professor sir john vane (1927–2004) f.r.s. nobel laureate “john vane has discovered prostacyclin...
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Professor Sir John Vane (1927–2004) F.R.S.
Nobel Laureate
“John Vane has discovered prostacyclin and has carried out detailed analyses of its
biological effects and function. In addition, Vane has made the fundamental discovery that antiinflammatory compounds such as
aspirin act by blocking the formation of prostaglandins and thromboxanes”
The Nobel Prize in Physiology or Medicine 19822
The discovery of prostacyclin (1976)
1. Moncada et al. Nature. 1976;263:663-665; 2. http://nobelprize.org/nobel_prizes/medicine/laureates/1982/press.html. Accessed May 2010
An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation
Nature, 19761
Anti-inflammato
ry
Anti-thrombotic
Vasodilatation
Anti-proliferativ
e
Pharmacological effects of prostacyclin
O
OH OH
HOOC
Prostacyclin
Gomberg-Maitland et al. Eur Respir J. 2008;31:891-901; Zardi et al. Int Immunopharmacol. 2005;5:437-459; Ghofrani et al. J Am Coll Cardiol. 2004;43:68S-72S; Humbert et al. J Am Coll Cardiol. 2004;43:13S-24S
Prostacyclin stimulates cAMP production
in platelets: Anti-thrombotic effects
0
5
10
15
20
25
3 10 30 100 300 1000 3000
PGI2 (nM)
Mu
ltip
les o
f b
asal cA
MP
Platelet-rich plasmaWashed platelets
Platelet disaggregation
Data are mean ±standard errorcAMP, cyclic adenosine monophosphate; IP, prostacyclin receptor; PGI2, prostacyclin
Adapted from Moncada et al. In: Westwick et al, eds. Mechanisms of Stimulus-Response Coupling in Platelets. 1985;159:337–358
• Elevation of platelet cAMP following 1 min incubation at 37°C with prostacyclin in human platelet-rich plasma or washed platelets
Prostacyclin inhibits adhesion of platelets exposed to blood vessel wall
Control1
+ PGI2
Platelets
GPIb, platelet glycoprotein 1b; GPIIb/IIIa, platelet integrin IIbβ3; PGI2, prostacyclin
Images from Brendan Whittle, Dept of Prostaglandin Research, Wellcome, Beckenham. With permission.
1. Dept of Prostaglandin Research, Wellcome, Beckenham; 2. Zardi et al. Int Immunopharmacol. 2005;5:437–459
• Prostacyclin analogues play an important role as regulators of endothelial function including maintaining vascular homeostasis of the microcirculation2
BP-lowering effects of prostacyclin in systemic and pulmonary circulation
PA
P (
mm
Hg
)
30
20
15
10
5
0
25
Time (min)
0 40 80 120 160 200 240 280 320
ControlU46619U46619 + prostacyclin (0.5 μg/kg)
PulmonaryRabbit perfused lung2
SystemicAnaesthetised rat1
Mean
fall in
dia
sto
lic B
P (
mm
Hg
)
Prostacyclin dose (µg/kg)
90
80
7060
50
0
0.250.5 2 8
Vasodilatory effects mediated through potassium channels
Intra-arterialIntravenous
Data are mean ±standard error. U46619 is a thromboxane-A2 mimeticBP, blood pressure; PAP, pulmonary arterial pressure
1. Adapted from Armstrong et al. Br J Pharmacol. 1978;62:125-130; 2. Adapted from Schermuly et al. Respir Res. 2007;8:4
410.125
40
3020
10n=6
Prostacyclin effect Cellular response Mechanism
Classical functions
Vessel tone ↑ Vasodilation ↑ cAMP, ↑ K+, ↓ Ca2+
Anti-proliferative↓ SMC proliferation↓ Fibroblast growth↑ Apoptosis
↑ cAMP, ↑ K+, ↓ ET-1↑ PPAR (?)
