Effets Vasculaires des Flavonoïdes Alimentaires et leurs Mécanismes

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Abel Potter
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Académie Nationale de Pharmacie Mercredi 27 janvier 21 Faculté de Pharmacie de Paris Effets Vasculaires des Flavonoïdes Alimentaires et leurs Mécanismes

1 Académie Nationale de Pharmacie Mercredi 27 janvier 21 Faculté de Pharmacie de Paris Effets Vasculaires des Flavonoïdes Alimentaires et leurs Mécanismes V.B. Schini-Kerth CNRS UMR 7213 Équipe Pharmacologie et Physiopathologie Cardiovasculaires Université de Strasbourg Faculté de Pharmacie Illkirch, France

2 Polyphenols decrease the Risk of Coronary Diseases Development of Atherosclerotic Lesions Myocardial Infarction Cerebral Ischemia Normal Artery Atherosclerotic Plaque Thrombus Peripheral Vascular Diseases

3 Polyphenols decrease the Risk of Coronary Diseases Development of Atherosclerotic Lesions Myocardial Infarction Cerebral Ischemia Normal Artery Atherosclerotic Plaque Thrombus Peripheral Vascular Diseases Determine the vasoprotective effect of polyphenols - Do they stimulate the formation of vasoprotective endothelial factors? - Do they prevent endothelial dysfunction?

4 Polyphenols and Vasoprotective Endothelial Factors in the Coronary Artery Endothelial cell IK Ca L-Arginine ens N PGI 2 AA Platelets SK Ca K + K + Jonction gap myoendothéliale EDHF N PGI 2 GTP sgc ATP AC Smooth muscle cell Hyperpolarization cgmp Relaxation Migration Proliferation camp

5 Polyphenols and Vasoprotective Endothelial Factors in the Coronary Artery Endothelial cell Polyphenols IK Ca? L-Arginine ens N PGI 2 AA Platelets SK Ca K + K + Jonction gap myoendothéliale EDHF N PGI 2 GTP sgc ATP AC Smooth muscle cell Hyperpolarization cgmp Relaxation Migration Proliferation camp

6 Red Wine Polyphenolic Extract (RWPs) French Red Wine (Corbières A..C.) 1 liter of wine produced 2.9 g of RWPs -Flavanol: - Catechin 8.6 mg/g - Epicatechin 8.7 mg/g -Anthocyanin: - malvidin-3-glucoside 11.7 mg/g - peonidin-3-glucoside.66 mg/g - cyanidin-3-glucoside.6 mg/g -Phenolic acid: - gallic acid 5. mg/g - caffeic acid 2.5 mg/g - caftaric acid 12.5 mg/g

7 Vascular Reactivity Studies Left anterior descending coronary artery Porcine heart Lüscher and Vanhoutte, 199, CRC Press

8 Red Wine Polyphenols cause Endothelium-Dependent Relaxations in the Porcine Coronary Artery (in the presence of indomethacin) RWPs -log (g/l) With endothelium Control N ω -nitro-l-arginine (L-NA) Charybdotoxin + apamin L-NA + Charybdotoxin + apamin Without endothelium 1 g 2.5 min Relaxation (%) U46619 (6 nmol/l) RWPs (µg/ml) Ndiaye et al., BBRC 23;31:

9 Grape Skin Extract and Concord Grape Juice cause Endothelium-Dependent Relaxations (in the presence of indomethacin) -1 Control N ω -nitro-l-arginine (L-NA) Charybdotoxin + apamin L-NA + Charybdotoxin + apamin Relaxation (%) (n = 7) -L-arginine (L-NA, n = 6) otoxin + apamin (n = 6) charybdotoxin + apamin (n = 5) GSE (µg/ml) CGJs (µg/ml) Madeira et al., J. Vasc. Res., 29;46: Anselm et al., Cardiovasc Res 27;73:44-413

10 Blackcurrant Juice causes Endothelium-Dependent Relaxations (in the presence of indomethacin) Blackcurrant juice (µg GAE/mL) Control L-NA (1 µm) Charybdotoxin (1 nm) + Apamin (1 nm) L-NA + Charybdotoxine + Apamin With Endothelium Without Endothelium Relaxation (%) 5 1 U g 2 min Blackcurrant (% v/v)

