DOI: 10.1161/CIRCULATIONAHA.113.006824
Assessment of Atherosclerosis in Chronic Granulomatous Disease
Running title: Sibley et al.; Atherosclerosis in CGD
Christopher T. Sibley, MD1; Tyra Estwick, RN2; Anna Zavodni, MD1,3; Chiung-Yu Huang, PhD4;
Alan C. Kwan, BA1; Benjamin P. Soule, MD2; Debra A. Long Priel, MS5;
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
Alan T. Remaley, MD, PhD6; Amanda K. Rudman Spergel, MD2; Evrim B. Turkbey, MD1;
Douglas B. Kuhns, PhD5; Steven M. Holland, MD7; Harry L. Malech, MD2;
Kol A. Zarember, PhD2; David A. Bluemke, MD PhD1; John I. Gallin, MD2
1
Dept of Radiology and Imaging Sciences, NIH Clinical Center, National Institutes of Health,
Bethesda,, MD;; 2Laboratory of Host Defenses, National Institute of Allergy and Infectious
Dise
Di
Diseases,
seas
se
ases
as
es,, NI
es
N
NIH,
H,, B
Bethesda,
e hesda, MD; 3Current-Depa
et
Current-Department
art
rtm
ment of Medica
Medical
al Im
Imaging,
mag
agin
ing, Sunnybrook Health
in
Sci
Sc
Sciences
ie
iences
Center,
Centeer,, U
University
nive
ni
vers
rsit
ityy of T
Toronto,
oron
or
onto
to, To
Toronto,
Toro
ront
n o, ON;
N;; 4Bi
Biostatistics
Bios
o taati
tist
sttic
icss Re
Research
ese
s ar
arch
ch B
Branch,
ranc
ra
nch,
h,, N
NIAID,
IAID
IA
ID,
5
NIH, Bet
Bethesda,
the
hesd
sda,, M
sd
MD;
D; Ap
Applied/Developmental
Appl
plie
ieed/
d/De
Devvelopm
De
pmentaal Re
pm
Research
eseear
arch
ch D
Directorate,
irr cto
irec
tora
raatee, Le
Leidos
eid
dos B
Biomedical
i medi
io
dica
di
caal
Re
Research,
, Inc.
I c. Frederick
In
Fredeeriick National
Natio
ona
nall Laboratory
Laborattorry for
fo
or Cancer
Canc
Ca
nceer
nc
er Research,
Reseearrch, Frederick,
Freedeeriick
ck, MD;
MD; 6N
National
atioonaal
Hear
He
Heart,
art,
t,, L
Lung,
ung,
ung
g, aand
nd B
Blood
lood Institute,
lood
Inssti
titu
tute
tu
te,, NIH,
NIH, B
Bethesda,
ethe
et
hesdda, MD;
he
MD; 7La
Laboratory
Labo
booraato
tory
ry ooff Cl
C
Clinical
lin
inic
in
icaal
al IInfectious
nffecti
ectiou
ouus
Diseases,
Dise
Di
seas
a es, NI
NIAI
NIAID,
AIID, N
NIH,
IH, Be
IH
Beth
Bethesda,
thes
esda
da, MD
Address
Addr
Ad
dres
esss for
for Correspondence:
Corr
Co
rres
espo
pond
nden
ence
ce::
John I. Gallin, MD
Laboratory of Host Defenses
National Institute of Allergy and Infectious Diseases, NIH
10 Center Drive, RM 6-2551, MSC 1504
Bethesda, MD 20892-1504
Tel: 301-496-4114
Fax: 301-402-0244
E-mail: jgallin@cc.nih.gov
Journal Subject Codes: Atherosclerosis:[134] Pathophysiology, Imaging of the brain and
arteries:[58] Computerized tomography and magnetic resonance imaging, Atherosclerosis:[150]
Imaging, Myocardial biology:[91] Oxidant stress
1
DOI: 10.1161/CIRCULATIONAHA.113.006824
Abstract
Background—Patients with Chronic Granulomatous Disease (CGD) suffer immunodeficiency
due to defects in the phagocyte NADPH oxidase (NOX2) and concomitant reduction in reactive
oxygen intermediates. This may result in a reduction in atherosclerotic injury.
Methods and Results—We prospectively assessed the prevalence of cardiovascular risk factors,
biomarkers of inflammation and neutrophil activation, and the presence of MRI and CT
quantified subclinical atherosclerosis in the carotid and coronary arteries of 41 CGD patients and
25 healthy controls in the same age range. Uni- and multivariable associations between risk
factors, inflammatory markers and atherosclerosis burden were assessed. CGD patients had
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
significant elevations in traditional risk factors and inflammatory markers compared with
g; hypertension,
yp
, hsCRP,, oxidized LDL,, and low HDL. Despite
p this,, CGD
controls,, including;
patients had a 22% lower internal carotid artery wall volume compared with con
ntr
trol
olls (3
(361
61.3
61
.3 ±
controls
(361.3
h
76.4 mm3 vs. 463.5 ± 104.7 mm3, p<0.001). This difference was comparable in p47phox
and
gp91phox deficient subtypes of CGD, and independent of risk factors in multivariate regression
analysis.
arterial
calcification
was
CGD
an
naly
alysis
ysis. In ccontrast,
ontr
tras
ast,
t, pprevalence
reva
re
vale
l ncce of ccoronary
orron
naryy ar
arte
eri
riaal cal
lci
cifi
fica
c tion
nw
ass ssimilar
imilarr bbetween
im
e ween
et
nC
G
GD
patients
pati
ien
e ts and controls
con
onttrolss (14.6%,
(14.
4.6%
6%, CGD,
6%
CGD
D, and
an
nd 6.3%,
6.33%, controls,
co
ont
ntrrols
lss, p=0.39).
p=
=0.
0.39
39)).
Conclusions—The
Conc
Co
ncclu
usionss—The ob
observation
bseervat
attio
ionn by
yM
MRI
RI ooff rreduced
RI
edduce
duceed ca
carotid
arotid
otid bu
but
ut nnot
ut
ott cor
coronary
oron
onar
on
aryy arte
aartery
rtery
ry
y
atherosclerosis
CGD
patients
the
athe
ero
roscle
lero
rosiss in
i C
GD pat
atieent
ntss despite
de
the high
high prevalence
prev
val
alen
ence
ce of
of traditional
trad
adit
itio
iona
n l risk
r sk ffactors
ri
a to
ac
t rs rraises
aiise
sess
questions
about
clinically
significant
q estions ab
qu
bou
outt th
thee ro
role
le ooff NO
NOX2
X2 iin
n the
th
he pathogenesis
path
pa
thog
th
ogen
og
e es
en
esis
is ooff cl
lin
iniica
call
llyy si
ll
sign
gnif
gn
ific
if
ican
ic
antt athe
aatherosclerosis.
the
hero
rosc
ro
sclerosis.
Additional high-resolution studies in multiple vascular beds are required to address the
therapeutic potential of NOX-inhibition in cardiovascular diseases.
Clinical Trial Registration Information—clinicaltrials.gov. Identifier: NCT01063309.
