e to

B-a

ctin

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eNOS mRNA

Controls IPAH HHT0.000.050.100.150.200.250.300.350.400.45

P=0.06 v. Control

n=16 n=16n=16

Expr

essi

on re

lativ

e to

B-a

ctin

ALK-1 ENGn=3 n=13

Effect on Gene ExpressionEffect on Gene Expression

EC survival signaling microvascular homeostasis

p38MAPK

P

?

BMPR-IBMPR-II

Regulation of EC growth, ECM and vessel structure?

ALK-I/5TβR-II

P

Endoglin

Smad-1/5/8

Smad-4

P

BMP TGFβ

Smad-2P

Smad-4

eNOSeNOS

Smad-1/5/8

Smad-4

P

EC apoptosis vascularpruning

PAHPAH

Abnormal vascular remodelingAV malformations

HHTHHT

End

othe

lial C

ells

/E

ndot

helia

l Cel

ls/ E

PCs

EPC

s

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e AcknowledgementsAcknowledgements

PPH Research Team– Yidan Zhao– Andrew Campbell– Saeid Babaei– Lakshmi Kugathasan– Liana Zucco– Malcom Robb– Yu Pu Deng– Judy Trogatis– Qiuwang Zhang

Collaborators/Co-Investigators– David Courtman– John Granton– Mike Kutryk– Mike Ward– Marie Faughnan– NicK Morrell

Clinical Research Team– Nancy Camack– Jan Mitchell

Supported by Northern TherapeuticsSupported by Northern Therapeutics

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p38MAPK

P

?

BMPR-IBMPR-II

Regulation of EC growth, ECM and Motility?

ALK-I/5TβR-II

P

Endoglin

Smad-1/5/8

Smad-4

P

BMP TGFβ

Smad-2P

Smad-4

eNOSeNOS

Smad-1/5/8

Smad-4

P


PAHPAH

Abnormal vascular remodelingAV malformations

HHTHHT

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eArteriolar Arteriolar ““discontinuitydiscontinuity”” in MCTin MCT--

induced PAH induced PAH

Han et al. Am J Han et al. Am J RespirRespir Cell Mol Cell Mol BiolBiol 35, 2006 ; in press35, 2006 ; in press

SMA FMA

DAPI


p38MAPK

P

?

BMPR-IBMPR-II


Smad-2

Aberrant remodeling AV malformations

ALK-I/5TβR-II

P

Endoglin

PSmad-1/5/8

Smad-4

P

BMP TGFβ

Smad-4

eNOSeNOS

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Effect on ApoptosisEffect on Apoptosis

Basal Basal Basal0

10

20

30

ControlsControls IPAHIPAH HHTHHT

n = 24 n = 27 n = 16TNFn = 15

*

SFn = 17

*

TNFn = 16

*

TNFn = 8

*

SFn = 8

*

≤≠ ≤*P 0.05 v. BasalP 0.05 v. Control

SFn = 16

*≠

≠

% o

f Cel

ls U

nder

goin

g A

popt

osis

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e

% o

f %

of A

nnex

inA

nnex

inV

posi

tive

cells

V po

sitiv

e ce

lls

0

2

4

6

8

10

12

14

16

FCSFCS SFSF BMPBMP--22 BMPBMP--77

**

††††

BMPsBMPs Inhibits Apoptosis in human Inhibits Apoptosis in human PAEC post serum withdrawalPAEC post serum withdrawal

Serumwithdrawal


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ePAH in transgenic mice PAH in transgenic mice

overexpressingoverexpressing dominant negative dominant negative BMRPR2 BMRPR2

…but lack of marked arteriolar remodeling?

