HDL-C concentration versus HDL particle function: What exactly does HDL

functionality mean?

Prof. Ulf LandmesserUniversity Hospital Zürich

Zürich, Switzerland

Acute coronary syndromes result most frequently from coronary plaques

without significant stenosis

Plaque in mid RCA Occluded RCA

2 Month later

Intensive vs. moderate statin therapy and coronary disease

5 trials (intense vs. moderate statin)39 612 individuals; median follow-up 5·1 years

21 trials (statin versus control)129 526 individuals; median follow-up 4·8 years

Lancet 2010 Nov 13;376(9753):1670-81.

Risk reduction up to 40-50 % possible

Low HDL remains a predictor of cardio-vascular risk in statin-treated patients

Barter P et al., N Engl J Med 2007; 357: 1301-10

HDL – a novel target in coronary disease ?

HDL

The “good“ cholesterol ?

Atherosclerosis development:Dysbalance between increased foam cell formation and impaired HDL-dependent

cholesterol efflux from lipid-laden macrophages

Heinecke J, New Engl J Med 2011

• Anti-atherogenic effects of HDL ?

• “HDL function“ in coronary disease ?

• HDL-targeted therapy ?

HDL-C concentration versus HDL particle function: What exactly

does HDL functionality mean?

A-I

Bile

Nascent HDL

A-I

FCCE FC

LCATMature HDL

HDL

CETPVLDL/ LDL

TGCE

CEFC

PLTPLDL-R

SR-BI Macrophage

ABCA1

ABCG1

SR-BI ?

HDL: proposed anti-atherogenic effects

1. HDL-mediated promotion of RCT (reverse cholesterol transport)

Besler C et al. & Lüscher T, Landmesser U. Curr Pharmacol Des 2010, 16: 1480-93

HDL-mediated regression of atherosclerosis in cholesterol-fed rabbits

Badimon JJ et al.; J Clin Invest. 1990 Apr;85(4):1234-41

60 days 90 days 90 days + HDL

Increased atherosclerosis in ABCA-1 /ABCG1 bone-marrow-deficient

mice

Yvan-Charvet L et al.; J Clin Invest. 2007;117(12):3900-8

No significant differences in HDL plasma levels

A-I

LiverBile

Nascent HDL

A-I

FCCE FC

Endothelial NO Production

Anti-apoptotic Effects

Anti-inflammatoryEffects

Endothelial Repair

Anti-thromboticEffects

LCATMature HDL

HDL

CETPVLDL/ LDL

TGCE

CEFC

PLTPLDL-R

SR-BI Macrophage

ABCA1

ABCG1

SR-BI?

HDL: proposed anti-atherogenic effects

2. Direct HDL-mediated endothelial-protective potential anti-atherogenic effects

1. HDL-mediated promotion of RCT (reverse cholesterol transport)

Besler C et al. & Lüscher T, Landmesser U. Curr Pharmacol Des 2010, 16: 1480-93

Vasculoprotective effect of HDL: role of nitric oxide (NO)

Nofer et al.; J. Clin. Invest. 2004

Yuhanna IS et al.; Nat Med 2001

S1P3

Oxy-sterols

ABCG1

Terasaka N et al.; Arterioscler Thromb Vasc Biol. 2010

AMPK

Li D et al. Arterioscler Thromb Vasc Biol. 2010

Terasaka N et al.; J. Clin. Invest. 2008

Caveolin

Mineo e al.; J Biol Chem. 2003

Endothelial NO production – Anti-atherogenic effects

Vascular smooth muscle cell

Monocyt

Inhibition of Leukocyte adhesion und -infiltration

Inhibition of VSMC proliferation

NO

Platelets

Inhibition of Thrombocyte adhesion and -aggregation

Endothel

Landmesser et al.; Circulation 2004

Development of atherosclerosis

Clinical events

Endothelial dysfunction, nitric oxide, and coronary atherosclerosis

Summarized in: Landmesser et al. Circulation 2004; 109(21 Suppl 1):II27-33

• Anti-atherogenic effects of HDL ?

• “HDL function“ in coronary disease ?

• HDL-targeted therapy ?

HDL-C concentration versus HDL particle function: What exactly

does HDL functionality mean?

Effect of HDL on endothelial cell nitric oxide production in CAD ?

HDL from Healthy subject

HDL from CAD patient

30 minutes

Endothelial effects of HDL in patients with stable

coronary disease or acute coronary syndrome as compared to healthy

subjects ?

