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Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Vascular endothelial growth factor
(VEGF), basic fibroblast growth factor (bFGF), and transforming growth
factor-
1 (TGF-1) mRNA increase in rat
skeletal muscle in response to a single acute exercise bout. Nitric
oxide (NO) is released locally by muscle vascular endothelium
and muscle fibers during exercise, contributes to the blood flow
response to exercise, and regulates mitochondrial respiration. We
hypothesized that a reduction in NO production, via NO synthase
inhibition, would demonstrate a link between NO and the VEGF, bFGF, and
TGF-1 gene responses to exercise. To investigate this
hypothesis, 9-wk-old female Wistar rats were divided into eight
treatment groups (n = 6 each): 1) saline + rest,
2) saline + exercise, 3) 30 mg/kg
N
-nitro-L-arginine methyl ester
(L-NAME, a known NOS inhibitor) + rest, 4) 30 mg/kg
L-NAME + exercise, 5) 300 mg/kg L-NAME + rest, 6) 300 mg/kg L-NAME + exercise, 7)
300 mg/kg N-nitro-D-arginine methyl
ester (D-NAME, inactive enantiomer of L-NAME) + rest, and 8) 300 mg/kg D-NAME + exercise. Exercise
consisted of 1 h of running at 20 m/min on a 10° incline. VEGF,
TGF-1, and bFGF mRNA from left gastrocnemius were
analyzed by quantitative Northern blot. Submaximal exercise for 1 h
increased VEGF mRNA 4.2-fold and TGF-1 mRNA 1.5-fold in
untreated rats but did not increase bFGF mRNA. The exercise-induced
increase in VEGF mRNA was attenuated ~50% by 30 and 300 mg/kg
L-NAME; the TGF-1 mRNA increase was
unaffected by 300 mg/kg L-NAME. In addition, 300 mg/kg
D-NAME had no effect on the exercise-induced increase in VEGF mRNA. Administration of 300 mg/kg L-NAME had no effect
on bFGF mRNA. These findings suggest that NO is important in the regulation of the VEGF gene response to exercise through increases in
VEGF transcription or by increases in the VEGF mRNA half-life.
vascular endothelial growth factor; transforming growth
factor-1; basic fibroblast growth factor; N-nitro-L-arginine methyl ester; N-nitro-D-arginine methyl ester
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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VASCULAR ENDOTHELIAL GROWTH factor (VEGF) is believed to be an important regulator of angiogenesis during embryonic development, wound healing, reproductive functions, and tumor growth (14). VEGF is a predominantly endothelial cell-specific, heparin-binding, 45-kDa homodimeric glycoprotein mitogen that increases vascular permeability, endothelial cell proliferation, and angiogenesis (11, 15, 24, 36, 43). Sequence analysis of the cDNA indicates that VEGF may exist as different molecular species and that alternative splicing of a single VEGF gene is the basis for this molecular heterogeneity (14).
Endurance exercise training is well known to produce adaptive changes
in skeletal muscle and the cardiovascular system. In skeletal muscle,
endurance training results in increases in oxidative enzymes and in the
number of capillaries (2, 7, 34). It has been demonstrated in intact
rats that exercise induces greater expression of genes known to promote
angiogenesis. Breen et al. (6) have demonstrated that 1 h of acute,
submaximal treadmill running promotes a three- to fourfold increase in
VEGF mRNA and more modest increases in transforming growth
factor-1 (TGF-1) and basic fibroblast
growth factor (bFGF) mRNA in the rat gastrocnemius. A sixfold increase
in VEGF mRNA has been demonstrated in anterior tibialis and extensor
digitorum longus muscles after 4 days of electrical
stimulation (21).
It is now well established that VEGF gene expression is increased in
response to hypoxia (6, 19, 25, 35). Several other factors have been
shown to regulate VEGF gene expression, including nitric oxide (NO).
Regulation of VEGF gene expression has been demonstrated in arterial
smooth muscle, in the lung, in carcinoma cells, and in renal mesangial
cells (8, 17, 41, 42). NO is of particular interest because of the
regulatory role of NO in endothelium-dependent vasodilation in skeletal
muscle during rest and exercise, as well as the role of NO as an
important intracellular signaling mechanism in the regulation of
oxidative metabolism (10, 18, 22, 37). In addition to the effect of NO
on VEGF gene expression, NO has been shown to regulate
TGF-1 and bFGF gene expression (12, 44). We therefore
hypothesized that, during acute exercise, NO is an important signaling
mechanism for the regulation of VEGF, TGF-1, and bFGF
gene expression. Specifically, we hypothesized that NO synthase (NOS)
inhibition by N-nitro-L-arginine
methyl ester (L-NAME) would inhibit the acute exercise-induced increases in VEGF, TGF-1, and bFGF mRNA.