West et al. Circ West et al. Circ ResRes 94: 94: ●●●●●●, 2004, 2004

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e Effect of Effect of ““GenotypeGenotype”” of EPC Migration of EPC Migration

Control Control IPAH HHT05

1015202530354045

rhVEGF50ng/ml - + + +

n = 3 n = 3 n = 2 n = 8

Cel

l num

ber p

er h

pf

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e Effect of Effect of ““GenotypeGenotype”” of EPC Migration of EPC Migration

Migration

Control Control IPAH IPAH HHT HHT05

1015202530354045

rhVEGF50ng/ml - + - + - +

n = 3 n = 3 n = 2 n = 2 n = 8 n = 8

Cel

l num

ber p

er h

pf

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e EPC DifferentiationEPC Differentiation

Total Cell Number

Controls IPAH FPAH CTEPH0

100

200

300

n = 19 n = 28 n = 5

P=NS

n = 5

Tota

l Cel

l Num

ber/

mm

2Lectin


25

50

75

100

n = 15 n = 25 n = 5 n = 5

*

*P≤0.01 v. Control

*

% o

f Cel

ls P

ositi

ve fo

r U

EA-1

VEGFR2


10

20

30

40

50

60

70

80

90

n = 20 n = 29 n = 5 n = 5

*P≤0.01 v. Control

**

% o

f Cel

ls P

ositi

ve fo

r VE

GFR

2

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e Effect of Effect of ““genotypegenotype”” on EPC apoptosison EPC apoptosis

TUNEL I - Basal Apoptosis Rates

Controls IPAH FPAH CTEPH 0

10

20

n = 24 n = 27 n = 3 n = 3

*

**P≤0.05 v. Control

% o

f Cel

ls U

nder

goin

g A

popt

osis

TUNEL II - TNFα/Serum Free Stimulated Apoptosis

Basal TNF Serum Free Basal TNF Serum Free0

10

20

30

Controls IPAHn = 24 n = 15 n = 27 n = 16 n = 16n = 17

** *

**P<0.05 vs. Basal≠ P≤0.05 vs. Control

≠

≠

% o

f Cel

ls U

nder

goin

g A

popt

osis

TUNEL III - BMP2/Serum Free Stimulated Apoptosis

Basal Serum Free BMP2 Basal Serum Free BMP20

10

20

Controls (n=6) PAH (n=5)

P=0.16 vs. Contro

% C

ells

Und

ergo

ing

Apo

ptos

is

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e Circulating Circulating ““EPCsEPCs””

FACS Analysis - IPAH

+

CD34

+

CD133 +

/CD13

3

+

CD34

+

CD34

+

CD133 +

/CD13

3

+

CD34

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Controls (n=10) IPAH (n=14)

P<0.05 v. Control*

% o

f MN

Cs

Posi

tive

for

Mar

ker

FACS Analysis - FPAH and CTEPH

+

CD34

+

CD133 +

/CD13

3

+

CD34

+

CD34

+

CD133 +

/CD13

3

+

CD34

+

CD34

+

CD133 +

/CD13

3

+

CD34

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Controls (n=3) CTEPH (n=3)FPAH (n=3)

P<0.001 v. Control*%

of M

NC

s Po

sitiv

e fo

r M

arke

r

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eMRI perfusion imagining of the MRI perfusion imagining of the

pulmonary vasculature pulmonary vasculature

Courtesy of Courtesy of EvangelosEvangelos MichelakisMichelakis, U of Alberta, U of Alberta

RA

PA

NormalNormal PAHPAH

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e

The problem The problem –– PAH through the PAH through the looking glasslooking glass

Archer and Rich: Circulation 102:2781, 2000Archer and Rich: Circulation 102:2781, 2000

RA

PA

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e Thy lesions of HHTThy lesions of HHT

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eHeterogenousHeterogenous response to BMPresponse to BMP--2 in 2 in EPCsEPCs

from patients with from patients with iPAHiPAH

a Tie2 CD34

VEGFR2 UAE-1

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e

CellCell--based gene therapybased gene therapy

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e Imbalance in growth regulation?Imbalance in growth regulation?

Vascular growth Vascular growth factorsfactors–– ETET--11–– FGFsFGFs–– PDGFsPDGFs–– VEGFVEGF–– othersothers

↓↓ Growth inhibitory Growth inhibitory pathwayspathways–– NONO–– PGI2PGI2–– BMPs/BMPRsBMPs/BMPRs–– othersothers

ET-1

GiaidGiaid …… Stewart NEJM, 1993Stewart NEJM, 1993

BMPR2Atkinson et al. Circulation 105:1672,2002Atkinson et al. Circulation 105:1672,2002

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eThe The plexiformplexiform lesion: role of lesion: role of

angiogenesis? angiogenesis?

