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Endothelial-specific FoxO1 depletion prevents obesity-related disorders by increasing vascular metabolism and growth

Research Article
Cite this article as: eLife 2018;7:e39780 doi: 10.7554/eLife.39780
10 figures, 2 tables and 3 additional files

Figures

Endothelial-specific depletion of Foxo1 induced in adult male mice effectively reduces FoxO1 levels in skeletal muscle and adipose microvascular beds.

(A) PCR of genomic DNA from multiple organs of control (Cre-; Foxo1f/f) and EC-FoxO1 KD mice using primers for the floxed and deleted (E1∆E3) alleles. (B–C) Gene expression analysis of microvascular EC and CD16/CD32+ cells isolated from white adipose tissue of Control (n = 6) and EC-FoxO1 KD (n = 3–5) mice. (D) Representative Western blot images and quantitative analysis of FoxO1 and β-actin levels in capillary fragments isolated from skeletal muscle (n = 3–4). Results are expressed relative to β-actin levels. Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, calculated with two-tailed unpaired t-test.

https://doi.org/10.7554/eLife.39780.003
Greater vascular density in visceral adipose tissue of normal chow-fed EC-FoxO1 KD mice.

(A) Pecam1 mRNA levels in various tissues of Control and EC-FoxO1 KD mice after 16 weeks of normal chow (NC) diet (Control n = 6, EC-FoxO1 KD n = 5). (B) Representative confocal images of adipose tissue whole-mount staining with BODIPY 493/503 (green) and G. simplicifolia lectin (red) (×20 magnification; scale bar = 100 μm). (C–E) Lectin area (C), capillary branch density (D) and microvessel diameters (E) were quantified from confocal images (Control n = 5, EC-FoxO1 KD n = 6). Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, calculated with two-tailed unpaired t-test.

https://doi.org/10.7554/eLife.39780.004
EC-Foxo1 depletion strongly induces vascular growth within adipose tissue in response to HF diet.

(A) Pecam1 mRNA levels in different adipose tissue depots of Control and EC-FoxO1 KD mice after 16 weeks of high-fat (HF) diet (Control n = 5–7, EC-FoxO1 KD n = 7). (B) Gene expression analysis of eWAT of HF-fed Control and EC-FoxO1 KD mice (Control n = 7, EC-FoxO1 KD n = 6–7). (C) Representative confocal images of adipose tissue whole-mount staining with BODIPY 493/503 (green) and G. simplicifolia lectin (red) (C - scale bar = 100 μm). (D,E and I). Lectin area (D) and capillary branch density were quantified from these images (Control, n = 6; EC-FoxO1 KD, n = 5 or 6). (F–G) G. simplicifolia lectin (green) and Wheat germ aggluttinin (red) staining of paraffin-sectioned adipose tissue (F - scale bar = 100 μm) was used to assess capillary to adipocyte ratio (G). (H) Representative confocal images of adipose tissue whole-mount staining with Isolectin alone (greyscale; scale bar = 20 μm). (I) Microvessel diameters were quantified from confocal images (Control, n = 6; EC-FoxO1 KD, n = 6). Data in all panels are expressed as mean ± SEM; *p < 0.05, ***p < 0.001, calculated with two-tailed unpaired t-test.

https://doi.org/10.7554/eLife.39780.005
EC-Foxo1 depletion also favors microvascular expansion in skeletal muscle under HF diet feeding.

(A) Pecam1 mRNA levels in liver and skeletal of HF-fed Control (n = 5–6) and EC-FoxO1 KD (n = 6–7) mice. (B) Images of EDL muscle stained with Isolectin-FITC to identify capillaries (scale bar = 50 μm). (C) Capillary to fiber (C:F) ratios were calculated from 3 to 4 independent fields of view per mouse (Control n = 6, EC-FoxO1 KD n = 6). (D) Representative EM images of capillaries within skeletal muscle from HF-fed Control and EC-FoxO1 KD mice (×6.5k magnification; scale bar = 2 μm). (E–F) EC cross-sectional area (E) and capillary luminal diameter were quantified from EM images from n = 4 mice per group, with individual capillary measurements shown (F). Data in all panels are expressed as mean ± SEM; *p < 0.05, calculated with two-tailed unpaired t-test.

https://doi.org/10.7554/eLife.39780.006
Figure 5 with 2 supplements
EC-FoxO1 KD mice exhibit a healthier adipose tissue expansion in response to HF diet.

