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