Abnormal growth of vascular endothelial cellsAbnormal growth of vascular endothelial cells

25X

Archer and Rich Circulation 102:2781, 2000Archer and Rich Circulation 102:2781, 2000

TuderTuder et al J et al J PatholPathol 195:367, 2001195:367, 2001

UpregulationUpregulation of VEGFof VEGF

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eExperimental studies: inhibition of Experimental studies: inhibition of growth factor signalinggrowth factor signaling

HypoxiaHypoxia HypoxHypox & SU5416& SU5416

Effect of VEGF receptorEffect of VEGF receptorantagonist (SU5416)antagonist (SU5416)

TarasevicieneTaraseviciene--Stewart et al. FASEB J. 15:427,2001Stewart et al. FASEB J. 15:427,2001

Effects of SU5416 reversed by zEffects of SU5416 reversed by z--ASPASP

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eEffect of VEGF gene transfer in MCT Effect of VEGF gene transfer in MCT

model of PAHmodel of PAH

Campbell et al. Circulation 2001;104:2242Campbell et al. Circulation 2001;104:2242--22482248

MCT + pcDNA

MCT + pVEGF

MCTMCTGTGT

-- ++ ++ ++

**

-- -- pcDNApcDNA pVEGFpVEGF

†† ††

††

0

10

20

30

40

50

60RVSP (mm Hg)RVSP (mm Hg)

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eVEGF inhibits MCTVEGF inhibits MCT--induced induced microvascularmicrovascular EC apoptosisEC apoptosis

MCT andMCT andpVEGFpVEGF

Day 14

MCT aloneMCT alone


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e

0

30

60

Mm

Hg

0

30

60

Mm

Hg

MCT alone MCT alone

MCT + ZMCT + Z--Asp Asp

Effect of ZEffect of Z--Asp on RVSP at day 21Asp on RVSP at day 21ZZ--Asp (2.5 mg/kg Asp (2.5 mg/kg i.pi.p.) administered 3.) administered 3--times/week times/week

0

10

20

30

40

50

60

70

80

90

100P < 0.0003 P < 00005

RS

VP

mm

Hg

Control MCT MCT+Z-Asp

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e Stopping Criteria for Dose EscalationStopping Criteria for Dose Escalation

1 pt with SAE 1 pt with SAE ““definitely relateddefinitely related””* to cell * to cell deliverydelivery2 pts with SAE 2 pts with SAE ““probably relatedprobably related””* to cell * to cell deliverydelivery3 pts with 3 pts with SAEsSAEs ““possibly relatedpossibly related””* to cell * to cell deliverydelivery

Go back to lower dose and complete Go back to lower dose and complete enrolenrol and and additional 3 pts to complete the trialadditional 3 pts to complete the trial

* Definition arrived at in consultation with the * Definition arrived at in consultation with the Safety Committee (D. Safety Committee (D. LanglebenLangleben, , MtlMtl; S. Mehta, ; S. Mehta, London)London)

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eSafety study in acute porcine PAH Safety study in acute porcine PAH

model model ––PVR (WoodPVR (Wood’’s units)s units)

Cell number = 200 million (cumulative)

U46819 infusion

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eFluorescent 3Fluorescent 3--dimensional dimensional

microangiographymicroangiography

MicroangiograpyMicroangiograpy with with fluorescent fluorescent microspheresmicrospheres

(0.2 (0.2 μμm) suspended inm) suspended inagaroseagarose solutionsolution

Confocal optical sectioning Confocal optical sectioning of thick (100 of thick (100 –– 200200μμm) m)

sections sections of lungof lung

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eDe novo angiogenesis from the De novo angiogenesis from the

pulmonary microvasculaturepulmonary microvasculature

AvascularMatrigel plug

Bronchial c. Pulmonary c.

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e The final product!The final product!