Anti-thrombotic↓ Platelet aggregation↓ Platelet adherence to vessel wall
↓ Thromboxane A2
↓ PDGF, ↓ AMs
Novel functions
Anti-inflammatory↓ Pro-inflammatory cytokines↑ Anti-inflammatory cytokines
↓ IL-1, IL-6↑ IL-10
Anti-mitogenic↓ ECM remodelling↓ Fibrosis
↓ MMP 2 & 9 ↓ TGF-, ↓ CTGF
Anti-tumourigenic↓ Tumour formation and metastasis
↑ PPAR
Prostacyclins have many diverse cellular functions
AM, adhesion molecule; Ca2+, calcium; cAMP, cyclic adenosine monophosphate; CTGF, connective tissue growth factor; ECM, extracellular matrix; ET-1, endothelin 1; IL, interleukin; K+, potassium; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; PPAR, peroxisome proliferator-activated receptor; SMC, smooth muscle cell; TGF, transforming growth factor
Gomberg-Maitland et al. Eur Respir J. 2008;31:891-901; Zardi et al. Int Immunopharmacol. 2005;5:437-459; Ghofrani et al. J Am Coll Cardiol. 2004;43:68S-72S; Humbert et al. J Am Coll Cardiol. 2004;43:13S-24S
Pathophysiology of PAH:Pathways of disease
Endothelin pathway
Nitric oxide pathway
Prostacyclin pathwayVessel
lumenEndothelialcells
L-arginine L-citrulline
Pre-proendothelin
Proendothelin
Arachidonic acid
Prostaglandin I2
Prostacyclin(prostaglandin
I2)Nitric oxideEndothelin-1
Endothelinreceptor A
Endothelinreceptor B
Phosphodiesterase type 5
cGMP
cAMP
Smooth muscle cells
Vasoconstriction and
proliferation
Vasodilatation and
antiproliferation
Vasodilatation and
antiproliferation
Phosphodiesterase type 5 inhibitor
Endothelin-
receptor antagonis
ts
+
+–
–
–
–Exogenou
s nitric oxide
Prostacyclin
derivatives
cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; PAH, pulmonary arterial hypertension
Humbert et al. N Engl J Med. 2004;351:1425-1436
Therapy targets for PAH:Prostacyclin pathway
cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; PAH, pulmonary arterial hypertension
Humbert et al. N Engl J Med. 2004;351:1425-1436
• Pulmonary hypertension is associated with a decrease in prostacyclin levels and an increase in thromboxane levels
Alterations between prostacyclin and thromboxane homeostasis in PAH
10,000
8000
6000
4000
2000
0
Control IPAH APAH PH-CVD11
-Deh
yd
ro-t
hro
mb
oxan
e B
2 (
pg/m
g o
f cr
eati
ne)
p<0.05†
n=14 n=20 n=8 n=6
Thromboxane
800
600
400
200
2,3
-Din
or-
6-k
eto
-PG
F1a (
pg/m
g o
f cr
eati
ne)
0
Control IPAH APAH PH-CVD
p<0.05*
n=9 n=20 n=5 n=2
Prostacyclin
Data are mean ±standard error. Statistical significance assessed using two-tailed Mann-Whitney test; *versus normal control; †versus other 3 groupsAPAH, associated pulmonary arterial hypertension; IPAH, idiopathic pulmonary arterial hypertension; PAH, pulmonary arterial hypertension; PGF, prostaglandin F; PH-CVD, pulmonary hypertension associated with collagen vascular disease
Adapted from Christman et al. N Engl J Med. 1992;327:70-75
Frequency of PGI2 synthase expression
Normal (n=7)
IPAH (n=12)
100
80
60
40
20
0
PG
I 2 s
yn
thase (
% p
osi
tive
vess
els
)
Large Medium Small
Pulmonary arteries
p=0.03
p=0.015
Prostacyclin synthase expression is reduced in PAH
Data are mean ±standard error. Statistical significance assessed using unpaired two-tailed t-testIPAH, idiopathic PAH; PAH, pulmonary arterial hypertension; PGI2, prostacyclin
Adapted from Tuder et al. Am J Respir Crit Care Med. 1999;159:1925-1932
*p<0.05 vs control2
Loss of IP receptor function may depress analogue efficacy in PAH
Data are mean ±standard error. Statistical significance assessed using Student’s t-testcAMP, cyclic adenosine monophosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, prostacyclin receptor; IPAH, idiopathic PAH; PAH, pulmonary arterial hypertension; SMC, smooth muscle cell; SPH, secondary pulmonary hypertension
Adapted from 1. Lai et al. Am J Respir Crit Care Med. 2008;178:188-96; 2. Murray F. Am J Physiol Lung Cell Mol Physiol. 2007;292:L294-L303
Whole lung1
0.6
0.4
0.3