Study design: Endothelial effects of HDL - endothelial bioassays

Isolation of HDL2/3(by sequential ultracentrifugation)

Endothelial Function

(Endothelial cell NO production and vasoreactivity)

ESR spectroscopyOrgan chamber

ESR spectroscopy

Patients with acute coronary syndrome (n=25)Patients with stable coronary disease (n=25)

Healthy control subjects (n=25)

Vascular effects

Anti-oxidanteffects

(Endothelial cell superoxide production)

Anti-inflammatoryeffects

(Endothelial cell inflammatory activation)

Monocyte adhesion VCAM-1 expression

Effects on Re-Endothelialization

Carotid artery injury model in nude mice

Anti-thromboticeffects

Tissue factor Arterial thrombosis

Characteristics of the study population

Healthy subjects(n=25)

Stable coronary

artery disease(n=25)

Acute coronary syndrome

(n=25)

Age (years) 54±5 56±6 54±6

Sex (male/female) 19/6 19/6 18/7

Body mass index (kg/m2) 26±5 27±4 26±3

Total cholesterol (mmol/l) 5.3±1.0 5.4±0.9 5.1±0.7HDL cholesterol (mmol/l) 1.4±0.4 1.2±0.3 1.2±0.3

LDL cholesterol (mmol/l) 2.9±0.9 2.7±1.0 2.8±0.6

Besler C et al. & von Eckardstein, Lüscher T, Landmesser U. J Clin Invest 2011;121(7):2693-708

HDL -endothelial effects in patients with coronary disease

(ESR spectroscopy measurement)

Healthy HDL

Stable CADHDL

ACS HDL-20

-15

-10

-5

0

5

10

15

20

25N

itric

oxi

de p

rodu

ctio

n (c

hang

es v

s. P

BS

-trea

ted

cells

, in

%)

P < 0.05 n.s.

P < 0.05

-150000

-100000

-50000

0

50000

100000

3450 3455 3460 3465 3470 3475 3480 3485

Magnetic field (G)

Arb

itrar

y U

nits

-150000

-100000

-50000

0

50000

100000

3450 3455 3460 3465 3470 3475 3480 3485

Magnetic field (G)

Arb

itrar

y U

nits

-150000

-100000

-50000

0

50000

100000

3450 3455 3460 3465 3470 3475 3480 3485

Magnetic fie ld (G)

AU

Besler C et al. & von Eckardstein, Lüscher T, Landmesser U. J Clin Invest 2011;121(7):2693-708

-20

-10

0

10

20

30

25 m g/ml 50 m g/ml 100 m g/mlHDL HDL HDL

Healthy

sCAD

ACS

P<0.025

D E

ndot

helia

l nitr

ic o

xide

prod

uctio

n[in

% o

f buf

fer-

treat

ed c

ells

]

HDL –effects on endothelial cell nitric oxide production in patients with

CAD

Besler C et al. & von Eckardstein, Lüscher T, Landmesser U. J Clin Invest 2011;121(7):2693-708

Baseline TNFαTNFα +

Healthy HDL

TNFα +Stable CAD

HDL

TNFα +ACS

HDL

0

5

10

15

20

25

30

35

Num

ber o

f GC

SF-la

bele

d m

onoc

ytes

pe

r hig

h po

wer

fiel

d

P < 0.05 P < 0.05n.s.

P < 0.05

Effects of HDL on vascular inflammation:monocyte adhesion on TNFα-stimulated

endothelial cells

Besler C et al. & von Eckardstein, Lüscher T, Landmesser U. J Clin Invest 2011;121(7):2693-708

Carotid injury model to examine in vivo endothelial repair capacity of HDL

4 mm4 mm

Evans blue staining

Sorrentino SA et al.; Circulation 2007; 116(2):163-73Sorrentino SA et al.; Circulation 2010; 121(1):110-22 Giannotti G et al.; Hypertension 2010; 55:1389-97

Effects of HDL on endothelial repair after arterial injury

PBS HDLHealthy

HDLStable CAD

HDLACS

0

5

10

15

20

25

30

Re-

endo

thel

ializ

ed a

rea

[in %

] P < 0.0001P < 0.05

Quantification of re-endothelialized area 3 days after induction of carotid injury by Evan`s blue staining

4 mm

Besler C et al. & von Eckardstein, Lüscher T, Landmesser U. J Clin Invest 2011;121(7):2693-708

HDL function(vascular effects)

Which changes of HDL are mediating

differences in HDL‘s vascular effects ?