In this report we demonstrate that the increase in VEGF mRNA levels
after acute exercise was attenuated (~50%) by NOS inhibition, suggesting that NO regulates VEGF mRNA through increased VEGF transcription or increased VEGF mRNA half-life. This attenuation was
not observed after the administration of 300 mg/kg
N-nitro-D-arginine methyl ester
(D-NAME), the inactive enantiomer of L-NAME.
The exercise-induced increase in TGF-1 mRNA was
unaffected by NOS inhibition. Neither exercise nor L-NAME
affected bFGF mRNA levels. These results suggest that NO is an
important signaling mechanism in the regulation of the exercise-induced
increase in VEGF mRNA.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study was approved by the University of California, San Diego,
Animal Subjects Committee. Female Wistar rats [68 ± 7 (SD) days
old, 220 ± 11 (SD) g body wt] were first familiarized with a
rodent treadmill (Omnipacer model LC-4, Omnitech, Columbus, OH) and
taught to run at 20 m/min on an incline of 10° for 5 min, 24-48 h before the experimental bout. Animals were housed in their cages and allowed standard rat food and water ad libitum before undertaking the study. The exercise bout consisted of 1 h of treadmill running at 20 m/min on a 10° incline. This speed represents 55% of
the speed required to attain maximal oxygen consumption (6). Initially,
four treatment groups were defined with six rats in each group:
1) saline + rest, 2) saline + exercise, 3) 300 mg/kg L-NAME (Sigma Chemical, St. Louis, MO) + rest, and
4) 300 mg/kg L-NAME + exercise. After the initial
results of 300 mg/kg L-NAME on the exercise-induced
increase in VEGF mRNA were obtained and to eliminate the potential for
nonspecific drug interactions, four additional groups of
animals were defined with six rats in each group: 1)
30 mg/kg L-NAME + rest, 2) 30 mg/kg
L-NAME + exercise, 3) 300 mg/kg D-NAME,
the inactive enantiomer of L-NAME (Sigma Chemical) + rest,
and 4) 300 mg/kg D-NAME + exercise. Animals were
injected intraperitoneally with saline, L-NAME,
or D-NAME 20 min before the start of rest or exercise.
After completing the 1 h of rest or exercise, animals were anesthetized
with pentobarbital sodium (50 mg/kg ip) while breathing 100%
O2 to avoid hypoxemia, which has been shown to stimulate
skeletal muscle VEGF mRNA (6). Within 20 min of the completion of
exercise and after topical administration of lidocaine, the left
carotid artery was catheterized for the measurement of mean arterial
pressure (MAP) to determine the effect of L-NAME on the
vasculature. After the measurement of MAP and within 40 min of the
completion of exercise, the left gastrocnemius muscles (both heads
combined) were removed for RNA isolation, frozen in liquid nitrogen,
and stored at 
80°C until further RNA analysis.
RNA isolation and Northern analysis.
Rat gastrocnemius muscles were removed, and total cellular RNA was
isolated from each sample by the method of Chomczynski and Sacchi (9).
These RNA preparations were quantitated by absorbance at 260 nm, and
RNA intactness and integrity were assessed by ethidium bromide staining
after separation by electrophoresis in a 6.6% formaldehyde-1% agarose
gel. Fractionated RNA was transferred by Northern blot to Zeta-probe
membrane (Bio-Rad, Hercules, CA). After transfer, RNA was cross-linked
to the membrane by ultraviolet irradiation for 1 min and stored at
4°C. The blots were then probed with oligolabeled
[
-32P]dCTP cDNA probes, which had a specific
activity >1 × 109
disintegrations · min
1 · µg
DNA1 (13). The rat VEGF probe is a
0.9-kb cDNA Pst I/Sma I insert cloned into pBluescript
II KS (+) vector (25). The rat TGF-1 cDNA probe is a
0.985-kb Hind III/Xba I insert cloned into pBluescript II KS (+) vector (31). The bFGF probe is a 1-kb Xho I fragment of human bFGF cDNA (23). Prehybridization and hybridization were
performed in 50% formamide, 5× saline-sodium citrate (SSC; 20× SSC is 0.3 M sodium chloride, 0.3 M sodium citrate),
10× Denhardt's solution (100× Denhardt's solution is 2%
Ficoll, 2% polyvinylpyrrolidone, 2% BSA factor V), 50 mM sodium
phosphate (pH 7.0), 1% SDS, and 250 µg/ml salmon sperm DNA at
42°C. Blots were washed with 2× SSC and 0.1% SDS at room
temperature and 0.1× SSC and 0.1% SDS at 55°C (bFGF and
TGF-1) or 65°C (VEGF). Blots were exposed to XAR-5
X-ray film (Eastman Kodak, New Haven, CT) for 2-3 (VEGF and bFGF)
or 7 days (TGF-1) with use of a Cronex Lightning Plus screen at 80°C. Autoradiographs were quantitated by
densitometry within the linear range of signals and normalized to 18S
rRNA levels.
