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Clinical Relevance: This study aimed to investigate the impact of zinc deficiency on
angiogenesis. We found that zinc deficiency impaired the rate of ischemia-induced
revascularization through enhanced oxidative stress rates in animal model. In addition,
serum zinc levels were positively associated with SPP in the CLTI patients. Thus, the
intake of zinc could be useful for the prevention and/or treatment of ischemic limb disease.
Circulating zinc level could be a useful marker for the assessment of atherosclerosisbased vascular disease such as limb ischemia. Possibly, nutritional improvement by zinc
intake could lead to prevention and treatment of ischemic vascular disease.
Before
surgery
Ischemic/Normal LDBF ratio
After
surgery
Day 3
Day 7
Day 14
Control
ZD
0.8
0.6
0.4
0.2
After
surgery
Day 28
Control
ZD
Control
ZD
14
21
28 (Day)
P<.05
100
Capillary density
(per field)
80
60
40
20
Control
P<.05
P<.05
1.2
Relative mRNA levels
(Fold change)
Capillaries/muscle fiber
1.5
0.5
ZD
0.8
0.6
0.4
0.2
Control
ZD
Control
ZD
Figure 1
P<.05
Control
d-ROMs (Carr units)
300
ZD
200
100
Control
ZD
P<.05
Relative mRNA levels
(Fold change)
Nitrotyrosine (nM)
Control
ZD
Control
ZD
Nox2
p22phox
p47phox
p67phox
Figure 2
10
12 (Week)
Hindlimb ischemic surgery
WT mice
Zinc deficient diet
Apocynin or vehicle
(300mg/kg/day)
LDBF analysis
Ischemic/Normal LDBF ratio
ZD+Vehicle
ZD+Apocynin
0.8
0.6
0.4
0.2
After
surgery
Capillary density
(per field)
80
14 (Day)
NS
60
40
20
ZD+Vehicle ZD+Apocynin
Figure 3
mmHg
50
r =.538
P <.01
SPP
40
30
20
10
50
100
150
Serum zinc concentration (mg/dL)
Figure 4
P<.05
Serum zinc level (μg/dL)
150
100
50
Control
ZD
Supplementary Figure 1
Alkaline phosphatase-positive cells
Supplementary Figure 2
Table I Patient characteristics
Characteristic
(n = 51)
Age (years)
71.4 ± 8.1
Male/Female
34/17
Body mass index (kg/m )
21.0 ± 4.4
Serum zinc concentration (µg/dL)
62.3 ± 16.9
Serum copper concentration (µg/dL)
129.3 ± 28.3
Alkaline phosphatase (IU/L)
292.8 ± 97.4
Albumin (g/dL)
3.4 ± 0.6
Hemoglobin (g/dL)
11.0 ± 2.0
C-reactive protein (mg/dL)
2.8 ± 3.5
Hypertension
39
Diabetes mellitus
33
Hemodialysis
19
Dyslipidemia
27
Smoker
40
Coronary artery disease
29
Cerebrovascular disease
10
Ankle Brachial index
0.45 ± 0.30
Skin perfusion pressure
18.7 ± 8.9
Drug
α-blocker
β-blocker
17
Ca-blocker
31
Angiotensin Ⅱ receptor blocker
18
Angiotensin-converting-enzyme inhibitor
Diuretic
Cilostazol
25
Clopidogrel
19
Aspirin
36
Warfarin
Direct oral anticoagulant
Statin
21
Insulin
Prednisolone
Categorical data are expressed as numbers. Continuous data are expressed as the means ± SD
Table II Patient classification and treatments
Characteristic
(n = 51)
Rutherford classification
5(10)
38(74)
8(16)
WIfI clinical stage
2(4)
8(16)
17(33)
24(47)
GLASS stage
2(4)
2(4)
47(92)
Treatments
Bypass surgery
34(67)
Endovascular surgery
2(4)
Bypass & Endovascular surgery
15(29)
Categorical data are expressed as numbers (%).