(A) Body weights during 16 weeks of HF feeding. (B) Summarized weight gain over the course of 0–14 weeks (Control n = 7, EC-FoxO1 KD n = 7). (C) Abdominal transverse micro-CT images of HF-fed Control (n = 5) and EC-FoxO1 KD (n = 6) mice (upper panel). Fat content (shown in white) was calculated as % of total trunk volume. (D) Representative hematoxylin and eosin-stained images of adipose tissue from the epididymal fat pad (scale bar = 100 μm). (E) Mean adipocyte cross-sectional area (Control n = 5 EC-FoxO1 KD n = 5). (F) mRNA for browning markers Ucp1 and Prdm16 relative to Hprt1 (Control n = 7, EC-FoxO1 KD n = 7). (G–H) Representative Western blot images (G) and quantitative analysis (H) of pSer473-Akt and total Akt levels in eWAT after ex vivo incubation in the absence or presence of insulin. Results are expressed relative to total Akt levels (Control n = 4, EC-FoxO1 KD n = 7). (I–J) mRNA for adipokines (I, Adipoq and Leptin) and angiogenic markers (J, Vegfa and Apln) in eWAT relative to Hprt1 (Control n = 6–7, EC-FoxO1 KD n = 5–7). Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, calculated with two-tailed unpaired t-test.

https://doi.org/10.7554/eLife.39780.007
Figure 5—figure supplement 1
Lower circulating and liver triglycerides in HF-fed EC-FoxO1 KD mice.

(A) Average weekly caloric intake of mice determined between diet weeks 4 to 11 from only single housed mice (Control n = 4, EC-FoxO1 KD n = 5). (B–C) Non-fasting serum levels of triglycerides (B) and glycerol (C) of Control (n = 7) and EC-FoxO1 KD (n = 7–8) mice. (D) Triglyceride concentration in the skeletal muscle (gastrocnemius) and liver (Control n = 6, EC-FoxO1 KD n = 7).

https://doi.org/10.7554/eLife.39780.008
Figure 5—figure supplement 2
EC-Foxo1 depletion has no impact on adipose mitochondrial content and respiration, or sensitivity to isoproterenol.

(A–B) Representative Western blot images (A) and quantitative analysis (B) of mitochondrial OXPHOS proteins in total homogenates from eWAT of HF-fed Control (n = 7) and EC-FoxO1 KD mice (n = 9). Results are expressed relative to β-actin. (C) Analysis of uncoupled (State 2) and ADP-stimulated (State 3) respiration rates in eWAT from HF-fed Control (n = 7) and EC-FoxO1 KD mice (n = 9) supported by Complex I (NADH from pyruvate/malate, PM, and glutamate, (G) and Complex II (FADH2 from succinate, (S). (D–E) Representative Western blot images (D) and quantitative analysis (E) of pSer563-HSL and total HSL levels in eWAT after ex vivo incubation in the absence or presence of 10 µmol/L isoproterenol for 30 min. Results are expressed relative to total HSL levels (Control n = 3, EC-FoxO1 KD n = 7). Data in all panels are expressed as mean ± SEM; *p < 0.05, ***p < 0.001, calculated with two-tailed paired t-test.

https://doi.org/10.7554/eLife.39780.009
Figure 6 with 2 supplements
EC-Foxo1 depletion improves glucose homeostasis in HF-fed mice.