11 CGJ induces Redox-sensitive Endothelium-Dependent Relaxations in the Porcine Coronary Artery (in the presence of indomethacin, charybdotoxin and apamin) Control MnTMPyP (1 µm) Superoxide dismutase (5 U/mL) Control PEG-catalase (5 U/mL) Catalase (5 U/mL) Concord Grape Juice (mg/l) Concord Grape Juice (mg/l) Anselm et al., Cardiovasc Res 27;73:44-413

12 Grape Skin Extract induces the Endothelial Formation of Reactive xygen Species in the Coronary Artery Fluorescent Probe: DCHF Control Grape Skin Extract (3 µg/ml, 1 min) PEG-SD MnTMPyP PEG-catalase + Endo + Endo + Endo + Endo + Endo - Endo Madeira et al., J. Vasc. Res., 29;46:46-416

13 CGJ-induced N-mediated Relaxations involve Activation of Srckinase and PI3-kinase/Akt in the Porcine Coronary Artery (in the presence of indomethacin, charybdotoxin and apamin) Control PP2 (1 µm) Control Wortmannin (3 nm) Relaxation (% %) 5 Relaxation (%) Concord Grape Juice (mg/l) Concord Grape Juice (mg/l) Anselm et al., Cardiovasc Res 27;73:44-413

14 Concord Grape Juice induces Phosphorylation of Src, Akt and ens in Coronary Artery Endothelial Cells CGJ (44 mg/l) p-src (Tyr418) Time (min) p-akt (Ser473) p-ens (Ser1177) ens Anselm et al., Cardiovasc Res 27;73:44-413

15 Role of Reactive xygen Species in the Concord Grape Juice-induced Phosphorylation of ens p-src (Tyr418) p-akt (Ser473) p-ens (Ser1177) CGJ (44 mg/l, 1 min) ens PEG-Catalase PEG-SD + MnTMPyP + Anselm et al., Cardiovasc Res 27;73:44-413

16 Polyphenols stimulate the Endothelial Formation of N Polyphenols Black tea polyphenols Resveratrol Green tea polyphenols RS [Ca 2+ ] i Src L-Arg PI3K ens P PIP 2 PIP 3 PIP 3 Akt Akt P PIP3 PDK1 ER L-Arg MAPK ens P p38 Cav-1 ERK 1/2 L-Arg ens Endothelial cell N Smooth muscle cell GTP sgc cgmp Relaxation

17 Characterization of the Active Polyphenols in the Red Wine Polyphenolic Extract 1 µg/ml for 1 minutes Control RWPs A B C D E F G H I p-akt (Ser473) Red Wine Phenolic extract p-ens (Ser1177) Sephadex LH2 chromatography Sephadex LH2 chromatography A B C D E F G H I ens p-akt (fold increase e) p-ens (fold increase) Ctrl RWPs A B C D E F G H I Auger et al., 29 Ctrl RWPs A B C D E F G H I

18 Characterization of the Active Polyphenols in the Red Wine Extract 1 µg/ml for 15 minutes Control F SF1 SF2 SF3 SF4 SF5 SF6 SF7 SF8 SF9 SF1 p-akt (Ser473) p-ens (Ser1177) Red Wine Phenolic extract ens A Sephadex LH2 chromatography Sephadex LH2 chromatography B C D E F G H I Preparative HPLC Preparative HPLC p-akt (fold increa ase) SF1 SF2 SF3 SF4 SF5 SF6 SF7 SF8 SF9 SF1 p-ens (fold increase) Ctrl F SF1 SF2 SF3 SF4 SF5 SF6 SF7 SF8 SF9 SF1 Auger et al., 29 Ctrl F SF1 SF2 SF3 SF4 SF5 SF6 SF7 SF8 SF9 SF1

