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ΑλέξανδροςΑλέξανδρος
ΔΔ. . ΤσελέπηςΤσελέπης, MD, MD, , PhDPhDΚαθηγητήςΚαθηγητής
ΒιοχημείαςΒιοχημείας
––
ΚλινικήςΚλινικής
ΧημείαςΧημείας
Τα
μικρά-πυκνά
σωματίδια
της
LDL (sdLDL) είναι
ο
κύριος
μεταφορέας
της
Λιποπρωτεϊνικής
Φωσφολιπάσης
Α2 (Lp-PLA2) στο
πλάσμα. Κλινική
σημασία
και
εργαστηριακή
διερεύνηση
ΠΑΝΕΠΙΣΤΗΜΙΟ
ΙΩΑΝΝΙΝΩΝΤΜΗΜΑ
ΧΗΜΕΙΑΣ
ΕΡΓΑΣΤΗΡΙΟ
ΒΙΟΧΗΜΕΙΑΣ
19.4%8.9%
43.0%
0.9%
27.8%
1 major risk factor
0 major risk factors
2 major risk factors
3 major risk factors
4 major risk factors
62.4% 0 to 1 major risk
factor
N=87,869
4 Major modifiable risk factors: hypertension, smoking,
hypercholesterolemia, diabetes
• Traditional risk factors are a useful first step in determining who could be at risk for a coronary event
• Exposure to one or more CHD risk factors is also highly prevalent in individuals who do not develop clinical CHD
• Less than 10% of patients have 3 or 4 major risk factor
Prevalence of major risk factors in men with CHD
Khot, et al. JAMA. 2003
• 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
A2
• Homocysteine
New CVD Biomarkers
Ο
Μηχανισμός
Παραγωγής
του
sdLDL
Mudd
JO, et al.
JACC 2007; 50: 1735–41
Σχηματισμός
μεγάλων
LDL σωματιδίων σε
νορμοτριγλυκεριδαιμία
2
TG
Σχηματισμός
sdLDL
σωματιδίων
σε υπερτριγλυκεριδαιμία
VLDL1
TG
Συσχέτιση
μεταξύ Τριγλυκεριδίων
και
μεγέθους
LDL
Rizzo et al, Eur
J Clin
Invest, 2003
B
Tg
CE
B
Tg
CE
Relative Relative AtherogenicityAtherogenicity
of Large and of Large and sdLDLsdLDL
ParticlesParticles
Mudd
JO, et al.
JACC 2007; 50: 1735–41
Mηχανισμοί
Αυξημένης
Αθηρογόνου
Δράσης
του
sdLDL
Εμφάνιση
του
Φαινότυπου
Β
σε
Μεσογειακό πληθυσμό
διαφορετικών
ηλικιών
Rizzo et al,
Eur
J
Clin
Invest, 2003
Interrelation BetweenInterrelation Between AtherosclerosisAtherosclerosisand Insulin Resistanceand Insulin Resistance
HypertensionHypertension
ObesityObesity
HyperinsulinemiaHyperinsulinemia
DiabetesDiabetes
HypertriglyceridemiaHypertriglyceridemia
Small, dense LDLSmall, dense LDL
Low HDLLow HDL
HypercoagulabilityHypercoagulability
InsulinResistance
InsulinInsulinResistanceResistance AtherosclerosisAtherosclerosisAtherosclerosis
The atherogenic triad of new metabolic risk factors
Importance of waist and fasting triglycerides as screening tools
The atherogenic triad of new metabolic risk factors
Importance of waist and fasting triglycerides as screening tools
InsulinInsulin Apo BApo B Small, denseLDL
Small, denseLDL
Waist> 90 cm (>40 yrs)
Waist> 90 cm (>40 yrs)
Triglycerides> 2.0 mmol/L
Triglycerides> 2.0 mmol/L
Circulation (2000) 102:179-184
LDL Subclass Phenotypes in LDL Subclass Phenotypes in Diabetes MellitusDiabetes Mellitus
Men*Men*DiabeticNondiabetic
Women**Women**DiabeticNondiabetic
** Selby JV et al. Circulation 1993; 88:381-387.
IntInt BB
* Feingold KR et al. Arterioscler Thromb 1992; 12:1496-1502.