(A–D) O2 consumption (A), CO2 production (B), Respiratory exchange ratio - RER (C) and daily activity (D) were measured during indirect calorimetry tests using a comprehensive laboratory animal monitoring system (CLAMS, Control n = 5, EC-FoxO1 KD n = 6). (E) Glucose tolerance of HF-fed Control and EC-FoxO1 KD mice was examined by intraperitoneal glucose tolerance test after 15 weeks of HF diet and 16 hr fasting. (F) Area under the curve (AUC, Control n = 7, EC-FoxO1 KD n = 7). (G) Insulin sensitivity of HF-fed Control and EC-FoxO1 KD mice was assessed by intraperitoneal insulin tolerance test after 14 weeks of HF diet and 4 hr fasting. (H) Area over the curve (AOC, Control n = 14, EC-FoxO1 KD n = 16). (I) Plasma glucose levels of HF-fed Control (n = 14) and EC-FoxO1 KD (n = 16) mice after 4 hr fasting. (J–K) eWAT gene expression analysis by qPCR (Control n = 6–7, EC-FoxO1 KD n = 6). Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, calculated with two-tailed unpaired t-test (F, I, J, K) or post hoc Bonferroni-corrected t-tests when a statistical significance was detected by two-way ANOVA model (A,C,D).

https://doi.org/10.7554/eLife.39780.011
Figure 6—figure supplement 1
EC-Foxo1 depletion has no effect on muscle insulin sensitivity.

Control and EC-FoxO1 KD mice fed NC or HF diet for 16 weeks were analyzed for muscle insulin sensitivity. Representative Western blot images (top) and quantitative analysis (bottom) of pSer473-Akt and total Akt levels in EDL muscle before and after in vivo insulin injection. Results are expressed relative to total Akt levels (NC-fed mice: Control n = 7, EC-FoxO1 KD n = 6; HF-fed mice: Control n = 6 and EC-FoxO1 KD n = 6). Data are expressed as mean ± SEM; *p < 0.05, calculated with two-tailed paired t-test.

https://doi.org/10.7554/eLife.39780.012
Figure 6—figure supplement 2
EC-Foxo1 depletion does not affect glucose homeostasis in NC-fed mice.

Glucose metabolism was assessed in Control and EC-FoxO1 KD mice after 16 weeks of NC diet. (A) Glucose tolerance of Control and EC-FoxO1 KD mice was examined by intraperitoneal glucose tolerance test after 15 weeks of NC diet and 16 hr fasting (Control n = 7 and EC-FoxO1 KD n = 6). (B) Area under the curve (AUC). (C) Insulin sensitivity of Control and EC-FoxO1 KD mice was assessed by intraperitoneal insulin tolerance test after 14 weeks of NC diet and 4 hr fasting. (D) Area over the curve (AOC, Control n = 7 and EC-FoxO1 KD n = 6). (E) Plasma glucose levels after 4 hr fasting (Control n = 7 and EC-FoxO1 KD n = 6). Data in all panels are expressed as mean ± SEM.

https://doi.org/10.7554/eLife.39780.013
FoxO1 is a critical regulator of glucose metabolism in EC.

(A, B, F) Gene expression analysis of EC fraction from adipose tissue from Control (n = 3–6) and EC-FoxO1 KD (n = 4–5) mice fed a HF diet for 7 weeks. (C–E) Increased glucose uptake (C) glucose consumption (D) and lactate production (E) in EC fraction from HF-fed EC-FoxO1 KD (n = 4) mice compared to Control (n = 4–5). Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, calculated with two-tailed unpaired t-test (A,B,C,F) or post hoc Bonferroni-corrected t-tests when a statistical significance was detected by two-way ANOVA model (D,E).

https://doi.org/10.7554/eLife.39780.014
Figure 8 with 1 supplement
Pharmacological inhibition of FoxO1 in SMEC reproduces in vitro the endothelial phenotype observed with EC-Foxo1 depletion.

