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A riddle wrapped in a mystery inside an enigma
Jacques Genest MD
Cardiovascular Research Laboratories
McGill University Health Center
Metabolism and Protective
Properties of HDL
Merck *
Pfizer
Novartis
AMGEN *
Roche *
AstraZeneca
Disclosure J. Genest MD 2013
Relevant disclosure: JUPITER, IMPROVE-IT, CANTOS , CAPREE steering Committees; REVEAL , ACCELERATE, AMG145 , Lilly Clinical Trials.
Advisory Board, Speaker’s Bureau, Consultant, Grants, Clinical Trials
Sanofi/Regeneron *
Lilly
Valeant
Genzyme *
Aegerion
Stock ownership: none;
Off label use: none
* Scientific Advisory
The Controversy
A low level of HDL-C is a major independent risk factor for atherosclerotic cardiovascular disease.
However, in randomized, controlled trials, high-dose niacin or inhibitors of cholesteryl ester transfer protein did not improve CV outcomes despite significantly increasing the HDL-C level.
Furthermore, genetic variants associated with HDL-C levels are often not associated with CVD.
These observations suggest that HDL cholesterol may not be causally associated with CVD
N Engl J Med 2014;371:2383-93.
HDL Metabolism
Complexity of HDL
HDL Biogenesis
HDL Metabolism
HDL Metabolism: The Simplified
View
Circ Res. 2014;114:171-182
Human Plasma Lipidome (Quenhenburger N Engl J Med 2011;365:1812-23)
Human Plasma Lipidome
Apolipoprotein AI
Adenosine Triphosphate Binding
Cassette Transporter A1 (ABCA1)
Full-transporter
Phospholipid and
(cholesterol) efflux to apoA-I
Early HDL maturation
LxR/RxR
- COOH
NH
2-
cholesterol
&
phospholipid
ATP ADP+Pi
apoA-I
ATP ADP+Pi
Iatan I et al. JLR 2011
HDL Biogenesis Model
HDL Biogenesis
Nascent HDL Particles
Sorci-Thomas JLR 2012
HDL Biogenesis
CE C
Two-D PAGGE Analysis of HDL
- 17.0
- 12.2
- 7.1
[nm]
- 9.5
a - LpA - I
pre - b 1 - LpA - I
O a pre - b
- 17.0
- 12.2
- 7.1
[nm]
- 9.5
- 17.0
- 12.2
- 7.1
[nm]
- 9.5
Apo AI-containing
lipoproteins
a - LpA - I
pre - b 1 - LpA - I
O a pre - b
Hafiane A. CLC 2013 R17; P18
Intestine
Liver
Endogenous Pathway
FFA
VLDL
ApoA-I, A-II ApoC-I, C-II, C-III Phospholipids Free cholesterol
ApoA-I, A-II ApoC-I, C-II, C-III Phospholipids Free cholesterol
Nascent HDL
Peripheral Cells
FFA
Free Cholesterol
HL
Liver
LCAT
HL Steroidogenic Cells
Exogenous Pathway Chylomicron
Chylo Remnant
HDL2 LDL
IDL
LPL
LPL
CETP PLTP
CE
Tg
HDL3
3 Liver
HL
Lipoprotein Metabolism
Feces
HDL-C factors:
◦ Environmental
◦ Genetic: heritability 50%
Novel genes: PCSK5, WWOX
Known genes:
º Mutations in several genes have
identified (ApoAI, ABCA1, LCAT )
Kathiresan S, Cupples L et al. Nat Genet (2009) 41(1): 56-65
Genes involved in HDL regulation
16
Cellular Cholesterol Efflux
Anti-oxidant
Anti-inflammatory
Vascular Endothelial function
Anti-thrombotic
HDL Functions
Figure 1 Pleiotropic biological functions of apolipoprotein A-I
Shah, P. K. (2011) Targeting endogenous apo A-I—a new approach for raising HDL Nat. Rev. Cardiol. doi:10.1038/nrcardio.2011.37
ABCA1 –Mediated Cellular Cholesterol Efflux
LxR/RxR
HDL Cholesterol efflux:
Biomarker of CVD
N Engl J Med 2014;371:2383-93.
