what’s new in hdl function testing?...modified after rosenson rs, brewer hb jr. davidson ws, fayad...

What’s new in HDL function testing?

Robert S. Rosenson, MD

Professor of Medicine (Cardiology)

Icahn School of Medicine at Mount Sinai

Director, Cardiometabolic Disorders

Mount Sinai Medical Center

New York, NY USA

Disclosures

Grant/Research Support: Amgen, Medicines Company, Novarits, Regeneron, NIH

Consultant/Advisor: Amgen, C5, CVS CareMark, Novartis, Regeneron, Sanofi, 89 Bio

Speaking fees (non-promotional): Amgen, Kowa, Regeneron

Royalties: Wolters Kluwer (UpToDate)

HDL-LHDL-VL HDL-VSHDL-SHDL-M

Particle size

Apolipoprotein compositionParticle shape

Discoidal

SphericalA-I HDL A-I/A-II HDL

Lipid-poor ApoA-I

E HDL

Adapted from Barter PJ. Atheroscler Suppl. 2002;3:39–47.

HDL is comprised of discrete subpopulations of particles

Proposed term Very large

HDL (HDL-VL)

Large HDL-V

(HDL-L)

Medium HDL

(HDL-M)

Small HDL

(HDL-S)

Very small

HDL (VS-HDL)

Density range, g/mL 1.063-1.087 1.088-1.110 1.110-1.129 1.129-1.154 1.154-1.21

Size range, nm 12.9-9.7 9.7-8.8 8.8-8.2 8.2-7.8 7.8-7.2

Density gradient

ultracentrifugation HDL2b HDL2a HDL3a HDL3b HDL3c

Density range, g/mL 1.063-1.087 1.088-1.110 1.110-1.129 1.129-1.154 1.154-1.170

Gradient gel

electrophoresis HDL2b HDL2a HDL3a HDL3b HDL3c

Size range, nm 12.9-9.7 9.7-8.8 8.8-8.2 8.2-7.8 7.8-7.2

2D gel

electrophoresis Alpha-1 Alpha-2 Alpha-3 Alpha-4 Preβ-1 HDL

Size range, nm 11.2-10.8 9.4-9.0 8.5-7.5 7.5-7.0 6.0-5.0

NMR Large HDL-P Medium HDL-P Small HDL-P

Size range, nm 12.9-9.7 9.7-8.8 8.8-8.2 8.2-7.8 7.8-7.2

Ion mobility HDL 2b HDL 2a + 3

Size range, nm 14.5-10.5 10.5-7.65

Classification of HDL by Physical Properties

Rosenson RS, et al., Brewer HB Jr, Chapman MJ, Fazio S, Hussain MM, Kontush A, Krauss RM, Otvos JD, Remaley AT, Schaefer EJ.HDL Measures, HDL Particle Heterogeneity, Proposed HDL Nomenclature and Relation to Atherosclerotic Cardiovascular Events. Clin Chem. 2011;57:392-410.

HDL Particles and Incident CVDStudy name Population Significant study findings

Veterans Affairs High-Density

Lipoprotein Intervention Trial (VA-HIT)1Nested case-control study of 364 men with a

new CHD event (nonfatal myocardial infarction

or cardiac death) during a median 5.1-year

follow-up and 697 age-matched controls.

HDL particles achieved with gemfibrozil were significant, independent predictors of

new CHD events. For total HDL particles, odds ratios

predicting CHD benefit was 0.71 (95% CI, 0.61 to 0.81).