19 Tentative Identification by HPLC-MS² of the Main Polyphenolic Compounds present in Active Sub-Fractions SF1 SF2 [M+H]+ (m/z) Fragments (m/z) Identification , 317 Petunidin-vinylphenol , 427, 31 Procyanidin Trimer , 443, 287, 577 Procyanidin Dimer Gallate , 443, 287, 577 Procyanidin Dimer Gallate , 49, 31 Procyanidin Dimer SF , 49, 31 Procyanidin Dimer SF4 SF5 SF6 SF7 SF8 SF9 SF , 49, 31 Procyanidin Dimer , 49, 31,291 Procyanidin Dimer , 49, 31,291 Procyanidin Dimer , 49, 31,291 Procyanidin Dimer , 49, 31,291 Procyanidin Dimer , 49, 31,291 Procyanidin Dimer , 165, 123 Procyanidin Dimer Malvidin-3--glucoside Malvidin-3--(6"-acetyl)-glucoside Peonidin-3--(6"-acetyl)-glucoside , 357 Malvidin-3--glucoside-8-ethyl-(epi)catechin Malvidin-3--(6"-coumaroyl)-glucoside Malvidin-3--glucoside Petunidin-(coumaroyl)-glucoside Malvidin-3--(6"-coumaroyl)-glucoside Quercetin-3--glucuronide Malvidin-3--glucoside Petunidin-3--(6"-coumaroyl)-glucoside Malvidin-3--(6"-coumaroyl)-glucoside Petunidin-3--(6"-coumaroyl)-glucoside Peonidin-3--(6"-coumaroyl)-glucoside Malvidin-3--(6"-coumaroyl)-glucoside Auger et al., 29

20 Tea Catechins and the Endothelial Formation of N H H (-)-Epicatechin (-)-Epigallocatechin H H A C B (-)-Epicatechin-3--gallate (-)-Epigallocatechin-3--gallate Auger et al., 29

21 ECg and EGCg are Potent Activators of ens p-akt (Ser473) 1 µm for 15 min Control EC EGC ECg EGCg p-ens (Ser1177) β-tubulin p-akt (fold increase) ) p-ens (fold increase) Control EC EGC ECg EGCg Auger et al., 29

22 EGCG-induced Endothelium-dependent Relaxations: Role of xidative Stress (in the presence of charybdotoxin and apamin, and indomethacin) Relaxation (%) 5 1 Control SD (5 U/mL) MnTMPyP (1 µm) B Relaxation (%) 5 1 Control Catalase (5 U/mL) PEG-catalase (5 U/mL) EGCg [M] EGCg [M] Auger et al., 29

23 Role of Major Enzymatic Sources of RS (in the presence of charybdotoxin and apamin, and indomethacin) Relaxati ion (%) 5 1 Control Apocynin (1 µm) Allopurinol (1 µm) Relaxati ion (%) 5 1 Control Sulfaphenazol (1 µm) KCN (1 µm) + Myxothiazol (.5 µm) + Rotenone (1 µm) EGCg [M] EGCg [M] Auger et al., 29

24 Auto-oxidation of Polyphenols FAD ox FAD red H A C B H A C B - Redox Cycling H A C B EGCg Semi-quinone Quinone 2 2-2? ens P? 2 - N Auger et al., 29

25 EGCg-induced N-mediated Relaxations are Dependent on Hydroxyl moieties (in the presence of charybdotoxin and apamin, and indomethacin) CH 3 CH 3 CH 3 CH 3 H H 3 C CH 3 H 3 C CH 3 CH 3 CH 3 EGCg 5M-EGCg 8M-EGCg CH 3 CH 3 CH 3 Relaxation (%) 5 1 EGCG 5M-EGCG 8M-EGCG Concentration [M] Auger et al., 29

26 EGCg and 5M-EGCg induce Intracellular Formation of RS in Endothelial Cells Fluorescent probe: DHE Control EGCg 5M-EGCg 8M-EGCg EGCg 5M-EGCg + + MnTMPyP MnTMPyP Auger et al., 29

27 Cardiovascular Diseases and Endothelial Dysfunction Red Wine Polyphenols? L-Arginine N Platelets Endothelial cell 2 - ens N GTP sgc Smooth muscle cell cgmp Relaxation Migration Proliferation

RWPs and Endothelial Dysfunction in Hypertension Hemodynamic parameters Vascular reactivity 7

28 RWPs and Endothelial Dysfunction in Hypertension Hemodynamic parameters Vascular reactivity Immunohistochemistry Angiotensin II (.4 mg/kg/day) Red Wine Polyphenols (15 mg/kg/day)

29 RWPs prevent Angiotensin II-induced Hypertension and Endothelial Dysfunction Control group RWP- treated group Ang II -treated group Ang II + RWP -treated group Ang II infusion Time of Ang II infusion (Days) Acetylcholine, log M Sarr et al., Cardiovasc. Res. 26, 71(4):794-82