CVD, HDL-C and Efflux Capacity
N Engl J Med 2014;371:2383-93.
Cholesterol Efflux and CVD
Khera AV. N Engl J Med 2011;364:127-35
Cholesterol Efflux and CVD
Arterioscler Thromb Vasc Biol. 2013;33:1696-1705
* * B
*
ACS1 ACS2 CAD Control 0
2
4
6
8
10
12
14 25
50
75 290
300
hsC
RP
(m
g/L)
A
Hafiane A. CLC 2013 P10
HDL-Mediated Cellular Cholesterol Efflux in CAS
*** ***
***
HDL-Mediated Cellular Cholesterol Efflux in CAS
Hafiane A. CLC 2013 P10
Antioxidant Properties of HDL
Antioxidant Properties of HDL
HDL
LDL-ox LDL
PL, FFA
PL-ox POVPC PGPC PAPC
Apo AI, J
LOOX CE-OOX
HOPDE HPETE
a-Toc
O2-
OH
Paraoxonase (PON) Glutathion peroxidase (GPX) PAF-acetyl hydrolase (PAF-AH) Lp-PLA2
12-Lipooxygenase
Generation of truncated phospholipids by oxidative fragmentation and chemical synthesis.
Salomon R G Circulation Research. 2012;111:930-946
Copyright © American Heart Association, Inc. All rights reserved.
POVPC activates aortic smooth muscle cell proliferation.
Salomon R G Circulation Research. 2012;111:930-946
Copyright © American Heart Association, Inc. All rights reserved.
Prothrombotic activities of oxidatively truncated phospholipids.
Salomon R G Circulation Research. 2012;111:930-946
Copyright © American Heart Association, Inc. All rights reserved.
Dehydrative cyclization of isoprostanoid hydroperoxy endoperoxides generates
epoxyisoprostanes.
Salomon R G Circulation Research. 2012;111:930-946
Copyright © American Heart Association, Inc. All rights reserved.
No consensus has yet emerged on the proper biomarker of HDL anti-oxidant function.
Antioxidant Properties of HDL
HDL exerts beneficial effects on VEC
Increase eNOS stability, mRNA and pNOS
Decrease in NF-kB-mediated inflammatory gene expression
Increases endothelial progenitor cells
Endothelial cells and HDL
Campbell S, Genest J, 2012
HDL- Vascular endothelial Cell Interaction
150
150
250
150
100
75
50
37
Loading controls (Ponceau S staining)
eNOS (pS1177: positive phosphorylation)
eNOS (pT495: negative phosphorylation) A
CS1
HD
L
AC
S2 H
DL
Me
diu
m 2
00
Hyp
o a
He
alth
y H
DL
HDL stimulation of Human Aortic Endothelial eNOS
Choi, H 2013 Unpublished
Bessler et al. JCI 2011. Confocal microscopy of GFP-anti RelA/p65
HDLhealthy prevents NF-KB nuclear translocation/activation
HDL and Endothelial Progenitor Cells
Sorrentino SA et al. Circulation 2010;121;110-122
Circ Res. 2014;114:171-182
Cholesterol efflux ABCA1, ABCG1 and SR-BI
Anti-infectious trypanosome lytic factor and LPS inactivation
Anti-apoptic recruitment of endothelial progenitor
cells (EPC)
Vasodilatory NO release, prostacyclin (PGI2) production and eNOS activation
Anti-inflammatory inhibit VCAM-1 expression and adhesion molecules
Antioxidative anti-oxLDL, inactivate LOOH
and oxPhospholipase
Anti-thrombotic inhibition of platelet aggregation and
prostacyclin (PGI2) production
HDL
Hafiane A. Cholesterol 2013
The HDL Proteome
Over 200 proteins have been identified as part of the HDL proteome
(http://homepages.uc.edu/~davidswm/HDLproteome.html
-0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4
serum_amyloid_A_isotype_1_protein
alanyl_(membrane)_aminopeptidase
leucine-rich_alpha-2-glycoprotein_1
complement_component_4B
complement_factor_B
complement_component_3
profilin_1
apolipoprotein_L_1
fibrinogen_alpha_chain
apolipoprotein_C-IV
hemopexin
alpha-1_antiproteinase_antitrypsin
integrin_alpha_2b