Women’s Health Study (WHS)2 27,673 healthy women; 26,658 without a CVD

event and 1015 with a CVD event after an 11

year follow-up period

CVD risk prediction associated with

lipoprotein profiles evaluated by NMR, including HDL-P was comparable but not

superior to that of standard lipids, apoB-100 or apoA-I

Heart

Protection Study (HPS)3Randomized trial of simvastatin versus placebo

(>5,000 vascular events during 5.3 years of

follow-up

among 20,000 participants)

After adjustment for LDL-P, the hazard ratios for major occlusive coronary event per

1-SD-higher level were 0.91 (95% CI, 0.86–0.96) for HDL-C and 0.89 (95% CI,

0.85– 0.93) for HDL-P. Other cardiac events were inversely associated with total

HDL-P (hazard ratio, 0.84; 95% CI, 0.79–0.90) and small (0.82; 95% CI, 0.76–0.89)

HDL-P but only very weakly associated with HDL-C (0.94; 95% CI, 0.88 –1.00).

Justification for the Use of statins in

Prevention: an Intervention Trial

Evaluating Rosuvastatin

(JUPITER)4

Randomized trial of 17,802 individuals; women

≥60 years and men ≥50 years without a previous

history of CVD or

diabetes mellitus who had LDL-C

Riggs KA, Joshi PH, Khera A, Singh K, Akinmolayemi O, Ayers CR, Rohatgi A. J Clin Med. 2019 Dec 3;8(12):2137.

Hazard Ratios of Quartiles of GlycA

Major Anti-Atherosclerotic Functional Roles of HDL With Available Clinical Measures

• Macrophage cholesterol efflux

• Anti-oxidative effects

• Anti-inflammatory effects

• Endothelial function

• Glucose homeostasis

Rosenson RS, Brewer Jr. HB, Ansell B, Barter PJ, Chapman MJ, Heinecke J, Kontush A, Tall A, Webb N. Circulation 2013 (in press)

Reverse Cholesterol Transport

Reverse cholesterol transport is comprised of

multiple components

➢ Macrophage-specific arterial wall efflux

➢ Non-macrophage arterial wall efflux

➢ Non-arterial wall efflux

➢ Lipoprotein transport

➢ Hepatobiliary excretion

➢ Fecal excretion

HDL-C is an inadequate surrogate for macrophage

cholesterol efflux

Fecal excretion of cholesterol is not (necessarily) a

pre-requisite for assessing the cholesterol efflux

from the arterial wallRosenson RS, Brewer HB Jr. Davidson WS, Fayad ZA, Fuster V, Goldstein J, Hellerstein M, Jiang X-C, Philips MC, Remaley A, Rader DJ, Rothblat GH, Tall AR, Yvan-Charvet L. Circulation 2012;125:1905-1919.

Plasma

αHDL

Cholesterol

Pool

Liver

75%

Percentage of HDL-C Synthesized by the Liver, Intestine,

and Peripheral Cells

< 5%SR-B1

Peripheral

Tissues

αHDL

(HDL-S,

HDL-M, &

HDL-L)

A-I

ABCG1

MacrophagesPreβ-HDL

A-I

A-I

αHDL (HDL-S,

HDL-M, & HDL-L)

Preβ-HDL

A-I

A-I

αHDL (HDL-S,

HDL-M, & HDL-L)

20%

Modified after Rosenson RS, Brewer HB Jr. Davidson WS, Fayad ZA, Fuster V, Goldstein J, Hellerstein M, Jiang X-C, Philips MC, Remaley A, Rader DJ, Rothblat GH, Tall AR, Yvan-Charvet L. Circulation 2012;125:1905-1919.

(HDL-VS)

(HDL-VS)

Intestine

Marina Cuchel et al. J. Lipid Res. 2017;58:752-762

Multicompartmental model used to determine rates of cholesterol transfer between different

cholesterol compartments.

Selected fractional transfer rates (per h) for cholesterol determined by multicompartmental

modeling of the nanoparticle cholesterol tracer data

Macrophage-

FC to HDL-FC

HDL-FC to

nonHDL-FC

HDL-FC to

HDL-CE

HDL-CE to

nonHDL-CE

Fractional transfer

(pools/h)0.028 ± 0.024 8.044 ± 3.728 0.249 ± 0.075 0.171 ± 0.116

Absolute transfer

(μmol/h)n.d. 9,050 ± 4,660 271 ± 79 568 ± 675

Pool size of originating

compartment (μmol)n.d. 1,145 ± 375 1,145 ± 375 3,107 ± 1,142

•Data are mean ±SD (n = 30). n.d., not determined. The transfer rates listed correspond to the following transfer rates in the model shown in Fig. 1: Macrophage-FC to HDL-FC (compartment 25 to 4); HDL-FC to nonHDL FC (compartment 4 to 5); HDL-FC to HDL-CE (compartment 4 to 9); HDL CE to nonHDL-CE (compartment 9 to 7).

