The role of endothelin in the The role of endothelin in the pathobiology of pulmonary arterial pathobiology of pulmonary arterial

hypertensionhypertension

Robert Naeije

Erasme Hospital, Faculty of Medicine, Free University of Brussels

•

Pulmonary arterial hypertension: a diseasePulmonary arterial hypertension: a diseaseof the small resistive arteriolesof the small resistive arterioles

After Heath & Edwards, Circulation 1958;18:533-47After Heath & Edwards, Circulation 1958;18:533-47Wagenvoort & Wagenvoort, Circulation 1970; 42: 1163-84Wagenvoort & Wagenvoort, Circulation 1970; 42: 1163-84

The historical notion of a vasoconstrictive factor The historical notion of a vasoconstrictive factor in PAHin PAH

Demonstration of a reversible component of PVR with acetylcholine and tolazaline as soon as with initial recognition of PAH as a clinical entity Dresdale et al, Am J Med 1951; 11: 686-705 Wood et al, Br Heart J 1957; 19: 279-292

Morphology suggestive of early reversibility in earlier stages of disease Heath & Edwards, Circulation 1958;18:533-47Heath & Edwards, Circulation 1958;18:533-47Wagenvoort & Wagenvoort, Circulation 1970;42:1163-84Wagenvoort & Wagenvoort, Circulation 1970;42:1163-84

Palevsky et al Circulation 1989; 80: 1207-1221

The pathogenesis of PAH: from vasospasms to The pathogenesis of PAH: from vasospasms to remodellingremodelling

EarlyMatrix and

fibrin

CellsVasoconstrictor factors

Detectionlimit

PVR

VasospasmsVasospasms RemodellingRemodelling

P Wood P Wood

Wood P. Pulmonary hypertension with special reference to the vasoconstrictive factor. Br Heart J 1958; 21: 557-70.

LateLate

EndothelinEndothelin

21-amino-acid peptide isolated from cultured porcine aortic endothelial cells1, first reported in 1988

Regulates pulmonary vascular tone, sodium balance, neural crest cell development and neurotransmission

Key mediator in pulmonary hypertension

C

ET-1NCysCys Ser

SerSer

Leu

TrpIleIleAspLeuHisCysTyrVal PheCysGlu

Met

Asp

Lys

Yanagisawa M, et al. Nature 1988;3 32:411-15.

Endothelin plasma levels are Endothelin plasma levels are increased in PAHincreased in PAH

Congenitalheart disease3

1Stewart DJ, et al. Ann Intern Med 1991;114:464-9.2Vancheeswaran R, et al. J Rheum 1994;21:1838-44.3Yoshibayashi M, et al. Circulation 1991;84:2280-5.

ET-L

I (P

V-R

V)

(pg/m

l)

Idiopathic PAH1

ET-1

(pg/m

l)

Non-PPH

PPH0

2

4

6

8

10

Non-PH PH0

1

2

3

4

5

Scleroderma2

ET-1

(pg/m

l)

4

6

8

10

Non-PAH

PAH

Expression of endothelin-1 is increased in the Expression of endothelin-1 is increased in the lungs of patients with pulmonary hypertensionlungs of patients with pulmonary hypertension

Increased immunostaining for ET-1 in small size pulmonaryarteries of PAH patients, in proportion to increased PVR

Normal Small size pulmonary artery and plexiform lesion in PAH

Giaid et al. N Engl J Med 1993;328:1732-9.

Galiè N, et al. Eur J Clin Invest 1996;26 (Suppl 1):A48.

Endothelin drives disease progression and Endothelin drives disease progression and determines patients‘ long-term outcomedetermines patients‘ long-term outcome

0 12 24 36 480.0

0.5

1.0

ET-1 4.3 pg/mlET-1 < 4.3 pg/ml

p = 0.04 (log rank)

Time (months)

Su

rviv

al

n=24 IPAH patients

S h a m P l a c e b o

B o s e n t a nA d v

E E L

I E L

L u m e n

M T

E D

% M T = 2 x M T / E D x 1 0 0

Rondelet B et al. Circulation 2003; 107:1329-35.

