Mitochondrial Aldehyde Dehydrogenase 2 Regulates
Revascularization in Chronic Ischemia
Potential Impact on the Development of Coronary Collateral Circulation
Xiangwei Liu, Xiaolei Sun, Hua Liao, Zhen Dong, Jingjing Zhao, Hong Zhu, Peng Wang,
Li Shen, Lei Xu, Xin Ma, Cheng Shen, Fan Fan, Cong Wang, Kai Hu, Yunzeng Zou, Junbo Ge,
Jun Ren, Aijun Sun
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Objective—Revascularization is an essential process to compensate for cardiac underperfusion and, therefore, preserves
cardiac function in the face of chronic ischemic injury. Recent evidence suggested a vital role of aldehyde dehydrogenase
2 (ALDH2) in cardiac protection after ischemia. This study was designed to determine whether ALDH2 regulates chronic
ischemia−induced angiogenesis and to explore the underlying mechanism involved. Moreover, the clinical impact of the
ALDH2 mutant allele on the development of coronary collateral circulation (CCC) was evaluated.
Approach and Results—Mice limb ischemia was performed. Compared with wild-type, ALDH2 deletion significantly
reduced perfusion recovery, small artery and capillary density, and increased muscle atrophy in this ischemic model.
In vitro, ALDH2-knockdown reduced proliferation, migration and hypoxia triggered endothelial tube formation of
endothelial cells, the effects of which were restored by ALDH2 transfection. Further examination revealed that ALDH2
regulated angiogenesis possibly through hypoxia-inducible factor-1α/vascular endothelial growth factor pathways. To
further discern the role of ALDH2 deficiency in the function of bone marrow stem/progenitor cells, cross bone marrow
transplantation was performed between wild-type and ALDH2-knockout mice. However, there was no significant
improvement for wild-type bone marrow transplantation into knockout mice. ALDH2 genotyping was screened in 139
patients with chronic total occlusion recruited to Zhongshan Hospital (2011.10–2014.4). Patients with poor CCC (Rentrop
0–1; n=51) exhibited a higher frequency of the AA genotype than those with enriched CCC (Rentrop 2–3; n=88; 11.76%
versus 1.14%; P=0 0.01). However, the AA group displayed less enriched CCC frequency in Logistic regression model
when compared with the GG group (odds ratio=0.08; 95% confidence interval, 0.009–0.701; P=0 0.026). Furthermore,
serum vascular endothelial growth factor level tended to be lower in patients with ALDH2 mutation.
Conclusions—This study demonstrated that ALDH2 possesses an intrinsic capacity to regulate angiogenesis via hypoxiainducible factor-1α and vascular endothelial growth factor. Patients with ALDH2-deficient genotype displayed a higher
risk of developing poor CCC. Therapeutic individualization based on ALDH2 allele distribution may thus improve
the therapeutic benefit, especially in the East Asian decedents. (Arterioscler Thromb Vasc Biol. 2015;35:2196-2206.
DOI: 10.1161/ATVBAHA.115.306012.)
Key Words: aldehyde dehydrogenase ◼ coronary occlusion ◼ endothelial progenitor cells
◼ hypoxia-inducible factor ◼ neovascularization
T
he reestablishment of blood vessels through the process of
angiogenesis, or formation of new blood vessels, is critical to protect the ischemic injury and promote the recovery
because of the essential role to deliver oxygen and nutrients.1–4
Specifically in cardiovascular system, a functional collateral
circulation may counter ischemia to preserve ventricular function and to improve the overall prognosis.5–8 Despite the high
prevalence of coronary collateral circulation (CCC) in coronary angiogram images from patients with coronary heart disease, particularly chronic total occlusion (CTO), the efficacy
of angiogenesis varies from one patient to the next, creating a
dramatic inconsistency in the clinical outcome. The underlying mechanisms responsible for such variability seems to be
complex and still remains unknown.1,9–12
Received on: June 11, 2014; final version accepted on: July 30, 2015.
From the Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital (X.L., H.Z., P.W., L.S., L.X., C.S., F.F., C.W., K.H., Y.Z., J.G., J.R., A.S.),
Institute of Biomedical Science (X.S., L.X., X.M., Y.Z., J.G., A.S.), Department of Cardiology, Huashan Hospital (Z.D.), Fudan University, Shanghai, P.R.
China; Center for Cardiovascular Research and Alternative Medicine, School of Pharmacy, University of Wyoming College of Health Sciences, Laramie
(X.L., J.R.); Dongfang Hospital, Tongji University, Shanghai, P.R. China (H.L.); and Department of Cardiology, First Affiliated Hospital, Sun Yat-sen
University, Guangzhou, P.R. China (J.Z.).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.115.306012/-/DC1.
Correspondence to Aijun Sun, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Fenglin R 180, Shanghai 200032,
P.R. China. E-mail [email protected]
© 2015 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
2196
DOI: 10.1161/ATVBAHA.115.306012
Liu et al ALDH2 Regulates Revascularization in Chronic Ischemia 2197
Nonstandard Abbreviations and Acronyms
ALDH2
CCC
CTO
EPC
HIF-1α
KO
VEGF
WT
aldehyde dehydrogenase 2
coronary collateral circulation
chronic total occlusion
endothelial progenitor cell
hypoxia-inducible factor-1α
ALDH2-knockout mice
vascular endothelial growth factor
wild-type
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Mitochondria aldehyde dehydrogenase (ALDH) or
ALDH2 is a key enzyme in eliminating toxic aldehydes
by catalyzing their oxidation to nonreactive acids.13–15
Accumulating data from our laboratory and others indicated
a key protective role for ALDH2 in ischemic injury.16–18 These
data suggest that ALDH2 is involved in regulation of the
microenvironment homeostasis under ischemia or hypoxia
condition. Notably, a loss-of-function allele of ALDH2 is
found in nearly half of East Asian or 8% of world populations,
which may be correlated with various pathological processes,
such as ischemia/reperfusion injury and cardiac remodeling.19–21 Nonetheless, it remains somewhat elusive whether
ALDH2 plays a role in angiogenesis in chronic ischemia.
