Κλασικοί και νεώτεροι βιοδείκτες καρδιαγγειακού κινδύνου

ΠΑΝΕΠΙΣΤΗΜΙΟ ΙΩΑΝΝΙΝΩΝ ΔΠΜΣ Ιατρική Χημεία Κλασικοί και νεώτεροι βιοδείκτες καρδιαγγειακού κινδύνου Αλέξανδρος Δ. Τσελέπης, MD, PhD Καθηγητής Βιοχημείας Κλινικής Χημείας

Prevalence of major risk factors in men with CHD 2 major risk factors 3 major risk factors 8.9% 4 major risk factors 0.9% 0 major risk factors 19.4% Traditional risk factors are a useful first step in determining who could be at risk for a coronary event 27.8% 43.0% 62.4% 0 to 1 major risk factor Exposure to one or more CHD risk factors is also highly prevalent in individuals who do not develop clinical CHD N=87,869 1 major risk factor Less than 10% of patients have 3 or 4 major risk factor 4 Major modifiable risk factors: hypertension, smoking, hypercholesterolemia, diabetes Khot, et al. JAMA. 2003

New CVD Biomarkers Non-HDL cholesterol Apolipoproteins (Apo) Apolipoprotein A1 (ApoA1) Apolipoprotein B (ApoB) Apolipoprotein B/A1 ratio C-reactive Protein (CRP) sdldl Lipoprotein (a) spla 2 Lipoprotein-Associated Phospholipase A 2 Homocysteine

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The JUPITER trial Ridker PM, et al. N Engl J Med 2008;359:2195-207

The JUPITER trial Ridker PM, et al. N Engl J Med 2008;359:2195-207

The JUPITER trial Ridker PM, et al. N Engl J Med 2008;359:2195-207

Ο Μηχανισμός Παραγωγής του sdldl Mudd JO, et al. JACC 2007; 50: 1735 41

Σχηματισμός μεγάλων LDL σωματιδίων σε νορμοτριγλυκεριδαιμία TG 2

Σχηματισμός sdldl σωματιδίων σε υπερτριγλυκεριδαιμία TG VLDL 1

Συσχέτιση μεταξύ Τριγλυκεριδίων και μεγέθους LDL Rizzo et al, Eur J Clin Invest, 2003

Relative Atherogenicity of Large and sdldl Particles B B CE CE Tg Tg

Mηχανισμοί Αυξημένης Αθηρογόνου Δράσης του sdldl Mudd JO, et al. JACC 2007; 50: 1735 41

Εμφάνιση του Φαινότυπου Β σε Μεσογειακό πληθυσμό διαφορετικών ηλικιών Rizzo et al, Eur J Clin Invest, 2003

Interrelation Between Atherosclerosis and Insulin Resistance Hypertension Obesity Insulin Resistance Hyperinsulinemia Diabetes Hypertriglyceridemia Small, dense LDL Low HDL Hypercoagulability Atherosclerosis

The atherogenic triad of new metabolic risk factors Importance of waist and fasting triglycerides as screening tools Insulin Apo B Small, dense LDL Waist > 90 cm (>40 yrs) Triglycerides > 2.0 mmol/l Circulation (2000) 102:179-184

LDL particle diameter and Prevalence of Pattern B in CAD Koba et al, Am Heart J, 2002

Increased sdldl associated with reduced CVD survival N = 2072 men without IHD at baseline;13-year follow-up 1.00 Survival probabilities 0.90 P < 0.001 0.80 0 2 4 6 8 10 12 Follow-up (years) Tertiles of LDL-C 255Å <1.07 mmol/l 1.07 1.86 mmol/l 1.86 mmol/l St-Pierre AC et al. ATVB. 2005;25:553-9

People with predominance sdldl (phenotype B patients) have a 3-fold increased risk of CVD (Austin MA, et al. JAMA, 1988) NCEP ATP III guidelines have adopted sdldl particles as an emerging risk factor for CVD

Lipoprotein Subclass Measurements Choice of techniques for the separation of LDL subclasses Commercial services Bench or Reference methods Commercial tests LipoScience (NMR Lipotest) Analytical and Density gradient UC Lipoprint (Tube gel EP) Berkeley HeartLab (GGE) Gradient gel electrophoresis (GGE) Precipitation assay (Randox) Atherotech (VAP-II cholesterol) Ion mobility & Electron microscopy

Lipoprint LDL subclass analysis A non-denaturing linear PAGE that separates LDL subfractions on the basis of size The only system approved from FDA

