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RESEARCH PROJECT AT LYON
HYPOXIA INDUCIBLE FACTOR (HIF) AND SOME OF ITS TARGET GENES EXPRESSION IN PULMONARY VASCULAR
BED DURING HYPOXIA
FACULTAD DE MEDICINA CGMC - CNRSUNIVERSIDAD DE CHILE UNIVERSITÉ CLAUDE BERNARD
Student: Emilio Herrera VMentors: Dr Catherine Godinot
Dr Jean Marc PequignotDr Anibal Llanos
HAPPOM - ALFAMEETING 2-4 DECEMBER, 2005
INTRODUCTION
PAP vs High-altitude
Moore et al, 1998 Monge & León-Velarde, 2004
INTRODUCTION
Contractility vs High-altitude
0 20 40 60 80 100 120
0
1
2
3
Tens
ion,
N/m
KCl, mM
HANB
LANB
Pulmonary small arteries
Herrera et al, 2004
p<0.05 * vs LANB
0,0
1,0
2,0
pD2
0,0
1,0
2,0
3,0
Tens
ión
N/m
LANB HANB
*
PulmonaryPulmonary circulationcirculation in in thethe NewbornNewborn
INTRODUCTION
Modified from Abman, 1999
Hem
PPHPT IN THE NEWBORNPPHPT IN THE NEWBORN
INTRODUCTION
Abman, 1999
INTRODUCTION
HIF-1
INTRODUCTION
Hochachka & Rupert, 2003
INTRODUCTION
Modified from Schofield & Ratcliffe, 2004
Remodelación
eNO
S, iNO
S
INTRODUCTION
PULMONARY HYPERTENSIONPULMONARY HYPERTENSION
coco
Modified from Strange et al, 2002
HYPOTHESISHYPOTHESIS
Hypoxia determines a pulmonary hypertension. This Hypoxia determines a pulmonary hypertension. This will be accompanied by HIF activation (expression) will be accompanied by HIF activation (expression) and an increased transcription of its regulated genes and an increased transcription of its regulated genes in the lung.in the lung.
PRIMARY AIMSPRIMARY AIMS
In the laboratory of Professor JeanIn the laboratory of Professor Jean--Marc Marc PequignotPequignot, from , from normoxicnormoxic and hypoxic rats we obtained lung tissue and and hypoxic rats we obtained lung tissue and different brain areas, dissected and rapidly frozen for different brain areas, dissected and rapidly frozen for further mRNA and protein studies. Separate samples were further mRNA and protein studies. Separate samples were collected for mRNA and protein analysis. collected for mRNA and protein analysis.
In the laboratory of Professor Catherine In the laboratory of Professor Catherine GodinotGodinot, mRNAs , mRNAs coding for HIF1a, HIF2a, HIF3 and coding for HIF1a, HIF2a, HIF3 and aHIFaHIF will be analysed by will be analysed by RTRT--PCR. Since, in most cases, the protein of HIF is PCR. Since, in most cases, the protein of HIF is degraded in degraded in normoxianormoxia and stabilized in hypoxia, it will be and stabilized in hypoxia, it will be important to measure some targets of HIF that should be important to measure some targets of HIF that should be induced by hypoxia. Our targets will be VEGF and NOS II induced by hypoxia. Our targets will be VEGF and NOS II ((iNOSiNOS).).
AIMS
SECONDARY AIMSSECONDARY AIMS
Test all the above in tissue (pulmonary) from sheep Test all the above in tissue (pulmonary) from sheep from lowlands and highlands, and submitted to a from lowlands and highlands, and submitted to a acute hypoxic episode.acute hypoxic episode.
AIMS
AIMS
PRIMARY AIMS?