Key words: atherosclerosis, inflammation, carotid artery, coronary, immune system
2
DOI: 10.1161/CIRCULATIONAHA.113.006824
Introduction
Chronic Granulomatous Disease (CGD) is an inherited immunodeficiency caused by mutations
in genes encoding the main components of the phagocyte NADPH oxidase (NOX2) ,gp91 phox,
p22 phox, p47 phox, p67 phox, and rarely p40 phox, resulting in impaired production of superoxide
anion and other reactive oxygen intermediates.1 Hemizygous mutations in gp91phox cause the
most common form, X-linked CGD (X-CGD), while autosomal CGD (A-CGD) is due to
mutations in the other subunits. 2 CGD manifests clinically with recurrent infections and
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
granulomatous complications. 3Lower levels of residual ROS production by neutrophils are
associated with earlier mortality. 2 Significantly elevated production of pro-inflammatory
mediators by CGD myeloid cells (e.g., IL-8 4, LTB4 5), and decreased neutrophil
neutrophiil ap
apoptosis
pop
pto
osi
siss 6ar
are
also thought to contribute to the excessive inflammation secondary or independent of infection
that
hatt is
is often
ofte
of
tenn seen
te
seen
n in
in CGD.
Beyond
nd a role
rolle inn immune
imm
mmuune
une defense,
defe
de
fennse
nse, increased
inncrreassed
d inflammation
innfl
nflamm
mat
atio
io
on with
with aassociated
sssoci
ociate
iatedd in
incr
increased
crreaased
sed
reactive
NOX
eac
acti
tiive
v oxygen
oxy
xyggen
gen species
sppec
eciies generated
g neera
ge
rate
tedd by
te
b a family
fam
amiilyy of
o N
OX
X pproteins,
rotteiins
ro
n , NOX2,
NOX2
NO
X22, NOX1
NO
OX1 and
and NOX4,
NOX
OX4,
4, have
haave
av
been implica
implicated
ateed inn the
the pathogenesis
patho
hoge
ho
g ne
nesi
s s off cardiovascular
si
car
ardi
diov
di
ovas
ov
a cu
as
cula
laar disease
dise
di
seas
se
asee and
as
and atherosclerosis.
athe
at
hero
he
rosc
ro
scle
sc
l ro
le
osi
sis.
s. 7 N
NADPH
ADPH
oxidases contribute to the differentiation and migration of vascular smooth muscle cells,
endothelial cell response to nitric oxide, and are highly expressed in atherosclerotic plaque.8-12
Reduced NADPH oxidase activity may reduce vascular inflammation and thereby decrease
susceptibility to atherosclerosis – a possibility that makes pharmacologic inhibition of NOX a
potential target of therapy for cardiovascular diseases. 13
Mouse models of NOX deficiency have yielded conflicting results on atherosclerosis
progression. 14-18 Pharmacologic inhibition of NOX in murine models has succeeded in reducing
atherosclerosis progression. 19 Studies in gp91 phox and p47 phox - deficient human CGD subjects
3
DOI: 10.1161/CIRCULATIONAHA.113.006824
demonstrated significant differences in cardiovascular function. Enhanced arterial dilatation and
vascular endothelial function following ischemia and reperfusion have been noted in CGD. 20, 21
Enhanced arterial dilatation was noted in male CGD patients and lower carotid intimal-medial
thickness in X-CGD patients and female carriers compared to healthy subjects20 22, suggesting
that even a 50% reduction in NOX2 function is sufficient for cardiovascular protective effects.
To date, no studies have reported the effects of NOX2 deficiency in the coronary circulation. We
investigated markers of inflammation and the prevalence of subclinical carotid and coronary
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
atherosclerosis using noninvasive MRI and CT techniques in normal controls and patients with
gp91 phox or p47 phox CGD.
Methods
Patients
Pati
tien
en
nts
phox
phox
Patients
P
Pati
a ien
e ts over
oveer 188 years
yeears
ears of
of age
age with
with a clinical
cli
linnic
ical
al diagnosis
dia
i gnnosis of
of CGD
CGD and
a d either
an
e th
ei
ther
err gp9
gp91
p911 phox
p9
o pp47
or
47 phox
de
deficiency
efi
fici
c en
ci
ncy
y and
and healthy
heaaltthy
y volunteers
volun
nteeer
erss in the
the
he same
sam
me age
agee range
ran
ange
ge w
were
eree en
enrolled
nro
olled
ed fr
from
rom 2010
rom
22010-2014
010--20
-20144 in
in aan
n
IRB
NIH
Clinical
RB approved
appr
ap
prov
pr
oved
ov
ed protocol
pro
roto
toco
to
coll (10-I-0029)
co
(100-I
(1
0-I-00
I-00
0029
29)) conducted
29
cond
co
nduc
nd
ucte
uc
tedd at the
te
the N
IH C
lini
li
nica
ni
call Center.
ca
Cent
Ce
nter
nt
er. All
er
All subjects
subj
su
bjec
bj
ects
ec
ts pprovided
rovi
ro
vide
vi
dedd
de
documented informed consent. CGD diagnoses were confirmed by genetic sequencing and/or
western blotting as well as quantitation of reactive oxygen species.2 Volunteers underwent
history and physical examination to confirm that they were free of clinical cardiovascular disease
or active systemic infection. Patients with fever, atrial fibrillation or contraindication to
gadolinium or MR imaging were excluded. Patients with contraindication to iodinated contrast
were eligible to undergo MR and non-contrast CT (calcium scoring). No CGD patients in this
study had received a bone marrow transplant.
Acquisition and Analysis of Carotid MR Imaging
Carotid wall volume was determined to assess the extent of atherosclerotic disease.23 MR
4
DOI: 10.1161/CIRCULATIONAHA.113.006824
imaging was performed on a 3 T clinical scanner (Verio, Siemens) using four-channel carotid
coils (Machnet). T1 pre & post contrast and T2 weighted fat-suppressed, ECG-gated black blood
images were obtained using double inversion recovery fast spin echo sequences. Post-contrast T1
weighted images were obtained 5 minutes after an intravenous dose of 0.1 mmol/kg
gadopentetate dimeglumine (Magnevist, Bayer HealthCare). Scan resolution was 0.50 mm x 0.50
mm x 2.0 mm, with 5 consecutive slices and no gap obtained in each internal carotid artery
(ICA) beginning at the carotid bifurcation.
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
ICA wall volume was quantified using QPlaque (version 1.0, Medis) by a blinded
observer. The area within the external boundary of the vessel and the arterial lumen were semiautomatically contoured on post-contrast T1 images, using pre-contrast T1 and T2
T2 weighted
w ig
we
ght
hted
ed
images
mages to confirm vessel boundaries in slices with flow artifact. 24, 25 The corresponding volume
was obtained
was
obta
ob
tain
ta
ined
in
ed byy multiplying
mu
the area in each ima
mage
ma
gee by the slice th
hic
i knnes
esss and summing the total
image
thickness
nnumber
um
mb of slic
mber
slices
ces oobtained.
btai
bt
aine
ai
need. W
Wall
all vvolume
olum
ol
umee wass ccalculated
um
alcuula
ulated
d bby
y su
subtracting
ubt
btra
racctin
ingg lu
lumen
umeen vo
volu
volume
lu
ume
me ffrom
ro
om to
ttotal
tal
vess
ve
vessel
ssel
ss
el vvolume.
olum
ol
ume.
um
e.
Acquisition and
and Analysis
Anal
An
allys
y iss of
of Ca
Card
Cardiac
r ia
rd
iacc CT A
Angiography
ngio
ng
io
ogr
grap
a hy
ap
Coronary artery wall volume and calcium score were determined using CT angiography as
measures of atherosclerotic disease.26 Pre- and post-contrast CT imaging was performed using a
320-detector row scanner with slice thickness 0.5 mm (Aquilion ONE, Toshiba Medical
Systems). Calcium scoring was performed with prospective ECG gating with 350 msec gantry
rotation, 120 kV tube voltage and 300 mA tube current and quantified by the Agatston method.27
CT angiography was performed after administration of intravenous iopamidol (Isovue 370,
Bracco Diagnostics) using 120 kV tube voltage and tube current 350-580 mA dependent on
BMI. Beta-blockers were administered if the resting heart rate was >60 beats per minute.
