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Dr Pierre BOUTOUYRIE Pr Stéphane LAURENT

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1 Pharmacogénétique : identification des cibles : Artères et maladies monogéniques
Dr Pierre BOUTOUYRIE Pr Stéphane LAURENT Hôpital Européen Georges Pompidou Service de Pharmacologie et INSERM EMI 0107

2 Maladies monogéniques de la paroi artérielle rares
étiologie connue mais pathogenèse mal définie approche mécanique

3 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

4 Description phénotypique précise

5 Description phénotypique précise
Comparaison aux autres pathologies Hypothèse sur la pathogenèse des complications

6 Description phénotypique précise
Comparaison aux autres pathologies Hypothèse sur la pathogenèse des complications Attitude thérapeutique Essai clinique

7 ? Monogenic Polygenic k.o. mice Marfan Hypertension Williams
Ehlers-Danlos ... k.o. mice Monogenic Hypertension End stage renal disease Aortic aneurism Aging SHR SHR-SP Polygenic ? Fibromuscular dysplasia Spontaneous dissection of Cervical arteries

8 Phénotype artériel Epaisseur Rigidité

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

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

11 Organisation 3D des artères élastiques

12 Dingemans KP et al. Anat Rec 2000
Ultrastructural study of human aorta Dingemans KP et al. Anat Rec 2000

13 Méthodes d’investigation non invasives Epaisseur Rigidité
Phénotype artériel Méthodes d’investigation non invasives Epaisseur Rigidité

14 Peripheral Resistance Peripheral Resistance
ARTERIAL COMPLIANCE Arterial Compliance Arterial Compliance Left ventricule Peripheral Resistance Left ventricule Peripheral Resistance SYSTOLE DIASTOLE

15 Carotid-femoral pulse wave velocity
PWV can be assimilated to arterial stiffness L Dt Triggered on EKG Feasible and validated With Walltrack system

16 Les systèmes d’échotracking sont 3 à 10 fois plus précis que l’imagerie bi-dimensionnelle pour mesurer l’EIM Signal averaging RF lines IMT 2 D TM RF Signal Spatial resolution µm 20-40 µm

17 Hypertension 2001, Hypertension 2002, Stroke 2003
Aortic stiffness is an independent predictor for cardiovascular events in essential hypertensives Adjusted* RR (for 4 m/s PWV) Total mortality P<0.01 CV mortality P<0.01 P<0.01 Coronary events P=0.02 Stroke * adjusted for age, previous CV disease, and diabetes; Multivariate analysis. Hypertension 2001, Hypertension 2002, Stroke 2003

18 Association between aortic stiffness and primary CHD in hypertensive patients
Boutouyrie P . et al. Hypertension 2002 1045 hypertensives, mean delay to 1rst CHD event : 6.5 yrs Adjusted relative risk of CHD Whole population PWV <10.0 m/s m/s >12.3 1.00 1.63 [ ] 2.66 [ ] * 1st and 2nd tertiles of Framingham risk score : i.e. absolute risk < 12% at 10 yrs

19 Association between aortic stiffness and primary CHD in hypertensive patients considered as at low risk * Boutouyrie P . et al. Hypertension 2002 1045 hypertensives, mean delay to 1rst CHD event : 6.5 yrs Adjusted relative risk of CHD Whole population Low risk* patients 1.00 2.43 [ ] 5.90 [ ] PWV <10.0 m/s m/s >12.3 1.00 1.63 [ ] 2.66 [ ] * 1st and 2nd tertiles of Framingham risk score : i.e. absolute risk < 12% at 10 yrs

20 ? Genetic factors  arterial stiffness structure of the arterial wall
age, BP, smoking, cholesterol, glycemia, ... structure of the arterial wall  arterial stiffness

21 Genetics of arterial stiffness
Heritability of arterial stiffness Offsprings of patients with CVD Family and twin studies Structural aspects 2.1. Candidate genes Genetic polymorphisms Monogenic diseases Animal models : k.o. mice 2.2. Gene expression profile