To this end, this study was designed to investigate the role
of ALDH2 in angiogenesis and the underlying molecular basis
behind, if any, chronic ischemia. It further prompted us to scrutinize the impact of the Glu504Lys mutation in the development of CCC in the clinical setting. Our results showed that
ALDH2 deficiency impairs the angiogenesis process possibly
by inhibiting the endothelial cell function and the hypoxiainducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling cascade. On the other side of the coin,
overexpression of ALDH2 restores the angiogenesis process.
The clinical data implicated that loss-of-function for ALDH2
because of genetic mutation may present as an unfavorable
contributing factor for revascularization in patients with CTO.
Therefore, targeting on ALDH2 activity might serve as a potential therapeutic strategy for individualized ischemic injury.
Materials and Methods
Materials and Methods are available in the online-only Data
Supplement.
Results
ALDH2 Deficiency Deteriorates Perfusion
Recovery and Revascularization in Chronic
Hindlimb Ischemia, Leading to Promoted
Gastrocnemius Muscle Atrophy
To examine the effect of ALDH2 on perfusion recovery ability in chronic ischemic injury, a mouse ischemic limb model
was used in ALDH2-knockout (KO) and wild-type (WT)
mice (Figure 1A). The perfusion was assayed using the
laser Doppler perfusion imaging on days 1, 7, and 21 after
the femoral artery ligation surgery (Figure 1B). Our results
indicated that ALDH2-KO mice displayed reduced perfusion
signals (red and orange) associated with more pronounced
nonperfusion signals (blue and green) in ischemic limbs
compared with those from WT mice on days 7 and 21 (but
not on day 1). Quantitatively, we calculated the ratio of perfusion between ischemic and nonischemic limbs for each
mouse (Figure 1C). Our data revealed that the perfusion ratio
achieved 82.2% at the 3-week point in WT group. In comparison, only 59.3% perfusion ratio was found in ALDH2-KO
group, whereas there were no significant difference noted
between the 2 groups on day 1, indicating a slower recovery
of limb circulation (P<0.05; Figure 1B and 1C). These observations demonstrate that the perfusion recovery of ischemic
hindlimb may be impaired by deficient ALDH2 expression.
To determine if deteriorated tissue perfusion was mediated, in part, through reduced revascularization, small artery
density was determined 21 days after hindlimb surgery
(Figure 1A and 1D). Using immunofluorescence staining,
small artery was detected by FITC labeled anti-α smooth
muscle actin antibody in injured limbs. Less small artery
density was found in the injured limbs of ALDH2-KO mice
compared with that from the WT mice (22.8±5.2 versus
12.1±5.6 number/field in WT and ALDH2-KO mice, respectively; P<0.05; Figure 1D and 1E). To assess the angiogenesis effect, capillary density was determined at the same
time (Figure 1A and 1F). Using immunofluorescence stain,
capillary density was detected by the anti-CD31 antibody in
hindlimbs. The micrographs showed that WT ischemic limb
displayed reduced capillary density (less red CD31 (+) signal) compared with WT nonischemic limb. ALDH2-KO ischemic limb displayed the lowest CD31 (+) cell density among
the 4 groups. The CD31 (+) cells were significantly less in
KO-ischemic group when compared with WT-ischemic group
(P<0.05), although the nonischemic limb of ALDH2-KO
mice showed equivalent amount of CD31 (+) cells compared
with WT mice (0.757±0.036 versus 0.580±0.008 CD31 (+)/
DAPI in (Figure 1E; P<0.05) WT nonischemic and ischemic
limb, and 0.766±0.028 versus 0.402±0.040 (P<0.05) in KO
nonischemic and ischemic limb.
Chronic ischemic insult is usually linked to permanent
damage to the limb, manifested as muscle atrophy and loss
of limb function.22 Therefore, gastrocnemius muscle atrophy
rate was assessed in ischemic limbs, shown as the ratio of
weight of ischemic and nonischemic gastrocnemius muscles.
Our data revealed somewhat comparable muscle atrophy in
ischemic and nonischemic gastrocnemius muscles. Despite
the perfusion recovered to >50% of that of the noninjured
limb at the 3-week time point. ALDH2-KO mice presented a
25.2% loss of gastrocnemius muscle weight when compared
with a 17.5% loss in muscle weight in WT mice (P<0.05;
Figure 1F).
Taken together, these data demonstrate that deficient
ALDH2 expression directly impedes the revascularization and
perfusion, leading to worsened tissue repair and functional
restoration in chronic ischemia.
ALDH2 Deficiency Impairs Tube-Like Construction
Formation, Migration, and Proliferation In Vitro
To further determine the regulatory role of ALDH2 in
angiogenesis in chronic ischemic microenvironment, the
2198 Arterioscler Thromb Vasc Biol October 2015
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Figure 1. Effects of aldehyde dehydrogenase 2 (ALDH2) knockout (KO) on perfusion recovery, revascularization, and muscle atrophy in
chronic hindlimb ischemia. A, Experimental protocol. B, Original laser Doppler perfusion images (LDPIs) displaying hindlimb perfusion 1,
7, and 21 days after excision of femoral artery. C, Perfusion recovery in wild-type (WT) mice and ALDH2-/- mice at 1, 7, and 21 days after
surgery. D, Representative original micrographs of hindlimb sections 21 days after hindlimb surgery; smooth α-actin (+)–stained cells are
shown in green; 4′,6-diamidino-2-phenylindole (DAPI)–stained nuclei are shown in blue. E, Quantitative analysis of small artery density
(small artery number per 1 mm2 hindlimb). F, Representative original micrographs of hindlimb sections 21 days after surgery; CD31 (+)
stained are shown in green; DAPI-stained nuclei are shown in blue. G, Quantitative analysis of capillary density (Ratio of CD31 (+) cells
and DAPI (+) cells. H, Quantitative analysis of gastrocnemius muscle atrophy. The atrophy rate was manifested as the ratio of ischemic
and nonischemic gastrocnemius muscle weight for each mouse 21 days after hindlimb surgery. Mean±SEM, n=6 to 10, *P<0.05.
endothelial cell function was evaluated. First, the tube-like
construction formation assay was performed in rat cardiac
microvascular endothelial cells (; Figure 2A). To mimic
WT and ALDH2-knockdown endothelium, scramble and
ALDH2-siRNA were transfected, respectively. Western blot
data showed that ALDH2-siRNA downregulated the protein
expression of ALDH2 by 70% compared with the control and
scramble-siRNA group (Figure 2B and 2C; P<0.05). Indeed,
comparable regulation of ALDH2 was noted in microvascular
genesis in vitro (Figure 2D and 2E). There was no significant
difference among control, scramble-siRNA, and ALDH2siRNA groups under normoxia. After a 6-hour hypoxia,
formation of capillary-like tubes by microvascular endothelial cells was increased by 2-fold compared with formation
Liu et al ALDH2 Regulates Revascularization in Chronic Ischemia 2199
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Figure 2. Roles of aldehyde dehydrogenase 2 (ALDH2) in endothelial tube-like construction formation, migration and proliferation in vitro.
