From HDL Structure to Function - How
Structural Information Can Help Address
Clinical Need
March 17, 2012
Teaching Objectives
There exists no definitive test for risk of
cardiovascular disease. Age and family history
remain the best indicators of cardiovascular disease
risk.
The ability of High Density Lipoproteins (HDL) to
mobilize cholesterol from the artery wall is the best
functional aspect of HDL in determining risk for
cardiovascular disease.
Adult Patient Population
81 million people have some form of
cardiovascular disease in the United
States (high blood pressure, CAD,
stroke and heart failure)
American Heart Association 2006 Annual Report
Problem : There Are no Accurate
Diagnostics for Coronary Artery Disease
(CAD) at the Individual Level
HDL-C and LDL-C are the “good” and “bad”
cholesterol, respectively
HDL-C is the best known independent risk
factor for CAD monitored in the clinic
HDL-C and LDL-C are indicative of CAD but
not sufficiently correlated with CAD to serve
as a diagnostic marker
Current HDL-C Measurements
Analogous to measuring flux by taking a
snapshot of the number of cars on a highway
A more appropriate approach is examining the
dynamic flow of cholesterol or by analogy, the
rate of traffic at the on ramps and off ramps
Existing HDL-C assays look at plasma
concentration or particle numbers (NMR) not flux
Current HDL-C Measurements
Female patients generally do not present as at
risk by this metric until after menopause
CAD is the leading cause of sudden death in
women above the age of 35
Children are a growing risk group for early onset
cardiovascular disease due to increased
incidence of obesity and diabetes (>30% are
obese - BMI > 95%)
Existing HDL-C assays do not provide a clear
indication of cardiovascular risk in women and
children
Contributors to Risk of CAD
Rate of Cholesterol Deposition (LDL-C)
Rate of Cholesterol Clearance (HDL
function)
Plaque stability (not well understood)
HDL Cholesterol Efflux Capacity A Critical Aspect of HDL Function
The cholesterol efflux potential of plasma is
diagnostic of risk for CAD
Dan Rader & George Rothblat found that the
ABCA1 mediated cholesterol efflux capacity
of plasma is strongly associated with CAD
status. Inverse correlation odds ratio of 0.75
(95% CI; p<0.002)
Khera, et al., (2011) N Engl J Med 364, 127-135.
Standard Panels
Total Cholesterol
LDL-C
HDL-C
Triglycerides
Lp(a)
VLDL (subfractions)
LDL (particle
distribution)
HDL (particle
distribution)
•
•
•
•
Homocysteine
apoE
Cyp2C19
Lp-PLA2
CRP
Fibrinogen
NT-proBNP
Hemoglobin A1c
The Ideal Assay of HDL Function
Small Volume
50 µl of Sample
Convenient Sample
Plasma or Serum
Highly Accurate
< 4% Deviation
Rapid
1-10 Minutes per Sample
Scaleable
Can be automated
Reverse Cholesterol Transport Pathway
Reverse Cholesterol Transport Pathway
Reverse Cholesterol Transport Pathway
Fluorescence Resonance Energy
Transfer
FRET-based Detection of ApoA-I Lipoprotein Exchange
5
:
1
ApoA-I is the most specific probe of
HDL known
HDL Oxidation impairs apoA-I
exchange
HDL Oxidation impairs cholesterol
efflux
Subjecting apoA-I to the MPO-H2O2-nitrite
system impairs an early step in HDL formation,
namely the interaction of apoA-I with ABCA1In
patients with CAD, chlorinated HDL is present in
atherosclerotic plaques.
Chlorination impairs the ability of apoA-I to exit
the HDL particle.
Electron Paramagnetic Resonance
Spectroscopy (EPR)
Well suited for the
examination of proteins with
dynamic structures
Reports the structural
environment (regional
flexibility and solvent
accessibility) and the
interaction distance
between spin labels.
