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Figure legends
Figure 1. Opg deficiency tends to suppress AngII-induced aortic aneurysms
(A) Aortas of AngII-infused ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice were categorized into three
groups based on diameter and the presence of visible hematoma (No Aneurysm, Aneurysm, and
Dissection groups). Scale bars indicate 1 mm. (B) External diameter of the SRA in AngII-infused
ApoE-/-Opg+/+ (gray, n=16) and ApoE-/-Opg-/- (white, n=16) mice. All measurements are shown as
box plots and each measurement is shown as a white circle (No Aneurysm), gray triangle
(Aneurysm), or black cross (Dissection). (C) The incidence (%) of aortic aneurysms is shown for
No Aneurysm (white), Aneurysm (gray), and Dissection (black) groups. Statistical significance:
p<0.05.
Figure 2. Opg deficiency tends to suppress AngII-induced aortic dilatation and promotes
adventitial thickening
(A) Representative cross sections of the aorta stained with HE, EVG, and AZAN. Areas selected
by boxes surrounded by a black dotted line are magnified below. Scale bars indicate 0.1 mm. (B)
Internal area of the suprarenal aorta (SRA) of ApoE-/-Opg+/+ (n=16) and ApoE-/-Opg-/- (n=16) mice.
Measurements for all samples are presented as box plots. Measurements for the three groups are
presented as bar graphs. n=4 and 8 for the No-aneurysm group, n=5 and 5 for the Aneurysm group,
and n=7 and 3 for the Dissection group in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice, respectively. (C)
Medial layer width of the SRA of AngII-infused mice in each group. N.S; not significant. (D)
Relative adventitial area of the SRA of AngII-infused mice in each group. Statistical significance:
p<0.05.
22
Figure 3. Collagen accumulation in aortas of ApoE-/-Opg-/- mice
(A) Representative immunofluorescence images of aortas of AngII-infused mice stained with an
anti-collagen I antibody. Boxed areas (with white dotted lines) are magnified below. Scale bars
indicate 100 μm. (B) Percent area of collagen I expression in aortas of ApoE-/-Opg+/+ (n=16) and
ApoE-/-Opg-/- (n=16) mice. Measurements for all samples are presented as box plots.
Measurements for the three groups are presented as bar graphs (n=4 and 8 for the No Aneurysm
group, n=5 and 5 for the Aneurysm group, and n=7 and 3 for the Dissection group in ApoE-/Opg+/+ and ApoE-/-Opg-/- mice, respectively). (C) Tgf-β1 mRNA expression in the SRA at days 0,
7, and 28 after initiation of AngII infusion in ApoE-/-Opg+/+ (n=4, 5, 5) and ApoE-/-Opg-/- (n=5, 5,
5) mice. Statistical significance: p<0.05.
Figure 4. Accumulation of myofibroblasts in adventitias of AngII-infused ApoE-/-Opg-/- mice.
(A) Representative double-immunofluorescence images of aortas after AngII infusion with antiSMA (green) and anti-vimentin (red) antibodies. Scale bars indicate 100 μm. Areas selected by
the white box (dotted line) are magnified below. Nuclei were stained with DAPI (blue). Scale bars
indicate 25 μm. Medial layer (m), Adventitial layer (a), hematoma (b). The two arrowheads show
borders of the medial layer. The white dotted line in the magnified panels indicates the border of
the medial and adventitial layers. (B) Percent area of SMA expression in adventitias of ApoE-/Opg+/+ (n=16) and ApoE-/-Opg-/- (n=16) mice. Measurements for all samples are presented as box
plots. Measurements for the three groups are presented as bar graphs. n=4 and 8 for the No
Aneurysm group, n=5 and 5 for the Aneurysm group, and n=7 and 3 for the Dissection group in
ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice, respectively. Statistical significance: p<0.05.
23
Figure 5. Trail upregulation in aneurysm tissue of AngII-infused ApoE-/-Opg-/- mice.
