FIGURE LEGENDS
Figure 1. CCN2 deficiency decreases mice survival
following angiotensin II (Ang II) administration as a result of aortic
aneurysm development and rupture. Wild-type (WT) and CCN2-KO mice were
infused with Ang II for 15 days. Survival, aneurysm generation and blood
pressure were analyzed. A , Kaplan-Meier survival curve showed a
dramatic increase in mortality in Ang II-infused CCN2-KO mice.B, representative images of clean whole aorta showing
thoracoabdominal aneurysms (TAAAs) generation in CCN2-KO mice infused
with Ang II at end of follow-up. C, Evaluation of aneurysm
appearance. Larger TAAA formation was found in 92 % of Ang II-infused
CCN2-KO mice and smaller abdominal aneurysm (AbA) was observed in 30%
and 8% of CCN2-KO and WT + Ang II mice respectively. D,Time-course of blood pressure changes in all mice groups since the first
tamoxifen injection (Day -21) showed that Ang II increased blood
pressure independently of the presence or absence of CCN2, as well as
CCN2 deficiency decreased normal systolic blood pressure. Data are
showed as box-and-whisker plots, with 75th and 25th percentiles; bars
represent maximal and minimal values. n=8-13 mice per group.
Figure 2. CCN2 deficiency induces early aneurysm
formation in mice in response to Ang II administration. Daily magnetic
resonance imaging (MRI) assessments allowed a time-course analysis
showing early aneurysm formation in Ang II-infused CCN2-KO mice. MRI
experiments were followed only until 96 hours to avoid excessive
mortality. Studies were done in a 1 cm aortic section proximal to the
superior mesenteric artery (SMA) origin. A, Representative
axial MRI images showing an increase in the total aortic area and a
reduction in the minimal aortic lumen 48 hours after Ang II
administration in CCN2-KO mice. Time-course quantification of Btotal aortic area (please note the
different scale for the 96h panel), C lumen area, Dmaximum total area and E minimum lumen area of the 1cm SMA
analyzed. n= 4 mice per group. Data are shown as mean ± SEM. * p
< 0.05 increased vs . WT; # p < 0.05
decreased vs . WT.
Figure 3. CCN2 deficiency predisposes to TAAA formation
in response to Ang II administration in mice. Ultrasound scanning was
performed in live WT and CCN2-KO mice infused or not with Ang II, and
evaluated at the start and the end point (0 and 15 days) of Ang II
administration. A, representative ultrasound images.B, measurements of maximal diameters of TAsA, TDsA and AbA from
all mice groups. Red dashed lines indicate the lumen boundary, and the
yellow dashed lines indicate the lumen diameter. n = 5 mice per group.
Data are showed as box-and-whisker plots, with 75th and 25th
percentiles; bars represent maximal and minimal values.
Figure 4. CCN2 deficiency promotes aortic structural and
composition changes which are exacerbated in response to Ang II
administration. A, Representative images of the aortic structure
evaluated by Hematoxilin/Eosin (HE) at 10X and their magnification at
40X. *Considering aneurysms size, a lower magnification (4X) image was
included in the CCN2-KO + Ang II group. Aneurysms were characterized by
elastic lamina rupture and aortic wall dissection with extravasation of
red blood cells outside the muscle layer forming a neo-lumen.An inflammatory cell infiltration in the border of the dissected aortic
wall and reduced cellularity of the muscular layer were also observed in
aneurysms. Figures show a representative picture of 6-8 mice per group.B, Representative images of aortic Van Gieson staining showing
internal elastic lamina disrupted zones in CCN2-KO mice with or without
Ang II infusion (Black arrows). C , Representative images of
transmission electron microscopy (TEM) (2000X) confirming aortic elastic
layers’ disruption in absence of CCN2, boxed in red and magnified at
15000X below. EL= Elastic layer. Scale bars: 100 μm at 10X objective and
20 μm at 40X in histology; 10 μm at 2000X and 1 μm at 15000X in MET.
Figure 5. CCN2 deletion increases vascular
matrix metalloproteinases (MMPs). MMPs activities or levels were
evaluated in total aortic protein lysates. A, Representative
MMP-2 and -9 gel zymography (upper panel) and quantification (lower
panel) showing elevated MMP activities in the CCN2-KO group and
increased activity following Ang II infusion. B . Representative
image (upper panel) and quantification (lower panel) of MMP-8 western
blot showing increased MMP-8 protein expression in total aortic protein
extracts from the CCN2-KO group both treated or not with Ang II.C. In situ MMP activity was evaluated in
paraffin-preserved aortas by the DQ-Gelatin fluorogenic substrate assay.
Aortas of CCN2-KO and Ang II-infused WT mice displayed similar
fluorescence intensity (green signal, observed between elastic layers),
whereas there was a broadly distributed green signal in CCN2-KO + Ang II
mice. n= 5-10 mice per group. Data are shown as mean ± SEM. * p
< 0.05 increased vs . WT; # p < 0.05
decreased vs . WT. † p <0.05 vs . CCN2-KO. $ p
<0.05 vs. CCN2-KO + Ang II.
Figure 6. Aorta transcriptomic study. A, Heat map
diagram showing the two-way hierarchical clustering of genes for each
group of mice. Each column represents the pooled mRNA obtained from 8-10
aortas per group. B, Heat map diagram showing the two-way
hierarchical clustering of genes comparing just CCN2-KO mice vs.WT.
Figure 7 . Mineralocorticoid receptor blockade by
spironolactone improves survival, aneurysm appearance rates and
ameliorates aortic vascular function changes observed in Ang II-infused
CCN2-KO mice. Spironolactone (SP) was started at the time of CCN2
deletion in the CCN2-KO + Ang II group. SP (50mg/kg/day) was
intraperitoneally injected in alternate days until the end of follow-up.A, Kaplan-Meier survival curve showed less mortality in the
CCN2-KO + Ang II group treated with SP compared to no SP treatment.B, SP decreased the percentage of aneurysm appearance in Ang
II-infused CCN2-KO mice. C, SP did not prevent Ang II-induced
blood pressure levels elevation. Data are showed as box-and-whisker
plots, with 75th and 25th percentiles; bars represent maximal and
minimal values. n=8-10 mice per group. D , Representative MMP-2
and 9 gel zymography (upper panel) and quantification (lower panel)
showing a mild not significant MMP2 and MMP9 activity reduction in the
CCN2-KO + Ang II group treated with SP. n= 7-10 mice per group.