22 Heritability of carotid artery stiffness
Bogalusa Heart Study : Peterson elastic modulus in yrs adolescents: * * Parental history of MI, D, or HT: Ep kPa Riley et al. Arteriosclerosis 1986;6:

23 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 Methods Maximum likelihood variance decomposition method (SOLAR) Covariates : Age, sex, diabetes, smoking, cholesterol, hypertension, BSA (51 % of SI variance, and 38% of AI variance) Stiffness index Ln (PP) x Ad / (As-Ad) with PP = local PP (transfer function) North KE et al. ATVB 2002

24 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 % phenotypic variance Methods Maximum likelihood variance decomposition method (SOLAR) Covariates : Age, sex, diabetes, smoking, cholesterol, hypertension, BSA (51 % of SI variance, and 38% of AI variance) Stiffness index Ln (PP) x Ad / (As-Ad) with PP = local PP (transfer function) Classical covariates Heritability North KE et al. ATVB 2002

25 Heritability of aortic stiffness in twin pairs
Methods Co-twin case-control analysis in 213 MZ and 556 DZ female twin pairs AI = augmentation index (Sphygmocor + transfer function) Quantitative genetic modeling technique : comparison of the variance-covariance matrices in MZ and DZ twin pairs Sneider H et al. Hypertension 2000

26 Heritability of aortic stiffness in twin pairs
% variance explained by heritability Methods Co-twin case-control analysis in 213 MZ and 556 DZ female twin pairs AI = augmentation index (Sphygmocor + transfer function) Quantitative genetic modeling technique : comparison of the variance-covariance matrices in MZ and DZ twin pairs Sneider H et al. Hypertension 2000

27 Heritability of aortic stiffness in twin pairs
% variance explained by heritability Methods Co-twin case-control analysis in 213 MZ and 556 DZ female twin pairs AI = augmentation index (Sphygmocor + transfer function) Quantitative genetic modeling technique : comparison of the variance-covariance matrices in MZ and DZ twin pairs NOT explained by MAP, HR and height Sneider H et al. Hypertension 2000

28 Genetic factors  arterial stiffness
age, BP, smoking, cholesterol, glycemia, ... structure of the arterial wall : which changes ?  arterial stiffness

29 Genetics of arterial stiffness
Heritability of arterial stiffness Offsprings of patients with CVD Twin studies Structural aspects 2.1. Candidate genes Genetic polymorphisms and animal models Monogenic diseases and k.o. mice Animal models and k.o. mice 2.2. Gene expression profile

30 Genetics of arterial stiffness
Heritability of arterial stiffness Offsprings of patients with CVD Twin studies Structural aspects 2.1. Candidate genes Genetic polymorphisms and animal models Monogenic diseases and k.o. mice Animal models and k.o. mice 2.2. Gene expression profile

31 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

32 Genotype of Ang II AT1-Receptor (A1166C) and arterial stiffness in essential hypertensive patients
AA AC CC _________________________ n Age SBP PWV * * ______________________________ Bénétos et al. Circulation 1996

33 Functional correlate of A1166C polymorphism ?
Genotype of Ang II AT1-Receptor (A1166C) and arterial stiffness in essential hypertensive patients AA AC CC _________________________ n Age SBP PWV * * ______________________________ Bénétos et al. Circulation 1996 Functional correlate of A1166C polymorphism ?

34 TT patients have higher plasma Ang II levels (11 to 39 %)
Angiotensinogen M235T polymorphism and carotid distensibility in essential hypertensives TT patients have higher plasma Ang II levels (11 to 39 %)

35 Angiotensinogen M235T polymorphism and carotid distensibility in essential hypertensives
TT patients have higher plasma Ang II levels (11 to 39 %) * Distensibility (kPa ) n = 61 53 23 Bozec E et al. J Hypertens 2004

36 Angiotensinogen M235T polymorphism and carotid distensibility in essential hypertensives
TT patients have higher plasma Ang II levels (11 to 39 %) Multivariate analysis MAP p< % age p< % M235T p< % BSA NS total r 2 = 0.39 * Distensibility (kPa ) n = 61 53 23 Bozec E et al. J Hypertens 2004