(A) Representative immunofluorescence images of aortic tissue sections from AngII-infused mice
stained with anti-Trail (green) antibody. Scale bars indicate 100 μm. Areas selected by the white
box (dotted line) in the Aneurysm group are magnified below. (B) Magnified
immunofluorescence images of aortic tissue sections from the AngII-infused Aneurysm group
stained with anti-Trail (green; a, f), anti-SMA (green; b, g), anti-vimentin (red; c, h), anti-Ki67
(red; d, i), and anti-F4/80 (red; e, j) antibodies in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice. Nuclei
were stained with DAPI (blue; c-DAPI, h-DAPI). Scale bars indicate 25 μm. Medial layer (m),
adventitial layer (a). The two arrowheads show borders of the medial layer. The white dotted line
in the magnified panels indicates the border of the medial and adventitial layers. Small arrows in
panel j indicate large round-shaped macrophages infiltrating the adventitia. Statistical
significance: p<0.05. (C) Percent area of Trail expression in medial and adventitial layers of
ApoE-/-Opg+/+ (n=16) and ApoE-/-Opg-/- (n=16) mice. Measurements for all samples are presented
as box plots. Measurements for the three groups are presented as bar graphs. n=4 and 8 for the
No Aneurysm group, n=5 and 5 for the Aneurysm group, and n=7 and 3 for the Dissection group
in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice, respectively. Statistical significance: p<0.05.
24
Bumdelger et al.
Figure 1
Aneurysm
Dissection
ApoE-/Opg +/+
No-aneurysm
Aneurysm
Dissection
p=0.21
2.5
ApoE -/- Opg +/+
2.0
ApoE -/- Opg -/-
1.5
Dissection
Aneurysm
No-aneurysm
1.0
0.5
p=0.09
100
3.0
AngII
% of Incidence
External diameter of SRA(mm)
ApoE -/Opg -/-
No-aneurysm
80
Dissection
Aneurysm
No-aneurysm
60
40
20
ApoE -/Opg +/+
ApoE -/Opg -/-
No-aneurysm
Aneurysm
ApoE -/- Opg +/+
Bumdelger et al.
Figure 2
HE
EVG
AZAN
Dissection
No-aneurysm
Aneurysm
ApoE -/- Opg
-/-
Dissection
Internal area of
SRA(mm2 )
0.5
1.0
1.5
p=0.04
ApoE -/- ApoE -/Opg +/+ Opg -/-
p=0.07
p=0.01
ApoE -/- ApoE -/Opg +/+ Opg -/-
N.S.
N.S.
N.S.
ApoE -/Opg -/-
N.S.
N.S.
p=0.13
ApoE -/Opg +/+
0.5
0.4
0.3
0.2
0.1
1.0
0.8
0.6
0.4
0.2
Width of medial
layer(mm)
Relative area of
adventitia
No-aneurysm
Aneurysm
Dissection
No-aneurysm
Aneurysm
Dissection
All samples
No-aneurysm
Aneurysm
Dissection
Bumdelger et al.
Figure 3
ApoE -/- Opg +/+
Aneurysm
Dissection
No-aneurysm
Aneurysm
Dissection
Collagen I
No-aneurysm
ApoE -/- Opg -/-
N.S.
p<0.01
p<0.05
p<0.05
60
All samples
No-aneurysm
Aneurysm
Dissection
40
20
ApoE -/Opg +/+
ApoE -/Opg -/-
TGF-b1 expression in SRA
% of Collagen I expression
area in aorta
2.52.5
p=0.05
p=0.01
N.S.
N.S.
2.02
1.51.5
1.01
0.50.5
00 0
0d
7d
28d
AngII
ApoE -/+/+
Opg
0d
7d
28d
AngII
ApoE -/Opg -/-
Bumdelger et al.
Figure 4
ApoE -/- Opg -/-
ApoE -/- Opg +/+
Dissection
Aneurysm
No-aneurysm
Aneurysm
Dissection
Merged
Vimentin
SMA
No-aneurysm
DAPI
% of SMA expression area
in the adventitial layer of aorta
N.S.
p<0.01
N.S.
p<0.05
30
All samples
No-aneurysm
Aneurysm
Dissection
20
10
ApoE -/Opg +/+
ApoE -/Opg -/-
Bumdelger et al.
Figure 5
ApoE -/- Opg -/-
ApoE -/- Opg +/+
Aneurysm
Dissection
No-aneurysm
Aneurysm
Dissection
Trail
No-aneurysm
ApoE -/- Opg -/-
ApoE -/- Opg +/+
Aneurysm
Trail
SMA
Vimentin
Trail
SMA
Vimentin
F·
K·
% of TRAIL expression area
in aorta
Ki67
Aneurysm
F4/80
DAPI
Ki67
p<0.05
p<0.01
N.S.
p<0.01
30
All samples
No-aneurysm
Aneurysm
Dissection
20
10
ApoE -/Opg +/+
ApoE -/Opg -/-
F4/80
DAPI
...