PhospholipidsTriglycerids

Esterified cholesterol

Apo A1Free cholesterol

> 1000 different lipids(Phospholipid species, Cholesterol Ester, Trigylcerides, …)

70 different proteins(ApoA1, PON-1, ApoA2, ApoCIII, ApoE, ApoH, ….)

Changes in composition and modification of both, lipids and proteins of HDL

in cardiovascular disease results in altered HDL „function“

CholesterolEster

PON-1

The complexity of the HDL-lipoprotein

HDL-associated paraoxonase activity and content

HealthyHDL

Stable CADHDL

ACSHDL

0

0.1

0.2

0.3

0.4

0.5

0.6

Para

oxon

ase-

1 ac

tivity

(U

nits

/50μ

g H

DL) n.s.

P < 0.05

Paraoxonase activity

HealthyHDL

stable CADHDL

ACSHDL

0

100

200

300

400

500

600

700

800

Para

oxon

ase-

1 co

nten

t o

f iso

late

d H

DL

(in %

)

n.s.

P < 0.05

Paraoxonase-1 content

Besler C et al. & von Eckardstein, Lüscher T, Landmesser U. J Clin Invest 2011;121(7):2693-708

HDL from paraoxonase-deficient micefails to stimulate endothelial NO production

-25

0

25

50

P<0.01

HDLWild type

HDLPON1 -/-D

End

othe

lial n

itric

oxi

depr

oduc

tion

[in %

of b

uffe

r-tre

ated

cel

ls]

Besler C et al. & von Eckardstein, Lüscher T, Landmesser U. J Clin Invest 2011;121(7):2693-708

Mechanisms leading to HDL dysfunction with respect to effects on endothelial nitric oxide

Mineo C & Shaul PW. J Clin Invest 2011

Effects of HDL on endothelial apoptosis are eNOS-

independent

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Riwanto M, Besler C et al. (submitted)

Phosphatidylinositol-glycan-specific phospholipase D OS=Homo sapiens GN=GPLD1

Endosialin OS=Homo sapiens GN=CD248

Aminopeptidase N OS=Homo sapiens GN=ANPEP

Sonic hedgehog protein OS=Homo sapiens GN=SHH

Beta-1A of Integrin beta-1 OS=Homo sapiens GN=ITGB1

IGK@ protein OS=Homo sapiens GN=IGK@

Integrin alpha-2 OS=Homo sapiens GN=ITGA2

Apolipoprotein F (APOF), mRNA OS=Homo sapiens

Angiotensinogen OS=Homo sapiens GN=AGT

Serum paraoxonase/arylesterase 1 OS=Homo sapiens GN=PON1

Serum paraoxonase/lactonase 3 OS=Homo sapiens GN=PON3

Prenylcysteine oxidase 1 OS=Homo sapiens GN=PCYOX1

Apolipoprotein D OS=Homo sapiens GN=APOD

Cathelicidin antimicrobial peptide OS=Homo sapiens GN=CAMP

Apolipoprotein A-II OS=Homo sapiens GN=APOA2

DPI of Desmoplakin OS=Homo sapiens GN=DSP

Hemoglobin subunit alpha OS=Homo sapiens GN=HBA1

Antithrombin-III OS=Homo sapiens GN=SERPINC1

Apolipoprotein L1 OS=Homo sapiens GN=APOL1

Alpha-2-macroglobulin OS=Homo sapiens GN=A2M

0 0.1 0.2 0.3 0.4 0.5 0.6

Reduced in ACS patients

Pulmonary surfactant-associated protein B OS=Homo sapiens GN=SFTPB

Heparin cofactor 2 OS=Homo sapiens GN=SERPIND1

Lipopolysaccharide-binding protein OS=Homo sapiens GN=LBP

Serum amyloid A protein OS=Homo sapiens GN=SAA1

Haptoglobin OS=Homo sapiens GN=HP

Vitronectin OS=Homo sapiens GN=VTN

Alpha-2-HS-glycoprotein OS=Homo sapiens GN=AHSG

Alpha-1-antichymotrypsin OS=Homo sapiens GN=SERPINA3

Apolipoprotein C-IV OS=Homo sapiens GN=APOC4

Anthrax toxin receptor 1 OS=Homo sapiens GN=ANTXR1

Complement component C9 OS=Homo sapiens GN=C9

HLA class I histocompatibility antigen, A-24 alpha chain OS=Homo sapiens GN=HLA-A