0.2
0.1
0.0
Donor IPAH SMC
IP/G
AP
DH
rati
o p<0.01
n=3 n=3 n=3
n=4
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0
Log [Beraprost] (M)cA
MP
(pm
ol/m
g p
rote
in)
0
50
100
150
200
250
Control
SPH
* *
IPAH
Role of PPAR in the prostacyclin signalling pathway
• Peroxisome proliferator-activated receptors (PPARs)– Family of nuclear transcription factors
PPAR, PPAR, PPAR/1,2
– Regulate genes involved in cellular proliferation, apoptosis, migration and inflammation1-3
• PPAR expression is frequently decreased in PAH3
– Loss of PPAR gives rise to apoptotic-resistant cells3
• Prostacyclin activates PPAR– IP-receptor dependent1
– cAMP independent1cAMP, cyclic adenosine monophosphate; IP, prostacyclin receptor; PAH, pulmonary arterial hypertension; PPAR, peroxisome proliferator-activated receptor
1. Falcetti et al. Biochem Biophys Res Commun. 2007;360:821-827; 2. Belvisi et al. Chest. 2008;134:152-157; 3. Ameshima et al. Circ Res. 2003;92:1162-1169
Adhesion molecules
• Endothelium• Leukocytes
PPAR
PPARPPARs andPGI2 analogues
Inhibition of cellularproliferation, migration and
apoptosis
PPAR
Proinflammatory mediators
• Macrophage• T-lymphocyte• Dendritic cells
PPAR,
PPAR,
, /
Key functions of PPARs in the lung
ECM, extracellular matrix; PGI2, prostacyclin; PPAR, peroxisome proliferator-activated receptor
Belvisi et al. Chest. 2008;134:152-157; Becker et al. Fundam Clin Pharmacol. 2006;20:429-447
Growth factors• Vascular cells• Fibroblasts
ECM remodelling• Smooth muscle• Fibroblasts
Control
Normal IPAH 2nd PH COPD
PPAR protein expression
PPAR in lung endothelium
Normal
Diseased
PPAR expression is diminishedin lung tissue of patients with PH
• 38 plexiform lesions from 9 patients with severe PH expressed little to no PPAR
2nd PH, secondary pulmonary hypertension; COPD, chronic obstructive pulmonary disease; IPAH, idiopathic pulmonary arterial hypertension; PH, pulmonary hypertension; PPAR, peroxisome proliferator-activated receptor
Ameshima et al. Circ Res. 2003;92:1162-1169
PPAR
PAH cell type Agent Channel Cellular response
HPASM1,2 – ↓ Kv1.5Cell depolarisation
↓ Kv current↓ Apoptosis
RPASM3
Rat lungHuman lung2
Anorexic agent
↓ Kv1.5Cell depolarisationVasoconstriction
↑ Incidence of PAH
Rat lung4 Hypoxia↓ Kv1.5 ↓ Kv2.1
Pulmonary remodelling
↑ PVR
Decreased expression of Kv channels disrupts pulmonary vascular tone
HPASM, human pulmonary arterial smooth muscle; Kv, voltage-gated potassium channel; PAH, pulmonary arterial hypertension; PVR, pulmonary vascular resistance; RPASM, rat pulmonary arterial smooth muscle
1. Yuan et al. Circulation. 1998;98:1400-1406; 2. Moudgil et al. Microcirculation. 2006;13:615-632; 3. Weir et al. Circulation. 1996;94:2216-2220; 4. Michelakis et al. Circulation. 2002;105:244-250
Control chow
Iloprost chow
Iloprost chow delayed
*
14
12
10
8
Tum
ou
r m
ult
iplicit
y
4
2
0
Wild type PPAR OE
6
*
****
**
Mice fed iloprost and PPAR OE transgenic mice develop fewer lung
tumours
Data are mean ±standard error. Statistical significance assessed using Student’s unpaired t-testOE, over-expressing; PPAR, peroxisome proliferator-activated receptor
Adapted from Nemenoff et al. Cancer Prev Res (Phila Pa). 2008;1:349-356
*p<0.05 vs wild-type control**p<0.001 vs wild-type control
Importance of prostacyclin in PAH pathophysiology
AC, adenylyl cyclase; AMP, adenosine monophosphate; cAMP, cyclic adenosine monophosphate; IP; prostaglandin receptor; P, arachidonic acid; PAH, pulmonary arterial hypertension; PDE, phosphodiesterase; PGH2, prostaglandin H2; PGI2, prostacyclin; PPAR, peroxisome proliferator-activated receptor
1. Humbert et al. N Engl J Med. 2004;351:1425-1436; 2. Ghofrani et al. J Am Coll Cardiol. 2004;43:68S-72S; 3. Mitchell et al. Exp Physiol. 2007;93:141-147
VasodilatationAnti-proliferation
Anti-thrombosis