2.5 x106 heNOS-Tx EPCs/ml

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e

CMTMRCMTMR--labeled labeled SMCsSMCsTransmigration through arteriolar endotheliumTransmigration through arteriolar endothelium


a b

c d

5 min 30 min

1h 18h

MCT aloneMCT alone

RV

SP

(mm

Hg)

RV

SP

(mm

Hg)

00

1010

2020

3030

4040

5050

6060

MCT/MCT/peNOSpeNOS = <0.005= <0.005****

****

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eReversal of PH by Reversal of PH by eNOSeNOS gene gene

transfertransfer

Cell-based GT1.5 x 106 cells

CellCell--based GTbased GT1.5 x 101.5 x 1066 cellscells

MCT (70 mg/kg)MCT (70 mg/kg)

DaysDays

†

p<0.05p<0.05

0

20

40

60

80

00 77 1414 2121 2828 3535RV

SP (

mm

Hg)

RVSP

(m

m H

g)

Control (n=6)Control (n=6)peNOSpeNOS (n=6)(n=6)

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eReversal of PAH: Reversal of PAH: evidence for evidence for pulmonary vascular regeneration?pulmonary vascular regeneration?

Normal MCT (3 wks)MCT (5 wks)/eNOS (3 wks)

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eComparison of Comparison of eNOSeNOS vsvs VEGF gene VEGF gene

therapy for reversal of MCT PHtherapy for reversal of MCT PH

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

Normal Normal (n=40)(n=40)

pcDNApcDNA(n=32)(n=32)

VEGFVEGF(n=20)(n=20)

RV

SP

(mm

Hg)

RV

SP

(mm

Hg)

* = P<0.05

**

Day 21Day 21

Day 35Day 35

eNOSeNOS(n=36)(n=36)

**

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eDe novo angiogenesis from the De novo angiogenesis from the

pulmonary microvasculaturepulmonary microvasculature

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e

NO and NeovascularizationNO and NeovascularizationAngiogenic factors (VEGF, Angiogenic factors (VEGF, bFGFbFGF, , TGFTGFββ) ) upregulateupregulate eNOSeNOS and and stimulate NO releasestimulate NO releaseVEGFVEGF--stimulated capillary stimulated capillary formation is prevented by inhibitors formation is prevented by inhibitors of NOS of NOS in vitroin vitro and and in vivoin vivo ((Hood Hood 1998 and 1998 and ZicheZiche 19971997))eNOSeNOS knockout mice have knockout mice have impaired neovascularization impaired neovascularization ((MuroharaMurohara 19981998))eNOSeNOS has been shown to has been shown to upregulateupregulate VEGF (VEGF (DulakDulak 2000, 2000, JozkowiczJozkowicz 20012001))

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eAbnormal vascular development in Abnormal vascular development in

E20 E20 eNOSeNOS--//-- fetal lungsfetal lungs

Han et al. Circ. Res. in press, 2004Han et al. Circ. Res. in press, 2004

WT

KO

WT KO

MCT 21d

normalnormal

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eExperimental Plan: persistence of Experimental Plan: persistence of

beneficial effects in the prevention model beneficial effects in the prevention model

SalineReversal

1 x 101 x 1066 EPCsEPCs (n=12)(n=12)

FBsFBs or Saline (n=13)or Saline (n=13)

1.5 x106 EPCs ± eNOS

PersistencePersistence

00 33 2121 35 Days35 Days

MCT/MCT/salinesaline

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ePersistence of the effect Persistence of the effect EPCsEPCstreatment on RVSP >60 daystreatment on RVSP >60 days

00

2020

4040

6060

8080

100100

120120

<d39<d39 d40d40--d49d49 d50d50--d59d59 >d60>d60

RVS

P (m

m H

g)R

VSP

(mm

Hg)

MCTMCT

MCT/EPCMCT/EPC

2020

4040

6060

8080

100100

120120

RVS

P (m

m H

g)R

VSP

(mm

Hg)

MCTMCT controlcontrolMCT/EPCMCT/EPC00

P<0.001

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eHistological Changes of kidney from Histological Changes of kidney from

MCT treated ratsMCT treated rats

100um

Control MCT-eNOS/EPC

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eComparison of Comparison of eNOSeNOS vsvs VEGF gene VEGF gene

therapy for reversal of MCT PHtherapy for reversal of MCT PH

00

0.10.1

0.20.2

0.30.3

0.40.4

Normal Normal pcDNApcDNA peNOSpeNOS pVEGFpVEGF

Perf

usio

n In

dex

Perf

usio

n In

dex

**

Day 21Day 21

†*

**

Day 35Day 35

** p<0.01 vs.normal; * p<0.05 vs. normal; †<0.05 vs. Day 21

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e

Reversal ProtocolReversal Protocol

1 x 106 EPCs

FBs or SalinePrevention

SalineSalineReversalReversal

1.5 x101.5 x1066 EPCsEPCs ±± eNOSeNOS

00 33 2121 35 Days35 Days

MCT/MCT/salinesaline

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control MCT-35 days

MCT-35 days MCT-35 days

100μ

FMA SMA

FlurorescentFlurorescent microangiographymicroangiography

EPC eNOS/EPC

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FMA FMA -- perfusion index (PI):perfusion index (PI):