2987
54543
2847
3485
2129
309
5124
366
LDL SubclassLDL Subclass
nn AAPercentPercent
LDL particle diameter and Prevalence of Pattern B in CAD
Koba
et al, Am Heart J, 2002
Increased
sdLDL
associated with reduced
CVD survival
St-Pierre AC et al. ATVB. 2005;25:553-9
N = 2072 men without IHD at baseline;13-year follow-up
1.00
0.90
0.80
Survival probabilities
Follow-up (years)0 2 4 6 8 10 12
P < 0.001
Tertiles
of LDL-C 255Å <1.07
mmol/l 1.07–1.86
mmol/l ≥1.86
mmol/l
NCEP ATP III
guidelines have
adopted
sdLDL particles as
an emerging risk factor for CVD
People with predominance sdLDL (phenotype
B patients)
have
a 3-fold increased risk of CVD
(Austin
MA,
et al.
JAMA, 1988)
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
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
A non-denaturing linear PAGE that separates LDL
subfractions
on the basis of size
The only system approved from
FDA
Lipoprint
LDL subclass analysis
Lipoprint
LDL phenotypes
Phenotype Α: Low CVD risk Phenotype Β: High CVD risk
Therapeutic modulation of sdLDL particles
Mediterranean dietLow-carbohydrate
diets
Hypolipidaemic drugsStatins, Fibrates, Niacin, Niacin combined
with
statins, Extended-release niacin (niacin ER)
Omega-3 fatty acidsHypoglycaemic drugs
PPAR ligands,
glitazones and thiazolidinediones (pioglitazone and rosiglitazone)
Insulin therapyAcarbose
Gazi
IF,
Expert Opin. Biol. Ther. 2007;
7:53-72
sdLDL και
Λιποπρωτεϊνική
Φωσφολιπάση
(Lp-PLA2)
O
O
OPO
O O N
O
long chainO
PUFA
-
+
O
O
OPO
O O N
O
long chainO
ox-Fatty Acid
-
+
O OOHO
OH
O
ox-Fatty Acid
O OOHOO
O
P
O
OO N
O
long chain
HO
-
+
Φωσφατιδυλοχολίνη
(PC)
Οξειδωμένη
PC
Λυσοφωσφατιδυλοχολίνη
(Lyso-PC)Οξειδωμένο
λιπαρό
οξύ
(oxFA)
Οξείδωση
Lp-PLA2
H Lp-PLA2
Υδρολύει
τα
Οξειδωμένα Φωσφολιπίδια
της
OxLDL
+
Tselepis AD, et al. Atheroscler Suppl. 2002
0102030405060708090
100
LDL HDL
90%
10%Lp-
PLA
2 ac
tivity
, % o
f tot
al
Τselepis AD, et al, ATVB 1995; 15: 1764-1773
Η
Lp-PLA2
μεταφέρεται
στο
πλάσμα
συνδεδεμένη
κυρίως
με
την
LDL
Total Plasma Lp-PLA2
LDL-Lp-PLA2
=
0
50
100
150
200
Controls NonFH HeteroFH HomoFH
PAF-
AH
act
ivity
(n
mol
/ m
l pla
sma
/ m
PLASMA (LDL) Lp-PLA2
ACTIVITY IN PATIENTS WITH PRIMARY HYPERCHOLESTEROLEMIA
Tsimihodimos et al, JLR 2002; 43: 256-263
P<0.01
P<0.01
P<0.01
0
20
40
60
80
100
120
140
160
VLDL+IDL LDL-1 LDL-2 LDL-3 LDL-4 LDL-5
PAF-
AH a
ctiv
ity
(nm
ol /
mg
prot
ein
/ min
)
ControlsNonFHHeteroFHHomoFH
Η
Lp-PLA2
συνδέεται
κυρίως
sdLDL
σωματίδια
§
§
§§§§
*
*
****
†
††
§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
240 245 250 255 260 265 270 275 280
mean LDL particle size (A)
27
33
40
46
53
59
65
72
79
85
92
PA
F-A
H a
ctiv
ity (n
mol
/ml/m
in)
r=-0.35p=0.0001
r= -0.32, P<0.001
Lp-
PLA
2 ac
tivity
(nm
ol/m
l/min
Mean LDL particle size (A)
-10 0 10 20 30 40 50 60 70 80 90
sdLDL-C mass (mg/dl)
27
33
40
46
53
59
65
72
79
85
92
PAF-
AH
act
ivity
(nm
ol/m
l/min
) r=0.392p=0.0001
r=0.36, P<0.001