Statistical treatment.
Quantitative densitometry was used to measure the mRNA levels for all
three growth factors. Normalization of growth factor densitometric
signals with the 18S rRNA was used to control for lane loading
variation. One sample was lost from the D-NAME + rest group
and was not replaced. A two-way ANOVA (3 × 2, drug × exercise level) was used to determine changes in MAP. A two-way ANOVA
(2 × 2, drug × exercise level) was used to
determine changes in mRNA. Bonferroni's test was used to determine
significance between conditions. For VEGF mRNA, 30 mg/kg
L-NAME, 300 mg/kg L-NAME, and 300 mg/kg D-NAME samples were compared with saline data,
respectively. Significance was established at P 
0.05 for all
statistical sets, and values are means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Injection of 30 mg/kg L-NAME, 300 mg/kg L-NAME,
and 300 mg/kg D-NAME resulted in significant systemic
hypertension as measured under anesthesia (133 ± 2, 151 ± 4, 155 ± 3, and 154 ± 4 mmHg for saline, 30 mg/kg
L-NAME, 300 mg/kg L-NAME, and
D-NAME, respectively) ~1 h and 40 min after the injection
of L-NAME or D-NAME (Fig. 1). There was no significant difference in
MAP between rest and exercise for any treatment group; thus these
values were combined. Figure 2 shows
representative Northern blots in which VEGF mRNA levels were examined
after the single 1-h submaximal exercise run. It is clear that VEGF
mRNA increased after exercise and that this increase was attenuated by
30 and 300 mg/kg L-NAME (Fig. 2, A and B),
whereas 300 mg/kg D-NAME did not alter the exercise-induced increase in VEGF mRNA (Fig. 2C).
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Figure 3 portrays the quantitative
densitometry for VEGF mRNA for 30 and 300 mg/kg L-NAME and
300 mg/kg D-NAME normalized to 18S rRNA. Figure 3,
A and B, demonstrates that exercise induced a 4.2-fold
increase in VEGF mRNA; whereas L-NAME did not completely inhibit this response, it did attenuate the response ~50%. There was
no difference between the VEGF mRNA values for saline + rest and
L-NAME + rest. With the same saline samples (rest and
exercise) used in Fig. 3, A and B, Fig. 3C
shows similar exercise-induced increases in VEGF mRNA between saline
and D-NAME groups. There was no difference between the VEGF
mRNA values for saline + rest and D-NAME + rest. For the
drug × exercise interaction effect, P = 0.009 for 30 mg/kg L-NAME, P = 0.002 for 300 mg/kg
L-NAME, and P = 0.79 for 300 mg/kg
D-NAME. Figure 4 demonstrates
close correlation between the saline + rest and saline + exercise
samples used for the 30 mg/kg L-NAME blot with the 300 mg/kg D-NAME blot (Fig. 4A) and for the 300 mg/kg
L-NAME blot with the 300 mg/kg D-NAME blot
(Fig. 4B).
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Representative Northern blots for the TGF-1 and bFGF
responses to exercise and 300 mg/kg L-NAME are presented in
Fig. 5. Exercise increased
TGF-1 but did not increase bFGF mRNA.