Lipoprint LDL phenotypes Phenotype Α: Low CVD risk Phenotype Β: High CVD risk

Therapeutic modulation of sdldl particles Mediterranean diet Low-carbohydrate diets Hypolipidaemic drugs Statins, Fibrates, Niacin, Niacin combined with statins, Extended-release niacin (niacin ER) Omega-3 fatty acids Hypoglycaemic drugs PPAR ligands, glitazones and thiazolidinediones (pioglitazone and rosiglitazone) Insulin therapy Acarbose Gazi IF, Expert Opin. Biol. Ther. 2007; 7:53-72

Markers of inflammation and myocardial damage Adapted from: Rader D. N Engl J Med. 2000

sdldl και Λιποπρωτεϊνική Φωσφολιπάση (Lp-PLA2)

H Lp-PLA 2 Υδρολύει τα Οξειδωμένα Φωσφολιπίδια της OxLDL Φωσφατιδυλοχολίνη (PC) Οξείδωση Οξειδωμένη PC Lp-PLA 2 + Οξειδωμένο λιπαρό οξύ (oxfa) Λυσοφωσφατιδυλοχολίνη (Lyso-PC) Tselepis AD, et al. Atheroscler Suppl. 2002

Lp-PLA2 activity, % of total Η Lp-PLA 2 μεταφέρεται στο πλάσμα συνδεδεμένη κυρίως με την LDL 100 90 80 70 60 50 40 30 20 10 0 90% LDL Total Plasma Lp-PLA 2 10% HDL = LDL-Lp-PLA 2 Τselepis AD, et al, ATVB 1995; 15: 1764-1773

PAF-AH activity (nmol / ml plasma / min) PLASMA (LDL) Lp-PLA 2 ACTIVITY IN PATIENTS WITH PRIMARY HYPERCHOLESTEROLEMIA 200 P<0.01 150 P<0.01 100 P<0.01 50 0 Controls NonFH HeteroFH HomoFH Tsimihodimos et al, JLR 2002; 43: 256-263

PAF-AH activity (nmol / mg protein / min) Η Lp-PLA 2 συνδέεται κυρίως sdldl σωματίδια 160 140 120 100 Controls NonFH HeteroFH HomoFH * 80 60 40 20 0 * * * VLDL+IDL LDL-1 LDL-2 LDL-3 LDL-4 LDL-5 * * P<0.001 as compared to values of the corresponding subfraction of all other groups *P<0.01 as compared to values of the corresponding subfraction of NonFH and Controls P<0.03 as compared to values of the corresponding subfraction of Controls Tsimihodimos et al, JLR 2002; 43: 256-263

Lp-PLA2 activity (nmol/ml/min Lp-PLA2 activity (nmol/ml/min) Τα επίπεδα της Lp-PLA 2 συσχετίζονται με αυτά των sdldl P A F -A H a c tiv ity (n m o l/m l/m in 92 85 79 72 65 59 53 46 40 33 27 r= 0.39 2 p=0.00 01 r=0.36, P<0.001-1 0 0 10 20 30 40 50 60 70 80 90 s dld L -C m a s s (m g /dl) sdldl-c mass (mg/dl) P A F -A H a ctivity (n m o l/m l/m in ) 92 85 79 72 65 59 53 46 40 33 27 r=-0.3 5 p=0.00 01 r= -0.32, P<0.001 2 4 0 2 4 5 2 5 0 2 5 5 2 6 0 2 6 5 2 7 0 2 7 5 2 8 0 m e a n L D L p a size (A ) Mean LDL particle size (A) Gazi I, et al. Clinical Chemistry 2005; 51:2264-2273

Lp-PLA 2 activity is a marker of atherogenic sdldl particles in subjects at median cardiovascular risk Gazi I, et al. Clinical Chemistry 2005; 51:2264-2273

Ο Ρόλος της Lp-PLA 2 στην Αθηροσκλήρωση LUMEN Adhesion molecules Monocytes Cytokines Plaque formation Oxidized LDL INTIMA Lp-PLA 2 Lyso-PC OxFA Macrophage Foam cell MEDIA

Η Lp-PLA 2 είναι εξειδικευμένος δείκτης αγγειακής φλεγμονής

Τα επίπεδα της Lp-PLA 2 αυξάνονται στη στεφανιαία κυκλοφορία μόνο όταν υπάρχουν αθηρωματικές πλάκες (με IVUS) στα στεφανιαία αγγεία Lavi S, et al. Circulation 2007

H Lp-PLA 2 Υπερεκφράζεται στις Ευάλωτες Πλάκες Corson MA, et al. Am J Cardiol 2008;101[suppl]:41F 50F

