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 - CNRS UNIVERSIDAD DE CHILE UNIVERSITÉ CLAUDE BERNARD Student: Emilio Herrera V Mentors: Dr Catherine Godinot Dr Jean Marc Pequignot Dr Anibal Llanos HAPPOM - ALFA MEETING 2-4 DECEMBER, 2005
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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.
- 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
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)