ANIMALS
- 3 groups- 4 normoxic Wistar rats- 4 acute hypoxemic rats (6H at 8%O2)- 4 prolonged hypoxemic rats (12H at 8%O2)
- Eutanasia and Disection- Weight of Heart and lungs- Left lung in RNA later – for RNA analysis- Right lung in liquid nitrogen – for protein analysis
MATERIAL & METHODS
MATERIAL & METHODS
NN22
PROOX-110 OO2 sensor sensor
RatsRats cagecage
Soda limeSoda lime% O2
MATERIAL & METHODS
MOLECULAR BIOLOGY- mRNA expression (RT-PCR)
HIF-1, HIF-2, HIF-3, aHIF, iNOS, VEGF, Flk1 vs 18sDesign primers
- Protein expression (WB)HIF-1, HIF-2, VEGF Antibodies
NormoxiaHypoxia 6hHypoxia 12h
RESULTS
Body weight
0
100
200
300
400
gr B
W
Organ weight
0,0
0,4
0,8
1,2
1,6
Heart Right Left
gr o
rgan
% Body weight
0,0
0,4
0,8
Heart/BW RL/BW LL/BW L/BW
% o
rgan
/BW
% weight Lungs (by order of dissection)
0,0
0,4
0,8
1,2
orga
n/B
W
Lung Weight
1 5 10 15Time (min)
RESULTS
ProteinProtein expressionexpression –– Western BlotWestern Blot
ProteinProtein extractionextraction -- quantificationquantificationMigrationMigration in SDSin SDS--PAGE PAGE –– separated by sizeseparated by sizeTransferedTransfered toto a a nitrocellulosenitrocellulose membranemembraneBlockBlock membranemembranePrimaryPrimary AB AB incubationincubationSecondarySecondary AB AB incubationincubationRevealReveal -- BlotBlot
RESULTS
ProteinProtein expressionexpression –– Western BlotWestern Blot
10.000x10.000x
3.000x3.000x
10.000x10.000x
DilutionDilution
600x600x
250x250x
250x250x
DilutionDilution
ECLECLGoatGoat antiantirabbitrabbit
VEGF (AVEGF (A--20): 20): scsc--152152SANTA CRUZ BIOTECH.SANTA CRUZ BIOTECH.VEGFVEGF
ECLECLRatRat antiantigoatgoat
EPASEPAS--1 (C1 (C--16): 16): scsc--87128712SANTA CRUZ BIOTECH.SANTA CRUZ BIOTECH.HIFHIF--22αα
SuperSupersignalsignal
GoatGoat antiantirabbitrabbit
HIFHIF--11SANTA CRUZ BIOTECH.SANTA CRUZ BIOTECH.HIFHIF--11αα
WB WB reagentreagent
SecondarySecondaryABAB
PrimaryPrimary ABABTargetTargetproteinprotein
ProteinProtein extractionextraction -- quantificationquantificationMigrationMigration in SDSin SDS--PAGE PAGE –– separated by sizeseparated by sizeTransferedTransfered toto a a nitrocellulosenitrocellulose membranemembraneBlockBlock membranemembranePrimaryPrimary AB AB incubationincubationSecondarySecondary AB AB incubationincubationRevealReveal -- BlotBlot
RESULTS
25 50 100 25 50 100 P53 8 μgHip Nor Control (50μg)
72
170130100
55
4033
1 sample N, 1 sample H, SN, 200x aVEGF-A
1 sample N, 1 sample H, 200x and 1000x aVEGF-A
•••
•••
•
•••
•
••
•
25 100 25 100 25 100 25 100 100 25 100 25 μg200x 1000x 1000x
Pellet Supernatant
N H N H N H N H HB
72
170
100
5540
33
72
55
40
33
•••
•
24 •
72
55
40
33
•••
•
24 •
H6 12 6 12 6 6 L NB HB6 HB12
Heated 50μg, 600x Ab
VEGF VEGF expressionexpression
L 5 5 5 2 2 2 C1 C2 L 1 5 2 6 3 7 4 8 5b
L 5 9 6 10 7 8 2b 5b 9b
RESULTS
N H N H N H N H HB
72
55
40
33
•••
•
24 •
72
55
40
33
•••
•
24 •
H6 12 6 12 6 6 L NB HB6 HB12
Heated 50μg, 600x Ab
VEGF VEGF expressionexpression
L 1 5 2 6 3 7 4 8 5b
L 5 9 6 10 7 8 2b 5b 9b
0
0,05
0,1
0,15
Normoxia Hypoxia 6h Hypoxia 12h
VEG
F (r
elat
ive
units
)
RESULTS