Marina Cuchel et al. J. Lipid Res. 2017;58:752-762

HDL Particle Subclasses And Cholesterol Efflux From Cholesterol-Loaded Cells

Rosenson RS, Brewer HB, Jr., Davidson WS, Fayad ZA, Fuster V, Goldstein J, Hellerstein M, Jiang XC, Phillips MC, Rader DJ, Remaley AT, Rothblat GH, Tall AR, Yvan-Charvet L. Circulation. 2012;125(15):1905-1919. Copyright © 2012, Wolters Kluwer Health.

Translational Measures of Macrophage Cholesterol Efflux in

Humans

1. Efflux of 3H-Cholesterol radiolabeled from J774

macorphages.

2. Efflux of 3H-Cholesterol radiolabeled from

RAW264.7 macrophages.

3. Fluorescent BODIPY-cholesterol

Khera, A.V., et al. N Engl J Med 364, 127-135 (2011).Li, X.M., et al. Arterioscler Thromb Vasc Biol 33, 1696-1705 (2013).

Holtta-Vuori, M., et al. BODIPY-cholesterol: a new tool to visualize sterol traffickingin living cells and organisms. Traffic 9, 1839-1849 (2008).

48h w/ 3H-Cholesterol labeled 1% FBS RPMI

+ ACAT inhibitor (2 mcg/mL)

J774 MACROPHAGE CELLS

4h w/ 2.8% HDL

(apoB-depleted

sera)% FC Efflux

15h w/ cAMP + 0.2% BSA

Cholesterol Efflux Capacity of Human Serum

Data from Adorni MP, et al. J Lipid Res. 2007;48(11):2453-2462.

The inhibitor assay was used to document the

efflux pathways expressed by J774 cells upon

treatment with cAMP

Cholesterol Efflux Capacityof Human Serum

Efflux from control and upregulated J774 cells to the serum

HDL fraction from a pool of human serum

Summary of 3 experiments

16

14

12

10

8

6

4

2

0

% E

fflu

x/4

h ABCA1

SR-BI

ABCG1

Aq Diffusion

– cAMP + cAMP

de la Llera-Moya M, et al. Arterioscler Thromb Vasc Biol. 2010;30(4):796-801.

Odds Ratios for CAD Accordingto the Efflux Capacity and Selected Risk Factors

Risk Factor Odds Ratio (95% CI) P Value

Diabetes 1.92 (1.26–2.93) 0.003

Hypertension 1.80 (1.31–2.47) < 0.001

Smoking 1.10 (0.95–1.73) 1.78

LDL cholesterol 1.01 (0.86–1.18) 0.93

HDL cholesterol 0.85 (0.70–1.03) 0.09

Efflux capacity 0.75 (0.63–0.90) 0.002

0.5 1.0 2.0 4.0

The logistic-regression model also was adjusted for age and sex.

Odds ratios for continuous variables are per 1-SD increase.

1. Khera AV, et al. N Engl J Med. 2011;364(2):127-135. Copyright © 2011 Massachusetts Medical Society.

2. de la Llera-Moya M, et al. Arterioscler Thromb Vasc Biol. 2010;30(4):796-801.

