ΠΑΝΕΠΙΣΤΗΜΙΟ ΙΩΑΝΝΙΝΩΝ ΤΜΗΜΑ ΧΗΜΕΙΑΣ ΕΡΓΑΣΤΗΡΙΟ ΒΙΟΧΗΜΕΙΑΣ Τα μικρά-πυκνά σωματίδια της LDL (sdldl) είναι ο κύριος μεταφορέας της Λιποπρωτεϊνικής Φωσφολιπάσης Α2 (Lp-PLA2) στο πλάσμα. Κλινική σημασία και εργαστηριακή διερεύνηση 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) Lipoprotein-Associated Phospholipase A 2 Homocysteine
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 of waist and and fasting triglycerides as 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 Subclass Phenotypes in Diabetes Mellitus LDL Subclass n A Int B Men* Diabetic Nondiabetic 29 87 28 47 Percent 21 29 51 24 Women** Diabetic Nondiabetic 54 543 34 85 30 9 36 6 * Feingold KR et al. Arterioscler Thromb 1992; 12:1496-1502. ** Selby JV et al. Circulation 1993; 88:381-387.
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
sdldl και Λιποπρωτεϊνική Φωσφολιπάση (Lp-PLA2)
H Lp-PLA 2 Υδρολύει τα Οξειδωμένα Φωσφολιπίδια της OxLDL O PUFA O O O O O P O - O long chain + N Φωσφατιδυλοχολίνη (PC) Οξείδωση O O O OOH ox-fatty Acid O O O O O P O- O long chain + N Οξειδωμένη PC Lp-PLA 2 O O long chain O O OOH ox-fatty Acid O OH + HO O O P O O - + N Οξειδωμένο λιπαρό οξύ (oxfa) Λυσοφωσφατιδυλοχολίνη (Lyso-PC) Tselepis AD, et al. Atheroscler Suppl. 2002
Η Lp-PLA 2 μεταφέρεται στο πλάσμα συνδεδεμένη κυρίως με την LDL 100 Lp-PLA2 activity, % of total 90 80 70 60 50 40 30 20 10 0 90% Total Plasma Lp-PLA 2 10% = LDL-Lp-PLA 2 LDL HDL Τselepis AD, et al, ATVB 1995; 15: 1764-1773
PLASMA (LDL) Lp-PLA 2 ACTIVITY IN PATIENTS WITH PRIMARY HYPERCHOLESTEROLEMIA 200 P<0.01 PAF-AH activity (nmol / ml plasma / m 150 100 50 P<0.01 P<0.01 0 Controls NonFH HeteroFH HomoFH Tsimihodimos et al, JLR 2002; 43: 256-263
Η Lp-PLA 2 συνδέεται κυρίως sdldl σωματίδια PAF-AH activity (nmol / mg protein / min) 160 140 120 100 80 60 40 20 0 Controls NonFH HeteroFH HomoFH * * * 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
Τα επίπεδα της Lp-PLA 2 συσχετίζονται με αυτά των sdldl PAF-AH Lp-PLA2 activity (nmol/ml/min) 92 85 79 72 65 59 53 46 40 33 27 r=0.392 p=0.0001 r=0.36, P<0.001-10 0 10 20 30 40 50 60 70 80 90 sdldl-c mass (mg/dl) sdldl-c mass (mg/dl) Lp-PLA2 activity (nmol/ml/min PAF-AH activity (nmol/ml/min) 92 85 79 72 65 59 53 46 40 33 27 r=-0.35 p=0.0001 r= -0.32, P<0.001 240 245 250 255 260 265 270 275 280 mean Mean LDL particle particle size (A) 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
ΟΡόλοςτηςLp-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 Lp-PLA 2 Net Production (ng/min) C oronary Atheros cleros is No Atheros cleros is p < 0.01 p = NS CRP Net Production (μg/min) Lp-PLA 2 CRP Lavi S, et al. Circulation 2007
H Lp-PLA 2 Υπερεκφράζεται στις Ευάλωτες Πλάκες Thick Fibrous Cap Thin Fibrous Cap Modest Lipid Pool Large Lipid Pool Lumen Lumen Lp-PLA 2 Lp-PLA 2 Stable Plaque Low Lp-PLA 2 content (dark staining) May have significant stenosis Thick fibrous cap / high collagen content Modes t lipid pool Few inflammatory cells Ruptured Plaque High Lp-PLA 2 content (dark staining) May have minimal stenosis Thin fibrous cap / low collagen content Large lipid pool Many inflammatory c ells 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 2.0 (stroke n=130, MI n=131) 1.97 Hazard R atio for C V E vents 1.5 1.0 0.5 1.00 1.40* 1.46** *p = 0.034 **p = 0.095 p = 0.001 0.0 - Met S ynd & Low Lp-PLA 2 - Met S ynd & High Lp-PLA 2 + Met S ynd & Low Lp-PLA 2 + Met S ynd & High Lp-PLA 2 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 P ercent reduction in Lp-P LA 2-10% -20% -30% -40% -50% -60% -13% -18% -29% -29% Statin Niacin Added To Statin -53% Tsimihodimos et al, ATVB 2002; 22: 306-311 Saougos VG, et al. ATVB 2007. Tsimihodimos et al, JLR 2003; 44: 927-934
EFFECT OF ATORVASTATIN ON Lp-PLA 2 OF sdldl PAF-AH activity (nmol / mg protein / min) 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
Effect of Ezetimibe on Lp-PLA 2 activity associated with LDL subfractions Lp-PLA2 activity (nmol/ml plasma/min) 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
Ioannina