Pharmacogénétique : identification des cibles : Artères et maladies monogéniques Dr Pierre BOUTOUYRIE Pr Stéphane LAURENT Hôpital Européen Georges Pompidou

Pharmacogénétique : identification des cibles : Artères et maladies monogéniques

Dr Pierre BOUTOUYRIE

Pr Stéphane LAURENTHôpital Européen Georges Pompidou

Service de Pharmacologie et INSERM EMI 0107

Maladies monogéniques de la paroi artérielle

rares

étiologie connue mais pathogenèse mal définie

approche mécanique

Maladies monogéniques de la paroi artérielle

rares

étiologie connue mais pathogenèse mal définie

approche mécanique

hypothèse pharmacodynamique pour une

meilleure prise en charge thérapeutique

modèles d’étude des composants de la paroi

artérielle

Description phénotypique précise


Comparaison aux autres pathologies

Hypothèse sur la pathogenèse des complications


Comparaison aux autres pathologies

Hypothèse sur la pathogenèse des complications

Attitude thérapeutique

Essai clinique

MarfanWilliamsEhlers-Danlos...

k.o. mice

Monogenic

Hypertension

End stage renal disease

Aortic aneurism

Aging

SHRSHR-SP

Polygenic

?

Fibromuscular dysplasiaSpontaneous dissection of

Cervical arteries…

Phénotype artériel

Epaisseur

Rigidité

Organisation 3D des artères élastiques- cylindres de lames élastiques et de cellules musculaires lisses artérielles - organisation des CML en spirales

Organisation 3D des artères élastiques- cylindres de lames élastiques et de cellules musculaires lisses artérielles - organisation des CML en spirales

Organisation 3D des artères élastiques

Ultrastructural study of human aortaDingemans KP et al. Anat Rec 2000

Phénotype artériel

Méthodes d’investigation non invasives

EpaisseurRigidité

SYSTOLE DIASTOLE

Arterial Compliance

Arterial Compliance

Peripheral Resistance Peripheral

Resistance

Left ventricul

e Left ventricul

e

ARTERIAL COMPLIANCE

Carotid-femoral pulse wave velocity

L

DISTdV

VdP

t

LPWV

1.

.

PWV can be assimilatedto arterial stiffness

t

Triggered on EKGFeasible and validated With Walltrack system

2 D TM RF Signal

IMT

Signal averaging 10-10 000 RF lines

Spatialresolution 200-400 µm 20-40 µm

Les systèmes d’échotracking sont 3 à 10 fois plus précis que l’imagerie bi-dimensionnelle pour mesurer l’EIM

Aortic stiffness is an independent predictor for cardiovascular events in essential hypertensives

Adjusted* RR 1 1.5 2.0 (for 4 m/s PWV)

* adjusted for age, previous CV disease, and diabetes; Multivariate analysis.

Hypertension 2001, Hypertension 2002, Stroke 2003

Total mortality

CV mortality

Coronary events

Stroke

P<0.01

P=0.02

P<0.01

P<0.01

Association between aortic stiffness and primary CHD in hypertensive patients

PWV

<10.0 m/s

10.0 -12.3 m/s

>12.3

Adjusted relative risk of CHD

Boutouyrie P . et al. Hypertension 2002

* 1st and 2nd tertiles of Framingham risk score : i.e. absolute risk < 12% at 10 yrs

1045 hypertensives, mean delay to 1rst CHD event : 6.5 yrs

1.00

1.63 [1.13-2.36]

2.66 [1.27-5.56]

Whole population

Association between aortic stiffness and primary CHD in hypertensive patients considered as at low risk *

PWV

<10.0 m/s

10.0 -12.3 m/s

>12.3

1.00

2.43 [1.49-3.96]

5.90 [2.22-15.68]

Adjusted relative risk of CHD

Boutouyrie P . et al. Hypertension 2002

* 1st and 2nd tertiles of Framingham risk score : i.e. absolute risk < 12% at 10 yrs

1045 hypertensives, mean delay to 1rst CHD event : 6.5 yrs

Low risk* patients

1.00

1.63 [1.13-2.36]

2.66 [1.27-5.56]

Whole population

arterial stiffness

Genetic factors

structure of the arterial wall

age, BP, smoking, cholesterol, glycemia, ...?

