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.