-10 0 10 20 30 40 50 60 70 80 90
sdLDL-C mass (mg/dl)
27
33
40
46
53
59
65
72
79
85
92
PAF-
AH
act
ivity
(nm
ol/m
l/min
) r=0.392p=0.0001
r=0.36, P<0.001
-10 0 10 20 30 40 50 60 70 80 90
sdLDL-C mass (mg/dl)
27
33
40
46
53
59
65
72
79
85
92
PAF-
AH
act
ivity
(nm
ol/m
l/min
) r=0.392p=0.0001
r=0.36, P<0.001
sdLDL-C mass (mg/dl)
Lp-
PLA
2 ac
tivity
(nm
ol/m
l/min
)
Gazi I, et al. Clinical
Chemistry 2005; 51:2264-2273
Τα
επίπεδα
της
Lp-PLA2
συσχετίζονται
με
αυτά
των
sdLDL
Gazi I, et al. Clinical
Chemistry 2005; 51:2264-2273
Lp-PLA2
activity is a marker of atherogenic sdLDL particles in subjects at median cardiovascular risk
CytokinesPlaque formation
Foam cell
Monocytes
Macrophage
LUMEN
MEDIA
INTIMA
Oxidized LDL
Adhesion molecules
Lyso-PCOxFA
Lp-PLA2
Ο Ρόλος της Lp-PLA2
στην
Αθηροσκλήρωση
Η Lp-PLA2 είναι εξειδικευμένος δείκτης αγγειακής φλεγμονής
Τα
επίπεδα
της
Lp-PLA2
αυξάνονται
στη
στεφανιαία
κυκλοφορία
μόνοόταν
υπάρχουν
αθηρωματικές
πλάκες
(με
IVUS)
στα
στεφανιαία
αγγεία
Lavi S, et al. Circulation
2007
No Atheros c leros is
C oronary Atheros c leros is
p = NSp < 0.01
Lp-P LA2 C R P
No Atheros c leros is
C oronary Atheros c leros is
No Atheros c leros is
C oronary Atheros c leros is
p = NSp < 0.01
Lp-P LA2 C R P
Lp-P
LA2
Net
Pro
duct
ion
(ng/
min
)
CRP
Net
Pro
duct
ion
(μg/
min
)
H Lp-PLA2
Υπερεκφράζεται
στις
Ευάλωτες
Πλάκες
S table Plaque• L ow L p-PL A2 c ontent (dark s taining)
• May have s ignific ant s tenos is
• Thic k fibrous c ap / high c ollagen c ontent
• Modes t lipid pool
• Few inflammatory c ells
R uptured Plaque• High L p-PL A2 c ontent (dark s taining)
• May have minimal s tenos is
• Thin fibrous c ap / low c ollagen c ontent
• L arge lipid pool
• Many inflammatory c ells
Modes t Lipid Pool Large Lipid PoolModes t Lipid Pool Large Lipid Pool
Lp-PL A2 Lp-PL A2Lp-PL A2 Lp-PL A2
Thin Fibrous C apThic k Fibrous C ap Thin Fibrous C apThic k Fibrous C ap
LumenLumen LumenLumen
Corson MA, et al. Am J Cardiol 2008;101[suppl]:41F–50F
Elevated Lp-PLA2
is consistently associatedwith 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 non-
vascular 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
1.97†
1.00
1.40* 1.46**
2.0
1.5
1.0
0.5
0.0
Haz
ard
Rat
io fo
r C
V E
vent
s
- Met S ynd &L ow L p-PL A2
- Met S ynd &HighHigh L pL p--PL APL A22
+ Met S ynd &L ow L p-PL A2
+ Met S ynd &HighHigh L pL p--PL APL A22
1.97†
1.00
1.40* 1.46**
2.0
1.5
1.0
0.5
0.0
Haz
ard
Rat
io fo
r C
V E
vent
s
- Met S ynd &L ow L p-PL A2
- Met S ynd &HighHigh L pL p--PL APL A22
+ Met S ynd &L ow L p-PL A2
+ Met S ynd &HighHigh L pL p--PL APL A22
1.00
1.40* 1.46**
2.0
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
Haz
ard
Rat
io fo
r C
V E
vent
s
- Met S ynd &L ow L p-PL A2
- Met S ynd &HighHigh L pL p--PL APL A22
+ Met S ynd &L ow L p-PL A2
+ Met S ynd &HighHigh L pL p--PL APL A22
†p = 0.001
*p = 0.034**p = 0.095
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.