A, Experimental protocol. B, Representative Western blot of rat cardiac microvascular endothelial cells transfected with the scramblesiRNA or ALDH2-siRNA. C, Quantitative analysis of ALDH2 expression after siRNA transfection. D, Original micrograph of tube-like construction formation after 6 hours of hypoxia; cells were transfected with ALDH2-siRNA to knockdown ALDH2 (bottom). E, Quantitative
analysis of tube-like construction formation displayed as the number per field counted at ×50 magnification. F, Original micrographs of
wound healing assay. G, Quantitative analysis of wound healing assay displayed as the distance of human umbilical vein endothelial cells
migrated. H, Quantitative analysis of CCK-8 assay (Absorption at 450 nm); cells were transfected with ALDH2-shRNA lentiviral or ALDH2cDNA lentiviral to knockdown or overexpression ALDH2, respectively in (G) and (H). Mean±SEM, n=6 to 10, *P<0.05.
of capillary-like tubes by normoxic cells in both control
and scramble-siRNA–transfected groups. Interestingly, the
hypoxia-induced tube formation of endothelial cells seemed
to be negated in ALDH2-siRNA–transfected group (P<0.05;
Figure 2D and 2E). The angiogenesis process is also committed to proliferation and migration of endothelial cells. Wound
healing assay was performed to evaluate the role of ALDH2 in
endothelium migration. To get long-term ALDH2-knockdown
and ALHD2-overexpression cell line, and also to reduce
mouse euthanization, the following experiment was performed in human umbilical vein endothelial cells. The above
row showed the wound at 0 hour, and the below row showed
the wound at 6 hour after normoxia or hypoxia (Figure 2F).
The average migration distance was measured. Quantitatively,
2200 Arterioscler Thromb Vasc Biol October 2015
promoted endothelia proliferation in negative control,
ALDH2-knockdown, and ALDH2-transfected groups; (2)
compared with negative control groups, ALDH2-knockdown
suppressed migration both in normoxia (54.70±4.32 versus 44.80±2.43; 1-way ANOVA; NS) and in hypoxia
(74.80±4.55 versus 52.80±2.15; 1-way ANOVA; P<0.05);
and (3) conversely, compared with negative control groups,
ALDH2 transfection promoted migration both in normoxia
(54.70±4.32 versus 57.30±3.44; 1-way ANOVA; NS) and in
hypoxia (74.80±4.554 versus 78.00±7.817; 1-way ANOVA;
NS). Taken together, our data indicated that ALDH2 activity
might be permissive in angiogenesis process because of its
regulatory role of ischemic endothelial functions, including
pseudotubes formation, migration, and proliferation.
ALDH2 Regulates HIF-1α and VEGF
Expression In Vitro and In Vivo
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Figure 3. Regulation of aldehyde dehydrogenase 2 (ALDH2) on
hypoxia-inducible factor (HIF)-1α and vascular endothelial growth
factor (VEGF) signaling pathway in vitro. A, Representative
Western blot of VEGF, HIF-1α, and ALHD2 in human umbilical
vein endothelial cells (HUVECs) with negative control or ALDH2shRNA lentiviral transfection, cultured in normoxia and 6 hours
of hypoxia. B, Quantitative analysis of ALDH2 expression in
ALDH2-shRNA lentiviral-transfected cells. C and D, Quantitative
analysis of HIF-1α and VEGF expression after hypoxia treatment.
E, Representative Western blot of VEGF, HIF-1α, and ALHD2 in
HUVECs with negative control or ALDH2-cDNA lentiviral transfection, cultured in normoxia and 6 hours of hypoxia. F., Quantitative analysis of ALDH2 expression in ALDH2-cDNA group.
G and H, Quantitative analysis of HIF-1α and VEGF expression in
ALDH2 overexpression ion cells. Mean±SEM, n=3 to 5, *P<0.05.
WT indicates wild-type.
(1) compared with normoxia, hypoxia promoted endothelial
cell migration in negative control, ALDH2-knockdown, and
ALDH2-transfected groups; (2) compared with negative control groups, ALDH2-knockdown suppressed migration both
in normoxia (WT, 62.03±2.570 versus ALDH2-knockdown,
32.62±2.695; 1-way ANOVA; P<0.05) and in hypoxia (WT,
87.16±0.4702 versus ALDH2-knockdown, 49.95±5.373;
1-way ANOVA; P<0.05); (3) Conversely, compared with negative control groups, ALDH2 transfection increased migration both in normoxia (62.03±2.570 versus 77.42±2.993;
1-way ANOVA; NS) and in hypoxia (87.16±0.4702 versus 110.7±6.216; 1-way ANOVA; P<0.05; Figure 6G).
Furthermore, CCK-8 assay was used to investigate the regulation of ALDH2 in endothelial proliferation (Figure 2H).
The result showed the same pattern as in the wound healing
assay. Quantitatively, (1) compared with normoxia, hypoxia
To further investigate the molecular mechanisms behind
ALDH2-regulated angiogenesis, the HIF-1α/VEGF pathway
was evaluated. Control, ALDH2 gain- and loss-function cell
models were established using lentiviral vectors to determine
the role of ALDH2 in this process. Cells were exposed to normoxia or to hypoxia for 6 hours before assessment of protein levels for HIF-1α and VEGF. The immunoblot analysis
depicted a significant increase in the levels of HIF-1α and
VEGF in the control group after 6-hour hypoxia, the effect
of which was abolished in ALDH2-KO group (P<0.05;
Figure 3A and 3C). In contrary, the HIF-1α and VEGF expression showed a significant increase in the ALDH2-transfected
group under normoxia. Quantitatively, there is a 2-fold
increase in the levels of HIF-1α and VEGF in the ALDH2transfected group compared with the control normoxic group
(equivalent to the control-hypoxia group). After 6-hour
hypoxia, the HIF-1α and VEGF expression was increased
slightly in ALDH2 overexpression hypoxic group compared
with ALDH2 overexpression normoxia group (Figure 3E, 3G,
and 3H). Taken together, these data indicate that ALDH2 is
required in HIF-1α/VEGF pathway and that the regulation
seemed to be hypoxia independently in vitro.