30 Red Wine Polyphenols prevent Ang II-induced Vascular Formation of Reactive xygen Species A A A A M E M A M E E M M A E Relative DHE fluorescence (% control) # Control Ang II RWPs RWPs + Ang II

31 Vascular NADPH oxidase Ang II 2 AT 1 R nox p22 phox nox p22 phox Cell membrane PLD PKC c-src EGFR PI3K rac p67 phox p47 phox rac p47 phox p67 phox p4 phox P P P -. 2 N p4 phox NADPH NADP + Resting Activated

32 RWPs Prevent Ang II-induced NADPH xidase Expression in the rat Aorta nox-1 expression p22 phox expression Control Ang II RWPs RWPs + Ang II Sarr et al., Cardiovasc. Res. 26, 71(4):794-82

33 Curative study RWPs and Endothelial Dysfunction in Ageing Vascular reactivity week Immunohistochemistry RWPs (1 mg/kg/day) Apocynin (1 mg/kg/day) Solvent

34 Mesenteric artery RWPs and Endothelial Dysfunction in Ageing (in the presence of indomethacin, charybdotoxin and apamin) 12-week old 55-week old 55-week old + RWPs week old + Apocynin Ach, log [M] Dal-Ros et al., 29

35 Preventive study RWPs and Endothelial Dysfunction in Ageing Vascular reactivity Immunohistochemistry 16 4 week Physical exercise capacity RWPs (25 or 75 mg/kg/day) Apocynin (1 mg/kg/day) Solvent

36 RWPs and Endothelial Dysfunction in Ageing Mesenteric artery, 4-week old rat (in the presence of indomethacin, charybdotoxin and apamin) Control RWPs RWPs Apocynin (25 mg/kg/day) (75 mg/kg/day) (1 mg/kg/day) ACh, Log [M] Dal-Ros et al., 29

37 RWPs and Endothelial Dysfunction in Ageing Mesenteric artery 12-week old rats 4-week old rats Solvent RWPs (25mg/kg/day) RWPs (75mg/kg/day) Apocynin RS ens Peroxynitrite Dal-Ros et al., 29

38 RWPs and Endothelial Dysfunction in Ageing Mesenteric artery 12-week old rats 4-week old rats Solvent RWPs (25mg/kg/day) RWPs (75mg/kg/day) Apocynin p22phox AT1R AT2R Dal-Ros et al., 29

39 RWPs and Endothelial Dysfunction in Ageing Treadmill at 5 m/min Endurance capacity (m min) w 12-week old rats 4w 4-week old rats # # Solvent RWPs (25 mg/kg/day) RWPs (75 mg/kg/day) Apocynin (1 mg/kg/day) Dal-Ros et al., 29

40 Protective Effects of Polyphenols on Blood Vessels Polyphenols RS CXs P PI3K/Akt Ang II Endothelial cell EDCF EDHF N RS Smooth muscle cell Vasodilation Vasoconstriction Platelet aggregation Vasoconstriction Pro-inflammatory responses Endothelial dysfunction Atherosclerosis

41 Department of Cardiovascular Pharmacology and Pathophysiology University of Strasbourg, France Pr V.B. Schini-Kerth E. Anselm C. Auger T. Chataigneau S. Dal-Ros N. Idris Khodja N. Etienne-Selloum J.H. Kim A. Walter J. Elbedoui V. Frota Madeira M.. Kane M. Ndiaye M.-H. ak M. Sarr M.-H. ak, Seoul K.W. Lee, Seoul H.J. Lee, Seoul R. Soares de Moura, Rio de Janeiro P.M. Vanhoutte,Hong Kong JC Stoclet, Strasbourg

43 Red Wine Polyphenols induce Redox-sensitive N Formation in Coronary Artery Endothelial Cells Electron Spin Resonance Spectroscopy: DETC Amplitude of th he ESR signal (relative un nits) RWPs (1 µg/ml) # # # # Bradykinin (1 µm) # Control L-NA (1 µm) MnTMPyPPEG-Cat (1 µm) (5 U/ml) NAC (1 mm) Control L-NA (1 µm) MnTMPyP (1 µm) Ndiaye et al., Faseb J 25;19:

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