00

0.10.1

0.20.2

0.30.3

0.40.4

0.50.5

0.60.6

0.70.7Pe

rfus

ion

Inde

xPe

rfus

ion

Inde

x

PreventionPrevention

MCT MCT EPCsEPCs EPCsEPCs//eNOSeNOS

ControlControl

* = p< 0.05 vs. control; † = p<0.05 vs. MCT-alone

†

* ReversalReversal

†

**

Zhao et al. Circ Res. 2005 (in press)Zhao et al. Circ Res. 2005 (in press)

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e Summary and ConclusionsSummary and Conclusions

CellCell--based gene therapy with based gene therapy with eNOSeNOS can prevent can prevent and reverse experimental PAH at least in part by and reverse experimental PAH at least in part by regeneration of pulmonary microvasculatureregeneration of pulmonary microvasculatureEPC alone given within 3 days of MCT prevent the EPC alone given within 3 days of MCT prevent the development of PAH, but did not reverse development of PAH, but did not reverse established disease when delivered at 3 weeksestablished disease when delivered at 3 weeksEPCsEPCs transfectedtransfected with with eNOSeNOS dramatically improved dramatically improved survival in established PAH suggesting that the survival in established PAH suggesting that the combination of regenerative cell and gene therapy combination of regenerative cell and gene therapy will be the most effective in the treatment of this will be the most effective in the treatment of this diseasedisease

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eEffect of EPC transplant on lung Effect of EPC transplant on lung

microvascularmicrovascular structure: structure: 21 Days post MCT21 Days post MCT

ControlControl MCTMCT--FBFB MCTMCT--EPCEPC

40 x40 x 40 x40 x 40 x40 x

FMA SMA

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e

NO and NeovascularizationNO and NeovascularizationAngiogenic factors (VEGF, Angiogenic factors (VEGF, bFGFbFGF, , TGFTGFββ) ) upregulateupregulate eNOSeNOS and and stimulate NO releasestimulate NO releaseVEGFVEGF--stimulated capillary stimulated capillary formation is prevented by inhibitors formation is prevented by inhibitors of NOS of NOS in vitroin vitro and and in vivoin vivo Hood Hood 1998 and 1998 and ZicheZiche 19971997eNOSeNOS knockout mice have knockout mice have impaired neovascularization impaired neovascularization MuroharaMurohara 19981998eNOSeNOS has been shown to has been shown to upregulateupregulate VEGF VEGF DulakDulak 2000, 2000, JozkowiczJozkowicz 20012001

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e

Dose escalationDose escalation

00

2020

4040

6060

8080

100100

120120

140140

160160

panel 1panel 1 panel 2panel 2 panel 3panel 3 panel 4panel 4 panel 5panel 5

Cel

l num

ber (

x 1

mill

ion)

Cel

l num

ber (

x 1

mill

ion) Day 3Day 3

Day 2Day 2Day 1Day 1

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a b

c d1 10 100 1000 10000

Time (log minutes)

0

20

40

60

80

100

Perc

ent T

ran s

mig

ratio

n

***

CMTMRCMTMR--labelledlabelled SMCsSMCsTransmigration through arteriolar endotheliumTransmigration through arteriolar endothelium


5 min 30 min

1h 18h

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eEffect of cellEffect of cell--based based eNOSeNOS gene gene

therapy: therapy: rat MCT model of PHrat MCT model of PH

Fisher 344 rats injected Fisher 344 rats injected with 70 mg/kg MCT s.c. with 70 mg/kg MCT s.c. and 500,000 and 500,000 transfectedtransfectedsmooth muscle cells via smooth muscle cells via the internal jugular veinthe internal jugular veinRVSP measured at 28 RVSP measured at 28 daysdaysanimals sacrificed and animals sacrificed and RV/LV ratio measured, RV/LV ratio measured, pulmonary histology pulmonary histology examined, RNA extractedexamined, RNA extracted

-- pcDNA3.1pcDNA3.1-- peNOSpeNOS

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e Prognosis of PPHPrognosis of PPH

average of 3average of 3--5 year survival after 5 year survival after diagnosis!diagnosis!