To investigate whether ALDH2 regulates HIF-1α/VEGF
signaling in vivo, expression of these proteins was evaluated
in ischemic quadriceps from both nonischemic and ischemic limbs in WT and ALDH2-KO mice, on days 1, 7, and
21 after hindlimb surgery (Figure 4A). In this immunoblot
analysis, the nonischemia quadriceps of each mouse was used
as control. The data revealed that there was no significant
increase of the level of HIF-1α/VEGF in WT mice on day 1
after hindlimb ischemia (Figure 4B, 4D, and 4E). However,
ALDH2-KO showed temporary increase of HIF-1α (≈2-fold;
P<0.05; Figure 4B and 4D) and VEGF (≈1.5 fold; P<0.05;
Figure 4B and 4E). However, interestingly, although there was
significant increase of HIF-1α (≈1.2 fold; P<0.05; Figure 4F
and 4H) and VEGF (≈1.5 fold; P<0.05; Figure 4F and 4I) in
WT mice at the day 7 after surgery, the ALDH2-KO mice
showed few accumulation of HIF-1α and VEGF (Figure 4F,
4H, and 4I). Finally, the expression of HIF-1α and VEGF
was comparable between WT and ALDH2-KO groups on day
21 (Figure 4J, 4L, and 4M). Several reports have suggested
Liu et al ALDH2 Regulates Revascularization in Chronic Ischemia 2201
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Figure 4. Regulation of aldehyde dehydrogenase 2 (ALDH2) on hypoxia-inducible factor (HIF)-1α, and vascular endothelial growth factor
(VEGF) signaling pathway in vivo. A, Experimental protocol. B, G, and L, Representative Western blot of PHD2, VEGF, HIF-1α, and ALHD2
in ischemic hindlimb tissue from wild-type (WT) and knockout (KO) mice at 1, 7, and 21 days after surgery. C, H, and M, Quantitative
analysis of ALDH2 expression at days 1, 7, and 21. D, I, and N, Quantitative analysis of HIF-1α expression at days 1, 7, and 21. E, J, and
O, Quantitative analysis of VEGF expression at days 1, 7, and 21. F, K, and P, Quantitative analysis of PHD2 expression at days 1, 7, and
21. Mean±SEM, n=3 to 5, *P<0.05.
that PHD2, as a cellular oxygen sensor, may regulate HIF-1α
protein level through promoting its degradation. Therefore,
PHD2 expression was explored in our study (Figure 4B, 4F,
4G, 4K, 4L, and 4P). However, we did not find any difference in PHD2 protein level among the 4 experimental groups
at days 1, 7, and 21, in vivo. These data demonstrated that
although ALDH2-KO promoted HIF-1α/VEGF expression
transiently early on, it later downregulated the level of HIF1α/VEGF, which was PHD2 independently, eventually leading to impaired angiogenesis, revascularization, tissue repair,
and preservation of function.
ALDH2 Influences the Mobilization But
Not the Planting and Repair Effects of
Endogenous Stem/Progenitor Cells
Revascularization of ischemic injury is thought to be driven,
in part, by the bone marrow stem or progenitor cells. We
therefore investigated the effects of ALDH2-KO on endogenous stem/progenitor cells repair and whether such effects
contributes to the difference of revascularization between
WT and ALDH2-KO mice. First, the mobilization of EPC
was measured by flow cytometry at day 7 after hindlimb ischemia, shown as the ratio of CD34+/Flk1+ cells to mononuclear cells in peripheral blood, and the ratio of CD34+/Flk1+
cells to bone marrow cells in bone marrow, of both WT and
ALDH2-KO mice. Interestingly, circulating EPC of KO mice
was significantly increased when compared with WT mice
(Figure 5A; P<0.05), and the bone marrow EPC was slightly
decreased in KO group (Figure 5B; NS). We speculated that
the enhanced mobilization of EPC in ALDH2-KO mice is a
kind of compensatory response to the worse blood supply in
ischemic hindlimb. To further test the role of ALDH2 in postischemia endogenous stem/progenitor cells repair, we next
performed the cross bone marrow transplantation between WT
and ALDH2-KO mice (protocol was shown as Figure 5C).
Assessment of blood perfusion recovery by laser Doppler at
day 21 after hindlimb ischemia showed that KO mice with
WT bone marrow cells (W2K) displayed worse perfusion
than WT mice with WT bone marrow cells (W2W), and KO
mice with KO bone marrow cells (K2K) displayed worse perfusion than WT mice with KO bone marrow cells (W2W).
There was no difference between WT mice with KO bone
marrow and WT mice with WT bone marrow, or between KO
mice with KO bone marrow and KO mice with WT bone marrow (Figure 5D and 5E). Collectively, these data indicated
that genotype of recipient was a major determinant affecting
the blood perfusion recovery, but not the genotype of bone
marrow donor. We also observed the recruitment and planting
of bone marrow−derived EPCs to ischemic area by immunofluorescence stain (protocol was shown as Figure 5C).