BODIPY (boron dipyrromethene difluoride)

J774 cells (48 wells plate)-24 h

Labeled cells BODIPY/CD+ ACAT inhibitor 1 h

Equilibration ± cAMP + ACAT inhibitor 16 h

Incubation with acceptors4 h

Fluorescence reading of efflux media after filtration

Labeling of J774 macrophages with BODIPY-cholesterol

BODIPY-cholesterol labeling medium: BODIPY-cholesterol (20%), cholesterol (80%) in

beta methyl CD (10 mmol, 1:20)

g/ml)

Apo A

-I (50

g/ml)

(5

0 3

HDL

2% PE

G

0

5

10

15-cAMP +cAMP

3H-cholesterol efflux

0 10 20 30 40 500.02.55.07.5

10.012.515.017.520.0

BODIPY efflux

3H efflux

Apo A-I (mg/ml)

% e

fflu

x/4

hBODIPY-CHOLESTEROL EFFLUX

VS.3H-CHOLESTEROL EFFLUX

Adorni MP, Zimetti F, Billheimer JT, Wang N, Rader DJ, Phillips MC, Rothblat GH.J Lipid Res. 2007 Nov;48(11):2453-62.

0 2 4 6 8 10 12 140

15

30

45

60

75BODIPY efflux

3H efflux

BODIPY-CHOLESTEROL EFFLUX VS.3H-CHOLESTEROL EFFLUX

% PEG supernatant

% e

fflu

x/4

h

Adorni MP, Zimetti F, Billheimer JT, Wang N, Rader DJ, Phillips MC, Rothblat GH.J Lipid Res. 2007 Nov;48(11):2453-62.

CORRELATION BETWEEN BODIPY-CHOLESTEROL

EFFLUX AND 3H-CHOLESTEROL EFFLUX

20 30 40 50 60 70

2.5

7.5

12.5r2=0.69

BODIPY-CHOLESTEROL EFFLUX / 4 H

3H

-CH

OL

ES

TE

RO

L E

FF

LU

X /

4 H

3H-Cholesterol vs. preßHDL

0 5 10 15 200

5

10

15

20

r2 = 0.20NS

preßHDL (mg/dl)

%3H

eff

lux/4

h

0 25 50 75 100 1250

5

10

15

20

r2 = 0.57P < 0.0001

% 3H-Cholesterol vs. HDL-C

HDL-C (mg/dL)

%3H

eff

lux/4

h

BODIPY-cholesterol vs. preßHDL

0 5 10 15 200

10

20

30

40

50

60

70

r2 = 0.58P = 0.0002

preßHDL (mg/dL)

% B

OD

IPY

eff

lux/4

h

BODIPY-cholesterol vs. HDL-C

0 25 50 75 100 1250

10

20

30

40

50

60

70

NS

HDL-C (mg/dL)

% B

OD

IPY

eff

lux/4

h

Humans with Atherosclerosis Have Impaired ABCA1 Cholesterol Efflux and Enhanced HDL Oxidation by

Myeloperoxidase

Shao B, et al. Circ Res 2014 [Epub ahead of print]

Levels of Met(O)148 in control subjects, CAD subjects, and ACS subjects.

Cholesterol Efflux Capacity of Serum HDL in Control, CAD and ACS Subjects

Odds Ratio (95% CI) P Value

Met(O)148 7.3 ( 1.8-30 ) 0.006

3-ChloroTyr192 4.1 ( 1.5-12 ) 0.008

Plasma MPO 1.1 ( 0.7-1.7 ) 0.62

LDL cholesterol 0.75 ( 0.4-1.3 ) 0.26

HDL cholesterol 0.29 ( 0.14-0.61 ) 0.001

ABCA1 efflux capacity 0.30 ( 0.14-0.66 ) 0.003

0.3 1 3 10

Serum HDL was obtained by PEG precipitation of serum derived from plasma. Cholesterol efflux was measured from serum HDL to ABCA1-expressing BHK cells

Shao B, et al Circ Res 2014 [Epub ahead of print]

Atherosclerotic Cardiovascular Disease Events, According to Models Based on HDL Cholesterol Level and