Genetics of arterial stiffness

1. Heritability of arterial stiffness

1. Offsprings of patients with CVD 2. Family and twin studies

2. Structural aspects

2.1. Candidate genes• Genetic polymorphisms • Monogenic diseases• Animal models : k.o. mice

2.2. Gene expression profile

Bogalusa Heart Study :Peterson elastic modulus in 10-17 yrs adolescents:

56

58

60

62

64

66

68

70

MI Diabetes Hypertension

yes

no

Parental history of MI, D, or HT:

Riley et al. Arteriosclerosis 1986;6:378-386

Ep

kP

a

* *

Heritability of carotid artery stiffness

Heritability of carotid artery stiffness Strong Heart Study : 13 American Indian communities

32 extended families, 1011 individuals

Methods1. Maximum likelihood variance

decomposition method (SOLAR)

2. Covariates : Age, sex, diabetes, smoking, cholesterol, hypertension, BSA (51 % of SI variance, and 38% of AI variance)

3. Stiffness index

Ln (PP) x Ad / (As-Ad)with PP = local PP (transfer function) North KE et al. ATVB 2002

Heritability : proportion of residual phenotypic variance due to the additive effects of genes, after accounting for covariates

Heritability of carotid artery stiffness Strong Heart Study : 13 American Indian communities

32 extended families, 1011 individuals

Heritability : proportion of residual phenotypic variance due to the additive effects of genes, after accounting for covariates

North KE et al. ATVB 2002

0

10

20

30

40

50

60

stiffness index AI

Classical covariates

Heritability

% phenotypic varianceMethods1. Maximum likelihood variance

decomposition method (SOLAR)

2. Covariates : Age, sex, diabetes, smoking, cholesterol, hypertension, BSA (51 % of SI variance, and 38% of AI variance)

3. Stiffness index

Ln (PP) x Ad / (As-Ad)with PP = local PP (transfer function)

Heritability of aortic stiffness in twin pairs

Methods

1. Co-twin case-control analysis in 213 MZ and 556 DZ female twin pairs

2. AI = augmentation index (Sphygmocor + transfer function)

3. Quantitative genetic modeling technique : comparison of the variance-covariance matrices in MZ and DZ twin pairs

Sneider H et al. Hypertension 2000

Methods





% variance explained by heritability

0

10

20

30

40

50

60

A Index MAP HR height


Methods





% variance explained by heritability

0

10

20

30

40

50

60

A Index MAP HR height


NOT explained by MAP, HR and height

arterial stiffness

Genetic factors

structure of the arterial wall : which changes ?

age, BP, smoking, cholesterol, glycemia, ...



1. Offsprings of patients with CVD 2. Twin studies


2.1. Candidate genes• Genetic polymorphisms and animal models • Monogenic diseases and k.o. mice• Animal models and k.o. mice






2.1. Candidate genes• Genetic polymorphisms and animal models • Monogenic diseases and k.o. mice• Animal models and k.o. mice


Genetic polymorphisms of renin-angiotensin system

• RAS and arterial wall fibrosis

• Reno-vascular hypertension in rats : pressure-independent increase in arterial stiffness

• ACEI pressure-independent reduction in carotid artery stiffness in SHR

Genotype of Ang II AT1-Receptor (A1166C) and arterial stiffness in essential hypertensive patients

AA AC CC

_________________________

n 186 109 16

Age 48 50 52

SBP 158 157 158

PWV 11.6 13.2* 15.3*

______________________________

Bénétos et al. Circulation 1996

AA AC CC

_________________________

n 186 109 16

Age 48 50 52

SBP 158 157 158

PWV 11.6 13.2* 15.3*

______________________________

Bénétos et al. Circulation 1996

Functional correlate of A1166C polymorphism ?

Genotype of Ang II AT1-Receptor (A1166C) and arterial stiffness in essential hypertensive patients

TT patients have higher plasma Ang II levels (11 to 39 %)

Angiotensinogen M235T polymorphism and carotid distensibility in essential hypertensives

n = 61 53 23

Dis

tens

ibili

ty

(kP

a-1.1

0-3)

Bozec E et al. J Hypertens 2004


0

4

8

12

16

MM MT TT

*


Multivariate analysisMAP p<0.001 13 %

age p<0.001 15 %

M235T p<0.001 6 %

BSA NS

total r 2 = 0.39

n = 61 53 23

Dis

tens

ibili

ty

(kP

a-1.1

0-3)



0

4

8

12

16

MM MT TT

*



… and mice


n = 61 53 23

Dis

tens

ibili

ty

(kP

a-1.1

0-3)

0

4

8

12

16

MM MT TT

* Mutant mice with 3 copies of Agt gene

Agt 1/2 mice have + 24% plasma Ang II

than Agt 1/1



… and mice


n = 61 53 23

Dis

tens

ibili

ty

(kP

a-1.1

0-3)