Μελέτη
Malmö: Τα
υψηλά
επίπεδα
της
Lp-PLA2
αυξάνουν
τον καρδιαγγειακό
κίνδυνο
σε
ασθενείς
με
Μεταβολικό
Σύνδρομο
Lp-PLA2 and incident CHD
in Type 2 Diabetes
Health Professionals
(HPFS) and Nurses’
Health Study (NHS)
Hatoum IJ, et al. Diabetes 59:1239–1243, 2010
Relative risk (95% CIs) of incident CHD by
tertiles of Lp-PLA2 among 1,517 diabetic subjects
-18%
-29%-29%
-53%
-13%
-60%
-50%
-40%
-30%
-20%
-10%
0%
Niacin Added
To S tatin
Per
cent
red
uctio
n in
Lp-
PLA
2
Ezetimibe2Average Statin2-5Fenofibrate2,5
Niacin + Statin6Omega 3FA1
S tatin
-18%
-29%-29%
-53%
-13%
-60%
-50%
-40%
-30%
-20%
-10%
0%
-18%
-29%-29%
-53%
-13%
-60%
-50%
-40%
-30%
-20%
-10%
0%
Niacin Added
To S tatin
Per
cent
red
uctio
n in
Lp-
PLA
2
Ezetimibe2Average Statin2-5Fenofibrate2,5
Niacin + Statin6Omega 3FA1
S tatin
Τα
υπολιπιδαιμικά
φάρμακα
μειώνουν
τα
επίπεδα της
Lp-PLA2
στο
πλάσμα
Tsimihodimos et al, ATVB 2002; 22: 306-311
Tsimihodimos et al, JLR 2003; 44: 927-934
Saougos VG, et al. ATVB
2007.
020406080
100120140160180
LDL-4 LDL-5 LDL-4 LDL-5
PAF-
AH a
ctiv
ity
(nm
ol /
mg
prot
ein
/ min
)
BASELINE
AFTER TREATMENT
Type IIA Type IIB
P< O.O1
P< O.OO1
P< O.O1
P< O.OO1
EFFECT OF ATORVASTATIN ON Lp-PLA2
OF sdLDL
Tsimihodimos et al, ATVB 2002; 22: 306-311
EFFECT OF FENOFIBRATE ON EFFECT OF FENOFIBRATE ON LpLp--PLAPLA22
OF sdLDLOF sdLDL
0
2
4
6
8
10
12
14
16
LDL-4 LDL-5 LDL-4 LDL-5 LDL-4 LDL-5
BEFORE TREATMENTAFTER TREATMENT
PRIMARYHYPERCHOLESTEROLEMIA
COMBINED HYPERLIPIDEMIA
HYPERTRIGLYCERIDEMIA
P<0.01P<0.01P<0.05P<0.05
P<0.05P<0.05P<0.05P<0.05
P<0.01P<0.01
P<0.01P<0.01
Tsimihodimos et al, JLR 2003; 44: 927-934
0
1
2
3
4
5
6
7
8
9
VLDL+IDL LDL1 LDL2 LDL3 LDL4 LDL5
Lp-P
LA2
activ
ity
(nm
ol/m
l pla
sma/
min
) Baseline EzetimibeEzetimibe
* * * * * * * *
* *
* *
Effect of Ezetimibe on Lp-PLA2
activity associated with LDL subfractions
*P<0.01
Μέθοδος
για
τη
μέτρηση
της
μάζας
της
Lp-PLA2
H
μοναδική
εγκεκριμμένη
από
το
FDA
εξέταση
αίματος
Olympus
HitachiRoche Modular P
ILab 650
Το
PLAC test
Recommendation for use of Lp-PLA2 testing
Davidson ΜΗ, et al. Am J Cardiol 2008;
101[suppl]:51F–57F
SB 480848 (Darapladib)Phase II, Phase III trials
AzetidinonesSpecific
Lp-PLA2
inhibitorsDarapladib
reduces
Lp-PLA2
activity in plasma
Mohler
ER
et al, JACC 2008; 51: 1632-1641
Serruys
PW et al, Circulation 2008; 118: 1172-1182
Effects of Darapladib on Coronary plaque
Ioannina