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…..