EPR Spin Coupling - A High Resolution
Atomic Ruler
> 20Å
< 20Å
EPR Spectral Line Shape is Indicative of
Secondary Structure
CONFIDENTIAL
Free Label
Beta Sheet
Random Coil
Alpha Helix
Scanning EPR
N
CONFIDENTIAL
C
Spin Label Solvent Accessibility Identifies
Secondary Structure
Beta Sheet
Accessibility (π)
Accessibility (π)
Alpha Helix
Residue #
CONFIDENTIAL
Residue #
Interpretation of Solvent Accessibility
CONFIDENTIAL
Interpretation of Solvent Accessibility
Utilize difference between lipid-free and
lipid-bound apoA-I to measure HDL
dynamics
CONFIDENTIAL
J. Lagerstedt et al., JBC, 2011
Key Transition Site for Assessment
of HDL Function
CONFIDENTIAL
Human EPR Measurements
Patient ID
Statin Treatment
(y/n)
Diagnosed as
Diabetic (y/n)
LDL
(mg/dL)
HDL
(mg/dL)
TG
(mg/dL)
BMI
N1
N
N
90
43
90
21.5
N2
N
N
100
41
110
24.3
D1
Y
Y
83
42
182
46.6
M1
N
N
138
40
130
47.5
CONFIDENTIAL
Human EPR Measurements
Patient N1
Patient M1
Patient N2
Patient D1
1.5 Min
Lipid-Free
CONFIDENTIAL
Human EPR Measurements
Patient N1
Patient M1
Patient N2
Patient D1
1.5 Min
4 Min
Lipid-Free
CONFIDENTIAL
Human EPR Measurements
Patient N1
Patient M1
Patient N2
Patient D1
1.5 Min
4 Min
6 Min
Lipid-Free
CONFIDENTIAL
Human EPR Measurements
Patient N1
Patient M1
Patient N2
Patient D1
1.5 Min
8 Min
CONFIDENTIAL
4 Min
6 Min
Lipid-Free
Human EPR Measurements
Patient N1
Patient M1
Patient N2
Patient D1
1.5 Min
8 Min
CONFIDENTIAL
4 Min
10 Min
6 Min
Lipid-Free
The EPR Assay of HDL Function
Patient ID
Statin Treatment
(y/n)
Diagnosed as
Diabetic (y/n)
LDL
(mg/dL)
HDL
(mg/dL)
TG
(mg/dL)
BMI
N2
N
N
100
41
110
24.3
CONFIDENTIAL
EPR Measurements of HDL
Function
Representative Sample Set
Summary
There exists no definitive test for risk of
cardiovascular disease
Existing tests are complex and evaluate multiple
variables that can yield contradictory results
The measure of HDL cholesterol efflux function in
plasma can yield insightful information on
cardiovascular risk and diabetic status
Future Objectives
Identify limitations of the assay (sample size,
material storage, etc)
Expand cohort analysis to include broader
spectrum of patients
Assess the predictive value of the assay in the
context of CAD, diabetes and Alzheimer’s disease
Test future medications and the utility of the assay
as a companion diagnostic
Acknowledgments
Oda Lab
UCLA
Giorgio Cavigiolio, PhD
Jing Zhao, PhD
Ioanna Pagani, PhD
Bodo Paradi, PhD
Braydon L. Burgess, MS
Andrea Agcaolli
Mandy Baker
Sajiv Chandradas
Nicole DeValle
Ethan G. Geier
Leah Goodman
Yumin He
Daiva Pleskyte
Shanna Tucker
Jake Lusis, PhD
Margarete Mehrabian, PhD
Accent Assays
Shawn Hayes, PhD
Univ. of British Columbia
Cheryl Wellington, PhD
Sophie Stukas
UC Davis
John C. Voss, PhD
Jens Lagerstedt, PhD
Madhu S. Budamagunta, PhD
Univ. of Washington
Jay Heinecke, MD
Baohai Shao, PhD
Jack Oram, PhD
Conclusions
ApoA-I binding rates to HDL correlate with
plasma’s efflux capacity, a potent diagnostic of
CAD
We are developing a powerful assay that can
identify individuals with reduced cholesterol
efflux capacity, even for individuals whose lipid
panels appear normal