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