Haptoglobin-related protein OS=Homo sapiens GN=HPR

Anthrax toxin receptor 2 OS=Homo sapiens GN=ANTXR2

GTP-binding protein SAR1a OS=Homo sapiens GN=SAR1A

Inter-alpha-trypsin inhibitor heavy chain H4 OS=Homo sapiens GN=ITIH4

Apolipoprotein A-IV OS=Homo sapiens GN=APOA4

Serum amyloid A-4 protein OS=Homo sapiens GN=SAA4

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Increased in ACS patients

Changes of HDL proteome in coronary disease

ApoCIIIClusterin

Riwanto M, Besler C et al. (submitted)

Effects of HDL from type-2 diabetics on endothelial NO-Production is impaired

HDL Healthy

Endo

thel

ial N

O P

rodu

ctio

n [%

of b

uffer

- tre

ated

cel

ls]

PBS HDLDiabetics

0

50

100

150

200

P < 0.0001 P < 0.0001

Sorrentino SA et al. Circulation 2010; 121:110-22

Impaired endothelial effects of HDL in patients with antiphospholipid syndrome

HDLMatched Control

HDL-APS

-20

-15

-10

-5

0

5

10

15

20

25En

doth

elia

l NO

pro

duct

ion

(cha

nges

vs.

PB

S-tr

eate

d ce

lls, i

n %

)P < 0.001

Charakida M, Besler C et al. JAMA 2009; 302(11):1210-7

Cholesterol efflux capacity of ApoB-depleted serum and coronary

disease

Khera AV et al.; N Engl J Med. 2011; 364(2):127-35

Ulf LandmesserZurich Univ.

Altered endothelial effects of HDL in cardiovascular

disease

Alan FogelmanUCLA

Novel approaches to stimulate HDL functions

Alan Tall Columbia Univ., New York

Molecular mechanisms of vascular effects of HDL

John DeanfieldUCL,London

Clinical implications of HDL dysfunction

Bart StaelsLille, Inserm

Novel molecular targets stimu-lating HDL functions

Jan A. KuivenhovenJohn Kastelein

Amsterdam Univ.Effects of HDL-genes

on HDL functions

Thomas LüscherZurich Univ.

Vasculareffects of HDL and

its alterations

Stanley Hazen Cleveland Clinic

Molecular mechanisms of HDL dysfunction

Leducq Transatlantic Network:HDL dysfunction in the pathophysiology in

cardiovascular disease and as a novel treatment target

Network Project Managment: Michaela Keel

• Anti-atherogenic effects of HDL ?

• “HDL function“ in coronary disease ?

• HDL-targeted therapy ?

HDL-C concentration versus HDL particle function: What exactly

does HDL functionality mean?

(1) apoA-I(lipid-free)

(2)ABCA1 ABCA1PLFC

PLFC

Nascent HDL

Mature HDL

Peripheral tissues

(including lipid-laden macrophages)

(3) LCAT

(5) SR-BI

CE

TG-rich lipoproteins

(e.g. VLDL) PLTP

apoB

apoB

LDLR

CE

TG

TG

VLDL/LDL

(4) CETP

FC

CEBile

BA

ABCG1

(2) ABCA1

(1) apoA-I(lipid-free)

HDL metabolism – potential targets ?

Besler C, Lüscher T, Landmesser U. EMBO Mol Med 2011 (in press)

LDL-Cholesterol associated SNPs and CAD

HDL in genome-wide association studies

Willer C et al; Nature Genetics, 2008; 40,161-169

HDL-Cholesterol associated SNPs and CAD

(1) apoA-I(lipid-free)

(2)ABCA1 ABCA1PLFC

PLFC

Nascent HDL

Mature HDL

Peripheral tissues

(including lipid-laden macrophages)

(3) LCAT

(5) SR-BI

CE

TG-rich lipoproteins

(e.g. VLDL) PLTP

apoB

apoB

LDLR

CE

TG

TG

VLDL/LDL

(4) CETP

FC

CEBile

BA

ABCG1

(2) ABCA1

(1) apoA-I(lipid-free)

HDL metabolism – potential targets ?

Besler C, Lüscher T, Landmesser U. EMBO Mol Med 2011 (invited review)

1. Intense statin-therapy can reduce cardiovascular events up to 40-50 %.

2. Low HDL-C levels are associated with an increased cardiovascular risk in patients with coronary disease and low LDL levels.Several potentially anti-atherogenic properties of HDL are impaired in patients with coronary disease or diabetes.

3. HDL-targeted therapy should therefore likely not only increase HDL-C levels, but also stimulate anti-atherogenic properties of HDL.

Summary