(A–B) Representative Western blot images (A) and quantitative analysis (B) of FoxO1 and β-actin levels in primary EC from skeletal muscle cultivated under low (5 mmol/L) or high (25 mmol/L) glucose conditions for 48 hr (n = 5). Results are expressed relative to β-actin levels. (C–E) Transcript analysis by qPCR of microvascular EC from skeletal muscle cultivated under low (5 mmol/L, n = 8) or high (25 mmol/L, n = 8) glucose conditions for 48 hr in the presence or absence of the FoxO1 inhibitor (1 μmol/L AS1842856, n = 7) in the last 18 hr. (F–I) Representative Western blot images and quantitative analysis of hexokinase II (HK2, (F,G), PFKFB3 (H, I) and β-actin levels in primary EC from skeletal muscle cultivated under high glucose (25 mmol/L) conditions and treated with 1 μmol/L AS1842856 for 24 hr (n = 6). Results are expressed relative to β-actin levels. (J) Glucose uptake after 18 hr treatment with 1 μmol/L AS1842856 of microvascular EC from skeletal muscle cultivated under high glucose conditions (n = 3). (K–L) Glucose consumption (K) and Lactate production (L) were assessed in SMEC in the absence or presence of 1 μmol/L AS1842856. Cells were pretreated with 1 μmol/L AS1842856 for 24 hr (n = 4). Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, calculated with two-tailed unpaired t-test (B,J), post hoc Bonferroni-corrected t-tests when a statistical significance was detected by two-way ANOVA model (C–E, K–L) or two-tailed paired t-test (G,I).

https://doi.org/10.7554/eLife.39780.015
Figure 8—figure supplement 1
High-glucose conditions induce the expression of FoxO1 target genes.

(A–B) Gene expression analysis of cultured skeletal muscle ECs in low (5 mmol/L, n = 8) and high glucose (25 mmol/L, n = 8) conditions. Data in all panels are expressed as mean ± SEM; **p < 0.01, ***p < 0.001 calculated with two-tailed unpaired t-test.

https://doi.org/10.7554/eLife.39780.016
EC-Foxo1,3 depletion increases vascular growth and upregulates endothelial glycolytic processes comparable to EC-Foxo1 depletion.

(A) PCR of genomic DNA from eWAT of Control (Foxo1,3f/f) and EC-FoxO1,3 KD mice using primers for the floxed and deleted alleles for each gene. (B) Foxo1 and Foxo3 mRNA levels in microvascular EC from white adipose tissue (Control n = 4, EC-FoxO1,3 KD n = 3). (C) Glucose levels after 14 weeks of HF feeding and 4 hr fasting (Control n = 6, EC-FoxO1,3 KD n = 6). (D) Endothelial cell marker Pecam1 mRNA level in skeletal muscle and eWAT of HF-fed Control and EC-FoxO1,3 KD mice (Control n = 5, EC-FoxO1,3 KD n = 5). (E–F) Gene expression analysis by qPCR of eWAT (E) and EC fraction from adipose tissue (F) of HF-fed Control (n = 3–5) and EC-FoxO1,3 KD (n = 3–5) mice. (G–H) Increased glucose uptake (G) and lactate production (H) in EC fraction from adipose tissue of EC-FoxO1,3 KD mice (n = 4) compared to Control counterparts (n = 4–5). Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, calculated with two-tailed unpaired t-test.

https://doi.org/10.7554/eLife.39780.017
Schematic depicting the influence of FoxO1 in controlling the response of ECs to a HF diet.

In wild-type mice, FoxO1 represses glycolysis, which prevents endothelial cell growth. This results in impaired angiogenesis during adipose tissue expansion as well as dysfunction of the adipose tissue, which consequently leads to decreased glucose tolerance and increased serum and intra-tissue levels of triglycerides. Conversely, when FoxO1 is depleted in endothelial cells, up-regulation of glycolytic genes accelerates glycolysis, which supports increased cellular metabolism, growth and proliferation. This, in turn, increases the nutrient demand of endothelial cells, resulting in higher uptake and consumption of glucose and an increased production of lactate. The accelerated endothelial cell growth ultimately preserves adipose tissue functions and promotes improved systemic glucose tolerance and lipid metabolism.

https://doi.org/10.7554/eLife.39780.018

Tables

Table 1
Tissue weights of Control and EC-FoxO1 KD mice after 16 weeks of HF diet
https://doi.org/10.7554/eLife.39780.010
ControlEC-FoxO1 KD
Body weight (g)44 ± 1.138 ± 2.2
eWAT (g)1.8 ± 0.21.6 ± 0.6
rWAT (mg)881.7 ± 86.1579.8 ± 98.7*
Subcutaneous adipose (g)2.1 ± 0.31.3 ± 0.2*
BAT (mg)189.2 ± 21.2144.3 ± 14.8
Liver (g)1.5 ± 0.11.2 ± 0.1*
Heart (mg)135 ± 4.2126.6 ± 3.4
Gastrocnemius (mg)130.9 ± 4.3115.5 ± 4.4*
Soleus (mg)8.3 ± 0.58.0 ± 0.5
Tibialis anterior (mg)44.5 ± 1.542.6 ± 1.4
  1. eWAT: epididymal adipose tissue; rWAT: retroperitoneal adipose tissue; BAT: brown adipose tissue