AC, adenylyl cyclase; AMP, adenosine monophosphate; Ca2+, calcium; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; ECE, endothelin converting enzyme; ET-1, endothelin-1; ETA/B, endothelin receptor; ETRA, endothelin receptor antagonists; GC, guanylyl cyclases; GMP, guanosine monophosphate; IP; prostaglandin receptor; IP3, inositol trisphophate; L-Arg, L-Arginine; NO, nitric oxide; P, arachidonic acid; PDE, phosphodiesterase; PDE-5I, phosphodiesterase type 5 inhibitor; PGH 2, prostaglandin H2; PGI2, prostacyclin; PPAR, peroxisome proliferator-activated receptor; Pro-Endo, pro-endothelin1. Humbert et al. N Engl J Med. 2004;351:1425-1436; 2. Ghofrani et al. J Am Coll Cardiol. 2004;43:68S-72S; 3. Mitchell et al. Exp Physiol. 2007;93:141-147
Prostacyclin pathways
Nitric oxide-cGMP pathway
Endothelin pathway
PPAR mediated activity
cAMP mediated activity
cGMP mediated activity
Anti-proliferation
Vasodilatation Anti-thrombosis
• Direct vasodilation of the pulmonary and systemic arterial vascular beds1
• Vasodilatory effects reduce right and left ventricular afterload and increase cardiac output and stroke volume (as demonstrated in animal studies)1
• Inhibits platelet aggregation1
• Inhibits proliferation of human pulmonary artery smooth muscle cells in vitro2
Mechanism of action
Prostacyclin pathway
Pulmonary artery in patient
with PAH3
Arachidonic acid Prostaglandin I2
Prostacyclin (PGI2)
cAMP
Prostacyclin derivatives
+
Vasodilation and anti-proliferation
Smooth muscle cells
cAMP = cyclic adenosine monophosphate
1. Remodulin® (treprostinil sodium) Summary of Product Characteristics, United Therapeutics Europe Ltd. April 2010; 2. Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201; 3. Humbert et al. N Engl J Med. 2004;351:1425-1436
Decay of prostacyclin at 37°C in vitro1
• Unstable at physiological temperatures and pH2
• Light sensitive2
• Hydrolysed to 6-oxo-PGF1α2
• Elimination half-life of approximately 3 minutes2
Epoprostenol
10
20
40
60
80
100
% C
on
trol
20 30
Incubation time (min)
Tyrode’s solution pH 7.7
Washed human platelets(2x108 ml-1)
Instability of epoprostenol
Data are mean ±standard errorPGF, prostaglandin F
1. Adapted from Whittle BJR; 1983. Actions of prostacyclin and thromboxanes: Products of the arachidonic acid cascade. In: Hormones and cell regulation, Volume 7. Eds Dumont, J.E., Nunez, J., Denton, R.M. Elsevier Biomedical Press; Amsterdam, pp 3-23; 2. Flolan® (epoprostenol sodium) Summary of Product Characteristics, GlaxoSmithKline. March 2006
Modifications to the prostacyclin
side chain for increased stability
O
OH OH
HOOC
Prostacyclin
OH
CH3IloprostCH3
Sta
bilit
y
COOHO
Treprostinil
Beraprost
OH
CH3
OH
COOH
OH
O
PGI2 (t½= 2 min)1
OH
COOH
OH
O
Treprostinil (t½= ~240 min)3Beraprost (t½= ~30 min)2
COO*
Na+O
CH3
HO OH
COOH
OHOH
Iloprost (t½= ~30 min)2
CH3
PGI2, prostacyclin; t1/2, half-life
1. Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201; 2. Gomberg-Maitland et al. Eur Respir J. 2008;31:891-901; 3. Remodulin® (treprostinil) US prescribing information; United Therapeutics Corp. January 2010
Prostacyclin analogues:Chemical structures and plasma half-
lives
Ki (nM) values of various PGI2
analogues for human prostanoid receptors1,2
Ligand DP IP FP EP1 EP2 EP3 EP4
Cicaprost1 >1340 17 >1340 >1340 >1340 255 44
Iloprost1 1035 11 619 11 1870 56 284
Beraprost2 16 110
Carbacyclin1 132 275 427 23 942 14 352
PGE21 307 119 9 5 0.3 0.8
Blank means low affinity with Ki >2000 nMRed= Ki in mouse
Ki, inhibition constant
1. Adapted from Abramovitz et al. Biochim Biophys Acta. 2000;1483:285-293; 2. Adapted from Kiriyama et al. Br J Pharmacol. 1997;122:217-224
p<0.05
0
25
50
75
100
Serum +PPAR
antagonist
+TRE
+PPAR
antagonist+
TRE
% C
ell p
rolife
rati
on
Stimulation of PPAR
Inhibition of proliferation
*p<0.001 vs control
0
1
2
3
4
Serum 10-7 10-6 10-5
Treprostinil [M]
Fold
in
cre
ase in
rela
tive
lucif
era
se a
