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

http://circres.ahajournals.org/content/vol98/issue11/images/large/5FF2.jpeg

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