Systemic-to-pulmonary shunting during 3 months in piglets Systemic-to-pulmonary shunting during 3 months in piglets induces pulmonary hypertension with medial hypertrophy, which induces pulmonary hypertension with medial hypertrophy, which

is partially prevented dual ETA/ETB blockade with bosentan is partially prevented dual ETA/ETB blockade with bosentan

Rondelet B et al. Circulation 2003; 107:1329-35, Circulation 2004,110:2220-5

0

20

40

60

2 5

Ppa

m (

mm

Hg)

Q ( L . min-1 . m-2 )

*†

0

10

20

30

0 150 300 450

External diameter (mm)

Med

ial t

hick

nes

s (%

) ShamPlaceboTreatment*†‡

*†‡ *†

*†‡

*†‡

r = 0.4521p < 0.05

0

20

40

10 20 30 40

Ppam (mmHg)

r = 0.6104p < 0.01

0

20

40

10 20 30 40

Ppam (mmHg)

< 7

5m p

ulm

onar

y a

rter

ym

edia

l thi

ckn

ess

(%)

ShamPlaceboTreatment

< 3

00m

pul

mo

nary

art

ery

med

ial t

hick

nes

s (%

)

Bosentan prevents overcirculation-induced PAH in pigletsBosentan prevents overcirculation-induced PAH in piglets

-40

-20

0

20

40

60

80

Bosentan (n = 144)Placebo (n = 69)

Baseline Week 4 Week 8 Week 16

p = 0.0002

62.5 mg/bid 125 or 250 mg/bid

W

alk

Dis

tan

ce(m

eter

s)

Mean ± SEM

Rubin LJ et al. N Engl J Med 2002; 346: 896-903.

Dual endothelin A and B receptor blockade by Dual endothelin A and B receptor blockade by bosentan improves PAH patientsbosentan improves PAH patients

Pre-pro-ET pro-ET

Endothlin-1

vasoconstriction

proliferation

ETA ETB

-

Endothelin receptorantagonists

L-arginine L-citrulline

vasodilatation

anti-proliferation

cGMP

PDE5

PDE5 inhibitors

-

-vasodilatation

anti-proliferation

AMPc

Prostacyclin (PgI2 )

Arachidonic acid PgI2

Prostacyclinderivatives

+Nitric oxide

Endothelin pathway Prostacyclin pathwayNO pathway

Pulmonary circulation: ET-1 as a key mediator in endothelial

maintainance of tone and structure

Biological theories of PAHBiological theories of PAH

Endothelium-derived vasoconstrictor/dilator imbalance (ET-1/PGI2-NO): increased expression of ET-1, decreased expressions of PGI2 and NO synthases, efficacy of targeted therapies Gaine SP, Rubin LJ. Lancet Gaine SP, Rubin LJ. Lancet 1998; 352: 719-7251998; 352: 719-725

Serotonin receptors (5HT 1B, 2B and 2A) and transporter (anorexigens) Eddahibi et al, J Clin Invest 2001; 108: 1141-50

Other: Decreased PASM potassium channels, increased PDE5, inflammation, misguided angiogenesis Humbert et al, JACC 2004; 43suppl: 13S-24S

Genetics

IPAH: > 6 % familial, autosomal dominant pattern of inheritance with reduced penetrance

Mutation in gene encoding for bone morphogenetic protein receptor 2 (BMPR2) in 16 of 27 IPAH families Deng et al, Am J Human Genet 2000;67:737-44, and Internat PPH Consortium Nat Genet 2000;26:81-4

BMP’s are members of the TGF superfamilyBMPR2-smad signalling inhibits proliferation

BMPR2 mutations as a cause of PAHBMPR2 mutations as a cause of PAH

Estimated < 10 – 20 % incidence of PAH in carriers of BMPR2 mutations

Not all familial PAH have detectable BMPR2 mutations Incidence of BMPR2 mutations 5 % in sporadic IPAH BMPR2 mutations uncommon in other PAH categories