Before the transplantation, bone marrow stem/progenitor
2202 Arterioscler Thromb Vasc Biol October 2015
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Figure 5. Repair of endogenous stem/progenitor cells in bone marrow transplantation mice. A, Quantitative analysis of circulating
endothelial progenitor cells (EPCs) at day 7 after hindlimb ischemia surgery. B, Quantitative analysis of bone marrow EPC at day 7 after
hindlimb ischemia surgery; in (A) and (B) EPC were measured by flow cytometry displayed as the ratio of CD34+/Flk1+ cells to mononuclear cells or bone marrow cells, respectively. C, Experimental protocol. D, Original Laser Doppler perfusion images displaying hindlimb
perfusion 21 days after ischemia surgery in mice performed cross bone marrow transplantation. E, Perfusion recovery in mice performed
cross bone marrow transplantation. F, Representative original micrographs of hindlimb sections 21 days after surgery; CD31 (+) stained
are shown in red; GFP (+) cells are labeled as bone marrow–derived cells in blue. G, Quantitative analysis of bone marrow–derived endothelium cells (number of CD31 (+) and GFP (+) cells normalized by the ratio of GFP positive rate cells in bone marrow). Mean±SEM, n=3 to
5, *P<0.05. ALDH indicates aldehyde dehydrogenase; BMC, bone marrow cell; DAPI, 4′,6-diamidino-2-phenylindole; KO, knockout; and
WT, wild-type.
cells purified from WT and ALDH2-KO mice were exposed
to GFP-lentiviral for 12 hours, which enabled at least 50% of
bone marrow cells to express GFP cont inuously. Therefore,
GFP can be used as a marker for bone marrow−derived cells.
Immunostaining of tissue from ischemic muscles showed
that the there was no significant difference on the number of
GFP+/CD31+ cells among the 4 groups, indicating that equivalent level of bone marrow−derived EPC was planted in the
ischemic area of these 4 groups, eventually. Taken together,
these data demonstrated that although ALDH2-KO mice
mobilized more EPC after hindlimb ischemia, the same level
of bone marrow−derived EPC was found in the ischemic
area, which might be caused by the worse ischemic microenvironments in KO mice. The blood perfusion recovery
seemed mostly dependent on the ALDH2 genotype of recipient but not of the bone marrow. Therefore, endogenous stem/
progenitor cell repair was barely the reason of the impaired
postischemia revascularization in case of ALDH2 deficiency.
Liu et al ALDH2 Regulates Revascularization in Chronic Ischemia 2203
ALDH2 Glu504Lys Polymorphism
Contributes to Poor CCC
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The correlation between development of CCC and ALDH2
polymorphism was evaluated in a subset of Chinese patients.
As described, collateral circulation by coronary angiography
from 139 patients with CTO were examined. The patients
were divided into 2 groups, namely poor and enriched CCC
(Table 1). There were little differences between the 2 groups
with regard to sex, age, left anterior descending coronary
artery, right coronary artery, type 2 diabetes mellitus, hypertension, smoking, drinking, and hyperlipidemia (Table 1). Of
the 139 patients, 51 (34.09%) patients had poor CCC and 88
(63.31%) displayed enriched CCC. Our result revealed that
ALDH2 AA genotype (mutant homozygotes) exhibited a
higher frequency in the poor group than in the enriched group
(Table 2; 11.76% versus 1.14%; P=0.01), using a 2-tailed
Fisher exact test. Furthermore, the ALDH2 AA homozygotes
displayed a significant lower rate in enriched CCC than those
carrying at least 1 functional ALDH2*1 allele (WT allele;
14.29% versus 65.91%), resulting in an odds ratio of 0.08
(95% confidence interval, 0.009–0.701; P=0.026) in the
logistic regression model. A marginal P value was detected in
the association between time duration and CCC, it appeared
that longer duration was positively correlated with good
CCC (Table 1; 82.35% versus 93.18%; P=0.047). This variant was added into the logistic regression analysis, resulting no significant effect (P>0.05). Therefore, these findings
depict that loss-of-function ALDH2 mutation contributes to
risk of poor CCC in patients with CTO independently. To
recapitulate our laboratory finding in the clinic setting, we
further assessed the correlation between the ALDH2 polymorphism and the serum VEGF levels in patients with CTO.
Our result revealed that serum VEGF levels displayed a nonsignificant increase in the enriched CCC group compared
with those in the poor CCC group (Figure 6A; 256.4±37.01
versus 233.3±27.79; P=0.618). As expected, patients with
ALDH2 mutation displayed a trend of lowered serum VEGF
levels compared with patients with WT ALDH2 phenotype
(Figure 6A; 209.9±20.98 versus 279.5±42.62; P=0.146).
These clinical data, therefore, indicated that there may be a
correlation between ALDH2 mutation and poor CCC possibly resulted from reduced serum VEGF levels in patients
with CTO (Table 3).
Discussion
The salient findings from this study revealed that global
ALDH2 deficiency led to unfavorable vascular sequelae
including worsened blood supply after ligation of femoral
artery accompanied with fewer numbers of capillary and
small arteries, overt muscular atrophy, and reduced capacity of endothelial cells to proliferation, migration, and form
tube-like structure. Our data further suggested that the loss
of ALDH2-regulated essential angiogenesis factors such as
HIF-1α/VEGF pathway may underscore such anomalies.
Results from bone marrow transplantation showed that the
impaired postischemia repair because of ALDH2 deletion
was not related to endogenous stem/progenitor cells. Clinical
observation provided compelling evidence in that patients
with ALDH2 deficiency were more vulnerable to poor CCC
development possibly associated with reduced serum VEGF
levels in patients with CTO. Our study demonstrated, for the
first time, that ALDH2 possesses an intrinsic capacity to regulate ischemia-/hypoxia-induced angiogenesis, involved in the
ischemic microenvironment homeostasis and organ function
preservation, via HIF-1α/VEGF cascade, and therefore, the
common ALDH2 Glu504Lys mutation exhibit a great clinical impact on the fate of CCC in patients with chronic heart
ischemia.
Chronically ischemic tissue, a hallmark for peripheral
and coronary artery disease, requires the remodeling from
vascular network to reconstitute and sustain its viability.23,24
Therefore, therapeutic angiogenesis remains an unmet medical need. However, a tremendous interindividual variability
exists in the degree of new collateral formation in patients
with coronary artery disease. The observed interindividual
heterogeneity may be because of environmental, genetic and
epigenetic causes, complications or the severity and duration
of disease.25–27 Still, the mechanism underneath this interindividual heterogeneity remains poorly understood. Among various potential contributing factors, genetic mutations not only
affect the ischemia-angiogenesis response process but also
limit the efficiency of revascularization promote therapeutics.