    Data are expressed as mean ± SEM, n = 7 per group

  2. Significance was established using unpaired t-test

    *P < 0.05 vs Control group

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain,
strain background
(M. musculus,
FVB/n)
Foxo1f/f
(FVB-Foxo1tm1Rdp)
From the
laboratory of Dr
Tara L. Haas
Generated by
crossing Foxo1,3,4f/f
mice (FVB background;
from the laboratory
of Dr. Ronald A.
DePinho)
with FVB/n mice.
Strain,
strain background
(M. musculus,
FVB/n)
Foxo1,3f/f
(FVB-Foxo1tm1Rdp;
Foxo3tm1Rdp)
From the laboratory
of Dr Tara L. Haas
Generated by
crossing Foxo1,3,4f/f
mice (FVB background;
from the laboratory of
Dr. Ronald A. DePinho)
with FVB/n mice.
Strain,
strain background
(M. musculus,
(C57BL/6 x CBA)F2)
Pdgfb-iCre
[B6-Tg(Pdgfb-icre
/ERT2)1Frut]
From the laboratory of
Dr. Marcus Fruttiger
The founder mouse was
a kind gift from
Dr. Marcus Fruttiger..
Strain,
strain background
(M. musculus,
FVB.B6)
Foxo1iEC-D
[FVB.B6-Tg
(Pdgfb-icre/ERT2)
1Frut; Foxo1tm1Rdp]
This paperMice were generated
by cross-breeding
Pdgfb-iCre with
Foxo1f/f mice,
followed by
backcrossing
offspring with
Foxo1f/f mice for
3 + generations
Strain,
strain background
(M. musculus,
FVB.B6)
Foxo1,3iEC-D
[FVB.B6-Tg
(Pdgfb-icre/ERT2)
1Frut; Foxo1tm1Rdp;
Foxo3tm1Rdp]
This paperMice were generated
by cross-breeding
Pdgfb-iCre with Foxo1,3f/f
mice, followed
by backcrossing
offspring with Foxo1,3f/f
mice for
3 + generations
Cell line
(primary
mouse adipose
derived
endothelial cells,
male)
Endothelial cell
fraction from
adipose tissue
This paperIsolated freshly for
each experiment
Cell line
(primary
mouse skeletal
muscle
endothelial cells,
male)
Cultured
microvascular
EC, SMEC
This paper
AntibodyBiotin Rat
Anti-Mouse CD31
BD Pharmingen553371Cell purification
AntibodyMitoProfile Total
OXPHOS
Rodent WB
Antibody Cocktail
Abcamab110413WB (1:500)
AntibodyMouse
Anti-β-actin
Santa Cruz
Biotechnology
sc-47778WB (1:5000)
AntibodyPeroxidase AffiniPure
Goat Anti-Mouse
Jackson
ImmunoResearch
115-035-003WB (1:10,000)
AntibodyPeroxidase AffiniPure
Goat Anti-Rabbit
Jackson
ImmunoResearch
111-035-003WB (1:10,000)
AntibodyPurified Rat Anti-Mouse
CD16/CD32
(Mouse BD Fc Block)
BD Pharmingen553141Cell purification
AntibodyPurified Rat
Anti-Mouse CD144
BD Pharmingen555289Cell purification
AntibodyRabbit
Anti-α/β-tubulin
Cell Signaling2148WB (1:1000)
AntibodyRabbit
Anti-Akt
Cell Signaling9272WB (1:1000)
AntibodyRabbit
Anti-HSL
Cell Signaling4107WB (1:1000)
AntibodyRabbit
Anti-Hexokinase
Cell Signaling2867TWB (1:1000)
AntibodyRabbit
Anti-PFKFB3
Cell Signaling13123SWB (1:1000)
AntibodyRabbit
Anti-phospho-Akt
(Ser473)
Cell Signaling4058WB (1:1000)
AntibodyRabbit
Anti- phospho-HSL
(Ser563)
Cell Signaling4139WB (1:1000)
Sequence-
based
reagent (oligonucleotide)
oFK1ckA:
GCT TAG AGC
AGA GAT GTT
CTC ACA TT
ThermoFisher
Scientific
NA
