Erreur innée Macromolécule conjonctive Erreur innée Métabolisme Remodelage tissulaire dégradation synthèse.

Présentation au sujet: "Erreur innée Macromolécule conjonctive Erreur innée Métabolisme Remodelage tissulaire dégradation synthèse."— Transcription de la présentation:

1 Hyperhomocysteinémie Homocystinurie maladie métabolique maladie conjonctive

2 Erreur innée Macromolécule conjonctive Erreur innée Métabolisme Remodelage tissulaire dégradation synthèse

3 Hyperhomocystéinémies
modérée intermédiaire sévère homocystinurie 15 20 30 40 60 80 100 First, a few word on homocysteine which is a thiol containing amino acid formed during the metabolism of methionine and is not constitutive of the structural backbone of proteins. Physiological, homocysteine (it) is found in plasma at concentration lower than 15 µM. Over 15 µM, we speak of mild hyperhomocysteinemia between 15 and 40 µM, high hyperhomocysteinemia between 40 and 80 µM and severe hyperhomocysteinemia often found in homocystinuria wich is the major form of hyperhomocysteinemia. A possible relation between homocysteine and atherosclerosis has emerged with the earliest descriptions in the 60’s of homocystinuria. Indeed, homocystinuric children who were mentally retarded and displayed marphanoïd syndrome with abnormalities of the connective tissues, had severe and premature (atherothrombotic disease) vascular accident that often lead to death before adulthood. Following these observations, many epidemiological and clinical studies were conduced and evidenced that even moderate hyperhomocysteinemia is as a risk factor for atherosclerosis. The physiopathological processes by which homocysteine could promote atherosclerosis are not fully elucidated. Several mechanisms are currently investigated. One of these mechanism is the destruction of the elastic laminae that was first described by Mc Cully in arteries from homocystinuric patients. µmol/L Asymptomatique Facteur de risque vasculaire Symptomatologie sévère Complications

4 Hyperhomocystéinémies
5,10-methylene THF MTHFR Hcy Met SAM SAH cystathionine Cys CBS Ser Hser 5-Me X Me-X MS Gly B6 B12 proteines Etiologies Déficits enzymatiques

5 Hyperhomocystéinémies
proteines Etiologies Ser Met THF Gly B6 SAM X 5,10-methylene THF MS X B12 Déficits enzymatiques Me-X SAH X MTHFR Déficit en CBS Homozygote: très sévère B6-sensible B6-résistant Hétérozygote: modérée, discrète Déficit en MTHFR Homozygote: sévère Hétérozygote: modérée Déficit méthylation B12 intermédiaire Hcy 5-Me THF Ser X CBS B6 cystathionine Hser Cys

6 Hyperhomocystéinémies
Etiologies Déficits vitaminiques Folates, B6, B12, Insuffisance Rénale Chronique Facteurs iatrogènes - Antifoliques Méthotrexate, anticonvulsivants, Sulfasalazine - Anti B6 Isoniazide, Procarbazine, Cyclosérine, Azauridine - Inhibiteur MS protoxyde d’azote - Oestroprogestatifs Excès d’apport en méthionine ?

7 Hyperhomocystéinémies
Modèles animaux Modèles génétiques CBS -/- survie limitée +/- avec régime riche en Met MTHFR +/- Modèles diététiques Régimes riches en Met poulet pendant la croissance rat adulte porc adulte

8 Homocystinurie Symptomatologie Lésions oculaires
Ectopie du cristallin : inférieure Myopie Luxation du cristallin Diagnostic différentiel Maladie de Marfan : ectopie supérieure ou externe

9 Homocystinurie Symptomatologie Lésions squelettiques et musculaires
Aspect marfanoïde Taille élevée Extrémités longues et fines Arachnodactylie Genu valgum Déformation du sternum Ostéoporose Scoliose Hypotonie musculaire

10 Homocystinurie Symptomatologie Lésions vasculaires
Artériosclérose Athérothrombose Atteintes neurologiques Retard mental Autres Lésions cutanées, peau fine, pores élargies Dépigmentation Pneumothorax

11 maladie métabolique maladie conjonctive
Clinique Histologique ? Biochimique ? Remodelages de la paroi artérielle