(anorexigens, CHD)

BMPR2 mutations neither necessary nor sufficient

Decreased BMPR2 protein in IPAH with BMPR2 Decreased BMPR2 protein in IPAH with BMPR2 mutations > IPAH > CHD or CTD - PAHmutations > IPAH > CHD or CTD - PAH

Atkinson et al, Circulation 2002;105: 1672-78 Atkinson et al, Circulation 2002;105: 1672-78

Signaling molecules in nonfamilial pulmonary Signaling molecules in nonfamilial pulmonary hypertension hypertension

Lingling Du et al, N Engl J Med 2003; 348: 500-509Lingling Du et al, N Engl J Med 2003; 348: 500-509

Increased expression of angiopoietin-1 in the lungs of patients with various forms of severe pulmonary hypertension: 22 CTEPH, 5 IPAH, 3 CTD-associated PAH, 3 Eisenmenger, 9 mitral valve disease-associated PH

Angiopoietin-1 shuts of the expression of BMPR1A, a transmembrane protein required for BMPR2 signalling

Lingling Du et al, N Engl J Med 2003; 348: 500-9.

r = 0.97 p ≤ 0.001

0 500 1000 1500 20000.0

0.2

0.4

0.6

0.8

1.0

Ang-1–Band Density

Pulmonary Vascular Resistance (dyn•sec•cm-5)

Central role for the angiopoietin-1 / BMPR2 Central role for the angiopoietin-1 / BMPR2 pathway in the pathogenesis of PAHpathway in the pathogenesis of PAH

BMPR2 receptorBMPR2 receptor

BMPR2 mutationBMPR2 mutation Angiopoietin-1Angiopoietin-1

Unrestricted PVSMC proliferationUnrestricted PVSMC proliferationPulmonary arterial hyprtensionPulmonary arterial hyprtension

Smad signallingSmad signalling

----

--

-- Other factorsOther factorsCytokines?Cytokines?

Expression of ET-1, ETExpression of ET-1, ETBB, angiopoietin-1 , angiopoietin-1

and BMPR2 in a piglet model of CHD-PAHand BMPR2 in a piglet model of CHD-PAH

Rondelet B et al. Circulation 2003; 107:1329-35, Circulation 2004; 110:2220-5.

0

10

0 10 20 30 40 50

R = 0.5675p < 0.01

00 10 20 30 40 50

R = 0.7074p < 0.001

20.103

0

70

0 10 20 30 40 50

ET-1

mR

NA

lung

conte

nt

(2-

Ct )

R = 0.5357p < 0.01

0

20

0 10 20 30 40 50

R = 0.3792p = NS

Ppam (mmHg)

Ppam (mmHg)

BM

PR

2 m

RN

A lung

conte

nt

(2-

Ct )

ET-B

mR

NA

lung

conte

nt

(2-

Ct )

Ang-1

mR

NA

lung

conte

nt

(2-

Ct )

Signalling molecules in experimental Signalling molecules in experimental overcirculation-induced PAHovercirculation-induced PAH

Endothelial imbalance: Increased expressions of ET-1 / ETB receptor, and PDE5

Angiogenesis/inflammation: Increased expressions of angiopoietin-1, VEGF, angiotensin-II, MCP-1 and ICAM

Serotonin: increased expression of 5HT1B Decreased expressions of BMPR2 and BMPR1A

Rondelet B et al. Circulation 2003; 107:1329-35, 2004; 110:2220-5.

Induction of pulmonary hypertension by an Induction of pulmonary hypertension by an angiopoietin-1/TIE2/serotonin pathwayangiopoietin-1/TIE2/serotonin pathwaySullivan CC et al, PNAS; 2004; 100: 12331-6Sullivan CC et al, PNAS; 2004; 100: 12331-6

Rodents engineerd to express angiopoietin-1 in the lung develop severe pulmonary hypertension (medial thickening)

Angiopoietin-1 stimulates pulmonary arteriolar endothelial cells through a TIE2 receptor pathway to produce serotonin

High levels of serotonin found in human and rodent pulmonary hypertensive lung tissue

Sullivan CC, et al. PNAS 2003; 100: 12331-6.