To this end, genetic predisposition may be considered as, perhaps, one of the major host factors crucial for the reestablishment of functional collateral circulations. Coronary CTO is
considered to be the last frontier of coronary interventional
medical specialties. The formation of CCC in patients with
CTO is essential to the maintenance of cardiac function.28,29
This study showed an inverse correlation between ALDH2
rs671 polymorphism and CCC formation in patients with
Table 1. Baseline Data
CCC
Parameters
Poor
Enriched
P Value
79 (89.77%)
0.12
Sex
Male
Female
Age
41 (80.39%)
10 (19.61%)
56.6±7.9
9 (10.23%)
58.1±7.9
0.28
Duration
≤3 mo
9 (17.65%)
6 (6.82%)
0.047
>3 mo
42 (82.35%)
82 (93.18%)
Diabetes mellitus
17 (33.33%)
24 (27.59%)
0.47
Hypertension
27 (52.94%)
44 (50.00%)
0.73
Drinking
12 (24.49%)
20 (24.20%)
0.17
Smoking
21 (42.00%)
39 (44.32%)
0.14
Risk factors
Blood lipid profile
TC
4.05±1.18
4.20±1.26
0.51
TG
2.06±1.31
1.91±1.03
0.47
HDL
0.99±0.24
1.04±0.26
0.27
LDL
2.24±1.00
2.32±1.16
0.68
Mean±SEM. CCC indicates coronary collateral circulation; HDL, high density
lipoprotein; LDL, low density lipoprotein; TC, total cholesterol; and TG, total
triglyceride.
2204 Arterioscler Thromb Vasc Biol October 2015
Table 2. Genotypes and CCC
Table 3. Logistic Regression
CCC
Parameters
Poor (%)
Enriched (%)
P Value
ALDH2 genotype
AA
6 (11.76)
1 (1.14)
GA
19 (37.25)
33 (37.50)
GG
26 (50.98)
54 (61.36)
0.02
Mean±SEM. A indicates ALDH2 mutant type; ALDH, aldehyde dehydrogenase;
CCC, coronary collateral circulation; and G, ALDH2 wild-type.
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
CTO. Also a nonsignificant correlation was noted between
ALDH2 mutation and serum VEGF levels in patients with
CTO. However, it is noteworthy that serum rather than
plasma samples were used in our study thus may have contaminated the actual plasma values of VEGF because of the
interference of platelets and fibrinogen in the serum. These
findings should shed some lights toward the interindividual
heterogeneity in collateral vessels formation and offer new
insights for clinical decision making in the face of ALDH2
mutant individuals.
Our earlier study suggested that mitochondrial isoform
of ALDH namely ALDH2 may reduce myocardial infarction injury through regulating the p53/JNK/4-HNE signaling cascade. Furthermore, ALDH2 is capable of mitigating
myocardial anomalies through regulating autophagy via a
HSP70/JNK/p53-dependent mechanism in doxorubicininduced dilated cardiomyopathy.30 Our data also revealed
that ALDH2 alleviates endoplasmic reticulum stressinduced myocardial anomalies through reduced myocardial apoptosis.31–34 These data showed significant protective
capacity of ALDH2 through a p53-dependent pathway
in cardiomyocytes. However, prolonged or more severe
hypoxia has been reported to induce the tumor suppressor
p53, which binds to HIF-1α, to promote its degradation and
to inhibit its transactivation properties.35,36 It was reported
that sustained cardiac pressure overload induced an accumulation of p53, resulting in the inhibited HIF-1 activity,
impaired cardiac angiogenesis, and ultimately systolic
function.37 This piece of evidence implied that ALDH2 may
participate in the angiogenesis progress. Furthermore, our
Figure 6. Correlation of aldehyde dehydrogenase 2 (ALDH2)
Glu504Lys polymorphism with coronary collateral circulation and
serum vascular endothelial growth factor (VEGF). A, Serum VEGF
level of patients with enriched or poor CCVs. B, Serum VEGF
level of patients with wild-type or mutant ALDH2. Mean±SEM,
n=139.
Effect
Odds Ratio Point
Estimates
95% Wald
Confidence Limits
P Value
Genotype AA vs GG
0.08
0.009
0.701
0.026
Genotype GA vs GG
0.836
0.402
1.741
0.082
Mean±SEM. A indicates ALDH2 mutant type; ALDH, aldehyde dehydrogenase;
and G, ALDH2 wild-type.
recent study suggested that host ALDH2 regulates transplanted MSC survival and therapy as a microenvironment
homeostasis mediator via local capillary density, energy
supply, and oxidative stress regulating after ischemia.38
Therefore, this study provided evidence in support of the
hypothesis that ALDH2 may be protective for cardiomyocyte through enhancing homeostasis of microenvironment
under ischemic conditions via promoting revascularization
and perfusion.
Angiogenesis is the process through which new blood
vessels form from pre-existing vessels. This is distinct from
vasculogenesis, and is responsible for majority of blood vessel growth during development and in pathological conditions.39,40 A hypoxia-inducible program, driven by HIF-1α,
renders endothelial cells responsive to angiogenic signals.41,42
Normally, when sufficient oxygen is available, HIF-1α may
be hydroxylated by the oxygen-sensing enzymes named proteins PHD1–3. And the hydroxylated HIF proteins would next
be degraded by proteasomal degradation. Although hypoxia
inactive the PHDs, the upregulation of HIF-1 correlates with
its combination to the hypoxia response element of its extensive target genes, mainly responsible for oxygen supply and
cell survival, such as VEGF, then subsequently initiates the
process of angiogenesis.43,44 Therefore, the use of HIF-1α
inhibitors to block tumor or ocular angiogenesis has received
attention, and conversely, HIF-1α gene transfer in mice or
activation of HIF-1α promotes ischemic tissue revascularization. In our study, ALDH2 was found to regulate hypoxia-/
ischemia-induced HIF-1α and then accumulation of its downstream angiogenetic protein VEGF. However, this regulation
was independent of PHD2. It seems that HIF-1α is not regulated only by the oxygen tension but also by various other
stimuli, such as transition metals, nitric oxide, reactive oxygen
species, growth factors, and mechanical stresses and the activation of signal proteins, such as p53, Akt, mTOR, MEK1/2,
Erk1/2, and so on.45
Significantly, ALDH2 activity would be strongly affected
by genetic mutation. For instance, the common human polymorphism in ALDH2, in which glutamate at amino acid 504
is replaced by lysine, displays a dramatically high prevalence (≈40%) in Asian populations.46,47 It is well conceived
that ALDH2 acts as a homotetramer or heterotetramer, and
all tetramers that contain at least 1 ALDH2*2 subunit being
inactive.48 Therefore, identification of its clinical phenotype
is rather important to the health impact of ALDH2 gene. This
interindividual heterogeneity may also help to explain the
variable angiogenic responses seen in other conditions, such
as diabetic retinopathy and solid tumors.