Elevated Lp-PLA 2 is consistently associated with a doubling of risk for CVD

Tsimikas S, et al. European Heart Journal (2009) 30, 107 115

Risk ratios for CAD, ischaemic stroke, and vascular and nonvascular mortality per 1 SD higher Lp-PLA2 activity or mass at baseline, adjusted for several risk factors The Lp-PLA2 Studies Collaboration. Lancet 2010; 375: 1536 44

Lp-PLA2 activity and mass each show continuous associations with risk of coronary heart disease, similar in magnitude to that with non-hdl cholesterol or systolic blood pressure in this population The Lp-PLA2 Studies Collaboration. Lancet 2010; 375: 1536 44

Μελέτη Malmö: Τα υψηλά επίπεδα της Lp-PLA 2 αυξάνουν τον καρδιαγγειακό κίνδυνο σε ασθενείς με Μεταβολικό Σύνδρομο 4,480 Non-Diabetics with 261 Major Adverse CV Events Over 10 Years (stroke n=130, MI n=131) Persson M, et al. Arterioscler Thromb Vasc Biol 2007.

Lp-PLA2 and incident CHD in Type 2 Diabetes Health Professionals (HPFS) and Nurses Health Study (NHS) Relative risk (95% CIs) of incident CHD by tertiles of Lp-PLA2 among 1,517 diabetic subjects Hatoum IJ, et al. Diabetes 59:1239 1243, 2010

Τα υπολιπιδαιμικά φάρμακα μειώνουν τα επίπεδα της Lp-PLA 2 στο πλάσμα 0% Omega 3FA 1 Ezetimibe 2 Average Niacin + Fenofibrate 2,5 Statin 2-5 Statin 6-10% -20% -13% -18% -30% -40% -29% -29% -50% -60% Tsimihodimos et al, ATVB 2002; 22: 306-311 -53% Saougos VG, et al. ATVB 2007 Tsimihodimos et al, JLR 2003; 44: 927-934

PAF-AH activity (nmol / mg protein / min) EFFECT OF ATORVASTATIN ON Lp-PLA 2 OF sdldl 180 160 140 120 100 80 60 40 20 0 BASELINE AFTER TREATMENT P< O.OO1 P< O.OO1 P< O.O1 P< O.O1 LDL-4 LDL-5 LDL-4 LDL-5 Type IIA Type IIB Tsimihodimos et al, ATVB 2002; 22: 306-311

EFFECT OF FENOFIBRATE ON Lp-PLA 2 OF sdldl 16 14 12 10 8 6 4 2 0 BEFORE TREATMENT AFTER TREATMENT P<0.05 P<0.05 LDL-4 LDL-5 LDL-4 LDL-5 LDL-4 LDL-5 PRIMARY HYPERCHOLESTEROLEMIA P<0.01 COMBINED HYPERLIPIDEMIA P<0.01 P<0.05 P<0.01 HYPERTRIGLYCERIDEMIA Tsimihodimos et al, JLR 2003; 44: 927-934

Lp-PLA2 activity (nmol/ml plasma/min) Effect of Ezetimibe on Lp-PLA 2 activity associated with LDL subfractions 9 8 7 6 5 4 3 2 1 0 * * Baseline Ezetimibe Ezetimibe *P<0.01 * * * * * * * * * * VLDL+IDL LDL1 LDL2 LDL3 LDL4 LDL5

Μέθοδος για τη μέτρηση της μάζας της Lp-PLA 2 H μοναδική εγκεκριμμένη από το FDA εξέταση αίματος

Το PLAC test Roche Modular P Hitachi ILab 650 Olympus

Recommendation for use of Lp-PLA 2 testing Davidson ΜΗ, et al. Am J Cardiol 2008; 101[suppl]:51F 57F

Azetidinones Specific Lp-PLA 2 inhibitors Darapladib reduces Lp-PLA 2 activity in plasma SB 480848 (Darapladib) Phase II, Phase III trials Mohler ER et al, JACC 2008; 51: 1632-1641

Effects of Darapladib on Coronary plaque Serruys PW et al, Circulation 2008; 118: 1172-1182

Lp-PLA2 activity and mass each show continuous associations with risk of CAD, similar in magnitude to that with non-hdl cholesterol or SBP in this population The Lp-PLA2 Studies Collaboration. Lancet 2010; 375: 1536 44

PAF-AH activity (nmol / ml plasma / min) PLASMA (LDL) Lp-PLA 2 ACTIVITY IN PATIENTS WITH PRIMARY HYPERCHOLESTEROLEMIA 200 P<0.01 150 P<0.01 100 P<0.01 50 0 Controls NonFH HeteroFH HomoFH Tsimihodimos et al, JLR 2002; 43: 256-263