37 Angiotensinogen M235T polymorphism and carotid distensibility in essential hypertensives … and mice
TT patients have higher plasma Ang II levels (11 to 39 %) Mutant mice with 3 copies of Agt gene * Agt 1/2 mice have + 24% plasma Ang II than Agt 1/1 Distensibility (kPa ) n = 61 53 23 Bozec E et al. J Hypertens 2004

38 Angiotensinogen M235T polymorphism and carotid distensibility in essential hypertensives … and mice
TT patients have higher plasma Ang II levels (11 to 39 %) NS * Distensibility (kPa ) n = 61 53 23 Bozec E et al. J Hypertens 2004

39 Angiotensinogen M235T polymorphism and carotid distensibility in essential hypertensives … and mice
TT patients have higher plasma Ang II levels (11 to 39 %) NS Adaptive mechanism: Down regulation of AT1 receptors Bozec E et al. J Hypertens 2004

40 Dingemans KP et al. Anat Rec 2000
Fibrillin-1 and load bearing Oxytalan fibers (FBN-1 + coll VI) Dingemans KP et al. Anat Rec 2000

41 Fibrillin-1 genotype and aortic stiffness
* Methods 145 CAD patients Aortic impedance - Aortic flow velocimetry - Aortic pressure : carotid tonometry + calibration on MBP/DBP 3 genotypes (2-2,2-4,2-3) account for 86% of the population * Medley TL et al. Circulation 2002

42 Genetic polymorphisms and arterial stiffness
Genotype Phenotype Correlation Reference (% explained var.) Renin-angiotensin aldosterone system AT1R 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 system Elastin Ser422Gly carotid dist. yes Hanon, 2001 Fibrillin-1 aortic impedance yes Madley, 2002 MMP-3 aortic impedance yes Madley, 2003 Endothelin system ETA-R -231A/G PWV C-F women (19%) Lajemi, 2001

43 Genetic polymorphisms and arterial stiffness
Genotype Phenotype Correlation Reference (% explained var.) Renin-angiotensin aldosterone system AT1R 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 system Elastin Ser422Gly carotid dist. yes Hanon, 2001 Fibrillin-1 aortic impedance yes Madley, 2002 MMP-3 aortic impedance yes Madley, 2003 Endothelin system ETA-R -231A/G PWV C-F women (19%) Lajemi, 2001 which percentage of variance ?

44 Genetics of arterial stiffness
Heritability of arterial stiffness Offsprings of patients with CVD Twin studies Structural aspects 2.1. Candidate genes Genetic polymorphisms and animal models Monogenic diseases and k.o. mice Animal models : k.o. mice 2.2. Gene expression profile

45 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

46 ED IV SVAS Marfan PXE Alagille Fabry

47 Arterial stiffness in monogenic diseases of the arterial wall
Art. Stiffness References Lacolley P et al. Clinical Science 2002 Jondeau G et al. Circulation 1999 Boutouyrie P et al Hypertension 2001 Germain D et al. ATVB 2003 Boutouyrie P et al. Hypertension 2000 Circulation 2004 J Med Genet 2001 Acta Pediatrica 2003 # #

48 Elastin fiber structure and assembly
other proteins tropoelastin monomers fibrillin MAGP elastin microfibrils alignment of elastin polypeptides into fibers structures stabilisation by cross-links (desmosines, isodesmosines) insoluble elastic fibers

49 Marfan syndrome Aorta 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 Bunton et al. Circ Res 2001

50 Reduced arterial distensibility is confined to the aorta
in Marfan syndrome NS Marfan Distensibility (kPa ) Matched controls * NS NS Jondeau G et al. Circulation 1999

51 Echographic measurements of aortic size
Marfan syndrome Echographic measurements of aortic size Diameter (mm) Marfan Co n= n=20 1.Annulus 2.Sinuses Valsalva * 3.Supra-aortic ridge 4.Aortic arch 4 3 1 2 LV LA

52 * Local PP is a major determinant
of ascending aorta dilatation in Marfan syndrome * Diameter (mm)