5
DOI: 10.1161/CIRCULATIONAHA.113.006824
Image analysis was performed on a Vitrea FX workstation (Version 6.1, Vital Images).
The left main coronary artery and proximal coronary artery segments were segmented according
to previously published definitions.28 The lumen and external vessel wall were semiautomatically contoured at 0.5 mm intervals. The area between the lumen and external wall was
multiplied by slice thickness and summed over the total number of slices per segment to
calculate coronary arterial wall volume (mm3). Coronary artery wall volume was indexed to
segment length to account for variability in coronary anatomy. The resulting value is reported as
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
the coronary plaque index (mm2). Inter-reader reproducibility for this method in a sub-sample of
10 consecutive participants was excellent (ICC=0.97).
Plasma Analytes
Glucose, lipids, and biomarker measurements were obtained after • 12 hour fasting. Biomarker
analysis
an
nal
alys
ysis
ys
is was
was
a on
on plasma
plaasma
pl
as prepared from heparinized
heparinize
zedd blood
ze
blood by 2 cen
centrifugation
ntr
t iffuggation
at
steps at 500 g for 10
min.
minn.
n. Plasma aliquots
aliq
al
iquootss were
iq
were
ere stored
stor
st
orred
d in
in the
the vapor
vapoor phase
phaasee off liquid
liq
iquuidd N2 freezer
fre
reez
ezeer prior
priior to
to analysis.
anal
anal
alyysiss. IL-1ȕ,
IL
IL-1
L 1ȕ,
IL-2,
L-2
-2,, IL
IL-4
IL-4,
-4,, IL
IL-5
IL-5,
-5,, IL
IL-8,
L-8
-8,, IL
IL-10,
L-1
-10,
0, IIL-12p70,
L-12
L12pp70
12
p70, IIFN-Ȗ,
FN-ȖȖ, an
FN
and
nd TNF-Į
TN
NFF-Į
Į were
wer
eree measured
meas
me
a ureed
as
ed with
witth the
th
he TH1/TH2
TH
H1//TH
H2
Ultrasensitive
vee 110-Plex
0-Pl
0Plex
Pl
ex ((Meso
Meeso S
Scale
cale
ca
le D
Discovery,
issco
cove
very
ve
ry
y, Ga
Gait
Gaithersburg,
i he
it
h rssbu
burg
rg,, MD
rg
MD)) wh
whil
while
ilee IL
il
IL-6
IL-6,
-6
6, IL
IL-1
IL-17,
-1
17, GM-CSF,
MIP-1Į MIP-1ȕ, RANTES, soluble TNF-RI, soluble TNF-RII, and soluble IL-6R were measured
with customized multiplex cytokine immunoassays (Meso Scale Discovery) using a SECTOR™
Imager 6000 reader (Meso Scale Discovery). Standard curves were analyzed using nonlinear 4parameter curve fitting and unknowns were calculated based on the best fit equation. For
multiplex cytokine assays, an internal control was run on each plate to monitor inter-assay
variability (mean CV% was 50.4%; range 16.3 – 111.4%). An aliquot of standard was spiked
into a control plasma sample to determine the recovery of the specific cytokine standards in the
sample matrix (mean recovery was 95.7%; range 78.2 – 119.8%). Commercial immunoassays
6
DOI: 10.1161/CIRCULATIONAHA.113.006824
were used for: oxidized LDL (Mercodia, Uppsala, Sweden), matrix metallopeptidase 9 (R&D
Biosystems, Minneapolis, MN), lactoferrin (Oxis International ,Portland, OR), Į-defensins
(Hycult Biotech, Plymouth Meeting, PA), and myeloperoxidase (Meso Scale Discovery). Plasma
levels of total nitrate/nitrite (NOx-) and nitrite (NO2-) were determined using a Total Nitric Oxide
and Nitrate/Nitrite kit (KGE001, R&D Systems, Minneapolis, MN).
Statistical Analysis
Continuous outcomes were summarized using means and standard deviations (SDs) or medians
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
and interquartile ranges (IQRs) as appropriate, while counts and percentage values were reported
for binary outcomes. Continuous outcomes of different subgroups were compared using t-tests
with unequal variances. Binary outcomes were compared by chi-squared test. Spearman’s
Sppearm
r an
rm
a ’s
correlation coefficient was used to summarize the correlation between two continuous outcomes.
Univ
Un
ivar
iv
aria
ar
iabl
ia
blee and
bl
andd multivariable
mu
m
dels were used
d to ev
valua
alu te the unadjusted and
Univariable
linear regression mo
models
evaluate
ad
dju
ust
s ed effects
effeccts of
of potential
pottent
po
ntia
iall risk
ia
rissk
ri
sk ffactors
acto
ac
torrs oon
n car
ccarotid
arotidd w
alll
ll vvolume
ollum
umee an
andd co
oro
rona
narry
ry pplaque
laqque
la
que
adjusted
wall
coronary
bu
urd
rden
en.. Two
en
Tw
wo multivariable
mulltiivar
mu
ivaria
iabble
ble linear
liine
n ar regression
reg
egre
reesssio
ionn mo
mode
deelss were
werre constructed:
cons
cons
nstr
truc
ucte
uc
ted:
te
d: a bbase
asse mo
m
dell adjusted
de
ad
dju
ust
sted
ed
d for
for
o
burden.
models
model
age and gender,
gend
der
er,, an
nd a second
seeco
ond model
mod
odel
ell inc
nclu
nc
lu
udi
ding
ng ttraditional
raadittio
i naal cl
cclinical
inic
in
ical
ic
a ccardiovascular
al
a di
ar
d ov
ovaasc
s ullar risk
rissk factors.
and
including
Coefficients of determination R2 were reported to summarize the predictive power of a
regression model. Carotid wall volume measures were logarithmically transformed (on a
common log scale) before analysis to reduce skewness. Data analyses were carried out using
STATA 10.1 (College Station, TX). All p values were two-sided and were not adjusted for
multiple comparisons given the exploratory nature of this study. Graphpad Prism version 6.0d
was used for ANOVA with a Holm-Šídák post-test 29. P values less than 0.05 were considered
statistically significant.
7
DOI: 10.1161/CIRCULATIONAHA.113.006824
Results
Patients
Twenty-two CGD patients with mutations in gp91phox, 19 patients with mutations in p47 phox and
25 age- matched healthy controls were enrolled. Demographic and clinical characteristics of the
study population are shown in Table 1. CGD patients were 32.5 ± 9.4 years old and 76% were
male. CGD patients had significantly elevated cardiovascular risk factors compared with
controls, including more prevalent hypertension, decreased HDL-c, as well as increased oxidized
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
LDL. Amongst CGD patients, those with p47 phox mutations had significantly greater residual
superoxide production than those with gp91phox mutations (3.0 ± 0.7 vs. 1.7 ± 1.8 nM superoxide/
1 x 106 neutrophils/ 60 min respectively, p<0.01), about
a
1.3 % and 0.7% of super
superoxide
erroxxid
idee produced
prod
pr
oduc
od
u ed
uc
by neutrophils from normal volunteers (226.29 ± 3.01 nM superoxide/1 x 106 neutrophils/60
mi
min)
n)..2
n)
min).