cti
vit
y
+ PPAR
*
**
─ PPAR
Treprostinil activates PPAR and inhibits proliferation
Data are mean ±standard error. Statistical significance assessed using One-way ANOVAPPAR, peroxisome proliferator-activated receptor; TRE, treprostinil
Adapted from Falcetti et al. Biochem Biophys Res Commun. 2007;360:821-827
• ~2.5-fold ↑ in PPAR in treprostinil-stimulated cells
• PPAR inhibition partially reverses anti-proliferative effects of treprostinil
Treprostinil cAMP generation through EP2
but not EP4 receptorsRat alveolar macrophages
p<0.001
Statistical significance assessed using ANOVA followed by Bonferroni correctioncAMP, cyclic adenosine monophosphate; ND, no data; TRE, treprostinil
Adapted from Aronoff et al. J Immunol. 2007;178:1628-1634
Vehicle Treprostinil Treprostinil+
EP2 antagonist
(AH-6809)
Treprostinil+
EP4 antagonist
(AE3-208)
N.D.0
2
4
6
8
10
12
14
16
cA
MP
(pm
ol/m
illio
n c
ells
)
Differential effects on cAMP production and cell proliferation in smooth muscle cells
cAMP generation
Data are mean ±standard error (of 6–12 determinations for first graph). Statistical significance assessed using one- or two-way ANOVAcAMP, cyclic adenosine monophosphate
Adapted from Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201
*p<0.02 vs treprostinil†p<0.01 vs iloprost
*†
n=5–12
TreprostinilBeraprostIloprostCicaprost
cA
MP
(pm
ol/m
g p
rote
in) 400
300
200
100
0
-12 -11 -10 -9 -8 -7 -6 -5
Prostacyclin analogue (log M)
Smooth muscle cell growth
% C
ell g
row
th
100
80
60
20
0
-12 -11 -10 -9 -8 -7 -6 -4
40
-5
Prostacyclin analogue (log M)
Differential effects on cAMP production
Treprostinil
Iloprost
Beraprost
Cicaprost
cA
MP
(pm
ol/m
g p
rote
in)
400
300
200
100
0
-12 -11 -10 -9 -8 -7 -6 -5
Prostacyclin analogue (log M)
Data are mean ±standard error of 6–12 determinationscAMP, cyclic adenosine monophosphate
Adapted from Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201
Differential effects on smooth muscle cell proliferation
Data are mean ±standard error. Statistical significance assessed using one- or two-way ANOVA
Adapted from Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201
Smooth muscle cell growth
% C
ell g
row
th
100
80
60
20
0
-12 -11 -10 -9 -8 -7 -6 -4
40
-5
Prostacyclin analogue (log M)
Treprostinil
Iloprost
Beraprost
Cicaprost
*p<0.02 vs treprostinil†p<0.01 vs iloprost
n=5–12
Prostacyclin inhibits proinflammatory cytokines and chemokines
*p<0.05 vs vehicle-treated cells
0
25
50
75
100
120
150
% o
f veh
icle
IL-12 p70TNF-α IL-1α IL-6 MIP-1α MCP-1
* * * * * * Indomethacin 40 nM
Iloprost 40 nM
Cicaprost 10 nM
Treprostinil 40 nM
Data are mean ±standard deviation of four experiments. Statistical significance assessed using unpaired Student t testcAMP, cyclic adenosine monophosphate; IL, interleukin; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NFB, nuclear factor kappa B; PGI2, prostacyclin; TNF, tumour necrosis factor
Adapted from Zhou et al. J Immunol. 2007;178:702-710
• PGI2 analogues neutralise proinflammatory proteins and promote anti-inflammatory proteins through NFB
• IP receptor dependent involving in part cAMP
Differential effects on IL-10 production
Indo Iloprost Cicaprost Treprostinil
40
0 n
M
0.4
nM
4 n
M
40
0 n
M
40
nM
0.1
nM
1 n
M
10
0 n
M
10
nM
0.4
nM
4 n
M
40
0 n
M
40
nM
% o
f veh
icle
0
100
200
300
400
500
600
p<0.05 compared to vehicle treated
Data are mean ±standard deviationIL, interleukin; Indo, indomethacin
Adapted from Zhou et al. J Immunol. 2007;178:702-710
• Prostacyclin analogues promote anti-inflammatory protein expression
Summary
• Prostacyclins are a heterogeneous class of agents with different half-lives and receptor specificities
• PPAR represents an important intracellular target for prostacyclins
• Non-classical effects suggest a broader clinical application
• Prostacyclin pathway remains a key target to modify PAH disease