GLASS = Global Limb Anatomic Staging System
Table III Correlation with skin perfusion pressure
Variable
Univariate
Multivariate
P-value
P-value
Serum zinc level
.538
.000
0.445
.001
Albumin
.302
.031
0.147
.265
Diabetes mellitus
.288
.041
0.240
.058
Hemoglobin
.255
.071
Age
- .195
.171
Body mass index
.194
.173
Antiplatelets
.193
.174
C-reactive protein
- .191
.179
Hemodialysis
.178
.212
Statin
.163
.254
Cerebrovascular disease
.158
.268
Antihypertensives
.147
.302
Hypertension
.082
.568
Female
.061
.671
Smoker
- .047
.743
Alkaline phosphatase
.042
.772
Coronary artery disease
.024
.865
Dyslipidemia
- .017
.904
Serum copper level
.008
.956
Figure Legends
Figure 1. Zinc-deficient mice showing reductions in the rates of perfusion recovery
and capillary vessel formation in ischemic limbs. A. Representative LDBF images in
the ischemic limb of zinc-deficient mice or control WT mice. A low perfusion signal (dark
blue) was observed in the ischemic hindlimb of zinc-deficient mice, whereas a high
perfusion signal (red) was detected in the control WT mice at postoperative days 14, 21,
and 28. B. Quantitative analysis of the ischemic to nonischemic LDBF ratio in the zincdeficient mice or control WT mice before surgery and at different time points after surgery.
Results are presented as the mean ± standard deviation (n=8 in each group). *P< .05 vs
Zinc-deficient mice. C. Fluorescence staining of ischemic tissues with anti-CD31
monoclonal antibody (red) on postoperative day 28. D and E. Quantitative analysis of
capillary density in zinc-deficient mice or the control WT mice on postoperative day 28.
Capillary density was expressed as the number of capillaries per high power field (×400)
(left) and capillaries per muscle fiber (right). F. mRNA levels of VEGF in the ischemic
muscle in the zinc-deficient mice or control WT mice on postoperative day 28. Levels of
mRNA were measured using the real-time polymerase chain reaction method (n=8 in each
group). All results are normalized to GAPDH. *P< .01 vs control. Results are presented
as mean ± standard deviation (n=8 in each group). LDBF, laser Doppler blood flow; WT,
wild type; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 2. Zinc deficiency increases the rate of oxidative damage in ischemic muscles.
A. Production of reactive oxygen species (ROS) was evaluated by immunostaining with
dihydroethidium on postoperative day 28 (×400 each field; red). B and C. Serum levels
of the derivatives of reactive oxygen metabolites (d-ROMs) (B) and nitrotyrosine (C) in
the zinc-deficient mice or the control WT mice after hindlimb surgery (n=8 in each group).
D. mRNA levels of Nox2, p22phox, p47phox and p67phox in the ischemic muscle in the zincdeficient mice or control WT mice on postoperative day 28. Levels of mRNA were
measured using the real-time polymerase chain reaction method (n=8 in each group). All
results are normalized to GAPDH. *P< .01 vs control. WT, wild type; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase
Figure 3. NADPH oxidase inhibitor, apocynin, restores ischemia-induced
angiogenesis in zinc-deficient mice. A. Schematic illustration of the experimental
protocol. Zinc-deficient and control mice were treated with both a zinc-deficient diet and
an NADPH oxidase inhibitor, apocynin (300 mg/kg/day), in drinking water from 3 weeks
of age. Then, the mice were subjected to unilateral hindlimb surgery at the age of 10
weeks. B. Quantitative analysis of the ischemic to nonischemic LDBF ratio in the zincdeficient mice treated with apocynin at different time points after surgery. C. the
quantitative analysis of capillary density of the ischemic muscle in the zinc- deficient
mice treated with apocynin at 14 days after surgery. Results are shown as the mean ±
standard deviation (n=8 in each group). *P< .05. NADPH, nicotinamide adenine
dinucleotide phosphate; LDBF, laser Doppler blood flow, NS, nonsignificant.
Figure 4. Association of serum zinc level with skin perfusion pressure (SPP) as an
index of tissue blood perfusion in patients with chronic limb-threatening ischemia
(CLTI). A total of 51 patients with CLTI who referred for de novo revascularization were
enrolled in this study. The results are presented as the mean ± standard error.
Table Legends
Table I The clinical characteristics of the patients are shown. The patients with CLTI
who underwent de novo revascularization were enrolled in this study (N=51).
Table II The classification and treatments of the patients are shown. The patients with
CLTI who underwent de novo revascularization were enrolled in this study (N=51).
Table III Univariate linear regression analysis to examine the relationship between SPP
and the clinical parameters of the patients with CLTI (N=51).
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