Plasma
P
las
asma
sm marke
markers
kerrs ooff in
infl
inflammation
fllam
mma
mati
tion
on iin
nC
CGD
GD
D
CGD
CG
GD su
ssubjects
bjec
bj
ects
ec
ts hhad
ad hhigher
ad
ig
gheer le
llevels
vels
ve
lss of cclassical
lasssic
la
ical
ic
al acute
acu
cute
cu
tee phase
pha
hasse rreactants
eacttan
ea
antts
ts ((hsCRP)
hsC
hs
CRP)
CRP
P) aand
nd iinnate
nnaate
nn
at defe
ddefense
efens
nsse
(myeloperoxidase,
neutrophil
proteins (my
yel
elop
oper
op
erox
er
o id
ox
i as
a e, llactoferrin,
a to
ac
ofe
f rr
rrin
i , an
andd Į-defensins)
Į-d
def
efen
en
nsi
s ns
ns)) suggesting
suugg
gges
esti
es
ting
ti
ng
g increased
in
ncr
crea
ease
ea
sedd ne
se
neut
utro
ut
roph
ro
p il
degranulation and systemic inflammation. Pro-inflammatory cytokines such as IL-6 and TNFĮ as
well as the chemokines CXCL8 (IL-8) and CCL4 (MIP1ȕ) were significantly elevated in CGD
(Table 1).
Subclinical Atherosclerosis
Internal Carotid Artery
Patients with impaired NADPH oxidase function had 22% lower internal carotid arterial (ICA)
wall volume than age-matched healthy individuals. The mean ICA wall volume in CGD patients
was 361.3 ± 76.4 mm3 , compared with 463.5 ± 104.7 mm3 in controls (p<0.001. To reduce
8
DOI: 10.1161/CIRCULATIONAHA.113.006824
skewness the data were log-transformed prior to the statistical analyses that follow. The mean ±
standard deviation of the common log-transformed ICA wall volume in CGD patients was 2.55 ±
0.02 log10 mm3, compared with 2.66 ± 0.02 log10 mm3 in controls (p=0.0001, Table 2 & Figure
1A). No participant in either group had evidence of atherosclerotic plaque that resulted in
significant carotid luminal stenosis.
There were significant univariate associations between ICA wall volume and CGD,
HDL-c, IL-6, IL-10, IL-12p70 and IL-13. (Table 3) Only CGD (regression coefficient, -0.14;
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
95% CI, -0.21 – -0.08; p<0.001) male gender (regression coefficient, 0.06; 95% CI, 0.01 – 0.12;
p=0.02) and hypertension (regression coefficient, 0.09; 95% CI, 0.02 – 0.15; p=0.01) remained
card
dio
iova
v sccul
ular
ar rrisk
is
isk
as significant predictors of ICA wall volume after controlling for traditional cardiovascular
factors in a multivariate linear regression model (R
R2 = 0.50).
Coro
Co
Coronary
rona
ro
nary
na
ry
yP
Plaque
laqu
la
qu
ue In
IIndex
dex and Coronary Arterial
Arteria
iall Calcium
ia
Calcium (CAC)
(CAC
AC
C)
C
Cardiac
arrdiac
rd CT ang
angiography
ngio
i grap
io
ap
phyy aand
nd ccalcium
alci
al
ciium sscoring
corinng
ng wer
were
re perf
performed
rfoorm
rf
ormed
med in
n 221
1 CG
CGD
D pa
pati
patients
tien
ti
ents
ts aand
nd 166 heal
he
healthy
eallthy
volu
vo
volunteers.
lunt
lu
ntee
nt
e rs
ee
rs.. Analyzable
A al
An
alyz
yzzab
ablle results
res
esullts
t were
wer
eree obtained
obbtaain
ineed
ed in
in 36
36 of 37
37 scans
scan
sc
an
ns (97.3%).
(9
97.
7 3%
3%)). Four
Fouur CGD
CGD patients
paatieents
ents and
annd
nd
one volunteer
volunteeer ha
hadd at
athe
atherosclerotic
heero
rosccle
lero
r tiic pl
ro
plaque
laq
a ue rresulting
essul
ulti
tiing iin
n <5
<50%
50% lluminal
um
min
inal
all sstenosis.
teno
te
nosi
no
sis.
si
s O
s.
One
ne C
CGD
G patient
GD
and no volunteers had plaque resulting in >50% luminal stenosis. The coronary plaque index,
including calcified and non-calcified plaque, was similar in CGD patients and controls (8.3 ± 2.1
and 7.8 ± 1.5 mm2 respectively, p=0.46).
Prevalent coronary calcification, defined as an Agatston score • 1, was present in one
healthy control and 6 CGD patients (6.3% and 14.6%, respectively, p=0.39). The median CAC in
CGD patients with measurable calcification was 173 (IQR 9-366) and CAC for the control
patient was 5.
Significant univariable associations with coronary plaque were present for age and
9
DOI: 10.1161/CIRCULATIONAHA.113.006824
hypertension but not CGD. (Table 4) A minimally adjusted multivariable linear regression
model including CGD, age and gender found significant associations between age and gender –
but not CGD – with coronary plaque. After correction for CGD and traditional cardiovascular
risk factors in a multivariable linear regression model, only hypertension emerged as an
independent predictor of coronary wall volume (coefficient of association=2.3; 95% CI, 0.5 –
4.1; p=0.02; overall r2 for model 0.52, p<0.01). ICA wall volume and coronary arterial wall
volume were not significantly correlated (Spearman’s rho = 0.18, p=0.33).
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
Subgroup Analyses
Amongst patients with CGD, those with deficiency in gp91 phox (X-linked CGD) were, as
expected, entirely male while the p47 phox patients were evenly divided between me
m
men
enn an
and
nd wo
wome
women
m n
(47%
47% male). TNF-Į levels were significantly lower in p47 phox patients. We otherwise observed
noo significant
sig
igni
nifi
ni
fica
fi
cant
ca
nt differences
dif
ifffe
fere
r nces in prevalent traditional
traditionaal ca
ardiovascular rrisk
i k fa
is
act
ctor
o s, lipid subfraction
cardiovascular
factors,
levels,
eveels
l , or markers
marrke
kerrs off systemic
syst
sy
stem
st
emic
em
ic inflammation
inf
nfla
lamm
la
mmaationn by
mm
by genotype.
gen
enotyp
en
ype.
yp
e. (Table
(Tabl
blee 55))
As eexpected,
xpec
xp
eccte
ted,
d, iin
n heal
hhealthy
ea th
hy co
cont
control
ntro
nt
roll su
subjects
ubj
bjec
e ts tthe
ec
he ccarotid
he
arot
arot
otid
id aartery
rter
rt
e y wa
er
wall
ll vo
volume
olum
olum
me wa
was
as ssignificantly
ign
gnif
gn
iffic
ican
antl
tlly
h
lower
ower in females
fem
mal
ales
e ccompared
es
ompa
om
pare
pa
r d wi
w
with
th
h males
mal
ales
es (Fig
(Fi
Figg 1B
1B).
). CGD
CGD
G gp91
gp9
p911 phox
ddeficient
de
fici
fi
cien
ci
entt pa
en
pati
patients
t en
ents
ts (all
(al
alll male) had
significantly reduced carotid artery wall volume compared with age matched control males
(Figure 1B). CGD p47 phox deficient male and female patients also had significantly reduced
carotid artery wall volume compared with age and sex matched controls (Figure 1B). There was
no significant relationship between residual superoxide production and the internal carotid artery
wall volume among all the CGD patients (not shown) nor was there a significant difference
between common log-transformed internal carotid artery wall volume of p47 phox deficient and
gp91 phox deficient patients (2.53 ± 0.02 vs. 2.57 ± 0.02 log10 mm3, p=0.19).
There was no difference between control subjects and CGD patients in coronary arterial
10
DOI: 10.1161/CIRCULATIONAHA.113.006824
plaque burden (p47 phox, 8.4 ± 2.5 vs. gp91 phox 8.1± 1.4 mm2, p=0.75) or the prevalence of
coronary arterial calcium (p47 phox 13.6% vs. gp91 phox 15.8% p=0.85).
As intraconazole has been demonstrated to affect HDL levels 30 we conducted a
sensitivity analysis excluding 16 patients on active itraconazole prophylaxis therapy. The
remaining 20 CGD patients still had a significant reduction in carotid atherosclerosis compared
to healthy controls (p=0.0012).
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
Discussion
We investigated the prevalence of subclinical atherosclerosis in the carotid and coronary arteries
of patients with CGD. Despite an adverse cardiovascular risk profile with signifi
significant
ficcant
n eelevations
nt
leva
le
vati
va
tion
ti
ons
inn multiple systemic markers of inflammation, the data demonstrate that CGD was associated
with
wi
th
h smaller
small
mall
ller
e ccarotid
er
arrot
otiid
id artery wall thickness, an established
esttabl
blished indicato
indicator
or of ssubclinical
ubcclinical atherosclerosis,
ub
co
compared
ompared
mp
to healthy
heal
he
a th
al
hy controls.
conntro
ntro
rols
lss. This
This effect
efffecct was
waas independent
inddepend
nddent
ent off traditional
traadiiti
tion
onal
a cardiovascular
car
arrdi
diov
ovas
ascu
as
cu
ula
larr risk
riskk
fa
factors.
act
ctor
orrs. IIn
n co
cont
contrast,
ntra
raastt, CT
T aangiography
ngio
ng
ogr
g ap
aphy
hy ooff co
coro
coronary
ronaary
ro
r aarteries
rtter
erie
i s di
ie
didd no
nott sh
show
how
o a ssignificant
igni
ig
n fi
ni
fica
cant
nt ddifference
ifffe
fere
r ncce
re
between CG
GD an
aand
d co
ccontrol
n ro
nt
r l su
subj
b ec
ects
t iin
ts
n th
thee pr
prev
eval
ev
a en
al
ence
ce ooff co
coro
rona
ro
nary
na
ry ath
ther
th
eros
er
oscl
os
cler
cl
e ossiss.
er
CGD
subjects
prevalence
coronary
atherosclerosis.
CGD offers an opportunity to study the clinical consequences of reduced NOX2 activity
on cardiovascular disease in humans. Violi and coauthors found a reduction in ultrasound carotid
intimal-medial thickness in male patients with CGD 20 and in female carriers of X-linked CGD.22
While X-CGD carriers are generally healthy, many face an increased frequency of autoimmune
disease 31 and, depending on the extent of lyonization in their myeloid cells, some face serious
CGD-like infectious complications. 32 Nevertheless, a reduction in carotid intimal-medial
thickness in both patients and carriers was associated with lower biomarkers of oxidative stress
and increased brachial arterial flow mediated dilation. These investigators also reported
11
DOI: 10.1161/CIRCULATIONAHA.113.006824
decreased isoprostane formation and increased NO generation in X-CGD.20 Increased flow
mediated dilation was reported in p47 phox -deficient CGD patients but intimal-medial thickness
did not differ from normal.33 These results provided the first evidence in vivo that NOX2 may
play a role in arterial tone and hypertension, and possibly contribute to the pathogenesis of
atherosclerotic disease.20, 22, 33 Although intimal-medial thickness is considered a useful surrogate
marker for atherosclerosis, hypertension alone is a primary driver of intimal thickening, and can
cause changes in carotid intimal-medial thickness that do not correspond histologically to
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
atherosclerotic injury.34 It is plausible that the observed reduction in carotid intimal-medial
thickness was driven by NOX2 related improvement in endothelial function in a process
independent
ndependent of atherosclerosis. Flow mediated dilation provides useful insights into
int
ntoo arterial
arrteeri
rial
al
endothelial function, but has not been demonstrated to predict future cardiovascular events
beyond
be
eyo
yond
nd ttraditional
raadi
diti
tion
nal rrisk
isk factors.35 The correlation
n bbetween
etween carotid
d an
aand
d co
cor
coronary
ro
ronary
atherosclerotic
bburden,
urd
den, whilee si
significant
ign
gniffic
i an
nt in ssome
ome po
ome
popu
populations,
puulaationns, ha
has
as iimportant
mp
porta
ortant
n llimitations
nt
im
mittat
atio
ionns 3366 , and
and
nd it
it remains
reema
main
ns
possible
poss
po
ssib
ss
ible
ib
l tthat
le
hatt th
ha
thee ob
observed
bse
serrveed
ed red
reduction
ed
duccti
tion
on iin
n ca
carotid
aro
rottidd in
iintimal-medial
timall-me
tim
meddial
diall tthickness
hiick
kne
n ss
ss m
may
ay
y nnot
o ttranslate
ot
rannsllate
ra
latee iinto
nto a
nto
protective effect
eff
ffec
ectt in
ec
n oother
th
her
e vvascular
a cu
as
c la
larr bbeds
e s co
ed
cons
consistent
nsis
ns
isste
tent
ntt w
with
i h pr
it
prev
previous
evio
ev
ious
io
us oobservations
bsserrva
vati
tion
ti
onss in a m
on
mouse
o se model.
ou
37
While associations between morphologic carotid atherosclerosis and coronary arterial disease
38, 39
incident cardiovascular events have been observed, the presence and strength of these
correlations varies importantly by population. 36
The present study demonstrates an association between both p47 phox and gp91 phox CGD
and lower carotid wall thickness using histologically validated high-resolution MRI techniques.
25, 40
Our analysis shows this difference in early vascular injury is independent of hypertension,
suggesting that it may be mediated through other NOX2 effects as discussed below. Importantly,
p47 phox is not only a cytosolic regulator of NOX2 activity but can also regulate NOX1. 41
12
DOI: 10.1161/CIRCULATIONAHA.113.006824
Deficiency in this protein may, therefore, alter multiple NADPH oxidases.
This is the first study, to our knowledge, to examine the prevalence of subclinical
coronary atherosclerosis in CGD. In contrast to the findings in the carotid arteries, noninvasive
quantification of wall thickening and coronary plaque burden showed no significant difference
between NOX2-deficient patients and controls. Quantification of coronary arterial calcium, a
finding representative of more advanced atherosclerotic lesions and a powerful independent
predictor of cardiovascular events, 35, 42 showed no reduction and a trend towards a greater
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
burden of calcified atherosclerosis in CGD patients. Physiologically, it is possible that NOX2related mechanisms play a lesser role in coronary atherosclerosis than in other arterial beds.
While there was a non-statistically significant trend towards greater coronary cal
alci
cifi
ficaati
fi
tion
on iin
n
calcification
CGD patients, the total volume of calcified and noncalcified plaque, as measured by coronary
pl
laq
aque
ue iindex,
ndex
nd
e , wa
ex
as nnearly
early identical to controls. Th
Thee rratio
atio of calcifi
fied
e too non-calcified
no
components
plaque
was
calcified
43-45
435
off atherosclerotic
ath
therosclerrot
otic
i plaque
ic
plaaquee varies
vaari
riees iin
n diff
ddifferent
iffer
ereent di
dis
seasse proc
oces
oc
esssess,
s, 43
aand
nd ppatients
atie
at
ieent
ntss with
with
h CGD
CGD
D may
may
y
disease
processes,
hav
ha
have
ve m
ve
more
oree ra
or
rapi
rapid
pidd pr
progression
rog
gresssio
ssionn off ccalcification
alci
al
c fi
ci
fica
cati
ca
tion
ti
on tha
than
hann fo
ha
found
ound
oun
nd iin
n typi
ttypical
ypi
pica
caal at
athe
atherosclerosis.
hero
he
rosc
scle
leero
osi
sis.
s.. W
Wee ca
cannot
ann
nnoot
exclude the po
poss
possibility
sssib
bil
ilit
i y th
it
tthat
att ssmall
mall
ma
ll ssample
ampl
am
plee si
pl
ssize
ze llimited
imit
im
ited
it
ed tthe
hee ppower
ower
ow
er tto
o de
ddetect
tect
te
ct a ssmaller
mall
ma
ller
ll
e m
er
magnitude
a nitude
ag
difference in coronary plaque burden between groups. Given the increases in the lifespan of
CGD patients since the advent of antimicrobial prophylaxis, further investigation of the aging
CGD population will likely reveal whether ROS play a role in calcified coronary atherosclerosis.
Our study of CGD patients also revealed significantly lower levels of HDL and a trend
toward elevated triglycerides despite normal cholesterol. This is in contrast to the reported
decrease in triglycerides in CGD mice. 14 Surprisingly, oxidized LDL was significantly higher in
CGD suggesting not only that NOX2 is not primarily responsible for the production of oxidized
LDL but possibly that NOX2 is involved in the catabolism of this lipid species. Importantly,
13
DOI: 10.1161/CIRCULATIONAHA.113.006824
CGD subjects are treated with prophylactic antibacterial and antifungal drugs including
itraconazole, which was been shown to reduce LDL and increase HDL in immunocompetent
men. 30 We did not detect any association between itraconazole prophylaxis or other antibiotic
therapies our patients were receiving and atherosclerotic burden or lipid profiles.
Despite the absence of overt signs of infection including normal white cell counts, plasma
from CGD subjects in this study contained significantly increased concentrations of clinically
recognized cardiovascular risk factors such as hsCRP 46 and MPO 47 as well as other
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
inflammatory biomarkers (e.g., TNFĮ, IL-6, GM-CSF). These elevations, which have not been
reported previously in CGD, may be due to unrecognized infection or inflammation but may also
elate to the direct regulatory role for reactive oxygen species in controlling infla
lamm
mmaatio
mm
ionn.
n. F
or
relate
inflammation.
For
example, ROS regulates the mRNA stability of IL8 4, the metabolism of leukotrienes resulting in
ac
ccu
umu
mullati
lati
tion
on off LT
LTB4 5, IL-1ȕ processing 48 andd tthe
he apoptosis off C
CGD
GD
D nneutrophils
e trophils 6. The role of
eu
accumulation
cytokines
cy
yto
oki
k nes as rregulators
egul
eg
u ator
orrs of iinflammation
nfla
nf
lamm
la
mm
matio
atio
on in ccardiovascular
arrdiova
vascuula
va
ular
ar ddisease
issea
easse hhas
as bbeen
een re
rev
reviewed
view
view
wed 49 aalthough
ltho
lt
hou
ough
specific
pec
ecif
ific
if
ic pathophysiologic
pat
atho
hoph
ho
phys
yssio
iolo
lo
ogiic roles
role
ro
l s and
le
and clinical
cliini
nica
call risk
ca
sk gguidelines
uiide
delline
ness have
ne
haave
v yyet
et tto
o bee eestablished.
stab
st
ab
bli
lish
sheed.
sh
ed
Interestingly,
nterestingly,
y a ccommon
y,
ommo
om
monn po
mo
polymorphism
oly
lymo
mo
orp
rphi
hism
hi
sm
m iin
n th
thee IL
IL-6
-6
6 rreceptor
e ep
ec
epto
to
or th
that
at rreduces
ed
duc
uces
es ffunction
unct
un
cttio
ionn wa
wass associated
d
in an 82-study meta-analysis with a decreased risk of coronary heart disease suggesting that
signaling by IL-6, which was also elevated in CGD, may be pathogenic.50 In parallel with MPO,
we also observed significant elevations in CGD patient plasma of other neutrophil-derived
factors including gelatinase, lactoferrin and defensins. Further work will be required to determine
whether or not the increases in these factors are due to mechanisms such as those controlling IL8 (see above) or reflect neutrophil degranulation due to increased inflammation in CGD.
The observation of reduced carotid but not coronary artery atherosclerosis in CGD
patients raises questions about the role of NOX2 in the pathogenesis of atherosclerosis.
14
DOI: 10.1161/CIRCULATIONAHA.113.006824
Importantly, this finding also raises questions about whether or not NOX2 inhibitors may be
beneficial in reducing all atherosclerosis or just that outside of the coronary circulation. Clearly,
further high-resolution studies of possible links between deficiencies in or inhibition of different
NOX proteins and atherogenesis in distinct anatomic vascular locations are indicated.
Acknowledgments: The authors gratefully acknowledge the participation of the CGD patients
and the clinical support teams of the Laboratory of Host Defenses, the Laboratory of Clinical
Infectious Diseases, and the Department of Radiology and Imaging Sciences.
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
Funding Sources: This work was supported by the Division of Intramural Research of the
National Institute of Allergy and Infectious Diseases and the National Institutes off He
H
alth
al
th
Health
Clinical Center.
Conf
nffli
lict
ct of
of Interest
Inteere
In
rest
st Disclosures: The content off this
thi
h s article does not necessarily
nece
ne
c ssarily reflect the
Conflict
vi
iew
ews or pol
olic
iciees of
ic
o tthe
hee D
epar
ep
artm
tm
ment
n ooff He
Heal
alth
th aand
ndd H
um
man S
e viices,
er
ces,, nnor
orr ddoes
oees me
ment
n io
nt
ionn of ttrade
rade
ra
d
de
views
policies
Department
Health
Human
Services,
mention
n mes, commerc
na
ciaal products,
prrodducts
tss, or
o organizations
org
rgan
aniz
izaatio
onss imply
im
mply endorsement
end
ndor
orse
or
seme
ment
nt by the
the U
.S
S. G
ov
vernm
ment. T
he
names,
commercial
U.S.
Government.
The
au
uth
thor
orss re
or
repo
port
po
rt nno
o conf
cconflict
onfflicct
ct ooff iinterest
ntter
eres
estt re
es
eleeva
vannt tto
o th
he co
ontten
entt off tthis
his ma
his
m
nusscri
scriipt
p.
authors
report
relevant
the
content
manuscript.
Refe
Re
fere
renc
nces
es::
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Table 1. Clinical and biomarker characteristics of study population.
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
Clinical Characteristics Age
Male, N (%)*
Hypertension*
Diabetes
Smoking
Family CVD history*
Total Cholesterol (mg/dL)
Lipid Levels
LDL (mg/dL)
HDL (mg/dL) ***
Triglycerides (mg/dL)
Oxidized LDL (mg/dL) ***
hsCRP (mg/L) ***
Other Biomarkers
Nitrite (NO2-, μM)
Nitrate (NO3-, μM)
Lactoferrin (ng/ml) ***
Neutrophil proteins
MPO (ng/ml) **
MMP-9 (ng/ml) ***
Į-Defensins (ng/ml)*
IFN-Ȗ (pg/ml)
Cytokines
Cyto
okines
TNF-Į(pg/ml)**
GM
M-C
CSF
S ((pg/ml)*
pg/m
pg
/m
ml)
l)**
GM-CSF
IL
L-1ȕ
-1ȕ(p
(pgg/m
g/ml)
ml)
IL-1ȕ(pg/ml)
IL-2
IL-2
- ((pg/ml)
pg
g/m
/ml)
l)
IL
L-4
4 ((pg/ml)
pg/m
/ml)
ml)
l
IL-4
IL
IL-5
L-5 ((pg/ml)
pg/ml)
pg
l)
IL-6
IL
6 ((pg/ml)**
pg/m
pg
/ l)
/m
l)**
**
IL
10 (p
(pg/
g/ml
ml))
IL-10
(pg/ml)
IL-12p70 (pg/ml)
IL-13 (pg/ml)
IL-17 (pg/ml)
CCL3 (MIP-1Į, pg/ml)
Chemokines
CCL4 (MIP-1ȕ, pg/ml)**
CCL5 (RANTES, ng/ml)
CXCL8 (IL-8, pg/ml)*
sL-selectin (ng/ml)
Soluble Selectins
sE-selectin (ng/ml)*
TNFR1 (pg/ml)
Soluble Receptors
TNFR2 (pg/ml) ***
IL-6R (pg/ml)
CGD (n=41)
32.5±9.3
31 (75.6%)
12 (29.3%)
3 (7.3%)
14 (34.2%)
22 (56.4%)
153.2±35.0
92.7±33.9
38.1±10.9
110.6±68.2
65.0±37.2
8.9±10.1
2.6±1.5
24.7±16.8
722.6±832.0
473.0±810.7
375.7±420.4
56.3±137.1
5.5±16.1
.1
10
10.7±5.7
0.7
. ±5
5.7
7
33.1±6.8
.1±
1±6.
1±
6.88
11.2±4.4
.22±4.
2±4 4
00.8±0.8
.88±0.88
00.2±0.5
.2±
2±0.
2±
0.55
1.8±
1.8±6.1
8±6.
61
10.3
10
3±1
±11.
1.00
1.
10.3±11.0
33.9±8.0
9±88 0
9±
10.0±56.7
2.2±4.4
12.6±19.1
38.8±42.2
178.7±226.5
25.9±19.2
19.6±37.6
1,163.9±245.6
39.8 ± 17.4
3,376.0±2434.9
3,986.5±2,170.6
18,223.8±4,780.8
Control (n=25)
32.1±7.1
12 (48.0%)
1(4.0%)
0 (0%)
7 (28.0%)
6 (24.0%)
162.4±28.1
84.7±21.7
60.2±14.7
87.8±50.9
23.4±18.6
2.4±3.8
2.8±1.0
22.8±17.6
225.
22
225.0±98.3
5.0±
0 98
0±
98.3
.3
1118.4±76.3
11
8.4±
8.
4±76
4±
76.3
76
.3
134.
13
4 5±
5±63
63.99
134.5±63.9
8.1±5.3
1.8±6.0
4.7±7.4
0.7±
0.
7±0.
7±
0.77
0.
0.7±0.7
0.4±
0.
±0.55
0.4±0.5
1.2±
1.
1.2±2.5
±2.55
0.
.3±
±0.
0.66
0.3±0.6
22.3±7.1
2.
3±
±7.
71
44.4±5.5
4.
4±5.5
10
3±3
±377 4
10.3±37.4
31.9±146.9
45.5±184.5
7.6±7.8
27.7±13.7
60.2±39.0
25.9±13. 3
5.5±6.6
1,152.5±187.5
31.3±13.8
2,914.8±1,256.7
2,389.4±652.2
18,695.1±5,162.2
Means ± SD. * denotes P ” 0.05, ** denotes P ” 0.01, and *** denotes P ” 0.001 for difference between CGD and
control patients respectively. Abbreviations: CGD, chronic granulomatous disease; LDL, low-density lipoprotein;
HDL, high-density lipoprotein; hsCRP, high sensitivity C-reactive protein; ESR, erythrocyte sedimentation rate;
MPO, myeloperoxidase; IL, interleukin; TNF, tumor necrosis factor; GM-CSF, granulocyte macrophage colony
stimulating factor; MIP, macrophage inflammatory protein; MMP, matrix metallopeptidase
21
DOI: 10.1161/CIRCULATIONAHA.113.006824
Table 2. Markers of subclinical atherosclerosis.
3
Internal carotid arterial wall volume (log10 mm )
Prevalent coronary arterial calcium (n,%)
Coronary plaque index (mm2)
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22
Control
2.66± 0.02
1 (6.3)
7.8 ± 1.5
CGD
2.55 ± 0.02
6 (14.6)
8.3 ± 2.1
p-value
0.0001
0.39
0.46
DOI: 10.1161/CIRCULATIONAHA.113.006824
Table 3. Internal Carotid Arterial Wall Volume in Univariate and Multivariable Linear Regression Analyses.
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
Univariate Model
CGD
age
male gender
hypertension
smoking
ing
family
y history
LDL
HDL
hsCRP
P
oxidized
zed LDL
ferri
rinn
lactoferrin
MPO
MMP-9
P-99
PTNF-Į
Į
GM-CSF
CSF
F
ȕ
IL-1ȕ
IL-6
IL-10
IL-12p70
p70
70
IL-13
IL-17
CCL3 (MIP-1Į)
CCL4 (MIP-1ȕ)
CCL5 RANTES
CXCL8 (IL-8)
TNFR1
TNFR2
IL-6R
coeff.
-0.11
0.001
0.05
0.03
-0.02
-0.01
0.08×10-3
2.0×10-3
-0.0×10-3
-0.6×10-3
-2
26×
6×10
1 -6
-26×10
-39×10
-39×
-3
39×
9 10
1 -6
-32×10
-32×
-3
2 10-6
2×
-0.2×10
-0.
0 2×100-6
1.6×10
1.6
. ×1
× 0-3
-3
--2.2×10
2.2×10-3
33.4×10
.4×1
× 0-3
×1
11.7×10
1.
7×
×10-33
00.4×10
.4
4×1
×10
10-33
00.3×10
.3×
3×10
10-33
00.5×10
5 10-33
-0.3×10-3
0.4×10-6
-0.7×10-6
-0.3×10-3
4×10-6
-8×10-6
2×10-6
SE
0.03
0.002
0.03
0.03
0.03
0.03
0.48 ×10-3
0.8×10-3
1.5×10-3
0.4×10-3
19×10-6
20×10-6
339×10
9×10
9×
100-6
00.1×10
0.
1×10
1×
10-66
3.4×10
3.4
.4×1
× 0-3
×1
33.9×10
.9×1
×10-33
11.3×10
.3×1
3× 0-3
0.5×10
0.5
5×1
×100-33
00.1×10
.11×1
×10
10-33
00.1×10
.11×1
× 0-3
00.8×10
8 10-33
0.4×10-3
72×10-6
0.8×10-6
0.4×10-3
7×10-6
8×10-6
3×10-6
p-value
<0.001
0.56
0.09
0.36
0.48
0.81
0.87
0.02
0.55
0.11
0.17
0.053
00.40
.400
.4
00.08
.08
0
00.64
.64
6
00.57
.57
.5
00.01
.01
01
00.002
.002
002
00.002
.002
002
00.002
.0002
00.58
58
0.43
0.99
0.37
0.50
0.54
0.29
0.43
Multivariable base model
(R2 = 0.38)
coeff.
SE
p-value
-0.13
0.02
<0.001
0.002
0.001
0.23
0.08
0.02
0.002
Coeff is the estimated regression coefficient. SE is the variance of the estimated regression coefficient.
23
Multivariable model 2
(R2 = 0.50)
coeff.
SE
p-value
-0.14
0.03
<0.001
0.001
0.001
0.70
0.06
0.03
0.02
0.09
0.03
0.01
-0.002
00.03
0.
03
00.94
.94
0.04
00.03
. 3
.0
00.09
0.
09
-3
-3
0.05×10
00.4×10
.44×1
×100
0.90
0.9
.90
1.0×10-3
11.0×10
.00×10
10-33
0.31
0.31
31
DOI: 10.1161/CIRCULATIONAHA.113.006824
Table 4. Coronary Plaque Index in Univariable and Multivariable Linear Regression Analyses
Univariate Model
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CGD
age
male gender
hypertension
smoking
family history
LDL
HDL
hsCRP
oxidized LDL
MPO
coeff.
0.44
0.09
1.10
3.2
-0.15
-0.10
-0.01
-0.03
0.03
0.01
-0.0006
SE
0.61
0.03
0.61
0.69
0.68
0.61
0.01
0.02
0.03
0.01
0.0005
p-value
0.47
0.01
0.08
<0.001
0.83
0.87
0.47
0.09
0.38
0.26
0.24
Multivariate Model 1
(R2 = 0.28)
coeff. SE p-value
0.14 0.55
0.80
0.09 0.03
0.007
1.23 0.57
0.04
Multivariate Model 2
(R2 = 0.52)
coeff.
SE p-value
-0.33
0.69
0.64
0.06
0.04
0.11
0.52
0.63
0.42
2.29
0.89
0.02
-0.21
0.59
0.72
-0.08
0.61
0.90
-0.02
0.01
0.08
-0.02
0.02
0.31
Coeff is the estimated regression coefficient. SE is the variance of the estimated regression coefficient.
24
DOI: 10.1161/CIRCULATIONAHA.113.006824
Table 5. Clinical and biomarker characteristics of CGD patients by genotype.
Clinical Characteristics
Lipid Levels
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Other Biomarkers
Neutrophil proteins
Cytokines
Cyto
okines
Chemokines
Soluble Selectins
Soluble Receptors
Age
Male, N (%)***
Hypertension
Diabetes
Smoking
Family CVD history
Total Cholesterol (mg/dL)
LDL (mg/dL)
HDL (mg/dL)
Triglycerides (mg/dL)
Oxidized LDL (mg/dL)
hsCRP (mg/L)
Nitrite (NO2-, μM)
Nitrate (NO3-, μM)
Lactoferrin (ng/ml)
MPO (ng/ml)
MMP-9 (ng/ml)
Į-Defensins (ng/ml)
IFN-Ȗ (pg/ml)
TNF-Į(pg/ml)
GM
M-C
CSF
F ((pg/ml)
p /m
pg
ml)
GM-CSF
IIL-1ȕ(pg/ml)
L-11ȕ(p
L(pg/
g/ml
ml)
IL
IL-2
L-2
2 (pg
(pg/ml)
pg
g/m
ml)
IL
-4
4 ((pg/ml)
pg/m
pg
/m
ml))
IL-4
IL-5
IL
-5 ((pg/ml)
pg/ml)
pg
l)
IL-6
IL
-6
6 ((pg/ml)
pgg/m
ml)
l
IL
10 (p
(pg/
g/ml
ml))
IL-10
(pg/ml)
IL-12p70 (pg/ml)
IL-13 (pg/ml)
IL-17 (pg/ml)
CCL3 (MIP-1Į, pg/ml)
CCL4 (MIP-1ȕ, pg/ml)
CCL5 (RANTES, ng/ml)
CXCL8 (IL-8, pg/ml)
sL-selectin (ng/ml)
sE-selectin (ng/ml)
TNFR1 (pg/ml)
TNFR2 (pg/ml)
IL-6R (pg/ml)
p47 (n=19)
34.8±8.0
9 (47.4%)
5 (26.3%)
3 (15.8%)
8 (42.1%)
13 (68.4%)
149.1 ± 24.4
85.9 ± 25.1
41.4 ±12.0
108.6 ± 57.1
57.4 ± 36.8
7.5 ± 9.4
2.8±1.4
21.3±12.7
538.9 ± 708.7
442.1 ± 819.7
248.2 ± 252.2
63.3 ± 187. 9
2.1 ± 2.0
2..0
8.66 ± 4.
4.2*
2*
11.4±
.4±
4± 22.2
.22
00.6
.66 ±0
±0.
.7
±0.7
00.8
.88 ±0.99
00.3
.33 ±0
±
.6
±0.6
0.88 ±0
±0.6
6
11.1±
11
± 14
14.1
.1
22.9
9 ± 11.8
8
1.2 ± 1.1
1.7 ± 3.5
11.0± 11.4
39.2 ± 48.5
128.9 ± 66.5
23.7± 9.7
13.9 ± 24.6
1108.4 ± 218.5
38.5 ± 15.3
3057.0 ± 1373.8
3514.7± 1204.8
17576.0 ± 3229.0
gp91 (n=22)
30.4±10.1
22 (100%)
7 (31.8%)
0 (0%)
6 (27.3%)
9 (45.0%)
156.8 ± 42.7
99.2 ± 40.1
35.2 ±9.2
112.3 ± 78.4
71.6 ± 37.1
10.1 ± 10.8
2.5±1.5
27.5±19.6
881.
88
881.3
1.33 ± 91
1.
911.
911.7
1.77
4499.7
499
99.7 ± 82
821.
12
1.
821.2
48
85.8±
8±
± 5504.7
04.7
7
485.8±
50.2 ± 74.0
8.4 ± 21.7
12.5 ± 6.2
44.6
.6 ± 88.9
.9
9
11.6
.6 ± 55.9
.9
9
00.8
.8
8±
±0.6
0.6
00.1
.1
1±
0.3
0.
3
±0.3
22.6
.6
6±
±8.3
83
8.
99.6
.6 ± 7.8
44.8
8 ± 10
8
10.8
17.6 ± 77.4
2.5 ± 5.1
13.9 ± 24.1
38.3 ± 37.2
221.8 ± 299.5
27.9 ± 24.8
24.6 ± 46.0
1211.8 ± 262.2
41.0± 19.3
3651.6 ± 3082.7
4393.9 ± 2712.0
18783.2 ± 5821.6
Means ± SD. * denotes P ” 0.05, ** denotes P ” 0.01, and *** denotes P ” 0.001 for difference between CGD
patients with deficiency p47phox and gp91phox deficiency.
25
DOI: 10.1161/CIRCULATIONAHA.113.006824
Figure Legend:
Figure 1. Comparison of Subclinical Coronary and Carotid Atherosclerosis in Healthy Controls
and CGD Patients. Bars denote mean and 95% CI of the mean and p-values are from t-tests
(Panels A, C) and ANOVA with a Holm-Šídák post-test (Panel B).
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26
Downloaded from http://circ.ahajournals.org/ by guest on October 2, 2016
Assessment of Atherosclerosis in Chronic Granulomatous Disease
Christopher T. Sibley, Tyra Estwick, Anna Zavodni, Chiung-Yu Huang, Alan C. Kwan, Benjamin P.
Soule, Debra A. Long Priel, Alan T. Remaley, Amanda K. Rudman Spergel, Evrim B. Turkbey,
Douglas B. Kuhns, Steven M. Holland, Harry L. Malech, Kol A. Zarember, David A. Bluemke and
John I. Gallin
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Circulation. published online September 19, 2014;
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2014 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
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http://circ.ahajournals.org/content/early/2014/09/19/CIRCULATIONAHA.113.006824
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