Endothelial Biology of PAH and HHT: A “Genotype”-Phenotype … · 2006-12-14 · Terrence...
Transcript of Endothelial Biology of PAH and HHT: A “Genotype”-Phenotype … · 2006-12-14 · Terrence...
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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
Circulation Research 2005Circulation Research 2005
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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)
Circulation Research 2005Circulation Research 2005
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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
Zhao et al. Circ Res. 2005; 96(4):442Zhao et al. Circ Res. 2005; 96(4):442--5050
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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
Zhao et al. Circ Res. 2005; 96(4):442Zhao et al. Circ Res. 2005; 96(4):442--5050
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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
Zhao et al. Circ Res. 2005; 96(4):442Zhao et al. Circ Res. 2005; 96(4):442--5050
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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
Controls IPAH HHT0.0
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
Controls IPAH HHT0.00
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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EC survival signaling microvascular homeostasis
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
EC apoptosis vascularpruning
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
EC survival signaling microvascular homeostasis
p38MAPK
P
?
BMPR-IBMPR-II
EC apoptosis vascularpruning
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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% 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
Circulation Research 2005Circulation Research 2005
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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
Controls IPAH FPAH CTEPH0
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
Controls IPAH FPAH CTEPH0
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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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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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
Campbell et al. Circulation 2001;104:2242Campbell et al. Circulation 2001;104:2242--22482248
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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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CMTMRCMTMR--labeled labeled SMCsSMCsTransmigration through arteriolar endotheliumTransmigration through arteriolar endothelium
Campbell et al. Circulation 2001;104:2242Campbell et al. Circulation 2001;104:2242--22482248
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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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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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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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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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
Campbell et al. Circulation 2001;104:2242Campbell et al. Circulation 2001;104:2242--22482248
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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The Pulmonary VasculatureThe Pulmonary Vasculature
Courtesy of Courtesy of EvangelosEvangelos MichelakisMichelakis, U of Alberta, U of Alberta
RA
PA
NormalNormal
PAHPAH
HumanHuman Rat MCT modelRat MCT model
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e The Normal Pulmonary VasculatureThe Normal Pulmonary Vasculature
Courtesy of Courtesy of EvangelosEvangelos MichelakisMichelakis, U of Alberta, U of Alberta
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e Pulmonary Pulmonary VascularityVascularity in PAHin PAH
RA
PA
Courtesy of Courtesy of EvangelosEvangelos MichelakisMichelakis, U of Alberta, U of Alberta
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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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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