Administration of 300 mg/kg L-NAME had no effect on
TGF-1 or bFGF mRNA levels. Figure
6 portrays the quantitative densitometry
for TGF-1 and bFGF mRNA normalized to 18S rRNA. Figure
6A demonstrates that exercise increased TGF-1
~1.5-fold and that L-NAME did not affect this
exercise-induced increase. Figure 6B shows that neither
exercise nor L-NAME affected bFGF mRNA levels.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The principal findings of the present study are as follows: 1)
VEGF and TGF-1 mRNA were increased after 1 h of
submaximal exercise; 2) L-NAME attenuated the VEGF
mRNA increase ~50%; and 3) L-NAME did not affect
rest or exercise TGF-1 or bFGF mRNA levels. Previously,
we demonstrated that skeletal muscle VEGF mRNA levels were increased
after 1 h of acute exercise, with this increase being intensity and
oxygen concentration dependent (6). This report is the first to
demonstrate that NO plays a significant role in the exercise-induced
increase in VEGF mRNA.
Exercise and VEGF. VEGF is a potent endothelial mitogen for arterial and venular endothelial cells (14). It has been shown that VEGF mRNA increases in skeletal muscle after 1 and 3 days of chronic nerve stimulation and immediately after a single exercise bout (6, 21, 39). Changes in mRNA are not necessarily followed by increases in protein, but VEGF protein is increased almost threefold after 3 days of chronic electrical nerve stimulation (3). Recent work has shown that VEGF mRNA is increased in humans in response to exercise (20, 33). Our data (Figs. 2 and 3) provide additional evidence supporting the hypothesis that VEGF plays an important role in exercise-induced skeletal muscle angiogenesis.
NO, exercise, TGF-1, and bFGF.
As is evident from Figs. 5 and 6, exercise produced at best only modest
increases in TGF-1 and bFGF mRNA, which were unaltered by NOS inhibition. Although NO has been shown to modify bFGF (12) and
TGF-1 (44) gene expression, we believe that the
relatively modest changes in bFGF and TGF-1 mRNA with
exercise and NOS inhibition suggest that these growth factors do not
play a significant role in the initial stages of exercise-induced
angiogenesis. In a previous report our group demonstrated a small, but
significant, increase in bFGF mRNA with exercise; in this report we do
not (6). One possible explanation for this apparent discrepancy may be
related to the delay required for the measurement of MAP before the
gastrocnemius samples were removed. Breen et al. (6) demonstrated that
the exercise-induced increase in bFGF mRNA returns to baseline within 2 h after the completion of exercise. Perhaps the 40 min required for the
measurement of MAP and gastrocnemius removal in this report represented
a sufficient amount of time for the bFGF mRNA to return to baseline.
NO and exercise-induced angiogenesis. One theory regarding exercise-induced angiogenesis in skeletal muscle suggests that prolonged imbalances between the perfusion capabilities of the blood vessels and the metabolic requirements of the tissue cells lead to modification of the vasculature to satisfy the tissue needs (see Ref. 1 for review). This theory suggests that decreased oxygenation resulting from this imbalance causes the tissues to become hypoxic and produces a variety of metabolites implicated in vessel growth, including adenosine, ADP, lactic acid, nicotinamide derivatives, and prostaglandins of the E series. The resulting increase in vascularity promotes oxygen delivery to the tissue cells by increasing the capillary-to-fiber surface area interface and increasing maximal blood flow. NO is known to be important in blood flow regulation during exercise and is a cellular signal regulating mitochondrial respiration, and within the physiological oxygen gradient, S-nitrosohemoglobin brings local blood flow into line with oxygen requirements in the brain (22, 37, 40). Given this influence of NO on blood flow and mitochondrial respiration, it appears reasonable that NO may be important for exercise-induced angiogenesis.
NO and VEGF. NO appears to be an important regulator of endothelial cell growth and angiogenesis. One such role for NO is to function in VEGF-induced angiogenesis as a cellular signal stimulated by VEGF mediating the mitogenic effect of VEGF in the coronary venular endothelium (29). In addition, NOS has been shown to lie downstream from VEGF during VEGF-induced angiogenesis (44).
It is now known that hypoxia increases VEGF transcription through hypoxia-inducible factor 1 (HIF-1) (16, 28) and stabilizes VEGF mRNA via the RNA-binding protein HuR (26). We have shown that hypoxic exercise produces a greater increase in VEGF mRNA levels than does exercise alone (6). In addition to HIF-1 regulation, the murine VEGF gene promoter region contains consensus binding sites for transcription factors special protein-1 (SP-1), activator protein-1 (AP-1), and activator protein-2 (AP-2), as well as nuclear factor-
B
(NF-B) (38). NO has been shown to inhibit VEGF upregulation through
inhibition of HIF-1 in aortic smooth muscle and pulmonary artery cells
or AP-1 in aortic smooth muscle cells (27, 41). In contrast, NO
increases VEGF mRNA via guanylate cyclase activity in human A-172
glioblastoma cells and human Hep G2 hepatocellular carcinoma cells and
increases VEGF mRNA stability in these same cells (8). The mechanism of
action for NO includes stimulation of soluble guanylate cyclase, which
converts GTP to the intracellular second-messenger cGMP, consistent
with the reported mechanism for AP-1 (30).