0

4

8

12

16

MM MT TT

*

0

20

40

60

80

Agt 1/1 Agt 1/2

NS



… and mice


0

20

40

60

80

Agt 1/1 Agt 1/2

NS


Adaptive mechanism:Down regulation of AT1 receptors

Fibrillin-1 and load bearing

Dingemans KP et al. Anat Rec 2000

Oxytalan fibers (FBN-1 + coll VI)

Fibrillin-1 genotype and aortic stiffness

Methods

1. 145 CAD patients

2. Aortic impedance

- Aortic flow velocimetry

- Aortic pressure : carotid tonometry + calibration on MBP/DBP

3. 3 genotypes (2-2,2-4,2-3) account for 86% of the population

Medley TL et al. Circulation 2002

*

0

1

2

3

4

Imput impedance Characteristicimpedance

2-2 gen

2-4 gen

2-3 gen

*

Genetic polymorphisms and arterial stiffness

Genotype Phenotype Correlation Reference (% explained var.)

Renin-angiotensin aldosterone systemAT1R A1166C PWVC-F yes Benetos, 1996

Aldost.CYP11B2 PWVC-F yes Pojoga, 1998

ACE I/D carotid compliance yes Balkestein, 2001

AGT M235T carotid distensibiity yes (15%) Bozec, 2004

Elastin systemElastin Ser422Gly carotid dist. yes Hanon, 2001

Fibrillin-1 aortic impedance yes Madley, 2002

MMP-3 aortic impedance yes Madley, 2003

Endothelin systemETA-R -231A/G PWV C-F women (19%) Lajemi, 2001

Genetic polymorphisms and arterial stiffness

Genotype Phenotype Correlation Reference (% explained var.)

Renin-angiotensin aldosterone systemAT1R A1166C PWVC-F yes Benetos, 1996

Aldost.CYP11B2 PWVC-F yes Pojoga, 1998

ACE I/D carotid compliance yes Balkestein, 2001

AGT M235T carotid distensibiity yes (15%) Bozec, 2004

Elastin systemElastin Ser422Gly carotid dist. yes Hanon, 2001

Fibrillin-1 aortic impedance yes Madley, 2002

MMP-3 aortic impedance yes Madley, 2003

Endothelin systemETA-R -231A/G PWV C-F women (19%) Lajemi, 2001

which percentage of variance ?





2.1. Candidate genes• Genetic polymorphisms and animal models • Monogenic diseases and k.o. mice• Animal models : k.o. mice


Monogenic diseases of the arterial wall

models for the study of arterial wall components

major cardiovascular complications

stenosis, ischemia of target organs

dissection, rupture

lack of preventive treatment

poorly defined pathogenesis

ED IV

SVAS

MarfanPXE

Alagille

Fabry

Arterial stiffness in monogenic diseases of the arterial wall

Protein Gene Syndrome

Elastic tissue

Elastin ELN Williams

Fibrillin-1 FBN-1 Marfan

Protein ABCC6 ( ?) ABCC6 Pseudoxanthoma Elasticum (PXE)

Collagen

Procollagen III COL3A1 Ehlers-Danlos Syndrome type IV

Lysosomes -galactosidase GLA Fabry

References

Lacolley P et al.Clinical Science 2002

Jondeau G et al.Circulation 1999

Boutouyrie P et alHypertension 2001Germain D et al.ATVB 2003

Boutouyrie P et al.Hypertension 2000Boutouyrie P et al.Circulation 2004

Germain D et al.J Med Genet 2001Germain D et al.Acta Pediatrica 2003

Art. Stiffness

#

#

Elastin fiber structure and assembly

tropoelastin monomers

elastin microfibrils

fibrillin MAGP other proteins

• alignment of elastin polypeptides into fibers structures• stabilisation by cross-links (desmosines, isodesmosines)

insoluble elastic fibers

Marfan syndrome

Connective tissue disorder

Autosomal dominant trait

Mutations in the genes encoding

fibrillin (FBN1, FBN2)

Abnormalities of elastic fibers

Microdissections,

fibrosis of the media

Major clinical feature :

- dilatation and dissection

of the ascending aorta

Calcifications

Ruptured internal elastic lamina

Aorta

Bunton et al. Circ Res 2001

0

5

10

15

20

25

30

35

40

Abd. Ao Carotid Femoral Radial

Est

Ouest

Reduced arterial distensibility is confined to the aorta in Marfan syndrome

Di s

t ens

ibil i

t y (

k Pa-1

.10-3

)

