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
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