Recently, Benoit et al. (5) demonstrated that nitroprusside, a direct
NO donor, and ACh, an NO donor via endothelial NOS, increased VEGF mRNA
in resting skeletal muscle. Our findings from NOS inhibition during
exercise are consistent with these findings. Given that NO is important
for vasodilation during exercise (22), it would appear that NO should
function to increase VEGF levels, rather than inhibit VEGF, as had been
demonstrated in aortic smooth muscle and pulmonary artery cells (27,
41). It is well recognized that different tissues can respond
differently to the same stimulus, as evidenced by the response to
hypoxia: in the lung, hypoxia causes vasoconstriction of the
vasculature, whereas in skeletal muscle, hypoxia vasodilates the
vasculature. It appears, therefore, that the regulation of VEGF gene
expression is different depending on the specific tissue under investigation.
Use of L-NAME and D-NAME. We used D-NAME, the inactive enantiomer of L-NAME, to control for nonspecific effects of L-NAME. The results from the Northern blots demonstrate that the attenuation of VEGF mRNA by L-NAME is not a result of nonspecific effects, as evidenced by no difference in the exercise-induced increase in VEGF mRNA levels between saline and D-NAME (Figs. 2 and 3). However, the increase in blood pressure with D-NAME is not consistent with previous reports in the literature (32). Although it has been shown previously that acute administration of 100 mg/kg iv D-NAME does not affect blood pressure (32), there are no known reports on the effects of acute administration of 300 mg/kg D-NAME. A recent report (4) demonstrated that chronic administration of D-NAME may increase resting blood pressure, suggesting that D-NAME may exhibit some activity similar to L-NAME.
In this report we have demonstrated that two different dosages of L-NAME produce similar results on VEGF gene expression. Figure 4 demonstrates that although the values for the increase in VEGF mRNA with exercise in the saline group are not exactly the same for the L-NAME and D-NAME blots, these values are in close agreement and that differences in these values result from technical issues inherent in comparisons of this type. Among the various NOS inhibitors, we selected L-NAME because of its nonspecific inhibition of all the NOS isoforms and because of its universal use as an NOS inhibitor. The majority of other NOS inhibitors, such as aminoguanidine and nitroindazole, which are chemically unrelated to L-NAME, demonstrate only isoform-specific inhibition. Although their use is unwarranted here, these isoform-specific inhibitors will be useful in the future to determine the NOS isoform responsible for the exercise-induced skeletal muscle VEGF gene expression. Our exercise model provides a unique opportunity to study growth factor regulation resulting from systemic, in vivo exercise. In this model it would be impossible to locally administer NO donors to the exercising skeletal muscle, inasmuch as this would require catheterizing the animal hindquarters, which would significantly impair running performance. It is also not possible to administer an NO donor systemically, inasmuch as this would result in undesirable side effects on blood pressure regulation. In addition, it would not be possible to measure local skeletal muscle NO release via a central catheter because of the short half-life of NO. On the basis of the work by Breen et al. (6), which demonstrated the largest increase in VEGF, TGF-1, and bFGF mRNA in animals killed immediately after exercise, we harvested our samples as
soon as possible after the measurement of MAP (6). Breen et al. also
demonstrated that the increase in VEGF mRNA with exercise in the rat
gastrocnemius is localized to the subsarcolemmal region of the muscle.
In summary, we have demonstrated that 1 h of acute exercise increases
VEGF and TGF-1 mRNA. We have also demonstrated that NOS
inhibition by L-NAME attenuates the exercise-induced
increase in VEGF mRNA ~50%, consistent with previous findings that
NO donors increase VEGF mRNA in resting skeletal muscle and that NO
increases VEGF mRNA half-life. Our data suggest that NO plays an
important role in exercise-induced VEGF gene regulation in skeletal muscle.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-17731. T. P. Gavin was supported in part by National Heart, Lung, and Blood Institute National Research Service Award HL-09624.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. P. Gavin, Dept. of Medicine, University of California, San Diego, La Jolla, CA 92093-0623 (E-mail: tgavin{at}ucsd.edu).
Received 6 October 1998; accepted in final form 10 November 1999.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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