Jondeau G et al. Circulation 1999

*

NS

NSNS

Marfan

Matched controls

Marfan syndrome Echographic measurements of aortic size

LV LA

1 23

4 Diameter (mm) Marfan Co n=20 n=20

1.Annulus 24.5 22.12.Sinuses Valsalva 43.4 31.7*3.Supra-aortic ridge 32.7 28.54.Aortic arch 22.9 20.8

Local PP is a major determinant of ascending aorta dilatation in Marfan syndrome

05

101520253035404550

As Ao Abd Ao Fem A Car A Rad A

Marfan Controls

Di a

met

er (

mm

)

*

Local PP is a major determinant of ascending aorta dilatation in Marfan syndrome

05

101520253035404550

As Ao Abd Ao Fem A Car A Rad A

Marfan Controls

Di a

met

er (

mm

)

*

Variable R R2 incr. P

yes Car PP 0.35 0.11 <0.001

yes age 0.43 0.14 <0.001

yes BSA 0.83 0.59 <0.001

no MBP 0.50

Jondeau G et al. Circulation 1999;99:2677-2681

cyclic stress

Pathogenesis of aortic enlargement in Marfan

abnormal fibrillin

Aortic PP Ao stiffness

cyclic stress


fatigue ofbiomaterials

fragmentation, disruption of elastic fibers

aorta enlargement

abnormal fibrillin


cyclic stress


fatigue ofbiomaterials

fragmentation, disruption of elastic fibers

aorta enlargement

abnormal fibrillin


MMP-2, MMP-9

Animal model for MFS : fibrillin-1 deficient mouse

Bunton et al, Circ Res 2001;88:37

Wild type Fbn-1 deficient

Disrupted elastic fibers

Lack of VSMC-elastin attachments

Smoothness of elastic fibers-VSMC interface

Animal model for MFS : fibrillin-1 deficient mouse

Bunton et al, Circ Res 2001;88:37

Wild type Fbn-1 deficient

Disrupted elastic fibers

Lack of VSMC-elastin attachments

Smoothness of elastic fibers-VSMC interface

Ehlers-Danlos syndrome type IV Ehlers-Danlos syndrome type IV, (EDS) the vascular type,

results from mutations in the gene for type III procollagen (COL3A1)

Transmission is autosomic dominant

The main cause of premature death is arterial rupture

Prognosis in ED IV syndrome

69%

28%3%

Arterial

Gastrointestinal

OrgansPepin et al. NEJM 1999

103 deaths caused by arterial rupture, 78 thoracic or abdominal vessels

9 CNS hemorrhage16 unspecified.

Median age of death# 40-50 years

EDS IV Controls

n=16 n=16

Age years 30 [14-51] 32 [16-56]

Sex ratio M / F 3/13 3/13

BSA m² 1.5 [1.3-1.7] 1.7 [1.4-2.0] *

Int. diameter mm 5.25 [4.70-6.11] 5.08 [4.41-6.22]

IMT µm 408 [257-513] 598 [416-968] *

Mean BP mmHg 79 [62-91] 81 [70-103]

W all stress kPa 69 [54-111] 48 [28-78] **

Mean ± SD ; * P<0.02 ; ** P<0.001

BP and carotid parameters in EDS patients

Boutouyrie et al. Circulation 2004IMT : intima-media thickness

Sujet sain EDS

Artères dans le syndrome d’Ehlers-Danlos

h, épaisseur intima-media

Régulation de l’épaisseur intima-media

Déterminants de la contrainte circonférentielle ?

R

h

circumferential wall stressP. R / h

-60

-30

0

30

60

Wall stress MBP. D / 2 IMT

Determinants of carotid wall stress

*

EDS type IV% change vs controls

* P<0.02 ; ** P<0.001

**

Boutouyrie et al. Circulation 2004

Conclusion

Au cours du syndrome d’Ehlers-Danlos vasculaire

la paroi artérielle n’est pas la capacité de normaliser la

contrainte pariétale

conduisant à une fragilité excessive de la paroi

artérielle et à sa rupture

Etude BBEST – PHRC 2001Investigateur principal : Dr Pierre Boutouyrie, HEGP, Paris

Objectif principal :

montrer une réduction des évènements CV

chez les patients EDSv

traités par un bêtabloquant avec propriétés

vasodilatatrices, le céliprolol,

par rapport aux patients n’en recevant pas

Onde observée

Pourquoi le céliprolol?