53 * Local PP is a major determinant
of ascending aorta dilatation in Marfan syndrome Jondeau G et al. Circulation 1999;99: Variable R R2 incr. P yes Car PP <0.001 yes age <0.001 yes BSA <0.001 no MBP * Diameter (mm)

54 Pathogenesis of aortic enlargement in Marfan
 Ao stiffness  Aortic PP  cyclic stress abnormal fibrillin

55 Pathogenesis of aortic enlargement in Marfan
 Ao stiffness  Aortic PP  cyclic stress abnormal fibrillin fatigue of biomaterials fragmentation, disruption of elastic fibers aorta enlargement

56 Pathogenesis of aortic enlargement in Marfan
 Ao stiffness  Aortic PP  cyclic stress abnormal fibrillin fatigue of biomaterials MMP-2, MMP-9 fragmentation, disruption of elastic fibers aorta enlargement

57 Animal model for MFS : fibrillin-1 deficient mouse
Wild type Fbn-1 deficient Disrupted elastic fibers Lack of VSMC-elastin attachments Smoothness of elastic fibers-VSMC interface Bunton et al, Circ Res 2001;88:37

58 Animal model for MFS : fibrillin-1 deficient mouse
Wild type Fbn-1 deficient Disrupted elastic fibers Lack of VSMC-elastin attachments Smoothness of elastic fibers-VSMC interface Bunton et al, Circ Res 2001;88:37

59 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

60 Prognosis in ED IV syndrome
Median age of death # years 103 deaths caused by arterial rupture, 78 thoracic or abdominal vessels 9 CNS hemorrhage 16 unspecified. Pepin et al. NEJM 1999

61 BP and carotid parameters in EDS patients
Mean ± SD ; * P<0.02 ; ** P<0.001 Boutouyrie et al. Circulation 2004 IMT : intima-media thickness

62 Artères dans le syndrome d’Ehlers-Danlos
Sujet sain EDS h, épaisseur intima-media

63 Régulation de l’épaisseur intima-media
Déterminants de la contrainte circonférentielle ? sq R h circumferential wall stress sq = P. R / h

64 Determinants of carotid wall stress
Wall stress = MBP. D / 2 IMT Boutouyrie et al. Circulation 2004 EDS type IV % change vs controls ** * * P<0.02 ; ** P<0.001

65 Au cours du syndrome d’Ehlers-Danlos vasculaire
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

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

67 Pourquoi le céliprolol?
Onde observée Moreover, tonometer allows the precise acquisition of pressure waveform, and thus permits the assessment of wave reflections

68 = Onde incidente Onde observée
Moreover, tonometer allows the precise acquisition of pressure waveform, and thus permits the assessment of wave reflections

69 + = Onde incidente Onde réfléchie Onde observée Onde réfléchie :
Moreover, tonometer allows the precise acquisition of pressure waveform, and thus permits the assessment of wave reflections Onde réfléchie : - rigidité aortique - sites de réflexion

70 + = Onde incidente Onde réfléchie Onde observée PAM
Moreover, tonometer allows the precise acquisition of pressure waveform, and thus permits the assessment of wave reflections PAM aténolol céliprolol

71 + = Onde incidente Onde réfléchie Onde observée b1 antag. b1 antag.
b2 ago. b1 antag. b2 0 Moreover, tonometer allows the precise acquisition of pressure waveform, and thus permits the assessment of wave reflections PAM aténolol céliprolol

72 Genetics of arterial stiffness
Heritability of arterial stiffness Offsprings of patients with CVD Twin studies Structural aspects 2.1. Candidate genes Genetic polymorphisms Monogenic diseases Animal models and k.o. mice 2.2. Gene expression profile

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

74 Target proteins for arterial stiffness
Extracell. matrix Basal Mb Focal contacts Cytoskeleton • collagen I, III • collagen IV • integrins • desmin • elastin • laminin • paxillin • a-actin • fibrillin • nidogen • tensin • vinculin • fibronectin • entactin • talin • SM-1 MHC, SM-2 proteoglycans arterial stiffness = elastic modulus + 3D organization of each compt cell-matrix interactions (elastic lamellae) organization of elastin and collagenous networks smooth muscle tone growth, differentiation