chromosome_1_open_reading_frame_56
phospholipid_transfer_protein
haptoglobin-related_protein
apolipoprotein_C-II
inter-alpha_(globulin)_inhibitor_H4
vitronectin
prenylcysteine_oxidase_1
complement_component_1s
fibronectin_1
afamin
orosomucoid_2
alpha-1_antiproteinase_antitrypsin
alpha-1-B_glycoprotein
beta-2-microglobulin
surfactant_protein_B
isocitrate_dehydrogenase_3_(NAD+)_alpha
orosomucoid_1
ceruloplasmin_(ferroxidase)
C1_protease_inhibitor
apolipoprotein_H
alpha-1-microglobulin/bikunin_precursor
cathelicidin_antimicrobial_peptide
apolipoprotein_E
albumin
serum_amyloid_A4_constitutive
apolipoprotein_A-I
apolipoprotein_C-I
coagulat ion_factor_II_(thrombin)
transthyret in
apolipoprotein_C-III
paraoxonase_1
transferrin
heparin_cofactor
vitamin_D_binding_protein
apolipoprotein_F
lecithin-cholesterol_acyltransferase
glycosylphosphatidylinositol_specif ic_phospholipase_D1
apolipoprotein_A-II
apolipoprotein_D
apolipoprotein_M
paraoxonase_3
retinol binding protein 4
lumican
alpha-2-HS-glycoprotein
angiotensinogen
gelsolin
inter-alpha_(globulin)_inhibitor_H2
alpha-2-glycoprotein_1_zinc-binding
peptidoglycan_recognit ion_protein_2
cystat in_C
antithrombin
apolipoprotein_A-IV
mult imerin_2
carbonic_anhydrase_I
clusterin
Enriched in ACS Enriched in controls
HDL Proteome in CVD
Riwanto M et al. Altered activation of endothelial anti- and proapoptotic pathways by
high-density lipoprotein from patients with coronary artery disease: role of high-density
lipoprotein-proteome remodeling. Circulation. 2013;127:891–904.
Independent confirmation of differentially expressed proteins by Western analysis or ELISA
Independent confirmation of differentially enriched proteins by Western analysis or ELISA - Controls
Electron paramagnetic resonance spectroscopy (EPR)
PLoS One. 2013 Aug 28;8(8):e71541. doi: 10.1371/journal.pone.0071541.
The HDL Lipidome
Specific lipid species may have a physiological role in HDL function
Kontush A et al. J. Lipid Res.
2013;54:2950-2963
Physicochemical properties and lipid class composition of HDL particle subpopulations.
Kontush A et al. J. Lipid Res. 2013;54:2950-2963
©2013 by American Society for Biochemistry and Molecular Biology
Specifically, the content of phosphatidylserine revealed positive correlations with all metrics of HDL functionality, reflecting enrichment of phosphatidylserine in small, dense HDL3.
Campbell S, Genest J, 2012
HDL- Vascular endothelial Cell Interaction
Cellular Cholesterol Efflux
Anti-oxidant
Anti-inflammatory
Vascular Endothelial function
Anti-thrombotic
The Challenge
Develop Biomarkers of HDL Function
That can be tested in outcome studies, be reproducible and brought to high throughput.
HDL Biomarkers presently limited in predicting outcomes
IVUS may be the least bad surrogate currently available
New imaging modalities (18FDG) and PET/CT scanning
High bar of regulatory agencies
The Short Term
Identify Responders, Sub groups
Need to identify new pathways of HDL biogenesis and metabolism
Study the structure of HDL in health and in disease (proteomics, lipidomics)
Design clinical trials that are highly focused (initially)
The Long Term
HDL is a Biologically SOUND Target
Points to Take Home
HDL particles are complex
Genes involved in HDL biogenesis may make better therapeutic targets than genes involved in HDL remodeling (Lipases, CETP)
New Gene, new pathways