In summary, ALDH2 deficiency compromises regenerative capacity of ischemic tissues as a result of impaired
Liu et al ALDH2 Regulates Revascularization in Chronic Ischemia 2205
angiogenesis via an HIF-1α-/VEGF-dependent mechanism.
Moreover, ALDH2 mutation displayed an inverse correlation coronary collateral vessel formation in patients. This
effect is of significant clinical implication as loss-of-function for ALDH2 represents the most popular human genetic
mutation. Our findings offer compelling evidence to support
that AA genotype may require more aggressive therapeutic
attention for vascular recanalization. Last but not least, our
investigation revealed therapeutic potential for ALDH2 in
the revascularization therapy in patients with coronary heart
disease.
Limitation
One obvious limitation noted for the clinical study is that we
had little information of the blood supply in patients with
CTO before the total occlusion period. Poor blood supply frequently occurs before coronary occlusion and might contribute to the poor CCC.
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
Sources of Funding
This work was supported by National Basic Research Program of
China (2011CB503905), National Natural Science Foundation of
China (30971250 and 81570224), and Program for New Century
Excellent Talents in University.
Disclosures
None.
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Significance
Our work described a novel finding that aldehyde dehydrogenase 2 regulates the angiogenesis and revascularization induced by chronic
ischemic injury in both mice and human subjects. This study is of particular clinical importance. First, this study may advance our understanding in that genetic variant serves as a hereditary regulatory factor in angiogenesis. Furthermore, it provides experimental evidence,
which explains possible heterogeneous clinical manifestation and outcomes in certain chronic ischemic disease. Finally, given the large
portion of aldehyde dehydrogenase 2 mutant carrier and the high prevalence of chronic ischemic disease, this study should shed some lights
in individualized revascularization therapy.
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Mitochondrial Aldehyde Dehydrogenase 2 Regulates Revascularization in Chronic
Ischemia: Potential Impact on the Development of Coronary Collateral Circulation
Xiangwei Liu, Xiaolei Sun, Hua Liao, Zhen Dong, Jingjing Zhao, Hong Zhu, Peng Wang, Li
Shen, Lei Xu, Xin Ma, Cheng Shen, Fan Fan, Cong Wang, Kai Hu, Yunzeng Zou, Junbo Ge,
Jun Ren and Aijun Sun
Arterioscler Thromb Vasc Biol. 2015;35:2196-2206; originally published online August 27,
2015;
doi: 10.1161/ATVBAHA.115.306012
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Aldehyde Dehydrogenase 2 (ALDH2) Regulates
Revascularization in Chronic Ischemia: Potential Impact on the
Development of Coronary Collateral Circulation
Materials and Methods
1. Animals
Male and female C57BL/6 mice (8 weeks old, weighing 20g-22g) were obtained
from the Shanghai Animal Administration Centre (Shanghai, China). Age- and
weight-matched ALDH2 knockout mice were generated as described previously1.
Mice were housed at room temperature under a 12/12 light/dark cycle with free
access to water and standard laboratory mouse chow. Adult male Wistar rats
weighting 200±10g were obtained from the Shanghai Animal Administration Centre
too, were used for rat cardiac microvascular endothelial cells (RCMECs) culture, and
operated as described2,3. Animal experiment protocols were approved by the Animal
Care and Use Committee of Fudan University. All experiments were performed
according to the guidelines of “the Guide for the Care and Use of Laboratory Animals”
Academy Press (NIH Publication No. 85–23, revised 1996).
2.
Hind-Limb Ischemia Model
Mice were anaesthetized with a single intraperitoneal ketamine injection (80μg/g
body weight). Chronic ischemia was generated in compliance with well-established
unilateral hind-limb surgery protocols4. In brief, following a 1 cm skin incision was
made at the medial thigh; the femoral artery was separated from the femoral vein and
nerve. The part proximal to the outlet of the profunda femoris artery and the distal end
(outlet of the saphenous artery) were ligated with 5–0 silk sutures. The femoral artery
was then excised between the ligations. Wound closures were conducted with
single-layer sutures with 4–0 Prolene threads.
3. Laser Doppler Perfusion Imaging
Limb perfusion was assessed by laser Doppler perfusion imaging (PeriScan
PIM 3 system, Perimed, Sweden). Before the assay, anesthetized mice were placed
on a heating pad at 37℃ for 5 min to maintain body temperature. Perfusion was
assessed on day 1, 14 and 21 after the surgery. Perfusion ratio of ischemic limb
versus non-ischemic hind-limb was quantified by averaging relative units of flux from
knee to toe using PIMsoft Software (Perimed med, Sweden).
4. Immunofluorescence (IF) Staining
Paraffin-embedded serial sections (6 um) obtained from skeletal muscles of the
ischemic hind limb of WT and KO mice were used for IF staining. To define small
arteries, capillary on day 21 after surgery, a primary antibody against α-smooth
muscle actin (Santa Cruz Biotechnology, Santa Cruz, CA) and a primary antibody
against CD31 (BD, United States) were used to detect smooth muscle cells and
endothelial cells, respectively. Tissues were mounted by the use of Vectashield DAPI
nuclear counterstain.
5. Cell Culture , Transfection and Hypoxia Model
Adult male Wistar rats weighting 200±10g were anesthetized by intraperitoneal
injection of 50mg/kg sodium thiopental. Under sterile conditions, a midline
thoracotomy was performed. The heart was cannulated, the left atrium, right atrium,
right ventricular was incised, and the left ventricular were flushed with 20ml of cold
PBS. Remove the endocardial and epicardial. Then cut the tissue into 1 mm3 pieces.