Sequence-
based
reagent (oligonucleotide)
oFK1ckB:
CCA GAG TCT
TTG TAT CAG
GA AAT AA
ThermoFisher
Scientific
NA
Sequence-
based
reagent (oligonucleotide)
oFK1ckC: CAA
GTC
CAT TAA TTC AGC
ACA TTG A
ThermoFisher
Scientific
NA
Sequence-
based
reagent (oligonucleotide)
oFK2ckA: ATT CCT
TTG GAA ATC
AAC AAA ACT
ThermoFisher
Scientific
NA
Sequence-based
reagent (oligonucleotide)
oFK2ckB: TGC TTT
GAT ACT ATT
CCA CAA ACCC
ThermoFisher
Scientific
NA
Sequence-
based
reagent (oligonucleotide)
oFK1ckC: AGA TTT
ATG TTC CCA CTT
GCT TCCT
ThermoFisher
Scientific
NA
Peptide,
recombinant
protein
Humalog
Insulin
LillyNA
Commercial
assay or kit
PureLink
Genomic DNA
Mini Kit
ThermoFisher ScientificK182001
Commercial
assay or kit
EnzyFluoTML-
lactate Assay Kit
BioAssay
Systems
EFLLC-100
Commercial
assay or kit
Glycerol
Assay Kit
Sigma-
Aldrich
MAK117
Commercial
assay or kit
Glucose (HK)
Assay
Sigma-
Aldrich
GAHK20
Commercial
assay or kit
Lactate-Glo
Assay
PromegaJ5021
Commercial
assay or kit
RNeasy Mini KitQiagen74106
Commercial
assay or kit
Triglyceride
Colorimetric
Assay kit
Cayman
Chemical
Company
10010303
Chemical
compound,
drug
AS1842856 FoxO1
inhibitor
EMD Millipore344355
Chemical
compound,
drug
IsoproterenolTocris1747
Chemical
compound, drug
TamoxifenSigmaT5648
Software,
algorithm
Image J Analysis
Software
National
Institutes of
Health
https://imagej.nih.gov/ij/download.html
Software,
algorithm
GraphPad Prism
Version 6.07
GraphPad
Software Inc.
https://www.graphpad.com/scientific-software/prism/
Other11 kcal% fat w/
sucrose Surwit Diet
Research
Diets
D12329
Other58 kcal% fat w/sucrose Surwit DietResearch
Diets
D12331
OtherBODIPY
493/503
ThermoFisher
Scientific
D3922
OtherDynabeadsThermoFisher
Scientific
14311D
OtherFast TaqMan
Master Mix
ThermoFisher
Scientific
4444963
OtherM-MLV reverse
transcriptase
New England
Biolabs
M0253
OtherStreptavidin
Particles Plus -
DM
BD IMag557812
OtherRhodamine
labeled Griffonia
(Bandeiraea)
Simplicifolia lectin
VectorLabsRL1102
OtherQIAzol Lysis
Reagent
Qiagen79306
OtherSuperSignal
West Pico
ThermoFisher
Scientific
34080
OtherType I
collagenase
ThermoFisher
Scientific
17100–017
OtherType II
collagenase
ThermoFisher
Scientific
17101–005

Data availability

All data generated or analysed during this study are included in the manuscript. Individual replicates are plotted as dot plots, with the average and the appropriate error bars indicated for all numerical data.

Additional files

Supplementary file 1

Supplementary Table 1.

Tissue weights of Control and EC-FoxO1,3 KD mice after 14 weeks of HF diet

https://doi.org/10.7554/eLife.39780.019
Supplementary file 2

TaqMan primer sets used in gene expression analysis

https://doi.org/10.7554/eLife.39780.020
Transparent reporting form
https://doi.org/10.7554/eLife.39780.021

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