12 Homocystinurie – Remodelage matriciel
Histological examination showed spectacular alteration of elastic structure in arteries from homocystinuric human « …extracellular matrix deposition, elastica degeneration, smooth muscle cell hyperplasia, and fibrosis. » (Gibson. 1964). « The arteriosclerotic plaques occuring in homocystinuria, whether caused by CBS deficiency, methyl transferase deficiency, or MTHFR deficiency, are typical fibrous plaques, characterized by smooth muscle cell hyperplasia, deposition of extracellular matrix and collagen, and degeneration and destruction of elastic fibers. » (McCully, 1983) … he described very early the alteration of extracellular matrix in arteries from homocystinuric child. The former observations reported fibrosis deposition of collagen, proteoglycanes degradation of the elastic structures Considering the whole matrix is a too large topic for this talk, I would like to foccus mainly on the last point : the degradation of elastic structure which was observed in many different models of hyperhomocysteinemia

13 hyperhomocysteinemie
Hyperhomocystéinémie modérée – Dégradation des structures élastiques artérielles Coronaire Porcs adultes Régime riche en Met Controle hyperhomocysteinemie (x 2) Hyperhomocysteinemie Evènement précoce (4 mois) majeur (~50 %) Here are the results in minipig 1. The degradation induced by a dietary induced hyperhomocysteinemia was characterized by a decrease in elastin content and the opening of gaps,called « fenestrae », in the elastic laminae 2. such a defect was also found again all over the atherosclerotic arteries 3. This event is early 4 This is an important event: from a quantitative point of vue, the decrease in elastin content was up to 50% in coronary arteries Moreover, Atherosclerose Orcein stained 6 µm transverse section

14 Défaut des structures élastiques artérielles
- conséquences tissulaires Fenestration SMCs migration Souris KO elastine (-/-) proliferation et migration SMCs formation neointima stenose (DY. Li, 2002) IEL renal artery Vue ‘‘en face’’, confocal Microscopy Young animal Old This is also importatnt due to the phenotypic and tissue consequences The increased fenestration open pathways through the elastic laminae, such as during aging. This allows SMCs migration Moreover, the lack of structuration af the arterial wall in elastin deficient mice resulted in proliferation and migration of SMCs, then intimal thickening and stenosis And hyperhomocysteinemia is also known to induce SMC hyperplasia and intimal thickening All together this is believed to accelerate the developpement of arterio-atherosclerotic lesions Hyperhomocysteinemie proliferation SMCs Épaississement intimal

15 qui peut accélérer le développement de l’athérosclérose
L’hyperhomocysteinemie induit une dégradation majeure des structures élastiques qui peut accélérer le développement de l’athérosclérose Défaut d’assemblage Dégradation des structures élastiques ? This lead to the next question Is it a defect in the assembly Or a degradation of the elastic structure

16 Structures elastiques
Fibre élastique microfibrilles fibrilline-1, -2 MAGP fibuline-1, -2 Composant amorphe: élastine Remember that the elastic fiber is composed of a core of elastin associated with mifibrilles, mainly fibrilline-1 and some other glycoproteins Several processes occure in the assembly of the elastic fiber and in the association of fibers to form the laminae

17 Hyperhomocysteinemie et élastogenèse
Synthèse et réticulation élastine : observations contradictoires Fibrillines In vivo : embryon de poulet Fib ↓ sans défaut des structures élastiques (Boot. 2004) poulet en croissance : Fib ↓  défaut structures élastiques (Hill. 2002) In vitro : Fib-1 : défaut de repliement domaines EGF (2005) Fib-1 : dysfonction (↓ calcium binding, Hubmacher. 2005) You know the former hypothesis about a defect of the crosslinking of elastin. There is some conflicting observations Anyway may be this occurs during the formation of elastic laminae in growing arteries, but this is not sufficisant in full grown arteries, due to the very low turn over of elastin Fibrillins were shown to be a target for homocysteine. A defect in fibrillin, quantitative defect or misfolding or dysfunction could led to abnormal arterial elastic structure (but in a different way than in Marfan syndrom) Proteoglycans are also involved in elastic fiber formation an abnormal aggregation of proteoglycans (in culture of fibroblasts from homocystinuric pati ents) could disturbe the elastic fiber formation Proteoglycans aggregation  perturbation interactions (McCully. 1993) Fibres élastiques anormales