The tissue was planted in the petri dish and put 2-3ml fetal bovine serum (FBS) then
were placed in 5% CO2 37℃ cell incubator. After 6 hour incubation, the tissues in
petri dish were added another 3-4 ml of DMEM with 20% FBS and then were placed
back to incubator. On culture day 72h, cells migrated out from the explants, then
removed explants and rinsed gently with the PBS. The passages of cells between 2
and 4 were used in our experiment. HUVEC cell line was obtained from Cell Bank of
Chinese Academy of Science (Shanghai, China). For hypoxia treatment, cells were
incubated in a hypoxic chamber for 6 hours. ALDH2-siRNA was used to inhibit
expression of ALDH2 in MMVECs. Lenti viral vectors (Hanyin Co, shanghai, China),
were employed to deliver ALDH2 or to inhibit the expression of ALDH2 in the HUVEC
cell line.
6. Endothelial Tube Formulation Assay, Wound Healing Assay and CCK-8
Assay
Endothelial Tube formulation assay was performed as previously described5.
Briefly, MMVECs from different groups were seeded on the top of 200 μl
pre-polymerization growth factor-reduced Matrigel (BD Biosciences, UK) in 24-well
culture plates and exposed to normal or hypoxia for 6 hours. At the end of experiment,
floating cells were removed by washing and endothelial networks were counted under
an inverted microscope.
Wound healing assay was performed as previously described6. Briefly, HUVECs
were seeded on 6-well plates grew to full confluence. Subsequently, cells were
wounded by pipette tips and washed twice with PBS to remove detached cells, and
photomicrographs of initial wounds were taken. After 6 hours hypoxia,
photomicrographs of final wounds were taken for each group. Initial and final areas
covers by cells were measured using Image J software, and difference between the
two was used to determine migration distance using the formula: Final cell area minus
initial cell area, then divided by the width.
CCK-8 assay was performed using Cell Counting Kit-8 (Dojindo Laboratories.
Japan).
7. Immunoblot Analysis
Immunoblot analysis was carried out as described previously. Proteins were
separated on 12 % SDS-polyacrylamide gel, transferred to polyvinylidene fluoride
membranes, and subsequently subjected to immunoblot analysis using appropriate
antibodies. Anti-ALDH2 antibody (1:2000) was purchased from Epitomics Inc.
(Burlingame, CA 94010-1303 U.S.A.). The anti-HIF-1α antibody (1:1000) was from
NOVUS Biological Inc. (Littleton, CO 80120 U.S.A.) and the anti-VEGF antibody
(1:2000) from Abcam Inc. (Cambridge, MA 02139-1517 U.S.A.). Immunoreactive
antigen was then detected using enhanced chemiluminescence detection.
8. Flow Cytometry Measurement
To assess the percentage of endothelial progenitor cells those were mobilized to
the circulation after hind-limb ischemia surgery, blood samples and bone marrow were
harvested from animals 7 days after injury. Red blood cells were lysed at room
temperature for 5 minutes with erythrocyte lysis buffer, followed by washing with PBS.
Cells were fixed with 4% paraformaldehyde and blocked with 1% BSA in PBS
followed by staining with antibodies, APC labeled antibody against Flk-1 and FITC
labeled antibody against CD34 (both were purchased from BD, United States) for 30
minutes at 4 degree, then washed 3 times with PBS. Cells were analyzed on a
FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
9. Bone Marrow Transplantation
Wild-type C57BL/6J and ALDH2-KO mice were sacrificed to harvest bone
marrow cells. Lineage-/- bone marrow cells were obtained using flow cytometry
sorting, then transfected with GFP-lentiviral. Wild-type C57BL/6J and ALDH2-KO
mice were lethally irradiated (8 Gy). On the same day, those mice were injected with
1*106 GFP labeled lin-/- bone marrow cells. Reconstitution of bone marrow was
determined by flow cytometry 1 month after transplantation. The average percent
reconstitution was determined to be between 90% and 95%, with about 30-50%
GFP–positive cells in the marrow 4 weeks after transplantation.
10. Patients
From October 2011 to April 2014, consecutive patients with angiographically
documented CTO were included using the inclusion criteria of 1) displaying CTO in at
least 1 major epicardial coronary artery (left main coronary artery, left anterior
descending artery or left circumflex artery), 2) as TIMI flow 0 for more than 6 weeks;
by the exclusion criteria of 1) undergoing acute myocardial infarction within 90 days, 2)
previous percutaneous coronary intervention or coronary artery bypass grafting, 3)
congenital heart diseases or valvular heart diseases, or over 70 years old. A total of
139 patients were recruited to make up the study cohort. The study protocol was
approved by the ethical review of Zhongshan Hospital, Fudan University, and all
patients provided written informed consent to participate in the study.
11. Patient Data Collection
For each patient, a data sheet was completed with the patient’s name,
identification number, age, sex, previous revascularizations, past medical history of
hypertension, diabetes, cigarette smoking, and HLP (hyperlipidemia). Coronary
angiography was performed by the use of standard techniques. CTO was defined by
the presence of a coronary artery stenosis resulting in complete interruption of
antegrade flow in a major epicardial coronary artery or minimal contrast penetration
through the lesion without distal vessel opacification7,8. Retrograde collateral filling of
the vessel distal to a CTO was assessed by experienced interventional cardiologists
blinded to other clinical and imaging data. The diameter and angiographic flow of
collateral vessels was semiquantitatively assessed by the use of the Rentrop
classification (class 0=no visible filling of collaterals, class 1=filling of side branches,
class 2=partial filling of epicardial segment of the occluded vessel, class 3=total filling
of epicardial segment)9. In the current study the grade 0 or 1 were taken as poor CCC
and grade 2 or 3 as rich CCC.
12. Genotyping
Genomic DNA was extracted from leukocytes, and the DNA sequence spanning
the ALDH2 polymorphism was amplified by PCR. Genotyping was performed by direct
sequencing, using an ABI GeneScanTM-500 (Genesky Biotechnologies
Inc.,Shanghai,China).
13. Statistical Analysis
The continuous variables were expressed as the means ± SEM and compared
with Student’s independent t test to analyze 2 groups or one-way ANOVA to analyze
multiple groups. The categorical variables were expressed as percentages, and the
χ2 test was applied for the determination of significance of an association. Lab data
were analyzed with GraphPad Prism 5 and SPSS version 16.0. In clinical study the
effect of the covariants was examined by multiple logistic regression with SAS version
9.2. The value of P<0.05 was considered statistically significant.
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