75 Cell-matrix communication/signalisation
Target proteins for arterial stiffness Extracell. matrix Basal Mb Focal contacts Cytoskeleton • collagen I, III • collagen IV • integrins • desmin • elastin • laminin • paxillin • a-actin • fibrillin • nidogen • tensin • vinculin • fibronectin • entactin • talin • SM-1 MHC, SM-2 proteoglycans arterial stiffness = elastic modulus + 3D organization of each compt cell-matrix interactions (elastic lamellae) organization of elastin and collagenous networks smooth muscle tone growth, differentiation Cell-matrix communication/signalisation Structure/motility

76 Gene expression profile of the human aorta 27 patients with CAD
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 Adjusted PWV (m/s) 30 20 10 Distensible Medium Stiff 9 samples met quality and quantity criteria after RNA extraction n = n = n = 3

77 Cell-matrix communication
Gene expression profile of the human aorta : DNA microarray 151 genes differentially expressed between stiff and distensible aortas Among genes, Affymetrix ® DNA chips Cell-matrix communication and signalisation 21% Cell division 3% unclassified 25% Cell structure and motility 16% cell/organism Metabolism gene/protein expression defense 11% 9% 13% Durier S et al. Circulation 2003

78 Cell-matrix communication
Gene expression profile of the human aorta : DNA microarray 151 genes differentially expressed between stiff and distensible aortas Among genes, Affymetrix ® DNA chips Cell-matrix communication and signalisation 21% Cell division Structure and 3D organisation 37 % 3% unclassified 25% Cell structure and motility 16% cell/organism Metabolism gene/protein expression defense 11% 9% 13% Durier S et al. Circulation 2003

79 3 selected sequences : proteoglycans
Gene expression level 16000 * Distensible aorta : low PWV Stiff aorta : high PWV 12000 8000 * 4000 * decorin dermatopontin osteomodulin

80 Genetics of arterial stiffness
Heritability of arterial stiffness Offsprings of patients with CVD Twin studies Structural aspects 2.1. Candidate genes Genetic polymorphisms Monogenic diseases Animal models and k.o. mice 2.2. Gene expression profile  candidate genes

81 Polygenic-multifactorial
Human disease Monogenic Polygenic-multifactorial Marfan Williams Ehlers-Danlos PXE Fabry Allagile... Hypertension, diabetes End stage renal disease Aortic aneurysm Aging Menopause Animal models Genetically selected animals SHR SHR-SP... Transgenic animals Environmental factors modulating gene expression

82 Polygenic-multifactorial
Human disease Monogenic Polygenic-multifactorial Marfan Williams Ehlers-Danlos PXE Fabry Allagile... Hypertension, diabetes End stage renal disease Aortic aneurysm Aging Menopause Identification of targets for drug development Animal models Genetically selected animals SHR SHR-SP... Transgenic animals Environmental factors modulating gene expression

83 Conclusions Part of arterial stiffness is genetically determined,
Target proteins include not only elastic components, but mainly proteins involved in the 3-D organization of the arterial wall. These proteins are encoded by genes involved in cell-matrix communication and structure/motility The structural control of arterial stiffness is likely a dynamic process, continuously modulated. Many targets are being identified for future intervention on arterial stiffness

84 Cardiology, Hôpital A. Paré, Paris
G. Jondeau Pharmacology Inserm E 107, HEGP, Paris Hypertension, HEGP P-F Plouin P. Corvol Y. Bezie S. Boumaza P. Boutouyrie E. Bozec P. Challande S. Durier C. Fassot P. Lacolley B. Laloux S. Laurent Genetics and molecular biology Inserm U36, HEGP D. Germain X. Jeunemaître Anatomopathology Inserm U430, HEGP P. Bruneval Inserm U367, Paris F Alhenc-Gelas Inserm U441, Pessac J-M Daniel-Lamazière

85


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