Inflammation, HDL function, and
atherosclerosis
ACR/ARHP Scientific Meeting
Chicago
November 8, 2011
Daniel J. Rader, MD
University of Pennsylvania
Daniel J Rader, MD
Disclosure of Financial
Relationships
D Rader owns equity in VascularStrategies and
consults for multiple companies developing new therapies
targeted toward HDL metabolism, reverse cholesterol
transport, inflammation, and atherosclerosis.
Inflammation, HDL function, and
atherosclerosis
Reverse cholesterol transport and effect of
inflammation
Systemic inflammation and atherosclerosis:
implications for new therapies
B
VLDL, LDL,
remnants,
Lp(a)
TG, CE
Lipoproteins and Atherosclerosis
B
VLDL, LDL,
remnants,
Lp(a)
HDL
A-I
TG, CE
CE
Lipoproteins and Atherosclerosis
Raising plasma HDL-C levels will
reduce CV events.
The HDL-C hypothesis
The HDL-C hypothesis has taken
some recent hits
Genetics:
• ABCA1 deficiency and variants
• LCAT deficiency and variants
• Endothelial lipase variants
Interventions:
• Torcetrapib (ILLUMINATE)
• Fenofibrate (ACCORD)
• Niacin (AIM-HIGH)
Couzin, Science 2008
HDL
Anti-oxidant
Anti-thrombotic
Anti-inflammatory
Cholesterol efflux and
reverse cholesterol transport
Anti-atherogenic HDL functions
NO-promoting
Reverse Cholesterol Transport
A-I
Liver
CE
CE
FC
LCAT
FC
Bile
SR-BI
A-I
ABCA1
Macrophage
FC
ABCG1
Quantitation of macrophage to feces reverse
cholesterol transport in vivo
Plasma
3
H-cholesterol,
AcLDL
3
H-Chol
Bile
Feces
3
H-FC
3
H-chol
3
H-BA
3
H-BA
3
H-BA
3
H-FC
3
H-FC
Macrophage
Impact of intervention on macrophage reverse
cholesterol transport
Plasma
3
H-cholesterol,
AcLDL
3
H-Chol
Bile
Feces
3
H-FC
3
H-chol
3
H-BA
3
H-BA
3
H-BA
3
H-FC
3
H-FC
Macrophage
Transgenics,
knockouts, Viral
vectors: cDNA,
siRNA
Pharmacologic
interventions
SR-BI knockout mice have elevated HDLlevels
but impaired RCT and increased
atherosclerosis
Liver
CE
FC
FC
LCAT
FC
Bile
SR-BI
A-I
ABCA1
Macrophage
CE
X
A-I
CE
A-I
Liver
CE
CE
FC
FC
LCAT
FC
Bile
SR-BI
A-I
ABCA1
Macrophage
CE
FC
SR-BI
HDL
RCT
atherosclerosis
SR-BI
HDL
RCT
atherosclerosis
Zhang, et al, J Clin Invest, 2004
Hepatic SR-BI is a regulator of macrophage
reverse cholesterol transport
Does systemic inflammation
impair macrophage cholesterol
efflux and reverse cholesterol
transport?
Systemic inflammation and effect on reverse
cholesterol transport
Plasma
3
H-cholesterol,
3
H-Chol
Bile
Feces
3
H-FC
3
H-BA
3
H-BA
3
H-BA
3
H-FC
3
H-FC
Macrophage
Endotoxin
(LPS)
Systemic inflammation markedly impairs
reverse cholesterol transport in mice at
all steps of the process
A-I
Liver
CE
CE
FC
LCAT
FC
Bile
SR-BI
A-I
ABCA1
Macrophage
FC
ABCG1
Plasma HDL-C concentration is
not reliably predictive
macrophage RCT or
atherosclerosis risk in mice.
Patient with high HDL-C but CAD
• 67 year old female
• s/p ACS with PCI and stent
• On high dose potent statin
• Fasting Lipid Profile:
– Total Chol 178 mg/dL
– Triglycerides 115 mg/dL
– LDL-C 68 mg/dL
– HDL-C 87 mg/dL
Patient with high HDL-C but CAD
• 67 year old female
• s/p ACS with PCI and stent
• On high dose potent statin
• Fasting Lipid Profile:
– Total Chol 178 mg/dL
– Triglycerides 115 mg/dL
– LDL-C 68 mg/dL
– HDL-C 87 mg/dL
Is her HDL functional?
Could individuals differ in the
ability of their HDL to promote
macrophage cholesterol efflux?
3
H-Cholesterol labeled
J774 MACROPHAGES
HDL
(apoB-depleted sera)
% cholesterol
efflux
cAMP
Measuring HDL Efflux Capacity
Is Serum Cholesterol Efflux Capacity
associated with Atherosclerotic CVD?