Better late than never?

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eThe future The future –– reversing reversing ““irreversibleirreversible””

PAH?PAH?

JAMA 284:3164,2000

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CellCell--based gene therapybased gene therapy

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e

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The Pulmonary VasculatureThe Pulmonary Vasculature


RA

PA

NormalNormal

PAHPAH

HumanHuman Rat MCT modelRat MCT model

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e The Normal Pulmonary VasculatureThe Normal Pulmonary Vasculature


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e Pulmonary Pulmonary VascularityVascularity in PAHin PAH

RA

PA


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Pulmonary Arterial Hypertension Pulmonary Arterial Hypertension (PAH)(PAH)

The pulmonary vasculature bed is a The pulmonary vasculature bed is a high flow, low impedance system high flow, low impedance system that accommodates entire cardiac that accommodates entire cardiac output with low arterial pressuresoutput with low arterial pressures

In PAH, there is a persistent In PAH, there is a persistent elevation in PA pressure due to elevation in PA pressure due to narrowing of arterioles and narrowing of arterioles and loss of loss of pulmonary pulmonary microvesselsmicrovessels

MicrovascilarMicrovascilarlossloss

ArteriolarArteriolarnarrowingnarrowing

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e

Stewart and Kutryk, 2002

Angiogenesis/Angiogenesis/arteriognesisarteriognesis

VascularVascularregressionregression

Regulation of vascular growth and Regulation of vascular growth and regressionregression

/ANG1?

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Ang1 gene transfer inhibits apoptosis in Ang1 gene transfer inhibits apoptosis in rat model of MCTrat model of MCT--induced PHinduced PH

Activated caspaseActivated caspase--3 3 immunoreactivityimmunoreactivity

0.000.00

0.0400.040

0.0800.080

0.1200.120

0.1600.160

0.2000.200

Abs

orba

nce

(405

nm)

1 week1 week 2 weeks2 weeksnormalnormal

AngAng--1+ MCT1+ MCT

MCTMCT

Zhao et al Circulation Research 92:984, 2003Zhao et al Circulation Research 92:984, 2003

RV systolic pressureRV systolic pressure

1wk1wk 2wk2wk 3wk3wk 4wk4wk

(N=3/group)(N=3/group)

00

1010

2020

3030

4040

5050

6060

7070

8080

RVS

P (m

mH

g)R

VSP

(mm

Hg)

Ang-1 + MCTAng-1 + MCT

MCTMCT

0wk0wk

*

**

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e Is AngiopoietinIs Angiopoietin--1 a mediator of PAH? 1 a mediator of PAH?

DuDu L. Signaling Molecules in L. Signaling Molecules in NonfamilialNonfamilial Pulmonary Hypertension. NEJM Pulmonary Hypertension. NEJM 2003;348:5002003;348:500--509.509.

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RudgeRudge and and YancopolousYancopolous, , RegeneronRegeneron

Expression of Expression of AngiopoietinAngiopoietin in Multiple in Multiple Organ Blots: Organ Blots: ““Of Mice and MenOf Mice and Men””

Murine

580bp

343bpLu

ng

Mus

cle

Live

rpA

ng1

Human

AngAng--11

GAPDHGAPDH

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AngAng--1 in PPH lungs1 in PPH lungs

Nor

mal

don

or

Lung

s

Surg

ical

con

trol

Hum

an p

Ang1

Surg

ical

Con

trol

PPH

Lun

gs

Standard curve

Angiopoietin-1

PPH

Normal

A.E. A.E. DutlyDutly et al. Robust expression of angiopoietinet al. Robust expression of angiopoietin--1 in human lung 1 in human lung -- PosterPoster

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e AngiopoietinAngiopoietin expression in PAHexpression in PAH