Hypolipidemic drugs reduce Lp-PLA 2 in plasma Statins Fibrates Ezetimibe

Hypolipidemic drugs significantly reduce Lp-PLA 2 plasma levels 0% Omega 3FA 1 Ezetimibe 2 Average Niacin + Fenofibrate 2,5 Statin 2-5 Statin 6-10% -20% -13% -18% -30% -40% -29% -29% -50% -60% Tsimihodimos et al, ATVB 2002; 22: 306-311 -53% Saougos VG, et al. ATVB 2007 Tsimihodimos et al, JLR 2003; 44: 927-934

Lp-PLA2 is not associated with all LDL particles ApoB/Lp-PLA 2 (-) ApoB/Lp-PLA 2 Gazi I, et al. Clinical Chemistry 2005; 51:2264-2273

ApoB, mg/dl The increase of apob levels in hypercholesterolemic patients is mainly attributed to the relative increase in the lipoprotein particles carrying the Lp-PLA 2 160 140 120 * ** ApoB ΑpoB/Lp-PLA2(-) ApoB/Lp-PLA2 100 80 # $ 60 40 20 * # 0 Baseline Postreatment Control Hypercholesterolemic Patients Tellis CC, et al. JLR 2013

Simvastatin preferentially reduces the apob lipoprotein particles carrying the Lp-PLA 2 * # Tellis CC, et al. JLR 2013

Conclusion The elevation in ApoB-containing lipoproteins in primary hypercholesterolemic patients is mainly attributed to the relative increase in the proatherogenic ΑpoB/Lp-PLA 2, while simvastatin reduces these particles to a higher extent compared with ApoB/Lp-PLA 2 (-) Tellis CC, et al. JLR 2013

Substituted Pyrimidones Specific Lp-PLA 2 inhibitors SB 480848 (Darapladib) Phase II studies, Phase III trials

Darapladib reduces Lp-PLA 2 activity in plasma Mohler ER et al, JACC 2008; 51: 1632-1641

Effects of Darapladib on Coronary plaque Serruys PW et al, Circulation 2008; 118: 1172-1182

IBIS-2 study CONCLUSIONS Despite adherence to a high level of standard-of-care treatment, the necrotic core continued to expand among patients receiving placebo In contrast, Lp-PLA2 inhibition with darapladib prevented necrotic core expansion, a key determinant of plaque vulnerability These findings suggest that Lp-PLA2 inhibition may represent a novel therapeutic approach Mohler ER, et al, (J Am Coll Cardiol. 2008;51:1632 41

Secretory phospholipase A 2 (spla 2 )

Cleavage of phospholipid by PLA2

Classification of spla2 isoforms Lambeau G, et al. Annu Rev Biochem 2008. 77:495 520

Τροποποίηση της LDL από την spla2 spla2

Ομοιότητες μεταξύ sdldl και LDL τροποποιημένης από την spla 2 IIA Ιδιότητες sdldl spla 2 IIA LDL Πυκνότητα (g/ml) 1,040 1,060 1,041-1,046 Μέγεθος (nm) 26,46 26,05 26,6 26,5 Περιεκτικότητα φωσφολιπιδίων Μειωμένη Μειωμένη Εμβαδόν λιπιδικής Μειωμένο Μειωμένο επιφάνειας Ευαισθησία στην οξείδωση Πρόσληψη από μακροφάγα Ικανότητα πρόσδεσης στον υποδοχέα της LDL Ικανότητα σύνδεσης με πρωτεογλυκάνες Αυξημένη Αυξημένη Μειωμένη Αυξημένη Αυξημένη Αυξημένη Μειωμένη Αυξημένη

spla2 in Inflammation and Atherosclerosis Boyanovsky BB, et al. Cardiovasc Drugs Ther (2009) 23:61 72

spla 2 and risk of CVD Koenig W, et al. Cardiovasc Drugs Ther (2009) 23:85 92

Kaplan-Meier curves showing the cumulative incidence of death or MI according to the tertile of spla2 activity Global Registry of Acute Coronary Events (GRACE) Mallat Z, et al. JACC (2005) 46:1249 1257

Varespladib sodium Effect in experimental animals PLASMA: Phospholipase Levels And Serological Markers of Atherosclerosis (PLASMA: NCT00455546) 1. Varespladib treatment markedly reduced median spla 2 mass by 69% to 96% in a dose-dependent manner. 2. Compared to placebo, varespladib reduced LDL-C by 8.0 to 11.9% primarily mediated by a reduction in small LDL particles 3. LDL particle size increased in the varespladib-treated subjects by 0.02 nm compared to a decrease in placebo=treated subjects (-0.17 nm), p = 0.015) 4. Oxidized LDL was reduced in varespladib treated patients (p = 0.011) Rosenson RS, et al. Cardiovasc Drugs Ther (2009) 23:93 101