Onde incidente

Onde observée

=

Onde incidente

Onde réfléchie

Onde observée

+

=

Onde réfléchie : - rigidité aortique- sites de réflexion

Onde incidente

Onde réfléchie

Onde observée

+

=

PAM

aténolol céliprolol

Onde incidente

Onde réfléchie

Onde observée

+

=

PAM

aténolol céliprolol

1 antag.2 0

1 antag.2 ago.





2.1. Candidate genes• Genetic polymorphisms • Monogenic diseases• Animal models and k.o. mice


Target proteins for arterial stiffnessExtracell. matrix Basal Mb Focal contacts Cytoskeleton • collagen I, III • collagen IV • integrins • desmin• elastin • laminin • paxillin • -actin • fibrillin • nidogen • tensin • vinculin• fibronectin • entactin... • talin • SM-1 MHC, SM-2• proteoglycans


arterial stiffness = elastic modulus + 3D organizationof each compt

• cell-matrix interactions (elastic lamellae)

• organization of elastin and collagenous networks

• smooth muscle tone

• growth, differentiation


arterial stiffness = elastic modulus + 3D organizationof each compt

• cell-matrix interactions (elastic lamellae)

• organization of elastin and collagenous networks

• smooth muscle tone

• growth, differentiation

Cell-matrix communication/signalisationStructure/motility

10

20

30

Distensible Medium Stiff

Adjusted PWV (m/s)

9 samples met quality and quantity criteria after RNA extraction n = 4 n = 2 n = 3

Gene expression profile of the human aorta 27 patients with CAD. Aortic biopsy during bypass

Adjustment of PWV on age, height, MBP, diabetes, HR.

Durier S et al. Circulation 2003

Gene expression profile of the human aorta : DNA microarray

151 genes differentially expressed between stiff and distensible aortas

unclassified

25%

Metabolism11%

gene/proteinexpression

13%

cell/organism defense

9%

Cell structure and motility

16%

Cell-matrix communication and signalisation

21%

Cell division3%

Among 12 000 genes, Affymetrix ® DNA chips


unclassified

25%

Metabolism11%

gene/proteinexpression

13%

cell/organism defense

9%

Cell structure and motility

16%

Cell division3%

Among 12 000 genes, Affymetrix ® DNA chips


Structure and

3D organisation 37 %

Cell-matrix communication and signalisation

21%

Gene expression profile of the human aorta : DNA microarray

151 genes differentially expressed between stiff and distensible aortas

3 selected sequences : proteoglycans

0

4000

8000

12000

16000

decorin dermatopontin osteomodulin

*

*

*

Gene expression level

Distensible aorta : low PWV

Stiff aorta : high PWV




2.1. Candidate genes• Genetic polymorphisms • Monogenic diseases• Animal models and k.o. mice

2.2. Gene expression profile candidate genes


Hypertension, diabetes


Aortic aneurysm

Aging

Menopause

MarfanWilliamsEhlers-DanlosPXEFabryAllagile...

Monogenic Polygenic-multifactorial

Environmental factors modulating gene

expression

Animal models Genetically selectedanimalsSHRSHR-SP...

Transgenic animals

Human disease

Hypertension, diabetes


Aortic aneurysm

Aging

Menopause

MarfanWilliamsEhlers-DanlosPXEFabryAllagile...

Monogenic Polygenic-multifactorial

Environmental factors modulating gene

expression

Animal models Genetically selectedanimalsSHRSHR-SP...

Transgenic animals

Human disease

Identification of targets for drug development

1. Part of arterial stiffness is genetically determined,

2. Target proteins include not only elastic components, but mainly

proteins involved in the 3-D organization of the arterial wall.

3. These proteins are encoded by genes involved in cell-matrix

communication and structure/motility

4. The structural control of arterial stiffness is likely a dynamic process,

continuously modulated.

5. Many targets are being identified for future intervention on arterial

stiffness

Conclusions

PharmacologyInserm E 107,HEGP, Paris Y. Bezie S. Boumaza P. Boutouyrie E. Bozec P. Challande S. Durier C. Fassot P. Lacolley B. Laloux S. Laurent

Cardiology, Hôpital A. Paré, Paris G. Jondeau

Hypertension, HEGP P-F Plouin P. Corvol

Genetics and molecular biologyInserm U36, HEGP D. Germain X. Jeunemaître

Inserm U367, Paris F Alhenc-Gelas

AnatomopathologyInserm U430, HEGP P. Bruneval

Inserm U441, Pessac J-M Daniel-Lamazière

Documents

Pharmacogénétique : identification des cibles : Artères et maladies monogéniques Dr Pierre BOUTOUYRIE Pr Stéphane LAURENT Hôpital Européen Georges Pompidou