0

250

500

750

1000

1250

1500

1750

2000

NormalNormal PPHPPH SPHSPH0

250

500

750

1000

1250

1500

1750

2000AngiopoietinAngiopoietin--1 tissue levels1 tissue levels

(ELISA, pg/ul)

0.0

0.5

1.0

1.5

2.0

2.5

3.0 *

Ang1Ang1 Ang2Ang2 Tie2Tie20.0

0.5

1.0

1.5

2.0

2.5

3.0

Relative Expression Relative Expression (qc RT(qc RT--PCR)PCR)

*Normal

PPH

SPH

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NormalNormal PPHPPH

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ePartial rescue of Tie2Partial rescue of Tie2--//-- mice by mice by doxdox--conditional targeted Tie2 expressionconditional targeted Tie2 expression

Jones et al. EMBO J 5:438, 2001Jones et al. EMBO J 5:438, 2001

E11.5-12.5

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eTie2 +/Tie2 +/-- mice develop spontaneous mice develop spontaneous

PAHPAH

+/++/+N=40N=40

+/+/--N=52N=52

0

5

10

15

20

25

30

35

RVSP (mmHg)RVSP (mmHg)

P < 0.05

Frequency Distribution (%)Frequency Distribution (%)

0

5

10

1520

25

30

35

<20 20-24 25-29 30-34 35-39 40-44 45+

RVSP Range (mmHg)RVSP Range (mmHg)

Tie2 +/+Tie2 +/+Tie2 +/Tie2 +/--

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e Effect of chronic Serotonin infusionEffect of chronic Serotonin infusion

P<0.05

0.00

10.00

20.00

30.00

40.00

50.00

ControlControl 55--HTHT

RVS

P (m

m H

g)R

VSP

(mm

Hg)

WTWT

Tie2+/Tie2+/--

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eConditional Endothelial AngiopoietinConditional Endothelial Angiopoietin--1 1

OverexpressionOverexpression

tTAtTA Tie-1 promoter

+ DOX

ββ --galgalAng1Ang1IRES

tTA+/Ang1tTA+/Ang1-- tTA+/Ang1+ tTAtTA+/Ang1+ tTA--/Ang1+ tTA/Ang1+ tTA--/Ang1/Ang1--

tTA+/Ang1tTA+/Ang1-- tTAtTA--/Ang1+/Ang1+

DriverDriver

ResponderResponder

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eConditional Endothelial AngiopoietinConditional Endothelial Angiopoietin--1 1

OverexpressionOverexpression: protein: protein

NBTNBT BTBT0.00.00.50.51.01.01.51.52.02.02.52.53.03.03.53.54.04.04.54.5

Ang

1 (n

g/m

l)A

ng1

(ng/

ml)

BB--Gal stainingGal staining AngAng--1 ELISA1 ELISA

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eEndothelial AngiopoietinEndothelial Angiopoietin--1 1

overexpressionoverexpression reduces hypoxic PAHreduces hypoxic PAH

0

10

20

30

40

50

60

NBTNBT AngAng--1 BT1 BT

RVS

P (m

m H

g)R

VSP

(mm

Hg) NormoxiaNormoxia (n=6)(n=6)

Hypoxia (n=6)Hypoxia (n=6)

P=0.06

LakshmiLakshmi KugathasanKugathasan –– MscMsc CandidateCandidate

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eAngiopoietinAngiopoietin--1 in pulmonary 1 in pulmonary hypertension: hypertension: Cause or cureCause or cure? ?

Promotes EC survival; Does Promotes EC survival; Does not induce mitosisnot induce mitosisReduces vascular permeabilityReduces vascular permeabilityStabilizes Stabilizes microvesselsmicrovessels by by pericytepericyte recruitment and recruitment and physiologicalphysiological muscularizationmuscularizationThought to be a key factor in Thought to be a key factor in the maintenance of postnatal the maintenance of postnatal vascular homeostasisvascular homeostasis

“ANG-el”!

Editorial comment: Rudge, Thurston and Yancopolous, Circ Res 92:947, 2003

Documents

Endothelial Biology of PAH and HHT: A “Genotype”-Phenotype … · 2006-12-14 · Terrence Donnelly Heart Centre Terrence Donnelly Heart Centre Endothelial Biology of PAH and HHT: