Note: Descriptions are shown in the official language in which they were submitted.
COMPOSITIONS AND METHODS FOR DIAGNOSIS OF PERIPHERAL ARTERIAL DISEASE
Field
The present invention relates to peripheral arterial disease (PAD). In
particular, the
present invention relates to compositions and methods for diagnosis of
peripheral arterial
.. disease.
Background
Fatty acid-binding protein 3 (FABP3), also known as heart type fatty acid-
binding protein
(hFABP), is a small cytoplasmic protein that is thought to participate in the
intracellular
trafficking and metabolism of long-chain fatty acids. Fatty acid-binding
protein 4 (FABP4), also
known as adipocyte Protein 2 (aP2) is a carrier protein for fatty acids that
is primarily expressed
in adipocytes and macrophages. Peripheral artery disease (PAD) is an abnormal
narrowing of
arteries other than those that supply the heart or brain.
U.S. Patent No. 8,062,857 describes a method for diagnosing myocardial
infarction in a
subject based on the determination of H-FABP and, optionally, myoglobin in a
sample of the
subject. U.S. Patent No. 7,754,436 describes a diagnostic assay for H-FABP or
B-FABP that
distinguishes between stroke and acute myocardial infarction. U.S. Patent
Application
Publication No. 2017/0219608 describes a kit for testing myocardial infarction
comprising a strip
capable of detecting three markers, namely, human myeloperoxidase (MPO), heart-
fatty acid
binding protein (FABP3) and cardiac troponin I (cTnI) simultaneously. Karbek
et al.
.. (Cardiovascular Diabetology 2011, 10:37) describe that serum H-FABP levels
could represent a
useful marker for myocardial performance in patients with diabetes. Otaki et
al. (BBA Clinical 4
(2015) 35-41) describe that the myocardial damage markers H-FABP and hsTnT
were
increased in PAD patients with CLI and could predict MACCEs in PAD patients.
Pritt et al.
describe FABP3 as a biomarker of skeletal muscle toxicity in the rat.
Despite this, there is a need to develop biomarkers and methods associated
with PAD.
Summary of the Invention
In accordance with an aspect, there is provided fatty acid-binding protein 3
(FABP3)
and/or FABP4 for diagnosing peripheral artery disease (PAD).
In accordance with an aspect, there is provided fatty acid-binding protein 3
(FABP3)
and/or FABP4 for staging peripheral artery disease (PAD).
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CA 3057013 2019-09-27
In accordance with an aspect, there is provided fatty acid-binding protein 3
(FABP3)
and/or for assessing revascularization status in a subject afflicted with
peripheral artery disease
(PAD).
In an aspect, the FABP3 and/or FABP4 is in combination with at least one other
biomarker.
In an aspect, the at least one other biomarker comprises high sensitivity
troponin,
troponin I (TnI), troponin T (TnT), FABP3, FABP4, or a combination thereof.
In accordance with an aspect, there is provided a panel of biomarkers for
assessing
peripheral artery disease (PAD), the panel comprising FABP3 and/or FABP4 and
at least one
additional biomarker.
In an aspect, the at least one additional biomarker comprises a biomarker
associated
with PAD.
In an aspect, the at least one additional biomarker comprises a biomarker
associated
with myocardial ischemia.
In an aspect, the biomarker associated with myocardial ischemia is high
sensitivity
troponin, troponin I (TnI), and/or troponin T (TnT).
In an aspect, the at least one additional biomarker comprises the other of
FABP3 and/or
FABP4.
In an aspect, a detected level of FABP3 protein in a patient sample of <0.6
ng/ml, <0.7
ng/ml, <0.8 ng/ml, <0.9 ng/ml, <1.0 ng/ml, <1.1 ng/ml, <1.2 ng/ml, <1.3 ng/ml,
<1.4 ng/ml, <1.5
ng/ml, <1.6 ng/ml, <1.7 ng/ml, <1.8 ng/ml, <1.9 ng/ml, <2.0 ng/ml, <2.1 ng/ml,
or <2.2 ng/ml is
suggestive that the subject is highly unlikely to have PAD.
In an aspect, a detected level of FABP3 protein in a patient sample of Ø6
ng/ml and
<4.5 ng/ml, such as 13.6 ng/ml, ng/ml, ng/ml, ?Ø9 ng/ml, ng/ml,
ng/ml,
ng/ml, ng/ml, ng/ml, ng/ml, ng/ml, ng/ml, ng/ml, .?1.9
ng/ml,
ng/ml, ng/ml, or .2.2 ng/ml, and <3.5 ng/ml, <3.6 ng/ml, <3.7 ng/ml,
<3.8 ng/ml, <3.9 ng/ml,
<4.0 ng/ml, <4.1 ng/ml, <4.2 ng/ml, <4.3 ng/ml, <4.4 ng/ml, and <4.5 ng/ml is
suggestive that
the subject is at moderate risk of having PAD.
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CA 3057013 2019-09-27
In an aspect, a detected level of FABP3 protein in a patient sample of
ng/ml and
<5.3 ng/ml, such as .3.5 ng/ml, ng/ml, ?.3.7 ng/ml, ng/ml, ng/ml,
2.4.0 ng/ml, ?.4.1
ng/ml, ?.4.2 ng/ml, ?.4.3 ng/ml, ?.4.4 ng/ml, or
ng/ml, and <4.4 ng/ml, <4.5 ng/ml, <4.6 ng/ml,
<4.7 ng/ml, <4.8 ng/ml, <4.9 ng/ml, <5.0 ng/ml, <5.1 ng/ml, <5.2 ng/ml, and
<5.3 ng/ml is
suggestive that the subject is at moderate-high risk of having PAD.
In an aspect, a detected level of FABP3 protein in a patient sample of .4.6
ng/ml,
ng/ml, ?.4.8 ng/ml, ng/ml, ng/ml, ?_5.1 ng/ml,
ng/ml, or .5.3 ng/ml is suggestive
that the subject is at high risk of having PAD.
In an aspect, a detected level of FABP4 protein in a patient sample of <15
ng/ml, <16
ng/ml, <17 ng/ml, <18 ng/ml, <19 ng/ml, <20 ng/ml, <21 ng/ml, <22 ng/ml, <23
ng/ml, <24 ng/ml,
or <25 ng/ml is suggestive that the subject has PAD.
In accordance with an aspect, there is provided an assay comprising the FABP3
and/or
FABP4 or the panel described herein.
In an aspect, the assay is a point of care assay.
In accordance with an aspect, there is provided a kit comprising the FABP3
and/or
FABP4 or the panel described herein.
In accordance with an aspect, there is provided a method for diagnosing
peripheral
artery disease (PAD) in a subject, the method comprising detecting the level
of fatty acid-
binding protein 3 (FABP3) and/or FABP4 in the subject; wherein an elevated
level of FABP3
and/or FABP4 is indicative of PAD in the subject.
In an aspect, the elevated level of FABP3 and/or FABP4 in the subject is
determined by
comparing the detected level of FABP3 and/or FABP4 to a control level of FABP3
and/or
FABP4.
In an aspect, the control level of FABP3 and/or FABP4 is a predetermined value
obtained from one or a pool of non-PAD patients or healthy patients.
In an aspect, the method further comprises detecting the level of at least one
additional
biomarker.
In an aspect, the at least one additional biomarker comprises the other of
FABP3 and/or
FABP4, high sensitivity troponin, Tnl, TnT, and/or creatinine.
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In an aspect, the method further comprises assessing the ABI of the subject.
In an aspect, the PAD is non-symptomatic (stage 0), mild PAD (stage 1),
moderate PAD
(stage 2), severe PAD (stage 3), early chronically threatened limb ischemia
(CTLI) (stage 4) or
advanced CTLI (stages 5-6).
In an aspect, the PAD is early or advanced CTLI.
In an aspect, the subject is free of clinical and/or biochemical evidence of
myocardial
ischemia.
In an aspect, the method further comprises detecting the level of high
sensitivity
troponin, troponin I (TnI) and/or troponin T (TnT) in the subject, wherein a
substantially normal
level of high sensitivity troponin, TnI and/or TnT in the subject is further
indicative of PAD in the
subject.
In an aspect, the substantially normal level of high sensitivity troponin, TnI
and/or TnT in
the subject is determined by comparing the detected level of high sensitivity
troponin, TnI and/or
TnT to a control level of TnI and/or TnT.
In an aspect, the subject is free of clinical and/or biochemical evidence of
kidney
dysfunction.
In an aspect, the method further comprises detecting the level of creatinine
in the
subject, wherein a substantially normal level of creatinine in the subject is
further indicative of
PAD in the subject.
In an aspect, the substantially normal level of creatinine in the subject is
determined by
comparing the detected level of creatinine to a control level of creatinine.
In an aspect, the subject is free of clinical and/or biochemical evidence of
acute stroke
and/or acute muscle toxicity.
In an aspect, the subject has a concurrent condition and optionally wherein
the detected
level of FABP3 and/or FABP4 and/or the control level of FABP3 and/or FABP4 is
optionally
adjusted for the concurrent condition.
In an aspect, the concurrent condition is kidney dysfunction, stroke,
diabetes, and/or
muscle toxicity.
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In accordance with an aspect, there is provided a method for staging
peripheral artery
disease (PAD) in a subject, the method comprising detecting the level of fatty
acid-binding
protein 3 (FABP3) and/or FABP4 in the subject; wherein an elevated level of
FABP3 correlates
with the stage of PAD in the subject.
In an aspect, the elevated level of FABP3 and/or FABP4 in the subject is
determined by
comparing the detected level of FABP3 and/or FABP4 to a control level of FABP3
and/or
FABP4, and wherein the size of the difference between the detected level of
FABP3 and/or
FABP4 and the control level of FABP3 positively correlates with the stage of
PAD in the subject.
In an aspect, the control level of FABP3 and/or FABP4 is a predetermined value
obtained from one or a pool of non-PAD patients or healthy patients.
In an aspect, the method further comprises detecting the level of at least one
additional
biomarker.
In an aspect, the at least one additional biomarker comprises the other of
FABP3 and/or
FABP4, high sensitivity troponin, TnI, TnT, and/or creatinine,
In an aspect, the method further comprises assessing the ABI of the subject.
In an aspect, the method comprises staging the PAD as asymptomatic (stage 0),
mild
PAD (stage 1), moderate PAD (stage 2), severe PAD (stage 3), early CTLI (stage
4) or late
CTLI (stage 5-6) based on the detected level of FABP3.
In an aspect, the subject is free of clinical and/or biochemical evidence of
myocardial
ischemia.
In an aspect, the method further comprises detecting the level of high
sensitivity
troponin, troponin I (TnI) and/or troponin T (TnT) in the subject, wherein a
substantially normal
level of high sensitivity troponin, TnI and/or TnT in the subject is further
indicative of PAD in the
subject.
In an aspect, the substantially normal level of high sensitivity troponin, TnI
and/or TnT in
the subject is determined by comparing the detected level of high sensitivity
troponin, TnI and/or
TnT to a control level of high sensitivity troponin, TnI and/or TnT.
In an aspect, the subject is free of clinical and/or biochemical evidence of
kidney
dysfunction.
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In an aspect, the method further comprises detecting the level of creatinine
in the
subject, wherein a substantially normal level of creatinine in the subject is
further indicative of
PAD in the subject.
In an aspect, the substantially normal level of creatinine in the subject is
determined by
comparing the detected level of creatinine to a control level of creatinine.
In an aspect, the subject is free of clinical and/or biochemical evidence of
acute stroke
and/or muscle toxicity.
In an aspect, the subject has a concurrent condition and wherein the detected
level of
FABP3 and/or FABP4 and/or the control level of FABP3 and/or FABP4 is
optionally adjusted for
the concurrent condition.
In an aspect, the concurrent condition is kidney dysfunction, stroke,
diabetes, and/or
muscle toxicity.
In accordance with an aspect, there is provided a method for assessing
revascularization in a subject with peripheral artery disease (PAD), the
method comprising
detecting the level of fatty acid-binding protein 3 (FABP3) and/or FABP4 in
the subject; wherein
a substantially normal level of FABP3 and/or FABP4 or a reduction in an
elevated level of
FABP3 and/or FABP4 is indicative of arterial revascularization in the subject.
In an aspect, the substantially normal level of FABP3 and/or FABP4 or the
reduction in
the elevated level of FABP3 and/or FABP4 is determined by comparing the
detected level of
FABP3 and/or FABP4 to a control level of FABP3 and/or FABP4.
In an aspect, the control level of FABP3 and/or FABP4 is a predetermined value
obtained from one or a pool of non-PAD patients or healthy patients.
In an aspect, the control level of FABP3 and/or FABP4 is a predetermined value
obtained from one or a pool of PAD patients.
In an aspect, the control level of FABP3 and/or FABP4 is the level of FABP3
and/or
FABP4 detected in the subject prior to revascularization treatment.
In an aspect, the method further comprises detecting the level of at least one
additional
biomarker.
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In an aspect, the at least one additional biomarker comprises the other of
FABP3 and/or
FABP4, high sensitivity troponin, Tnl, TnT, and/or creatinine.
In an aspect, the method further comprises assessing the ABI of the subject.
In an aspect, the PAD is asymptomatic (stage 0), mild PAD (stage 1), moderate
PAD
(stage 2), severe PAD (stage 3), early CTLI (stage 4) or advanced CTLI (stages
5-6).
In an aspect, the PAD is early or advanced CTLI.
In an aspect, the subject is free of clinical and/or biochemical evidence of
myocardial
ischemia.
In an aspect, the method further comprises detecting the level of high
sensitivity
troponin, troponin I (Tn1) and/or troponin T (TnT) in the subject, wherein a
substantially normal
level of high sensitivity troponin, TnI and/or TnT in the subject is further
indicative of
revascularization in the subject.
In an aspect, the substantially normal level of high sensitivity troponin, TnI
and/or TnT in
the subject is determined by comparing the detected level of high sensitivity
troponin, TnI and/or
TnT to a control level of high sensitivity troponin, TnI and/or Tnt.
In an aspect, the subject is free of clinical and/or biochemical evidence of
kidney
dysfunction.
In an aspect, the method further comprises detecting the level of creatinine
in the
subject, wherein a substantially normal level of creatinine in the subject is
further indicative of
revascularization in the subject.
In an aspect, the substantially normal level of creatinine in the subject is
determined by
comparing the detected level of creatinine to a control level of creatinine.
In an aspect, the subject is free of clinical and/or biochemical evidence of
acute stroke
and/or muscle toxicity.
In an aspect, the subject has a concurrent condition and wherein the detected
level of
FABP3 and/or FABP4 and/or the control level of FABP3 and/or FABP4 is
optionally adjusted for
the concurrent condition.
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CA 3057013 2019-09-27
In an aspect, the concurrent condition is kidney dysfunction, stroke,
diabetes, and/or
muscle toxicity.
In accordance with an aspect, there is provided a method for predicting
whether a
subject with peripheral artery disease (PAD) is likely to progress to CTLI,
the method comprising
detecting the level of fatty acid-binding protein 3 (FABP3) and/or FABP4 in
the subject; wherein
the extent of elevation of FABP3 and/or FABP4 is correlated with the
likelihood of the subject
progressing to CTLI.
In an aspect, the extent of elevation of FABP3 and/or FABP4 in the subject is
determined by comparing the detected level of FABP3 and/or FABP4 to a control
level of
FABP3 and/or FABP4.
In an aspect, the control level of FABP3 and/or FABP4 is a predetermined value
obtained from one or a pool of non-PAD patients or healthy patients.
In an aspect, the method further comprises detecting the level of at least one
additional
biomarker.
In an aspect, the at least one additional biomarker comprises the other of
FABP3 and/or
FABP4, high sensitivity troponin, Tnl, TnT, and/or creatinine.
In an aspect, the method further comprises assessing the ABI of the subject.
In an aspect, the subject is free of clinical and/or biochemical evidence of
myocardial
ischemia.
In an aspect, the method further comprises detecting the level of high
sensitivity
troponin, troponin I (TnI) and/or troponin T (TnT) in the subject, wherein a
substantially normal
level of high sensitivity troponin, TnI and/or TnT in the subject is further
indicative of PAD in the
subject.
In an aspect, the substantially normal level of high sensitivity troponin, TnI
and/or TnT in
the subject is determined by comparing the detected level of high sensitivity
troponin, TnI and/or
TnT to a control level of high sensitivity troponin, TnI and/or TnT.
In an aspect, the subject is free of clinical and/or biochemical evidence of
kidney
dysfunction.
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In an aspect, the method further comprises detecting the level of creatinine
in the
subject, wherein a substantially normal level of creatinine in the subject is
further indicative of
PAD in the subject.
In an aspect, the substantially normal level of creatinine in the subject is
determined by
comparing the detected level of creatinine to a control level of creatinine.
In an aspect, the subject is free of clinical and/or biochemical evidence of
acute stroke
and/or muscle toxicity.
In an aspect, the subject has a concurrent condition and wherein the detected
level of
FABP3 and/or FABP4 and/or the control level of FABP3 and/or FABP4 is
optionally adjusted for
the concurrent condition.
In an aspect, the concurrent condition is kidney dysfunction, stroke, diabetes
and/or
muscle toxicity.
In an aspect, the FABP3 and/or FABP4 is detected in whole blood, plasma,
urine, saliva,
oral fluid, cerebrospinal fluid, amniotic fluid, milk, colostrum, mammary
gland secretion, lymph,
sweat, lacrimal fluid, gastric fluid, synovial fluid, mucus, or combinations
thereof.
In an aspect, the FABP3 and/or FABP4 is detected as protein, DNA, RNA, or a
combination thereof.
In an aspect, the subject is an adult.
In an aspect, the subject is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, or 80
years of age.
In an aspect, a detected level of FABP3 and/or FABP4 protein in a patient
sample of
<0.6 ng/ml, <0.7 ng/ml, <0.8 ng/ml, <0.9 ng/ml, <1.0 ng/ml, <1.1 ng/ml, <1.2
ng/ml, <1.3 ng/ml,
<1.4 ng/ml, <1.5 ng/ml, <1.6 ng/ml, <1.7 ng/ml, <1.8 ng/ml, <1.9 ng/ml, <2.0
ng/ml, <2.1 ng/ml,
or <2.2 ng/ml is suggestive that the subject is highly unlikely to have PAD.
In an aspect, a detected level of FABP3 and/or FABP4 protein in a patient
sample of
?_0.6 ng/ml and <4.5 ng/ml, such as 4.6 ng/ml, ng/ml, ng/ml, 4.9 ng/ml,
ng/ml,
ng/ml, ng/ml, ?.1.3 ng/ml, ng/ml, ng/ml, ng/ml,
ng/ml, ?.1.8 ng/ml,
?.1.9 ng/ml, ng/ml, ng/ml, or
ng/ml, and <3.5 ng/ml, <3.6 ng/ml, <3.7 ng/ml, <3.8
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ng/ml, <3.9 ng/ml, <4.0 ng/ml, <4.1 ng/ml, <4.2 ng/ml, <4.3 ng/ml, <4.4 ng/ml,
and <4.5 ng/ml is
suggestive that the subject is at moderate risk of having PAD.
In an aspect, a detected level of FABP3 and/or FABP4 protein in a patient
sample of
ng/ml and <5.3 ng/ml, such as ?.3.5 ng/ml, ng/ml, ng/ml, ng/ml,
ng/ml,
?_4.0 ng/ml, 4.1 ng/ml, a4.2 ng/ml, ?_4.3 ng/ml, ng/ml, or .4.3 ng/ml, and
<4.4 ng/ml, <4.5
ng/ml, <4.6 ng/ml, <4.7 ng/ml, <4.8 ng/ml, <4.9 ng/ml, <5.0 ng/ml, <5.1 ng/ml,
<5.2 ng/ml, and
<5.3 ng/ml is suggestive that the subject is at moderate-high risk of having
PAD.
In an aspect, a detected level of FABP3 and/or FABP4 protein in a patient
sample of
ng/ml, ng/ml, ng/ml, _?_4.9 ng/ml, ?.5.0 ng/ml, ng/ml,
ng/ml, or .?..5.3 ng/ml
is suggestive that the subject is at high risk of having PAD.
In an aspect, a detected level of FABP4 protein in a patient sample of <15
ng/ml, <16
ng/ml, <17 ng/ml, <18 ng/ml, <19 ng/ml, <20 ng/ml, <21 ng/ml, <22 ng/ml, <23
ng/ml, <24 ng/ml,
or <25 ng/ml is suggestive that the subject has PAD.
In an aspect, the method further comprising treating the subject based upon
the
outcome of the method.
In accordance with an aspect, there is provided a method of treating a subject
with
peripheral artery disease, the method comprising carrying out at least one
method described
herein and treating the subject based upon the outcome of the method.
In accordance with an aspect, there is provided a use of FABP3 and/or FABP4
for
diagnosing peripheral artery disease (PAD) in a subject, wherein an elevated
level of FABP3
and/or FABP4 is indicative of PAD in the subject.
In accordance with an aspect, there is provided a use of FABP3 and/or FABP4
for
staging peripheral artery disease (PAD) in a subject, wherein an elevated
level of FABP3 and/or
FABP4 correlates with the stage of PAD in the subject.
In accordance with an aspect, there is provided a use of FABP3 and/or FABP4
for
assessing revascularization in a subject with peripheral artery disease (PAD);
wherein a
substantially normal level of FABP3 and/or FABP4 or a reduction in an elevated
level of FABP3
is indicative of arterial revascularization in the subject.
CA 3057013 2019-09-27
In accordance with an aspect, there is provided a use of FABP3 and/or FABP4
for
predicting whether a subject with peripheral artery disease (PAD) is likely to
progress to CTLI,
wherein the extent of elevation of FABP3 and/or FABP4 is correlated with the
likelihood of the
subject progressing to CTLI.
The novel features of the present invention will become apparent to those of
skill in the
art upon examination of the following detailed description of the invention.
It should be
understood, however, that the detailed description of the invention and the
specific examples
presented, while indicating certain aspects of the present invention, are
provided for illustration
purposes only because various changes and modifications within the spirit and
scope of the
invention will become apparent to those of skill in the art from the detailed
description of the
invention and claims that follow.
Brief Description of the Drawings
The present invention will be further understood from the following
description with
reference to the Figures, in which:
Figure 1. An algorithm to diagnose PAD based on the Plasma levels of FABP3.
The
exemplary cut-off points were established using 486 patients.
Figure 2. Receiver Operating Characteristics (ROC) analyses on for FABP3 in
plasma in
486 patients. Unadjusted ROC analysis for FABP3 in plasma samples obtained
from 105 non-
PAD controls and 381 PAD patients with an area under curve (AUC) of 0.8234
(95% Cl,
0.7818 to 0.8651) is represented by the solid line. When compared to non-PAD
control, our
ROC analysis confirms the use of FABP3 in plasma as an excellent biomarker of
PAD with large
area under curve.
Figure 3. Receiver Operating Characteristics (ROC) analyses for FABP3 in
plasma of
patients with CTLI. Unadjusted ROC analysis for FABP3 AUC 0.80 (95% Cl, 0.65
¨ 0.87) is
represented by the dotted line. The ROC analysis for FABP3 after adjusting for
confounding
factors is represented by the dashed line. The AUC for FABP3 improved to 0.92
(95% Cl, 0.79 ¨
0.97) after adjusting for confounding factors.
Figure 4. FABP3 levels positively correlated with the severity of PAD. A bar
chart
representing mean values with confidence intervals for FABP3 levels in 250
controls and PAD
patients. Levels of FABP3 demonstrated an increase in FABP3 levels as the
Rutherford
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=
classification increase. The (*) denotes statistical difference with P value
50.05 between the
experimental group and control cohort.
Figure 5. Levels of Troponin I (TnI) in healthy, Non-PAD, CTLI and acute
coronary
syndrome (ACS) patients. The level of TnI was measured in a large cohort of
PAD, CTLI, ACS
and control patients. 50 CTLI patients were matched to 25 non-PAD patients and
15 ACS
patients. 15 healthy patients without risk factors were used as a negative
control. Relative to
healthy and non-PAD patients, increased levels of troponin I was only observed
in the ACS
group, and not in the CTLI group. The (*) denotes statistical difference with
P value 5Ø05.
Figure 6. Increased FABP3 expression levels in the skeletal muscles of CTLI
patients in
.. comparison to non-PAD controls. A) Western blot demonstrating FABP3 and
GAPDH
expressions in the skeletal muscle of non-PAD controls (n=3) and CTLI patients
(n=4). B) The
histogram shows a quantitative representation of the levels of protein
obtained from a
densitometry analysis of four independent experiments. Each value represents
the mean
standard error of the mean. A significant difference of comparison was
determined by t-test as
indicated by asterisk (*). P-value <0.05 versus control skeletal muscles. AU=
absolute units.
Figure 7. Immunohistochemistry (10X) of muscles obtained from non-PAD and CTLI
patients. Hematoxylin-eosin (A,B), 0D68 (CD), Masson's Trichrome staining
(E,F) and FABP3
(G.H) were used to assess cellular histology, Macrophages, muscular
pathology/fibrosis, and
localization of FABP3 in non-PAD and CTLI patients, respectively. This figure
demonstrates that
skeletal muscles is a source of expression of FABP3 in CTLI patients.
Figure 8. Receiver Operating Characteristics (ROC) analyses for FABP3 in
urine.
Unadjusted ROC analysis for FABP3 in urine samples obtained from 41 non-PAD
controls and
101 PAD patients with an area under curve (AUC) of 0.8644 (95% Cl, 0.7939 to
0.9348) is
represented by the solid line.
Detailed Description
Definitions
Unless otherwise explained, all technical and scientific terms used herein
have the same
meaning as commonly understood by one of ordinary skill in the art to which
this disclosure
belongs. Definitions of common terms in molecular biology may be found in
Benjamin Lewin,
.. Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9);
Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science
Ltd., 1994 (ISBN
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CA 3057013 2019-09-27
0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and
Biotechnology: a
Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-
56081-569-
8). Although any methods and materials similar or equivalent to those
described herein can be
used in the practice for testing of the present invention, the typical
materials and methods are
described herein. In describing and claiming the present invention, the
following terminology will
be used.
It is also to be understood that the terminology used herein is for the
purpose of
describing particular aspects only and is not intended to be limiting. Many
patent applications,
patents, and publications may be referred to herein to assist in understanding
the aspects
described. Each of these references is incorporated herein by reference in its
entirety.
In understanding the scope of the present application, the articles "a", "an",
"the", and
"said" are intended to mean that there are one or more of the elements.
Additionally, the term
"comprising" and its derivatives, as used herein, are intended to be open
ended terms that
specify the presence of the stated features, elements, components, groups,
integers, and/or
steps, but do not exclude the presence of other unstated features, elements,
components,
groups, integers and/or steps. The foregoing also applies to words having
similar meanings
such as the terms, "including", "having" and their derivatives.
It will be understood that any aspects described as "comprising" certain
components
may also "consist of" or "consist essentially of," wherein "consisting of" has
a closed-ended or
restrictive meaning and "consisting essentially of" means including the
components specified
but excluding other components except for materials present as impurities,
unavoidable
materials present as a result of processes used to provide the components, and
components
added for a purpose other than achieving the technical effect of the
invention. For example, a
composition defined using the phrase "consisting essentially of" encompasses
any known
acceptable additive, excipient, diluent, carrier, and the like. Typically, a
composition consisting
essentially of a set of components will comprise less than 5% by weight,
typically less than 3%
by weight, more typically less than 1%, and even more typically less than 0.1%
by weight of
non-specified component(s).
It will be understood that any component defined herein as being included may
be
explicitly excluded from the claimed invention by way of proviso or negative
limitation.
In addition, all ranges given herein include the end of the ranges and also
any
intermediate range points, whether explicitly stated or not.
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CA 3057013 2019-09-27
=
Terms of degree such as "substantially", "about" and "approximately" as used
herein
mean a reasonable amount of deviation of the modified term such that the end
result is not
significantly changed. These terms of degree should be construed as including
a deviation of at
least 5% of the modified term if this deviation would not negate the meaning
of the word it
modifies.
Although methods and materials similar or equivalent to those described herein
can be
used in the practice or testing of this disclosure, suitable methods and
materials are described
below. The abbreviation, "e.g." is derived from the Latin exempli gratia and
is used herein to
indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous
with the term "for
example." The word "or" is intended to include "and" unless the context
clearly indicates
otherwise.
As used herein, the term "biomarker" is intended to encompass a substance that
is used
as an indicator of a biologic state and includes genes (and nucleotide
sequences of such
genes), mRNAs (and nucleotide sequences of such mRNAs) and proteins (and amino
acid
sequences of such proteins). A "biomarker panel" includes a plurality of
biomarkers, the
expression of each of which is measured in order to provide a quantitative or
qualitative
summary of the expression of one or more biomarkers in a subject, such as in
comparison to a
standard or a control.
The terms "increased" or "increased expression" and "decreased" or "decreased
expression", with respect to the expression pattern of a biomarker(s), are
used herein as
meaning that the level of expression is increased or decreased relative to a
constant basal level
of expression of a household, or housekeeping, protein, whose expression level
does not
significantly vary under different conditions. A nonlimiting example of such a
household, or
housekeeping, protein is GAPDH. Other suitable household, or housekeeping,
proteins are well-
established in the art. In other aspects, these terms refer to an increase or
decrease in the level
of expression as compared to that observed in a control population, such as a
subject or pool of
subjects who have not experienced recent limb ischemia. In more typical
aspects, these terms
refer to an increase or decrease in relative concentrations in relation to the
mean values of the
sample in question.
The term "subject" as used herein refers to any member of the animal kingdom,
typically
a mammal. The term "mammal" refers to any animal classified as a mammal,
including humans,
other higher primates, domestic and farm animals, and zoo, sports, or pet
animals, such as
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=
dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Typically, the
mammal is human. In
specific aspects, the biomarkers and methods described herein can be used in
non-human
animals. It will be understood that the biomarkers may not be completely
conserved between
the human versions described herein and equivalent animal versions, however,
given the
descriptions and examples provided here in it is understood that a skilled
person could modify
the biomarkers to be suitable for a desired animal population.
"Peripheral artery disease" (PAD) is an abnormal narrowing of arteries other
than those
that supply the heart or brain. Peripheral artery disease most commonly
affects the legs, but
other arteries may also be involved. Many patients with PAD are asymptomatic;
the classic
symptom PAD patients usually experience is calf pain while walking known as
intermittent
claudication. This pain resolves with rest,. Other symptoms of advanced PAD
include skin
ulcers, bluish skin, cold skin, or abnormal nail and hair growth in the
affected leg. Up to 50% of
people with PAD do not have symptoms.
"Chronic limb threatening ischemia" (CLTI), also known as critical limb
ischemia (CLI), is
an advanced stage of peripheral artery disease (PAD). Compared earlier stages
of PAD
involving intermittent claudication, CTLI has a negative prognosis within a
year after the initial
diagnosis, with 1-year amputation rates of approximately 12% and mortality of
50% at 5 years
and 70% at 10 years.
Biomarkers and Panels
Fatty acid-binding protein 3 (FABP3), also known as heart type fatty acid-
binding protein
(hFABP), is a small cytoplasmic protein that is thought to participate in the
intracellular
trafficking and metabolism of long-chain fatty acids. Fatty acid-binding
protein 3 (FABP4), also
known as adipocyte Protein 2 (aP2) is a carrier protein for fatty acids that
is primarily expressed
in adipocytes and macrophages. Described herein is evidence that FABP3 is
released from
skeletal muscle tissue and is found in elevated levels in skeletal muscle,
blood, and urine in
subjects suffering from PAD. Similar findings with respect to FABP4 are also
shown herein.
Thus, described herein is FABP3 and/or FABP4 for diagnosing or staging PAD.
Further
described herein is FABP3 and/or FABP4 for assessing revascularization status
in subject
afflicted with PAD. FABP3 and/or FABP4 may be used alone as a biomarker
associated with
PAD or it may be combined with other PAD biomarkers, such as the other of
FABP3 and/or
FABP4.
CA 3057013 2019-09-27
=
FABP3 may also be combined with a biomarker associated with myocardial
ischemia,
such as troponin, for example, high sensitivity troponin, troponin I (TnI)
and/or troponin T (TnT).
In this way, it can be determined whether an elevation in FABP3 and/or FABP4
is due to
myocardial ischemia or PAD. For example, if FABP3 and/or FABP4 is elevated and
troponin is
within normal range, then it can be concluded that the FABP3 and/or FABP4 is
likely elevated
due to PAD and not myocardial ischemia. If, on the other hand, FABP3 and/or
FABP4 and
troponin are both elevated, then it is likely that the patient has more than
one possible source of
FABP3 and/or FABP4 release and more testing may be desired in order to
determine if PAD is
also present.
The biomarkers described herein may be assessed independently of one another
or they
may be assessed collectively in a panel. For example, a single blood or urine
sample, for
example, may be assessed in a single test to determine the levels of FABP3
together with
FABP4 and/or high sensitivity troponin, and/or TnI and/or TnT, or any other
desired biomarkers
or combinations thereof.
In order to determine whether any given biomarker measurement is in a normal
or
elevated range, typically a cut-off or control level is used. The control
levels may be, for
example, based on one or a pool of healthy subjects not known to be afflicted
by PAD or other
pathologies such as myocardial ischemia. Alternatively or additionally, the
control levels may be,
for example, based on one or a pool of subjects afflicted with PAD or a
specific stage of PAD
but not myocardial ischemia. Alternatively or additionally, the control levels
may be, for example,
based on one or a pool of subjects afflicted with both PAD and myocardial
ischemia.
Combinations of these controls may be used in order to determine suitable
ranges for
comparison between detected levels of biomarkers and a given disease state or
stage.
As an example, certain exemplary cut-offs are shown in Figure 1. In this
example, if
FABP3 is measured as being less than 1.0 ng/ml, the subject is determined to
be highly unlikely
to have PAD. If FABP3 is measured as being between 1.0 ng/ml and 4.2 ng/ml,
the subject is
determined to have a moderate risk of PAD. If FABP3 is measured as being
between 4.2 ng/ml
and 4.9 ng/ml, the subject is determined to have a moderate-high risk of PAD.
Finally, if FABP3
is measured as being higher than 4.9 ng/ml, the subject is determined to be
highly likely to have
PAD. As noted above, these cut-offs can be combined with other biomarker
measurements and
cut-offs to rule out myocardial ischemia as the source of FABP3 and/or to
corroborate the
conclusion with respect to risk of PAD.
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CA 3057013 2019-09-27
While Figure 1 shows exemplary specific cut-offs, it will be understood that
these may
vary depending upon the controls selected and the size of the pool chosen as
the control. For
example, reaching a conclusion that a subject is highly unlikely to have PAD
may be based on a
cut-off of FABP3 of, for example, <0.6 ng/ml, <0.7 ng/ml, <0.8 ng/ml, <0.9
ng/ml, <1.0 ng/ml,
.. <1.1 ng/ml, <1.2 ng/ml, <1.3 ng/ml, <1.4 ng/ml, <1.5 ng/ml, <1.6 ng/ml,
<1.7 ng/ml, <1.8 ng/ml,
<1.9 ng/ml, <2.0 ng/ml, <2.1 ng/ml, or <2.2 ng/ml.
Similarly, reaching a conclusion that a subject is at moderate risk of having
PAD may be
based on a cut-off range of FABP3 of, for example, ?.Ø6 ng/ml and <4.5
ng/ml, such as ?Ø6
ng/ml, ng/ml, ng/ml, ng/ml, ?.1.0 ng/ml,
?1.1 ng/ml, ng/ml, ng/ml, ?_1.4
ng/ml, ng/ml, ng/ml, ,7 ng/ml, ?1.8 ng/ml,
?1.9 ng/ml, ng/ml, ?.2.1 ng/ml, or
ng/ml, and <3.5 ng/ml, <3.6 ng/ml, <3.7 ng/ml, <3.8 ng/ml, <3.9 ng/ml, <4.0
ng/ml, <4.1
ng/ml, <4.2 ng/ml, <4.3 ng/ml, <4.4 ng/ml, and <4.5 ng/ml, for example.
Similarly, reaching a conclusion that a subject is at moderate-high risk of
having PAD
may be based on a cut-off range of FABP3 of, for example, ?.3.5 ng/ml and <5.3
ng/ml, such as
ng/ml, ?-3.6 ng/ml, ?.3.7 ng/ml, ng/ml, ng/ml,
2.4.0 ng/ml, ?Al ng/ml, ng/ml,
N1.3 ng/ml,
ng/ml, or ?.4.3 ng/ml, and <4.4 ng/ml, <4.5 ng/ml, <4.6 ng/ml, <4.7 ng/ml,
<4.8
ng/ml, <4.9 ng/ml, <5.0 ng/ml, <5.1 ng/ml, <5.2 ng/ml, and <5.3 ng/ml, for
example.
Similarly, reaching a conclusion that a subject is at high risk of having PAD
may be
based on a cut-off of FABP3 of, for example ?_4.6 ng/ml, _?.4.7 ng/ml,
ng/ml, ?.4.9 ng/ml, ?.5.0
.. ng/ml, .?_5.1 ng/ml, ?.5.2 ng/ml, or ?.5.3 ng/ml.
In aspects, reaching a conclusion that a subject is at risk of having PAD may
be based
on a cut-off of FABP4 of, for example, <15 ng/ml, <16 ng/ml, <17 ng/ml, <18
ng/ml, <19 ng/ml,
<20 ng/ml, <21 ng/ml, <22 ng/ml, <23 ng/ml, <24 ng/ml, or <25 ng/ml.
It will be appreciated that these cut-off measurements are based on plasma
FABP3
and/or FABP4 protein. It will be understood, as described below, urine FABP3
and/or FABP4 as
well as RNA or DNA could be measured instead and there will be likely changes
in these cut-off
values, which could be calculated by a skilled person based on the teachings
herein.
Typically, it is the protein biomarker that is measured. It is also possible
to measure
mRNA using known methods. Typically, the protein biomarkers are measured using
antibodies,
.. for example, in an ELISA or Luminex-based method. Methods for detecting and
measuring the
biomarkers are known to a skilled person and certain typical methods are
exemplified herein.
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For example, the expression pattern in blood, serum, urine etc. of the
biomarkers
provided herein is obtained. The quantitative data associated with the
biomarkers of interest can
be any data that allows generation of a useful result, including measurement
of DNA or RNA
levels associated with the markers but is typically protein expression
patterns. Protein levels can
be measured via any method known to those of skill in the art that generates a
quantitative
measurement either individually or via high-throughput methods as part of an
expression profile.
For example, a blood-derived patient sample, e.g., blood, plasma, or serum, or
a urine-derived
sample may be applied to a specific binding agent or panel of specific binding
agents to
determine the presence and quantity of the protein markers of interest.
The quantitative data associated with the biomarkers of interest typically
takes the form
of an expression profile. Expression profiles constitute a set of relative or
absolute expression
values for a number of biomarker products corresponding to the plurality of
markers evaluated.
In various embodiments, expression profiles containing expression patterns of
at least about 2,
3, 4, 5, 6, 7, 8 or more markers are produced. The expression pattern for each
differentially
expressed component member of the expression profile may provide a particular
specificity and
sensitivity with respect to predictive value, e.g., for diagnosis, prognosis,
monitoring treatment,
etc.
Numerous methods for obtaining expression data are known, and any one or more
of
these techniques, singly or in combination, are suitable for determining
expression patterns and
profiles in the context of the present disclosure.
For example, DNA and RNA (mRNA, pri-miRNA, pre-miRNA, miRNA, precursor hairpin
RNA, microRNP, and the like) expression patterns can be evaluated by northern
analysis, PCR,
RT-PCR, Taq Man analysis, FRET detection, monitoring one or more molecular
beacon,
hybridization to an oligonucleotide array, hybridization to a cDNA array,
hybridization to a
polynucleotide array, hybridization to a liquid microarray, hybridization to a
microelectric array,
cDNA sequencing, clone hybridization, cDNA fragment fingerprinting, serial
analysis of gene
expression (SAGE), subtractive hybridization, differential display and/or
differential screening.
These and other techniques are well known to those of skill in the art.
The present disclosure includes nucleic acid molecules, typically in isolated
form. As
used herein, a nucleic acid molecule is to be "isolated" when the nucleic acid
molecule is
substantially separated from contaminant nucleic acid molecules encoding other
polypeptides.
The term "nucleic acid" is defined as coding and noncoding RNA or DNA. Nucleic
acids that are
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complementary to, that is, hybridize to, and remain stably bound to the
molecules under
appropriate stringency conditions are included within the scope of this
disclosure. Such
sequences exhibit at least 50%, 60%, 70% or 75%, typically at least about 80-
90%, more
typically at least about 92-94%, and even more typically at least about 95%,
98%, 99% or more
nucleotide sequence identity with the sequences for the biomarkers disclosed
herein, and
include insertions, deletions, wobble bases, substitutions, and the like.
Further contemplated are
sequences sharing at least about 50%, 60%, 70% or 75%, typically at least
about 80-90%, more
typically at least about 92-94%, and most typically at least about 95%, 98%,
99% or more
identity with the biomarker sequences disclosed herein
Specifically contemplated within the scope of the disclosure are genomic DNA,
cDNA,
RNA (mRNA, pri-miRNA, pre-miRNA, miRNA, hairpin precursor RNA, RNP, etc.)
molecules, as
well as nucleic acids based on alternative backbones or including alternative
bases, whether
derived from natural sources or synthesized.
The present disclosure further provides fragments of the disclosed nucleic
acid
molecules and/or proteins. As used herein, a fragment of a nucleic acid
molecule refers to a
small portion of the coding or non-coding sequence. The size of the fragment
will be determined
by the intended use. For example, if the fragment is chosen so as to encode an
active portion of
the protein, the fragment will need to be large enough to encode the
functional region(s) of the
protein. For instance, fragments which encode peptides corresponding to
predicted antigenic
regions may be prepared. If the fragment is to be used as a nucleic acid probe
or PCR primer,
then the fragment length is chosen so as to obtain a relatively small number
of false positives
during probing/priming.
Protein expression patterns can be evaluated by any method known to those of
skill in
the art which provides a quantitative measure and is suitable for evaluation
of multiple markers
extracted from samples such as one or more of the following methods: ELISA
sandwich assays,
flow cytometry, mass spectrometric detection, calorimetric assays, binding to
a protein array
(e.g., antibody array), or fluorescent activated cell sorting (FACS).
In one embodiment, an approach involves the use of labeled affinity reagents
(e.g.,
antibodies, small molecules, etc.) that recognize epitopes of one or more
protein products in an
.. ELISA, antibody-labelled fluorescent bead array, antibody array, or FACS
screen. Methods for
producing and evaluating antibodies are well known in the art.
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A number of suitable high throughput formats exist for evaluating expression
patterns
and profiles of the disclosed biomarkers. Typically, the term high throughput
refers to a format
that performs at least about 100 assays, or at least about 500 assays, or at
least about 1000
assays, or at least about 5000 assays, or at least about 10,000 assays, or
more per day. When
.. enumerating assays, either the number of samples or the number of markers
assayed can be
considered.
Numerous technological platforms for performing high throughput expression
analysis
are known. Generally, such methods involve a logical or physical array of
either the subject
samples, or the protein markers, or both. Common array formats include both
liquid and solid
phase arrays. For example, assays employing liquid phase arrays, e.g., for
hybridization of
nucleic acids, binding of antibodies or other receptors to ligand, etc., can
be performed in
multiwell or microtiter plates. Microtiter plates with 96, 384 or 1536 wells
are widely available,
and even higher numbers of wells, e.g., 3456 and 9600 can be used. In general,
the choice of
microtiter plates is determined by the methods and equipment, e.g., robotic
handling and
.. loading systems, used for sample preparation and analysis. Exemplary
systems include, e.g.,
xMAPO technology from Luminex (Austin, Tex.), the SECTOR Imager with MULTI-
ARRAY
and MULTI-SPOT technologies from Meso Scale Discovery (Gaithersburg, Md.),
the ORCATM
system from Beckman-Coulter, Inc. (Fullerton, Calif.) and the ZYMATETm systems
from Zymark
Corporation (Hopkinton, Mass.), miRCURY LNATM microRNA Arrays (Exiqon, Woburn,
Mass.).
Alternatively, a variety of solid phase arrays can favorably be employed to
determine
expression patterns in the context of the disclosed methods, assays and kits.
Exemplary
formats include membrane or filter arrays (e.g., nitrocellulose, nylon), pin
arrays, and bead
arrays (e.g., in a liquid "slurry"). Typically, probes corresponding to
nucleic acid or protein
reagents that specifically interact with (e.g., hybridize to or bind to) an
expression product
corresponding to a, member of the candidate library, are immobilized, for
example by direct or
indirect cross-linking, to the solid support. Essentially any solid support
capable of withstanding
the reagents and conditions necessary for performing the particular expression
assay can be
utilized. For example, functionalized glass, silicon, silicon dioxide,
modified silicon, any of a
variety of polymers, such as (poly)tetrafluoroethylene,
(poly)vinylidenedifluoride, polystyrene,
polycarbonate, or combinations thereof can all serve as the substrate for a
solid phase array.
In one embodiment, the array is a "chip" composed, e.g., of one of the above-
specified
materials. Polynucleotide probes, e.g., RNA or DNA, such as cDNA, synthetic
oligonucleotides,
and the like, or binding proteins such as antibodies or antigen-binding
fragments or derivatives
CA 3057013 2019-09-27
thereof, that specifically interact with expression products of individual
components of the
candidate library are affixed to the chip in a logically ordered manner, i.e.,
in an array. In
addition, any molecule with a specific affinity for either the sense or anti-
sense sequence of the
marker nucleotide sequence (depending on the design of the sample labeling),
can be fixed to
the array surface without loss of specific affinity for the marker and can be
obtained and
produced for array production, for example, proteins that specifically
recognize the specific
nucleic acid sequence of the marker, ribozymes, peptide nucleic acids (PNA),
or other
chemicals or molecules with specific affinity.
Microarray expression may be detected by scanning the microarray with a
variety of
laser or CCD-based scanners, and extracting features with numerous software
packages, for
example, IMAGENETm (Biodiscovery), Feature Extraction Software (Agilent),
SCANLYZETM
(Stanford Univ., Stanford, Calif.), GENEPIXTM (Axon Instruments).
High-throughput protein systems include commercially available systems from
Ciphergen Biosystems, Inc. (Fremont, Calif.) such as PROTEIN CHIPTM arrays,
and
FASTQUANTTm human chemokine protein microspot array (S&S Bioscences Inc.,
Keene, N.H.,
US).
Quantitative data regarding other dataset components, such as clinical
indicia, metabolic
measures, and genetic assays, can be determined via methods known to those of
skill in the art.
Various analytic processes for obtaining a result useful for diagnosing or
staging PAD
are described herein, however, one of skill in the art will readily understand
that any suitable
type of analytic process is within the scope of this disclosure.
Methods
In aspects, the biomarkers described herein such as FABP3 and/or FABP4 find
use in
different aspects associated with diagnosing or staging PAD. Figure 1 shows
one exemplary
embodiment by which FABP3 may be used clinically. In Figure 1, an asymptomatic
patient or a
patient with lower limb pain presents to the clinic and FABP3 levels are
measured from, for
example, a blood or urine sample. Depending upon the measured amount of FABP3,
the
likelihood of the patient having PAD is estimated and further referrals,
monitoring, or other
testing is recommended. Exemplary cut-off values are shown in Figure 1,
however, it will be
understood that these are not limiting and other values may be used, as
explained above.
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Described herein are methods for diagnosing PAD in a subject. The method
typically
comprises detecting the level of FABP3 in the subject; wherein an elevated
level of FABP3
and/or FABP4 is indicative of PAD in the subject.
Also described herein are methods for staging PAD in a subject. Typically, the
method
comprises detecting the level of FABP3 and/or FABP4 in the subject; wherein an
elevated level
of FABP3 and/or FABP4 correlates with the stage of PAD in the subject.
Further described herein are methods for assessing arterial revascularization
in a
subject with PAD. Typically, the method comprises detecting the level of FABP3
and/or FABP4
in the subject; wherein a substantially normal level of FABP3 and/or FABP4 or
a reduction in
pre-operative elevated level of FABP3 and/or FABP4 is indicative of arterial
revascularization in
the subject.
Further described herein are methods for predicting whether a subject with PAD
is likely
to progress to CTLI. Typically, the method comprises detecting the level of
FABP3 and/or
FABP4 in the subject; wherein the extent of elevation of FABP3 and/or FABP4 is
correlated with
the likelihood of the subject progressing to CTLI.
In the methods described herein, typically the level of FABP3 and/or FABP4 is
assessed
as being elevated, normal, or reduced, by comparing the detected level of
FABP3 and/or
FABP4 to a control level of FABP3 and/or FABP4. For example, typically, the
control level of
FABP3 and/or FABP4 is a predetermined value obtained from one or a pool of non-
PAD
patients or healthy patients.
When assessing arterial revascularization, the control level of FABP3 and/or
FABP4
may be the level of FABP3 and/or FABP4 that was detected in the subject prior
to
revascularization treatment. Thus, in aspects, the methods may comprise
diagnosing and/or
staging PAD by measuring FABP3 and/or FABP4 levels in the subject, initiating
revascularization in subjects diagnosed with PAD, and then subsequently
assessing the
success and/or extent of the revascularization achieved in the subject by
again measuring
FABP3 and/or FABP4 levels in the subject and comparing the levels at diagnosis
with the levels
after treatment. If the levels have reduced over this time period, it can be
concluded that
revascularization is likely to have taken place to some extent. FABP3 and/or
FABP4 may be
measured in an ongoing manner over time to assess vascularization and/or PAD
in the subject.
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It will be understood that in the methods described herein, the PAD that may
be
diagnosed, staged, and/or treated may be non-symptomatic (stage 0), mild PAD
(stage 1),
moderate PAD (stage 2), severe PAD (stage 3), early CTLI (stage 4), or
advanced CTLI (stages
5-6). Typically, the PAD is asymptomatic, symptomatic or advanced CTLI.
Many subjects afflicted with or suspected of being afflicted with PAD may
suffer from
other concurrent disorders. In aspects, the subject is free of clinical and/or
biochemical evidence
of myocardial ischemia, which may be determined by detecting the level of high
sensitivity
troponin, TnI and/or TnT in the subject. A substantially normal level of high
sensitivity troponin,
TnI and/or TnT in the subject suggests that the subject is free of myocardial
ischemia and is
further indicative of PAD in the subject. Typically, the level of high
sensitivity troponin, TnI
and/or TnT in the subject is determined as being substantially normal by
comparing the
detected level of high sensitivity troponin, TnI and/or TnT to a control level
of high sensitivity
troponin, TnI and/or TnT.
In additional or alternate aspects, the subject is free of clinical and/or
biochemical
evidence of kidney dysfunction, which may be determined by detecting the level
of creatinine in
the subject. A substantially normal level of creatinine in the subject
suggests that the subject is
free of kidney dysfunction and is further indicative of PAD in the subject.
Typically, the level of
creatinine in the subject is determined as being substantially normal by
comparing the detected
level of creatinine to a control level of creatinine.
Likewise, the subject being assessed using the methods described herein may be
free of
clinical and/or biochemical evidence of acute stroke and/or acute muscle
toxicity.
On the other hand, as noted above, the subject may have a concurrent condition
that
would typically be expected to confuse the diagnosis of PAD. However, it has
been found herein
that FABP3 and/or FABP4 is still a good predictor of PAD despite the presence
of these
conditions, even without adjusting the detected level of FABP3 and/or FABP4
and/or the control
level of FABP3 and/or FABP4. In these cases where concurrent conditions may
exist in the
subject, the detected level of FABP3 and/or FABP4 and/or the control level of
FABP3 and/or
FABP4 is optionally adjusted for the concurrent condition. Typically, the
concurrent condition is
kidney dysfunction, stroke, diabetes, and/or muscle toxicity.
It will be understood that the biomarkers described herein may be detected in
any bodily
fluid or tissue in which it is expressed. For example, the biomarkers may be
detected in whole
blood, plasma, urine, muscle tissue, saliva, oral fluid, cerebrospinal fluid,
amniotic fluid, milk,
23
CA 3057013 2019-09-27
colostrum, mammary gland secretion, lymph, sweat, lacrimal fluid, gastric
fluid, synovial fluid,
mucus, or combinations thereof. Typically, the biomarkers are detected in
blood, urine, or a
biopsy sample.
Likewise, as described above, the biomarkers described herein may be detected
in any
form, including protein, DNA, RNA, or a combination thereof. Typically, the
biomarkers are
detected as protein.
It will be understood that the biomarkers may be assessed in any known age
group
suspected of being afflicted with PAD. Typically, the subject is an adult and
is typically at least
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 years of age.
The methods described herein also encompass treating the subject based upon
the
outcome of the method. For example, if the subject is diagnosed with early
CTLI, the subject
may be further screened and referred for treatment. Thus, also described
herein are methods of
treating a subject with peripheral artery disease. The methods of treatment
comprise carrying
out a diagnostic or staging method described herein and treating the subject
based upon the
diagnosis or stage of disease.
The above disclosure generally describes the present invention. A more
complete
understanding can be obtained by reference to the following specific examples.
These
examples are provided for purposes of illustration only and are not intended
to be limiting unless
otherwise specified. Thus, the invention should in no way be construed as
being limited to the
following examples, but rather, should be construed to encompass any and all
variations which
become evident as a result of the teaching provided herein.
The following examples do not include detailed descriptions of conventional
methods,
such as those employed in the construction of vectors and plasmids, the
insertion of genes
encoding polypeptides into such vectors and plasmids, or the introduction of
plasmids into host
cells. Such methods are well known to those of ordinary skill in the art and
are described in
numerous publications including Sambrook, J., Fritsch, E. F. and Maniatis, T.
(1989), Molecular
Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory
Press, which is
incorporated by reference herein.
Without further description, it is believed that one of ordinary skill in the
art can, using the
preceding description and the following illustrative examples, make and
utilize the compounds
of the present invention and practice the claimed methods. The following
working examples
24
CA 3057013 2019-09-27
therefore, specifically point out the typical aspects of the present invention
and are not to be
construed as limiting in any way the remainder of the disclosure.
Examples
Example 1: Fatty Acid-Binding Protein 3 (FABP3): A Novel Biomarker for the
Diagnosis of
Peripheral Arterial Disease
Abstract
Of the 200 million people that suffer from peripheral artery disease (PAD)
worldwide, 5-
25% progress to chronically threatened limb ischemia (CTLI), which has
devastatingly high
rates of lower limb amputations and mortality. Unfortunately, the diagnosis of
CTLI is often
delayed, which results in an increased risk of limb loss, morbidity, and
mortality. The purpose of
this study was to identify circulating blood biomarker(s) for use alongside
clinical assessments
to diagnose PAD including patients with CTLI .
In this study, ELISA experiments were conducted on non-PAD (n=40) and CTLI
patients
(n=50) to investigate the levels of Fatty Acid Binding protein 3 ( FABP3),
also referred to as
heart type FABP (hFABP). Binary logistic regression analysis was also
conducted while
controlling for confounding factors. Receiver operating characteristic (ROC)
curves, alongside
the non-parametric estimate of the area under the curve (AUC), and their
corresponding 95% Cl
were calculated. Our data demonstrated that FABP3 is significantly up-
regulated in CTLI
patients when compared to the non-PAD control group, even after adjusting for
confounding risk
factors. FABP3 has a large effect size for CTLI when compared to non-PAD
patients, an
associated OR of 1.8, an AUC of 0.8, as well as a significant reduction in its
plasma levels after
arterial revascularization. Lastly, FABP3 levels were noted to increase with
worsened PAD
severity with the absence of clinical and biochemical evidence of myocardial
ischemia.
Our findings demonstrate that FABP3 is a potential biomarker that can be used
to
diagnose patients with PAD as well as CTLI. This will significantly curtail
the morbidity and
mortality associated with this disease.
Introduction
Lower extremity peripheral arterial disease (PAD) is a common presentation of
atherosclerosis!. Over 200 million people are reported to have the disease-Z,
and the global
prevalence of PAD is estimated to range from 3-12%1,1. However, despite its
prevalence, PAD
goes undiagnosed commonly, with studies suggesting that physicians fail to
detect and
CA 3057013 2019-09-27
diagnose PAD in 51% of their patients. This lack of PAD awareness among
healthcare
providers and patients results in the failure of medical therapy initiation,
atherosclerosis risk
factor modification, and deferred referral to specialists. Moreover, it also
leads to a delay in
surgical intervention, which puts patients at significant risk of morbidity,
disability, mortality and
major limb amputation.
Furthermore, it is estimated that over a period of five years, 5-25% of PAD
patients
experience disease progression to the most severe form of PAD, known as
critical limb
ischemia (CTLI)-. CTLI manifests as rest-pain (early stage CTLI), or non-
healing ischemic
ulcers with gangrenous tissue loss in the lower extremities (late stage CTLI).
Currently,
treatment options for CTLI are limited to medical management in addition to
arterial
revascularization or major limb amputation. However, despite our best efforts,
patients with
CTLI still face a high mortality and disability risk. The Trans-Atlantic Inter-
Society Consensus for
the Management of Peripheral Arterial Disease (TASC II) report estimates a 25%
mortality rate
at one year for patients diagnosed with CTLI. Similarly, a review of 20,464
patients who
underwent a major amputation secondary to CTLI demonstrated that a significant
portion of
patients had no surgical attempts performed on them in the year prior to
amputationil.
One major reason for these high rates of lower limb amputation and deaths is
delayed
diagnosis. This is exemplified in a recent study which showed that primary
amputation was the
first treatment performed for 67% of Medicare patients with CTLIn. However,
several studies
have suggested that a significant portion of lower limb amputations can been
delayed or
prevented, especially through early diagnosis and routine screening13-15.
However, identifying patients in the early stages of CTLI is a major challenge
faced by
many physicians and primary healthcare providers. The ratio of the brachial
artery blood
pressure to the ankle blood pressure - ankle brachial index (ABI) ¨ serves as
a cost-effective
screening tool for PADn, E. However, it fails to reliably diagnose PAD in many
patients as well
as patients with CTLIn' -12. Moreover, ABI values of diabetic patients, with
and without PAD, are
usually falsely elevated due to incompressible calcified vesselsn. Thus, the
ABI ratio is not a
reliable tool in being able to distinguish patients with PAD from within a
large overall proportion
of patients with suspected lower limb pain. Furthermore, while a few risk-
prediction models for
PAD have been deve10ped20-23, these models currently lack rigorous external
data validation
and at best have demonstrated modest predictive abilities. As a result, they
are not widely used
26
CA 3057013 2019-09-27
in clinical practice24. Circulating biomarkers such as 8-2-microglobulin and C-
Reactive Protein
(CRP) have also been proposed to be indicative of PAD status, however, they
lack the
appropriate specificity and sensitivity required to diagnose PAD or CTLIE.
Consequently, distinguishing patients with PAD still remains a major
diagnostic
challenge for clinicians today. This is especially true for physicians with
limited access to
medical specialists and diagnostic imaging. Therefore, a purpose of this study
was to
characterize the proteomic plasma profile of patients with PAD as well as CTLI
in order to
identify a biomarker(s) that can assist physicians in diagnosing PAD and CTLI.
Material and Methods
Study design, setting, and data sources
We conducted a case-control study at St. Michael's Hospital (Toronto, Canada)
with
PAD and CTLI patients serving as cases and non-PAD patients serving as
controls.
Ethics approval
This study was approved by the research ethics board at St. Michael's Hospital-
University of Toronto in Ontario, Canada. Informed consent was obtained from
all participants.
All the experiments performed were conducted in accordance to the relevant
guidelines and
regulations.
Patient selection
The PAD status was defined clinically as per the Rutherford Classification
Criteria of
chronic limb ischemiaa. Patients with PAD referred to vascular surgery
ambulatory clinics or
emergency department at St. Michael's Hospital from June 2017 through March
2018 were
asked to participate in this study. We excluded all patients on
anticoagulants, chemotherapy or
biological anti-inflammatory agents. Patients diagnosed with sepsis,
systematic inflammatory
disease or with active/history of any cancer or deep vein thrombosis (DVT)
were excluded as
well. Moreover, patients with an acute or 6 month history of acute coronary
syndrome, heart
failure, or uncontrolled arrhythmia as defined by American College of
Cardiology, also failed to
meet the inclusion criteria of this study18-23. The non-PAD control cohort was
defined as patients
with cardiovascular risk factors alongside a normal arterial US of the lower
limbs, palpable distal
pulses and without a significant clinical history of claudication.
27
CA 3057013 2019-09-27
Baseline measurements
In all subjects, a thorough physical exam and complete medical history was
obtained
from each patient by an independent PAD expert. Medical history including
details of any
previous acute coronary syndrome, hyperlipidemia, arterial arrhythmia,
arterial hypertension,
.. renal disease, congestive heart failure, history of stroke or transient
ischemic attack (TIA),
history of cancer, diabetes, and smoking status. Each patient received lower
limb arterial
imaging (arterial ultrasound (US), computed tomography angiogram (CTA) or
angiogram) as
part of the PAD assessment. Arterial US findings including ankle brachial
index (ABI) were
recorded for each patient. Blood samples were drawn into vacutainer tubes
containing EDTA.
.. Plasma was then extracted from this blood via centrifugation at 3000 rpm
for 10 min (4 C),
which was then aliquoted and stored at -80 C. Plasma samples that had
previously been
thawed were not utilized for this study.
Study Size and Bias
In this study, plasma samples were collected from 40 non-PAD controls and 50
CTLI
.. patients (Table 1). This sample size allowed us to detect a difference
between non-PAD
controls and CTLI patients of 0.50 standard deviation units in FABP3 with 80%
power at a two-
sided alpha of 0.05. Since our analyses were exploratory, alpha levels were
not adjusted for
multiple comparisons.
Table 1: Baseline Characteristics for the patient Cohort by subgroup (N = 90).
Baseline Characteristics
Non-PAD CTLI
p-value
Number of patients 40 50
Age, (yrs) 62 70
<0.001*
Male (%) 70 69
0.563
Hypertension CYO 72 88
0.014*
Hypercholesterolemia (%) 62 85
0.003*
Diabetes mellitus (%) 46 57
0.106
Renal Insufficiency (%) 2 12
0.061
Smoking (%) 58 85
0.010*
Congestive heart failure (%) 0 7
0.086
Coronary artery disease (%) 16 52
<0.001*
Stroke/TIA CYO 6 16
0.122
Ankle pressure (mmHg) 109 38
<0.001*
Significant values are noted with an (*) for P value 5_0.05
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CA 3057013 2019-09-27
Outcomes
A primary outcome of this study was to confirm FABP3 a potential biomarker for
PAD
and CTLI that could be used for diagnostic purposes. Secondary outcomes
included identifying
the source of this biomarker, as well as its effects after arterial
revascularization, among others.
Patient selection for proteomics protein discovery
The non-PAD control cohort were defined as patients with cardiovascular risk
factors
including hypertension, hyperlipidemia, diabetes, smoking, or family history
of heart disease,
alongside a normal arterial US of the lower limbs and palpable distal pulses,
but without a
significant clinical history of PAD claudication. The PAD patients were
defined clinically as per
the Rutherford Classification Criteria of chronic limb ischemiaa. Asymptomatic
patients had no
clinical symptoms of PAD however these patients had radio-graphical evidence
of PAD on
ultrasound (stage 0). Mild PAD patients had mild claudication and were able to
complete a
treadmill exercise test (five minutes walking on a treadmill at 2 mph on a 12%
incline) with ankle
pressure (AP) of > 50 mm Hg after exercise. However, the AP needed to be at
least 20 mmHg
lower than resting value (stage 1). The moderate PAD patients had moderate
claudication and
were between stages 1-3 (stage 2). The severe PAD patients had disabling
claudication and
were not able to complete the treadmill exercise with an AP after exercise of
< 50 mmHg (stage
3). In contrast, the early CTLI patients (stage 4) were defined clinically as
patients with the
presence of ischemic rest pain and the absence of ischemic ulcers or
gangrenous tissue. These
patients had a resting AP of < 40 mmHg with a toe pressure (TP) of < 30 mmHg,
as well as an
evidence of severe arterial disease documented on angiogram or CTA. The
advanced CTLI
patients had evidence of tissue loss as well as AP of < 40 mmHg with a TP of <
30 mmHg (
stages 5-6). Lastly, we recruited a group of healthy patients, which served as
the negative
control in some experiments. This group was defined as participants without
any cardiovascular
risk factors, alongside a normal arterial US of the lower limbs.
The following patients were excluded:
1) Patients on chemotherapy or biological anti-inflammatory agents.
2) Patients diagnosed with sepsis, inflammatory disease or with
active/history of
any cancer.
3) Patients with an acute or 6 month history of acute coronary syndrome,
heart
failure, or uncontrolled arrhythmia as defined by American College of
Cardiology29-32.
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CA 3057013 2019-09-27
4) Patients diagnosed with acute limb ischemia.
5) Patients with tissue loss/gangrene (late CTLI Rutherford stages 5-6)
were
excluded from this study, as these patients have advanced disease and tissue
ischemia.
FABP3 and troponin multiplex assay
This experiment was carried out in order to simultaneously assess FABP3 and
Troponin
I (TnI) levels in 90 non-PAD and CTLI patients. Patients' arterial status was
classified as
described above. We also recruited patients that presented to our cardiac care
unit with
documented acute coronary disease (ACS) on ECG and angiogram to serve as a
positive
control cohort.
Plasma samples were analyzed in duplicate using MILLIPLEX MAP Human
Cardiovascular Disease (CVD) Magnetic Bead Panel 1 (EMD-Millipore; Billerica,
MA) to
determine the concentrations of FABP3 and Tnl. Analysis was completed as
described by the
manufacturer. All sample analyses were completed on the same day to eliminate
inter-assay
variability. Sample intra-assay and inter-assay CV were both <10%. The MagPix
analyzer
(Luminex Corp; Austin, Texas) was calibrated prior to analysis using Fluidics
Verification and
Calibration bead kits (Luminex Corp). A minimum of 50 beads for each targeted
biomarker were
acquired using Luminex xPonent software and analyzed using Milliplex Analyst
software (v.5.1;
EMD-Millipore).
Furthermore, in order to study the relationship between FABP3 and PAD, FABP3
levels
were measured in a 486 non-PAD and PAD patients. Lastly, in order to study the
relationship
between FABP3 and PAD severity status, FABP3 levels were measured in a group
of 250
patients stratified by their PAD status (PAD and CTLI) along with non-PAD
patients who served
as a negative control. Once again, samples were measured in duplicate using
M1LLIPLEX MAP
CVD Magnetic Bead Panel 1 kit, as described above
Plasma levels of biomarkers in CTLI before and after arterial
revascularization
In the CTLI cohort, twelve patients who had undergone infra-inguinal bypass
arterial
revascularization agreed to provide blood samples pre-operatively and post-
operatively. The
post-operative sample was collected twelve weeks after surgery. Blood samples
were
processed as aforementioned and used to assess the biomarker before and after
surgery via an
protein multiplex.
CA 3057013 2019-09-27
Muscle homogenate preparation and Western blot analysis
To localize our biomarker within skeletal muscles, gastrocnemius muscle was
obtained
from patients with end stage CTLI undergoing major limb amputation
(experimental group n=4)
and healthy non-PAD patients undergoing elective orthopedic surgeries
(negative control group
n=3). lmmunoblots were carried out using homogenate skeletal muscle protein
obtained both
groups. All snap frozen muscle samples were homogenized in a lysis buffer
(Cell Signaling
Technology, Beverly, MA, USA) using a ultrasonic homogenizer (Biologics Inc,
Manassa,
Virginia, USA). Protein concentration was measured in duplicate by the
bicinchoninic acid (BCA)
assay (Pierce, Rockford, IL USA). For the Western blot studies, aliquots
equivalent of
approximately 80pg of protein were separated on gradient (10-15%)
polyacrylamide sodium
dodecyl sulphate gels. After electrophoresis, proteins were electro-
transferred to a nitrocellulose
membrane. The membrane was blocked with skimmed milk powder in TBST (0.05%
Tween 20,
100 mM NaCl, 10 mM Tris¨HCI pH 7.8) for 30 minutes and then incubated with the
primary
antibody FABP3 (Abcam, Toronto, ON, Canada) overnight at 4 C. Secondary
antibody
polyclonal goat anti-mouse horseradish peroxidase (HRP) conjugated (Cedarlane,
Burlington,
ON, Canada) was added. Blots were developed in ECL detection reagents
(Annersham, ECL
Western Blotting Detection Reagents; GE Healthcare) and the chemiluminescence
emitted from
immune complexes was visualized with a ChemiDoc image system (Bio-Rad,
Mississauga, ON,
Canada). The images were quantified by Image J software.
Immunohistochemistry of FABP3 biomarkers
Immunohistochemistry was performed on 5 pm formalin-fixed, paraffin-embedded
muscles obtained from lower limb amputations performed to treat CTLI (n = 4).
Non-ischemic
gastrocnemius muscle was obtained from non-PAD patients undergoing elective
orthopedic
surgeries not related to CTLI (n = 3). Hematoxylin-eosin staining and Masson's
Trichrome were
conducted according to manufacturer's instructions (Sigma, St. Louis, MO,
USA). For detection
of FABP3 and CD68, sections were stained using anti-human mouse polyclonal
antibody
(Thermo Fisher Scientific, Massachusetts). These sections were incubated at 4
C, and then
incubated again with HRP-conjugated secondary antibodies according to the
immunostaining
procedure.
Statistical methods
Demographics and baseline measurements were recorded for each patient.
Baseline
data were expressed as means with standard deviations (SD) or as percentages.
Evaluations of
baseline characteristics were done using independent t-tests or Mann-Whitney U
test for
31
CA 3057013 2019-09-27
continuous variables. Fisher's exact test or chi-square test was used for
categorical variables.
ANOVA was used in experiments where more than two group differences needed to
be
analyzed. Cohen's d was used to compute the effect size for the comparison
between two group
means. Treatment outcomes across the groups or according to specific
biomarkers were
analyzed with logistic regression analyses. A stepwise binary logistic
regression analysis using
the backward elimination procedure was performed to study the impact of
potential
confounders. Confounding variables were identified to be age, gender, smoking,
diabetes
mellitus, coronary artery disease, hypertension, hypercholesterolemia and
statins, as per our
literature review-L1.5-' 3- . For significantly associated biomarkers,
receiver operator
characteristic (ROC) curves were estimated, which served as a visual means to
describe the
ability of the model to correctly classify `CTLI or PAD' and `non-PAD'
patients. Non-parametric
estimate of the area under the curve (AUC), Youden index and their
corresponding 95% Cl
were calculated SPSS software version 23 (SPSS Inc., Chicago, Illinois, USA)
was used for
data entry and analysis, while Prism 7 (Graphpad, San Diego, CA, USA) was used
for various
graphical illustrations and volcano plots. All analyses were carried out at a
5% two-sided
significance level.
Results
ELISA confirmation of candidate biomarkers
To confirm the up-regulation of FABP3 in patients with PAD, protein levels
were
measured in CTLI and non-PAD patients (Table 1). We identified significant
differences in the
demographics of both patient groups with regards to age, hypertension,
hypercholesterolemia,
history of smoking, coronary arterial disease and ABI values. After conducting
in-depth
statistical analysis, FABP3 had a large effect size with a corresponding value
of 1.02 as well as
a large mean difference of 2.51 ng/ml (95% Cl 1.17¨ 3.86) (Table 2). In order
to study the
association between FABP3 and CTLI, we calculated the odds ratio (OR).
Furthermore, to
account for the effect of confounding factors, we conducted binary logistic
regression analysis
accounting for age, gender, smoking, diabetes mellitus, hypertension,
hypercholesterolemia,
coronary arterial disease, chronic kidney disease and statin usage (Table 3).
FABP3 was found
to have a large OR (1.88, 95% 01 1.45 ¨ 2.37), which remained significant even
after adjusting
for confounding factors (1.42, 95% Cl 1.04 ¨ 1.93).
Table 2: Protein multiplex studies confirming the up-regulated of FABP3 in
CTLI patients.
Biomarker Effect Size Mean Difference (ng/ml) 95% CI
FABP3 1.02 2.51* 1.17 ¨
3.86
Significant values are noted with an (*) for P value 50.05
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CA 3057013 2019-09-27
Table 3: Unadjusted and adjusted odds ratio after conducting logistic
regression analysis.
Binary Logistic regression reference category is non-PAD. Adjusted regression
analysis were
conducted for the following confounding variables: age, gender, smoking,
diabetes mellitus,
hypertension, hypercholesterolemia, coronary artery disease, chronic kidney
disease and
statins.
Unadjusted Odds Ratios Adjusted Odds Ratio
OR 95% Cl OR 95% Cl
FABP3 1.875 1.454 ¨ 2.373 1.418 1.042 ¨ 1.929
Plasma levels of FABP3 in CTLI before and after arterial revascularization
For deeper insights on the biochemical effects of arterial revascularization
on the
FABP3, the levels of each protein were measured in twelve CTLI patients prior
to surgery and
three months following surgery via protein multiplex (Table 4). Our data shows
that arterial
revascularization of the ischemic limb causes a significant down regulation in
circulatory levels
of FABP3.
Table 4: Biomarker level in the same CTLI patients before and after arterial
revascularization.
Plasma levels of FABP3 were measured in twelve CTLI patients before and twelve
weeks after
re-establishing blood flow.
Effect Size Mean difference (ng/ml) 95% Cl
FABP3* 0.96 1.2* 0.26 ¨
2.20
(*) P value <0.05.
Receiver operating characteristic (ROC) analysis
FABP3 was selected for further statistical analysis as a potential biomarker
for PAD and
CTLI as this protein had a large OR, large effect size, as well as level
normalization after arterial
revascularization. Two predictive models were compared in this ROC analysis.
First, a ROC
curve was estimated for FABP3 as a single predictor for PAD in a group of 486
patients, which
demonstrated an area under curve (AUC) of 0.8234 (95% Cl, 0.7818 to 0.8651) is
represented
by the solid line (Figure 2). When compared to non-PAD control, our ROC
analysis confirms the
use of FABP3 in plasma as an excellent biomarker of PAD with large area under
curve. Second,
a ROC curve was estimated for FABP3 as a single predictor of CTLI, which
demonstrated an
AUC of 0.80 (95% Cl 0.65 ¨ 0.87) (Figure 3). However, to further understand
the prediction
capabilities of FABP3, another ROC curve was estimated using probability
estimates from a
fitted model featuring CTLI status regressed on FABP3 and previously validated
confounding
factors (age, gender, smoking, diabetes mellitus, hypertension,
hypercholesterolemia, coronary
artery disease, chronic kidney disease and statins). The AUC value for FABP3
was improved
after adjusting for confounding risk factors (0.92, 95% Cl 0.79 ¨ 0.97)
(Figure 3).
33
CA 3057013 2019-09-27
FABP3 levels in progressive PAD disease
The relationship between worsening PAD severity and FABP3 was also
investigated, as
our ROC analysis demonstrated a significant true positive rate (TPR) for
FABP3, indicating its
potential as a diagnostic biomarker for PAD and CTLI. In this experiment,
FABP3 levels were
measured in non-PAD, PAD and CTLI patients (n=250). An increase in FABP3
levels was
observed as the severity of PAD increased (Figure 4). FABP3 also had a
significantly high mean
value of 4.6ng/mL, SE 0.2ng/mL) in CTLI patients when compared to other
clinical stages (p-
value <0.0001), as per our ANOVA analysis.
FABP3 levels increase due to CTLI without evidence of myocardial injury or
renal failure
Although patients with ACS were excluded from this study, we wanted to assess
for
biochemical evidence of cardiac injury, as FABP3 is linked to cardiovascular
disease. Hence,
the presence of myocardial ischemia was investigated on a molecular level via
protein multiplex,
by simultaneously measuring the levels of troponin I and FABP3 in our control
and experimental
groups. Patients presenting to our hospital with ACS served as the positive
control (n=15),
whereas fifteen healthy patients without any cardiovascular risk factors or
PAD represented the
negative control group. After analyzing our data, there was no significant
difference found in
levels of troponin I in the CTLI cohort in comparison to the control group
(Figure 5). However, as
expected, the positive ACS control group had elevated levels of TnI relative
to healthy, non-PAD
and CTLI patients. Given the absence of clinical signs of myocardial ischemia,
in addition to
similar troponin levels in the CTLI group and the negative control group, the
increase in FABP3
levels observed in CTLI patients is likely not associated with myocardial
injury. This experiment
favours the hypothesis that the increased FABP3 plasma levels observed in PAD
and CTLI
patients is not a result of myocardial injury. Lastly, we did not observe any
statistical difference
in creatinine levels between non-PAD, PAD and CTLI cohorts (p-value = 0.153),
indicating that
the increase in FABP3 levels in the PAD and CTLI cohort is independent of
kidney dysfunction.
Localization of FABP3 within lower limb skeletal muscles
In this part of the study, gastrocnemius skeletal muscles were investigated as
a possible
source of FABP3 in patients with CTLI. Using Western blot studies, FABP3
expression was
investigated in samples obtained from control group (non-PAD patients
undergoing elective
orthopedic surgeries) and experimental group (CTLI patients undergoing lower
limb
amputation). Quantitative analysis revealed over two-fold increase in
expression of FABP3 in
muscle tissue obtained from CTLI patients, when compared to controls (Figure
6). Next,
34
CA 3057013 2019-09-27
immunohistochemistry was performed on experimental and control skeletal
muscles to further
confirm the immune-blot findings. Higher levels of FABP3 as well as 0D68
(marker of
macrophages) were observed within ischemic muscles in relation to control
samples.
Furthermore, the Masson's trichrome stain demonstrates increased levels of
tissue fibrosis
within muscles isolated from CTLI patients relative to the control group.
Along with the western
blot findings, this data favors the hypothesis that skeletal muscles might be
a potential source of
FABP3 in patients with CTLI (Figure 7).
Discussion
In this study, we sought to confirm FABP3 as a diagnostic marker for PAD and
CTLI. To
achieve this, non-PAD patients, PAD and CTLI patients were investigated for
the levels of
FABP3 in plasma. Our data demonstrated that, relative to non-PAD controls,
patients with CTLI
had a large FABP3 effect size and high FABP3 OR. Moreover, FABP3 levels were
observed to
increase with severity of PAD, but also decrease after successful arterial
revascularization.
Thus, given these novel findings, FABP3 appears to be a robust biomarker for
identifying
patients with PAD including patients with CTLI.
FABP3, also known as heart-type FABP, belongs to a family of multigene fatty
acid-
binding proteins. It is primarily expressed in the heart, where it constitutes
4-5% of all cellular
proteins, but is also expressed in the brain and skeletal muscle among other
organs and
tissues. Within muscle cells, FABP3 is primarily responsible for mediating the
uptake of
intracellular fatty acids as well as their transport toward the mitochondrial
8-oxidation system1 .
Moreover, elevated levels of FABP3 have been reported in patients with
diabetes, muscle
toxicity, among other conditionsL. 2.12. Recent studies have also alluded to
the capabilities of
FABP3 in serving as a serum biomarker for the early diagnosis of stroke and
acute myocardial
ischemia40-42. Similarly, the findings from this study suggest that FABP3 also
serves as a
biomarker for the diagnosis of PAD as well as CTLI, after adjusting for
confounding factors,
including those conditions that lead to elevated levels of FABP3 (such as
diabetes). This has
immense clinical utility, especially in ambiguous patient cases where
diagnosis of PAD is
suspected, but uncertain. For instance, studies have shown that diagnosing
diabetic patients
with PAD or CTLI can be challenging due to neuropathy, which masks the
ischemic rest pain
associated with CTLI. Consequently, this makes it harder for physicians to
decide if a high-risk
intervention is needed or not, as these patients are only suspected to have
CTLI. Thus, having
a clinical biomarker for PAD and CTLI eliminates the ambiguity surrounding
patient cases, as it
CA 3057013 2019-09-27
can concretely indicate to physicians if an intervention is needed or not.
Moreover, many CTLI
patients report experiences of prolonged wait times. Subsequently, another
clinical advantage
that comes with having a biomarker for CTLI is that it significantly reduces
the wait time, as it
can diagnose CTLI within minutes and subsequently lead to earlier
intervention.
Other studies have also explored the use of circulating protein biomarkers for
CTLI, but
reached vastly different conclusions in comparison to the findings from this
study. For instance,
Li et al., identified Siglec 5 as a biomarker of CTLP. However, our data does
not demonstrate
the overexpression of Siglec 5 in CTLI patients when compared to our control
non-PAD
patients. One reason for this disparity in findings could be due to
differences in patient cohort
selections. Li etal., only recruited diabetic patients with ABI>0.9, whereas
we recruited both
diabetic and non-diabetic CTLI patients with an ankle pressure of <40 mm Hg
and toe pressure
of < 30 mm Hg. We used Rutherford's criteria of ankle and toe pressures to
classify PAD
patients over ABI, as the ABI values of diabetic patients with and without
CTLI are usually
falsely elevated due to incompressible calcified vessels-13. Consequently, we
did not deem the
ABI ratio to be a reliable tool in being able to diagnose diabetic patients
with CTLI.
Furthermore, Li et al., used a targeted "246 protein based chip assay" to
identify their
biomarker4-c. In our study, not only did we recruit both diabetic and non-
diabetic patients, but we
were also the first to identify FABP3 as a candidate protein biomarker for PAD
and CTLI
patients. Similarly, another study by Hung and authors found over 50
differentially expressed
plasma proteins for CTLI. However, they were also limited in their patient
cohort as they only
recruited hemodialytic diabetic patients with and without CTLI4:1.
With regards to its source of expression, studies have reported that FABP3 is
mainly
expressed in myocardial and skeletal muscles'''. Upon conducting western blots
and
immunohistochemistry, our data was consistent with past research findings and
demonstrated
that ischemic skeletal muscles are the likely source of FABP3 expression in
CTLI patients,
suggesting a relationship between the two.
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Example 2: Detection of FABP3 in Urine and Plasma
As shown in Figure 8, we conducted ROC analysis for FABP3 in urine in non-PAD
and
PAD patients. When compared to non-PAD control n=41, our data confirms the
presence of
FABP3 in urine. Additionally, the ROC analysis demonstrates the excellent
potential use of
FABP3 in urine as a robust biomarker of PAD with large area under curve.
Example 3: A Comparison of Multiple Biomarkers in Non-PAD Subjects, PAD
Subjects,
and ACS Subjects
We assessed the levels of Tnl, FABP4, and FABP3 and assessed ABI in subjects
that
do not have PAD (n=114), those that do have PAD (n= 391), and those that have
ACS (n=15).
As shown in Table 5, FABP4 and FABP3 were both elevated in subjects with PAD
and those
with ACS. However, TnI was only elevated in ACS and, therefore, can act as a
useful secondary
biomarker to distinguish between PAD and ACS. FABP4 has been shown to be
released from
adipocytes while associating with lipolysis and possibly acting as an
adipokine. Elevation of
circulating FABP4 levels is associated with atherosclerosis, and
cardiovascular events.
CA 3057013 2019-09-27
Table 5: Comparison of multiple biomarkers in non-PAD, PAD and ACS.
Non-PAD PAD Acute coronary syndrome (ACS)
Troponin pg/mL 343 361 11.55
FABP4 ng/mL 8.61 21.85 38.50
FABP3 ng/mL 2.33 4.852 31.48
ABI 1.2 0.62 1.0
From Table 5, it can be seen that FABP3 or FABP4 can independently be used to
diagnose patients with PAD, as levels are elevated compared to controls but
not to the extent
seen in ACS. As shown in table 5, there was a 2.5 fold increase in FABP4
levels in PAD
patients relative to non-PAD patients. Troponin is a useful secondary marker
to assist in ruling
out ACS as the reason for the elevation. Combinations of all three of three
protein markers,
"FABP3 or FABP4" alone or in combination with troponin, increase the
sensitivity and specificity
for detection of PAD.
41
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