18 Hyperhomocysteinemie – dégradation des structures élastiques
In vivo, animal adulte Souris ApoE -/- mice + régime riche en Met (Hoffman. 2001) ↑ elastolyse dans aorte Rats + régime riche en Met (Zulli. 1995) disparition des lames élastiques aorte Porcs + régime riche en Met : fenestration des lames élastiques  ↓ contenu en élastine aorte abdominale et coronaire (Charpiot. 1998) On another hand, in full-grown arteries the elastin defect was described as a degradation process. This was observed inanimals fed a met-rich diet: apoE deficeint mice as well as rats as well as pigs Degradation structures élastiques artérielles

19 Proteolytic breakdown
Hyperhomocysteinemia induces a major degradation of the elastic structure in full grown arteries Proteolytic breakdown ?

20 Hyperhomocysteinemie
Processus Elastolytique In vivo 12 Porc + régime riche en Met * 8 Hyperhomocysteinemie modérée 4 mois 4 Aorte abdominale Elastin content (Vv, %) Control Hyperhcy C H Ex vivo * Culture d’explants artériels + Hcy To investigate this point we developped an exvivo model reproducing the alteration observed in vivo in pigs 8 72 h 4 Aorte abdominale 50 µm Hcy 10 µM 100 µM 10 100 µM Hcy

21 Processus Elastolytique
In vivo 200 * Porc + régime riche en Met 100 Hyperhomocysteinemie modérée 4 mois +Phe Aorte abdominale C H C H Specific elastolytic activity (AU / mg protein) * Ex vivo +Phe (phenanthroline) Inhibiteur de metalloproteinases Culture d’explants artériels + Hcy 40 72 h Aorte abdominale +Phe 10 100 10 100 µM Hcy

22 Processus Elastolytique MMP-dépendant
GM6001, inhibiteur large spectre des MMPs previent la degradation structurale induite par Hcy 12 * 8 Elastin content (Vv, %) 4 prevent Hcy 10 µM 100 µM 100 µM + GM6001 10 100 100 + GM6001 µM Hcy

23 Specific activity (AU / mg protein)
Balance MMP/TIMP MMP-9 92 kDa - - 92 kDa Specific activity (AU / mg protein) MMP-2 72 kDa - - 72 kDa 68 kDa - - 68 kDa 10 50 100 µM Hcy 10 50 100 µM Hcy Gelatin-zymographie 10 50 100 µM Hcy Western Blot TIMPs TIMP-1 ↑ MMP-2 et MMP-9 élastolytiques Inhibiteurs tissulaires inchangés 26 kDa - 22 kDa - TIMP-2 10 50 100 µM Hcy Reverse zymographie

24 Hyperhomocysteinemie – dégradation des structures élastiques
In vivo, the degradation of the arterial elastic structures induced by hyperhomocysteinemia is more marked in the inner part of the media. - In vivo in human (Mc Cully. 1983) « ...disruption of the elastic laminae, usually more marked toward the innermost elastic membrane,… » - In vivo in animal (Charpiot. 1998) Haemodynamic strains ? Endothelium ?

25 Hyperhomocysteinemie – dégradation des structures élastiques
Ex vivo, explants arteriels en culture + homocysteine  media The destruction of the elastic structure is more marked in the inner part of the media, even in culture free from haemodynamic strains (the microphotographs represent the inner half of the media) 50 µm Hcy 10 µM 100 µM 6 µm transverse section. (+) catechine- stained elastic structure

26 … mediated by MMPs/TIMPs imbalance
Hyperhomocysteinemia induces a major degradation of the elastic structure in full grown arteries… … mediated by MMPs/TIMPs imbalance in favour of elastinolytic MMP-2 and MMP-9 Localization ?