Carotid IMT (n~400)
Angiographic CAD (n~800)
Khera A, Cuchel M, et al. N Engl J Med 2010
Risk Factor
Odds Ratio
(95%CI)
value
P
Diabetes
2.27 (1.46 – 3.53) <0.001
Hypertension
1.91 (1.38 – 2.65) <0.001
Smoking
1.24 (0.90 – 1.71)
0.18
LDL Cholesterol
1.01 (0.86 – 1.19)
0.90
BMI
0.87 (0.74 – 1.03)
0.11
HDL Cholesterol
0.85 (0.70 – 1.04)
0.11
Efflux Capacity
0.74 (0.62 – 0.89)
0.002
Multivariable odds ratios for CAD according to selected risk factors.
Angiographic CAD efflux capacity study:
Predictors of CAD Status
Khera A, Cuchel M, et al. N Engl J Med 2010
Do individuals with systemic
inflammation have reduced
HDL efflux capacity?
Inflammation and cholesterol efflux
capacity
-12 0h 24h
48h
Lipid +
lipoprotein
Serum efflux
capacity
Whole blood
and adipose
isolation
Cytokines,
Inflam &
oxidant
markers
Insulin
sensitivity
Lipid +
lipoprotein
Serum efflux
capacity
Whole blood
and adipose
isolation
Cytokines,
Inflam &
oxidant
markers
Insulin
sensitivity
di
sc
harg
e
Saline LPS
(3 ng/kg IV)
Ad
m
it
to
G
CR
C
Experimental endotoxemia in
humans as a model of inflammation
1
2
3
**
C
A
1
d
e
p
e
n
d
e
n
t
e
ff
lu
x
(%
E
ff
lu
x
/4
h
)
Experimental endotoxemia in humans alters HDL and
impairs its capacity to efflux cholesterol
Cholesterol efflux capacity
-24
-18
-12
-6
0
6
12
18
24
0
20
40
60
80
100
Time post LPS (h)
S
p
L
A
2
m
as
s
(n
g
/m
l)
***
***
***
SPLA2
***
***
200
400
600
ndo
th
e
li
a
l
li
p
a
s
e
(ng
/m
l)
Endothelial lipase
SAA
Systemic inflammation alters HDL
composition and function
A-I
Liver
CE
CE
FC
LCAT
FC
Bile
SR-BI
A-I
ABCA1
Macrophage
FC
ABCG1
A-I
CE
SAA
EL
sPLA2
MPO
CV Disease in Psoriasis
Outcome
Hazard Ratio
(adjusted for CV risk
factors)
MI
1
1.58
Stroke
2
1.43
CV Death
3
1.57
MACE
4
1.53
Figure. Abuabara K, et al. Br J Dermatol. 2010 Sep;163(3):586-92.
1. Gelfand, JM et al. JAMA. 2006;296:1735-1741.
2. Gelfand, JM et al. J Invest Dermatol .2009; 129:2411-2418.
3. Mehta, NN et al. Eur Heart J. 2010;31:1000-6
4. Mehta, NN et al. Am Journal Of Medicine . 2011; 776: 1-7.
Raising plasma HDL-C levels will
reduce CV events.
The HDL-C hypothesis
X
The HDL flux hypothesis
Promoting cholesterol efflux and
RCT will reduce CV events.
Cholesteryl ester transfer protein (CETP)
transfers cholesterol out of HDL
A-I
Liver
CE
CE
FC
FC
LCAT
FC
Bile
SR-BI
A-I
ABCA1
Macrophage
CE
B
LDLR
VLDL/LDL
CETP
CE
TG
FC
CETP Deficiency is Associated with
Markedly Increased HDL-C Levels
Liver
CE
FC
FC
LCAT
FC
Bile
SR-BI
A-I
ABCA1
Macrophage
CE
B
LDLR
VLDL/LDL
CETP
CE
TG
A-I
CE
FC
X
CETP Inhibition as a Strategy to
Raise HDL
A-I
Liver
CE
CE
FC
FC
LCAT
FC
Bile
SR-BI
A-I
ABCA1
Macrophage
CE
B
LDLR
VLDL/LDL
CETP
CE
TG
FC
X
CETP inhibitor
Promoting Reverse Cholesterol
Transport
A-I
Liver
CE
CE
FC
LCAT
FC
Bile
SR-BI
A-I
ABCA1
Macrophage
FC
ABCG1
• LXR agonism
• apoA-I infusion
• apoA-I upregulation
• LCAT infusion/activation
• miR-33 antagonism
DNA variants
HDL-C
Slide courtesy of Dr Sek Kathiresan
DNA variants
HDL efflux capacity
HDL
Anti-oxidant
Anti-thrombotic
Anti-inflammatory
Cholesterol efflux and
reverse cholesterol transport
Anti-atherogenic HDL functions
NO-promoting
Inflammation, HDL function, and
atherosclerosis
Reverse cholesterol transport and effect of
inflammation
Systemic inflammation and atherosclerosis:
implications for new therapies
Atherosclerosis is in part an
inflammatory disease
DNA Variants
Coronary disease
N ATU RE GEN ETI CS VO LU M E 43 | N U M BER 4 | APRIL 2011 3 3 3 We performed a meta-analysis of 14 genome-wide association
studies of coronary artery disease (CAD) comprising 22,233 individuals with CAD (cases) and 64,762 controls of European descent followed by genotyping of top association signals in 56,682 additional individuals. This analysis identified 13 loci newly associated with CAD at P < 5 × 10−8 and confirmed the association of 10 of 12 previously reported CAD loci. The 13 new loci showed risk allele frequencies ranging from 0.13 to 0.91 and were associated with a 6% to 17% increase in the risk of CAD per allele. Notably, only three of the new loci showed significant association with traditional CAD risk factors and the majority lie in gene regions not previously implicated in the pathogenesis of CAD. Finally, five of the new CAD risk loci appear to have pleiotropic effects, showing strong association with various other human diseases or traits. It has been estimated that heritable factors account for 30%–60% of the inter-individual variation in the risk of coronary artery disease (CAD)1. Recently, genome-wide association studies (GWAS) have identified several common variants that associate with risk of CAD2. However, in aggregate, these variants explain only a small fraction of the heritability of CAD, probably partly due to the limited power of previous studies to discover effects of modest size. Recognizing the need for larger studies, we formed the transatlantic Coronar y ARter y DIsease Genome wide Replication and Meta-analysis (CARDIoGRAM) consortium3. We perfomed a meta-analysis of 14 GWAS of CAD comprising 22,233 cases and 64,762 controls, all of European ancestry (Supplementary Table 1a–c and Supplementary Fig. 1). We then genotyped the lead SNPs within the most promising previously unidentified loci as well as a subset of previously reported CAD loci in up to 56,682 additional subjects (approximately half cases and half controls) (Supplementary Table 2a,b). Lastly, we explored potential mechanisms and intermediate pathways by which previously unidentified loci may mediate risk.
Nine of the twelve loci previously associated with CAD through individual GWAS achieved genome-wide significance (P < 5 × 10−8) in our initial meta-analysis (Table 1 and Supplementary Table 3). We were, however, unable to test the previously reported associa-tion with a haplotype and a rare SNP in LPA in our GWAS data4,5, but we observed robust association with the rare LPA variant in our replication samples through direct genotyping (Table 1).
Thus, 10 of the 12 loci previously associated with CAD at a genome-wide significance level surpassed the same threshold of significance in CARDIoGRAM.
We selected 23 new loci with a significance level of P < 5 × 10−6 in the meta-analysis for follow up (Online Methods and Supplementary Note). Taking the number of loci into consideration, our replication study had >90% power to detect effect sizes observed in the GWAS meta-analysis. Of the 23 loci, 13 replicated using our a priori defini-tion of a validated locus, that is, showing independent replicadefini-tion after Bonferroni correction and also achieving P < 5 × 10−8 in the combined discovery and replication data (Table 2, Fig. 1 and Supplementary Figs. 2 and 3). Results for all loci from the replication phase are shown in Supplementary Tables 4 and 5.
The 13 new loci had risk allele frequencies ranging from 0.13 to 0.91 and were associated with a 6% to 17% increase in the risk of CAD per allele (Table 2). Out of the 13 new loci, the additive model appeared most appropriate for 6 whereas the recessive model performed best at 5 and the dominant model at 2 loci (Supplementary Table 6).
In sub-group analyses, 20 out of 22 loci with P < 5 × 10−8 (known and new loci combined; for one locus, age subgroups were not available) had higher odds ratios for early onset than for late onset CAD (P = 1.2 × 10−4 for observed versus expected; Supplementary Table 7). The CAD loci showed consistent associations irrespective of case definition, although the odds ratios for most individual SNPs tended to be slightly greater for cases with angiographically proven CAD than for cases with unknown angiographic status (P = 0.019 for observed versus expected) (Supplementary Table 8). In contrast, sub-group analyses in males and females revealed no sex-specific effects for any risk alleles (Supplementary Table 7) or for their observed versus expected pattern of association (P = 0.4).
Among 7,637 CAD cases and 7,523 controls for whom we had individual level genotype data, the minimum and maximum number of risk alleles observed per individual was 15 and 37, respectively, when considering 23 CAD susceptibility loci. The mean weighted risk score was significantly higher for cases than for controls (P < 10−20). Furthermore, being in the top tenth percentile or lowest tenth percen-tile of the weighted score was associated with an odds ratio for CAD of 1.88 (95% CI 1.67–2.11) and 0.55 (95% CI 0.48–0.64), respectively, compared to the fiftieth percentile. The change in odds ratio for CAD across a broader spectrum of categories of the weighted score is shown in Supplementary Figure 4.
Large-scale association analysis identifies 13 new
susceptibility loci for coronary artery disease
* A full list of authors and affiliations appears at the end of the paper. Received 10 August 2010; accepted 10 February 2011; published online 6 March 2011; doi:10.1038/ng.78 4
L E T T E R S © 2 0 11 N atu re A m eri ca , In c . A ll rig hts r es erv ed.