Cholesterol Efflux Capacity

Rohatgi A et al. N Engl J Med 2014;371:2383-2393

Kaplan–Meier Curves and Hazard Ratios for Cardiovascular Events, According to Quartile of Cholesterol Efflux Capacity


Cholesterol Efflux Capacity and Traditional Risk Factors for Atherosclerotic CVD


Correlation of HDL Cholesterol Level, HDL Particle Concentration, and Cholesterol Efflux Capacity with Lipoprotein and Metabolic Variables

and Inflammatory Markers


Cardiovascular Events and Hazard Ratios, According to Quartile of Cholesterol Efflux Capacity


Cholesterol Efflux Capacity and Incident CHD Events in EPIC-Norfolk

Nested case-control sample within a prospective study of 25,639 individuals aged 40-79 years examined in 1993-1997 and followed up to 2009

Efflux capacity was quantified in 1,895 incident CHD cases and 2,474 control participants free of any cardiovascular disorders

Validated ex vivo radiotracer assay that involved incubation of J774 macrohages with apoB-depleted serum from study participants

Saleheen D, Scott R, Javad S, et al. Lancet Diabetes Endocrinol 2015;3:507-13

Association of Cholesterol Efflux and Incident CHD Events – EPIC Norfolk

Saleheen Saleheen D, Scott R, Javad S, et al. The lancet Diabetes & endocrinology 2015;3:507-13

Validation of HDL Functional Measures in the Evaluation of Atherosclerosis and Cardiovascular Events

Biomarker of HDL Functionality

Surrogate Measure of

Atherosclerosis

Atherosclerosis

Volume and Composition

SurrogateMeasure of

Cardiovascular Events

Cardiovascular

Events

Biomarker ofHDL

Functionality

Rosenson RS, Brewer Jr. HB, Ansell B, Barter PJ, Chapman MJ, Heinecke J, Kontush A, Tall A, Webb N. Circulation 2013;128:1256-67

HDL/ABCA1/G1 Suppress Macrophage Inflammatory Cytokines That Promote Monocyte

Production

TLRs

myd88

Sterols

Lipid rafts

HDL HDL-FC

ABCG1ABCA1NF-κB

Macrophages in spleen

↑ production of monocytes and neutrophils in BM

M-CSF/MCP-1/G-CSF

Slide courtesy of Alan Tall, MD.

ABCA1

ABCG1

LL

LLipid

rafts

IL-3Rβ

Proliferation

HSPCL

C

CC

MonocytosisC

L

Data from Yvan-Charvet L, et al. Science. 2010;328(5986):1689-1693.

HDL

C

HDL

CC

ApoA-I/

HDL Atherosclerosis

L = lipid rafts; C = cholesterol; HSPC = hematopoietic stem and progenitor cell

Direct Link Between the CholesterolEfflux Pathway and Hematopoiesis

Formation of Dysfunctional ApoA-I

• Lipid-poor apoA-I in atheroma is cross-linked and oxidized resulting in impaired ABCA1 interaction and reduced ABCA1-mediated macrophage cholesterol efflux

• Oxidation of Try72 on apoA-I is a site-specific target for MPO-dependent oxidation that results in the formation of oxindolyl alanine (2-hydroxyl-tryptophan, or 2-OH –Trp) moiety

Huang Y, et al. Nat Med. 2014;20:193-203.

Formation of a Dysfunctional ApoA-I


An Abundant Dysfunctional ApoA-I in Human Atheroma


HDL Functionality

• Evaluation of HDL function begins with measures of

HDL particle concentration, and expression of specific

functions per particle.

• Macrophage cholesterol efflux is the most important

aspect of “reverse cholesterol transport”. The most

widely used tests include radiolabeled cholesterol and

BODIPY.

• Anti-oxidant and anti-inflammatory properties of HDL

are available with a functional assay and static assay.

• Ongoing investigations are evaluating specific HDL

protein and lipid modifications (structure) with

specific HDL functions..

what’s new in hdl function testing?...modified after rosenson rs, brewer hb jr. davidson ws, fayad...

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