27 Hyperhomocysteinemie – dégradation des structures élastiques
MMPs – Expression tissulaire ↑ expression MMP-2 et MMP-9 dans la partie interne de la média… MMP -2 MMP-9 media What about MMPs ans TIMPs? The expression of MMP-2 and -9 was increased in the innerpart of the media… Hcy 10 µM 100 µM 10 µM 100 µM Immunohistochimie 6 µm transverse sections

28 Hyperhomocysteinemie – dégradation des structures élastiques
TIMPs – Expression tissulaire expression et distributionTIMP-1 et TIMP-2 inchangées TIMP -1 TIMP-2 …whereas timp expression was nearly unchanged Hcy 10 µM 100 µM 10 µM 100 µM Immunohistochimie 6 µm transverse sections

29 …more marked in the inner part of the media, even ex vivo
Hyperhomocysteinemia induces a major degradation of the elastic structure in full grown arteries… …mediated by MMPs/TIMPs imbalance, in favour of elastinolytic MMP-2 and MMP-9,… …more marked in the inner part of the media, even ex vivo contribution of endothelium ?

30 Potentiel protéolytique des cellules endothéliales
200 MMP-9 * * * Activity (%) 100 92 kDa - (µM) 10 50 100 250 (µM) 10 50 100 250 MMP-2 200 * * * 72 kDa - Activity (%) 100 (µM) 10 50 100 250 (µM) 10 50 100 250 Gelatin Zymography (Lamy. 2004)

31 Potentiel protéolytique des cellules endothéliales
-Casein Zymography - 75 kDa - 27 kDa Western Blot 10 50 100 µM Homocysteine 250 27 kDa - 10 50 100 250 µM Homocysteine (1) Absence d’inhibiteur (2) Inhibiteur des métalloprotéases (1,10 phenanthroline) (3) Inhibiteur des serine protéases (PMSF) (4) Inhibiteur des cystéine protéases (Iodoacetamide) (5) Inhibiteur des aspartate protéases (Pepstatine A). 1 2 3 4 5 27 kDa - Caseinolytic serine protease Human tissue Kallikrein (hK1) For this study we cultured arterial explants with physiopathological concentrations of homocysteine. We choose this ex vivo model because : It maintains cell-matrix interaction contrary to cell culture It make it possible to control homocysteine concentrations used in experiment which is difficult with in vivo model And lastly it’s a model free of haemodynamic strains In practice, we cultured 5-mm segments of pig abdominal aorta in NCTC medium during 48 hours with physiopathological concentrations of homocysteine going from physiological homocysteinemia with 10 µM to severe hyperhomocysteinemia with 100 µM.

32 Dégradation des structures élastiques artérielles
métabolisme Problème métabolique Hyperhomocysteinemie Activation de systèmes protéolytiques - déséquilibre MMPs/TIMPs : MMP-2, MMP-9 - serine protease(s?) activatrice de MMPs remodelage Dégradation des structures élastiques artérielles clinique Artériosclérose, athérosclérose

33 Endothelial cells may participate through
Hyperhomocysteinemie induit une dégradation majeure induces a major elastic structure degradation, facilitating the development of atherosclerosis,… …mediated by MMPs/TIMPs imbalance, in favour of elastinolytic MMP-2 and MMP-9,… …more marked in the inner part of the media. Endothelial cells may participate through - elastinolytic MMPs : MMP-2 and MMP-9 - MMPs activating serine proteases : hK1

34 Hyperhomocystéinémie Remodelage matriciel
Atteinte structures élastiques artère, cristallin (?), peau (?) Elastolyse vs défaut d’élastogenèse dans: - hyperhomocysteinemies acquises - hyperhomocysteinemies innées défaut d’assemblage des fibres élastiques  ↑ sensibilité à la dégradation protéolytique ?  ↑ expression/activité des MMPs ? Et les collagènes ? os, artère, peau (?) First it is necessary to confirm th key role of MMPs in the arterial elastic degradation in vivo, by using MMPs inhibitors in Hhcy animals 2nd point What are the respective relevance of on one hand elastolysis and on the other hand elastogenesis defect The hcy-induecd misfolding of Fib-1 led to an increased sensitivity of Fib to proteolysis Could induce an increased expression or activity of MMPs, as shown in most of the inherited elastopathies.

35 Laboratoire de Biochimie UMRs-608.
Faculté de Pharmacie – Université de la Méditerranée. Marseille. Thierry Augier André Barlatier Alexandrine Bertaud Raymond Calaf Claire Cerini Corinne Chareyre Edouard Lamy Cécile Genovesio Philippe Charpiot Laboratoire de Physiopathologie des Tissus non Minéralisés Faculté de Chirurgie Dentaire– Université René Descartes – Montrouge Gaston Godeau Sylvie Igondjo-Tchen Karim Senni