N H P53 WTH
72
170
130
100
1 sample N, 1 sample H, SN, 250x aHIF-1α
1 sample N, 1 sample H, heated, 250x aHIF-1α
••
•
•
50 100 100 50 μg
72
170
100
•
•
••
130
HIFHIF--11αα expressionexpression
L 2 5
L 5 5 2 2
0
0,02
0,04
0,06
0,08
Normoxia Hypoxia 6h
HIF-
1 (re
lativ
e un
its)
RESULTS
mRNA expression – RT-PCRmRNAmRNA extractionextractionRTRTPCRPCRMigrationMigration by by sizesize en en AgaroseAgarose gelgel--BETBETUV UV RevealReveal
RESULTS
mRNA expression – RT-PCR
11211240405252ººTAAGTCCCTGCCCTTTGTAAGTCCCTGCCCTTTGTATATGAACCCCATTCGTGATTGAACCCCATTCGTGAT
18S18S
22022040406060ººCTCACTGGGACTGCACCTCACTGGGACTGCACAGAAAGAACAGAGGACCCAGAGACCAGAGGACCCAGAGACAAGCAAGC
iNOSiNOS
24524540405656ººGCAAGAGCAGAGACACGCAAGAGCAGAGACACTCTTCTCTTCCTGGCATCATAAGGCACTGGCATCATAAGGCAAGCGAGCG
FLKFLK--11
18618640406666ººCGCCTTGGCTTGTCACCGCCTTGGCTTGTCACATCTGCATCTGCCCCGGTTTAAATCCTGCCCGGTTTAAATCCTGGACGCGGACGCG
VEGFVEGF--AA
26026040406565ººCAGCCCTAGCTCCCTCCAGCCCTAGCTCCCTCCACAATGTCTGATGTGTCCCATGTCTGATGTGTCCCCAAATGCAAATG
aHIFaHIF
30130140405656ººAGAGAACGGAGTGGTGAGAGAACGGAGTGGTGCTGTCTGTATCAGCCGGAAGAGGAATCAGCCGGAAGAGGACTTTCTTT
HIFHIF--33
36936940406666ººGCGACAATGACAGCTGGCGACAATGACAGCTGACAAGGAGACAAGGAGGTCCCATGAACTTGCTGTCCCATGAACTTGCTGATGTTTGATGTTT
HIFHIF--22αα
15015040406565ººGGACAAGTCACCACAGGGACAAGTCACCACAGGAGAGGAGAAAATCAAGTCGGGAGAAAATCAAGTCGTGTG
HIFHIF--11αα
ProductProductsizesize ((bpbp))
CyclesCyclesAnnealingAnnealingTTºº
PrimersPrimerssequencesequence
TargetTargetmRNAmRNA
RESULTSmRNA expression
18S
L 1 1 2 3 4 6 8 9 10 C1 C2 B
L N N N N N H6 H6 H12 H12 C1 C2 B
L 1 1 2 3 4 6 8 9 10 C1 C2 B
L N N N N H6 H6 H12 H12 C1 C2 B
aHIF
RESULTSmRNA expression
HIF2α HIF1α
2 3 4 6 8 9 10 B L 2 3 4 6 8 9 10 B
L 2 3 4 6 8 9 10 B
N N N H6 H6 H12 H12 B L N N N H6 H6 H12 H12 B
L N N N H6 H6 H12 H12 B
??
HIF318S 18S
18S
RESULTS
iNOS FLK-1
2 3 4 6 9 10 C B L 2 3 4 6 9 10 C B
N N N H6 H12 H12 C B N N N H6 H12 H12 C B
VEGF-A
L 1 2 3 4 6 8 9 10 B L 1 2 3 4 6 8 9 10 C1 C2 B
L N N N N H6 H6 H12 H12 B
mRNA expression
L N N N N H6 H6 H12 H12 C1 C2 B
18S 18S
18S 18S
RESULTSmRNA expression
0
0,5
1
1,5
2
2,5
3
3,5
Normoxia Hypoxia 6h Hypoxia 12h
HIF
-2α
/18S
0
0,5
1
1,5
2
2,5
3
Normoxia Hypoxia 6h Hypoxia 12h
HIF
-3/1
8S
00,20,40,60,8
11,21,41,61,8
2
Normoxia Hypoxia 6h Hypoxia 12h
iNO
S/18
S
0
0,5
1
1,5
2
2,5
Normoxia Hypoxia 6h Hypoxia 12h
FLK
/18S
0
0,4
0,8
1,2
1,6
Normoxia Hypoxia 6h Hypoxia 12h
VEG
F/18
S
HIF
-2H
IF-3
VE
GF
iNO
SFL
K
0
0,5
1
1,5
2
2,5
3
3,5
Normoxia Hypoxia 6h Hypoxia 12h
aHIF
/18S
aHIF
RESULTS
RT-PCR in the sheep?
M L B M L B M L B M L B M L B18S HIF3 HIF2 iNOS VEGF-A
M L B M L B M L B M L BHIF1 HIF2 iNOS VEGF-A
18S, HIF-2, HIF-3, VEGF
HIF-1, iNOS ?
SamplesSamples: Abdominal : Abdominal MuscleMuscle, , LeucocytesLeucocytes
6330 mts6240 mts
FUTURE PLANSFUTURE PLANS
0
5
10
15
20
25
30
35
PAP
(mm
Hg)
H y p o x i a
Caquena´s Bofedal (4600 mts)
AcknowledgementsCatherine GodinotJean-Marc PequignotSilvia AranedaHelene SimmonetElodie de la PlancheEric HervouetJoselyne DemontPascal SanchezLaurent BezinManuela Molina Naira NaouarJoel Potier
and many other lyonesses…..