Contribution of the angiopoietin-1/Tie2 pathway Contribution of the angiopoietin-1/Tie2 pathway to pulmonary artery smooth muscle hyperplasia to pulmonary artery smooth muscle hyperplasia

in IPAH in IPAH Dewachter L et al, PATS 2005; 2: A710Dewachter L et al, PATS 2005; 2: A710

Lung tissue from 14 patients with IPAH, 9 patients with SPH, and 15 controls

RT-PCR, immunohistology, Western blotting Cultured pulmonary artery endothelial and

smooth muscle cells Treatment of the endothelial cells with human

recombinant angiopoietin-1, and mesurement of supernatant ET-1, 5HT, EGF and PDGF

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

CT IPH SPH

Rel

ati

ve

Tie

-2 p

rote

in e

xp

ress

ion

(Tie

-2/b

-act

in)

**

0

0,5

1

1,5

2

2,5

3

CT IPH SPH

Tie

-2 m

RN

A l

evel

s

(arb

itra

ry u

nit

s)

*

140 kD

42 kD

Tie2

- Actin

CT

SP

H

IPH

IPH

SP

H

CT

A B

Overexpression of Tie2 in IPAHOverexpression of Tie2 in IPAH

**

A B

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

CT PAH

Tie

2 m

RN

A l

evel

s(a

rbit

rary

un

its)

*

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

4,00

CT PH

Rel

ati

ve

Tie

-2 p

rote

in e

xp

ress

ion

(Tie

-2/b

-act

in)

*

Tie2 receptor in cultured pulmonary endothelial from IPAHTie2 receptor in cultured pulmonary endothelial from IPAHand SPH patientsand SPH patients

0

0,5

1

1,5

2

2,5

3

3,5

4

Base 1 5 10 100

Angiopoietin-1 (ng/ml)

5-H

T (

pg

/ml)

A B

0,00

1,00

2,00

3,00

4,00

5,00

Base 1 5 10 50 100

Angiopoietin-1 (ng/mL)

EG

F C

on

cen

trati

on

(p

g/m

L)

C D

Pulmonary artery endothelial cells treated with angiopoietin-1Pulmonary artery endothelial cells treated with angiopoietin-1

BMPR2 / TIE2 : loss of endothelial control of BMPR2 / TIE2 : loss of endothelial control of inflammation-angiogenesis - related remodellinginflammation-angiogenesis - related remodelling

BMPR2 receptorBMPR2 receptor

BMPR2 mutationBMPR2 mutation

Unrestricted PVSMC proliferationUnrestricted PVSMC proliferationPulmonary arterial hypPulmonary arterial hypeertensionrtension

Smad signallingSmad signalling

----

--

5HT, ET-15HT, ET-1

Tie2 receptorTie2 receptor

ET-1ET-1, MCP1, ICAM, VEGF, MCP1, ICAM, VEGFangiopoietin-1, angiotensin IIangiopoietin-1, angiotensin IIdecreased PGI2 and NOdecreased PGI2 and NO

++

ConclusionsConclusions

The pathobiology of PAH remains mysterious Central: abnormal BMPR2 and Tie2 signaling,

with excess endothelial-derived ET-1 + 5HT and insufficient endothelium-derived PGI2 + NO possibly triggered by injury-repair reaction (inflammation-angiogenesis)

Restoration of endothelial equilibrium by the dual ET-1 receptor blocker bosentan partially prevents experimental PAH, and is effective palliative therapy in PAH patients

AcknowledgmentsAcknowledgments

ULB INSERM U651, CréteilBenoît Rondelet Laurence DewachterSerge Brimioulle Saadia EddahibiRonald Van Beneden Serge AdnotSophie Motte Marc HumbertIsabelle SalmonMyriam RemmelinkVUBIves Hubloue MarseilleFrançois Kerbaul