Therapeutic Effect of Rapamycin on Aortic Dissection in Mice.

Aortic dissection (AD) is a serious clinical condition that is unpredictable and frequently results in fatal outcome. Although rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), has been reported to be effective in preventing aortopathies in mouse models, its mode of action has yet to be clarified. A mouse AD model that was created by the simultaneous administration of β-aminopropionitrile (BAPN) and angiotensin II (AngII) for 14 days. Rapamycin treatment was started either at day 1 or at day 7 of BAPN+AngII challenge, and continued throughout the observational period. Rapamycin was effective both in preventing AD development and in suppressing AD progression. On the other hand, gefitinib, an inhibitor of growth factor signaling, did not show such a beneficial effect, even though both rapamycin and gefitinib suppressed cell cycle activation in AD. Rapamycin suppressed cell cycle-related genes and induced muscle development-related genes in an AD-related gene expression network without a major impact on inflammation-related genes. Rapamycin augmented the activation of Akt1, Akt2, and Stat3, and maintained the contractile phenotype of aortic smooth muscle cells. These findings indicate that rapamycin was effective both in preventing the development and in suppressing the progression of AD, indicating the importance of the mTOR pathway in AD pathogenesis.

Supplemental Figure S2. Rapamycin treatment was performed either in the preventive intervention or in the therapeutic intervention. In the preventive intervention, intraperitoneal administration of rapamycin or vehicle (DMSO) was started on the day of the implantation of BAPN and AngII pumps, and continued throughout the 14-day observational period. In the therapeutic intervention, both the vehicle group and the rapamycin group received vehicle on the day of BAPN pump and AngII pump implantation and continued to day 7, when rapamycin administration was started, and continued for the rest of the observational period.
Supplemental Figure S5. The diagrams depict, by double arrows, the pair-wise comparisons in three transcriptome data sets; GSE116434 [21], GSE147078 [8], and GSE138558. All of the samples were thoracic aortas of the same AD model as the current study, with different experimental interventions. GSE116434: normal vs. high salt challenge and wild type vs. Il17a deletion. GSE147078: wild type vs. smooth muscle-specific Socs3 deletion. GSE138484: vehicle vs. rapamycin. Each experimental group contained three biological replicates (mice), and in total 48 aortic samples were used to construct the gene expression network in the AD model. Twelve comparisons (C1-C12) for GSE116434, and 4 comparisons (C1-C4) each for GSE147078 and GSE138558 were made to extract the genes with significant changes in expression.
Supplemental Figure S6. Volcano plots for the comparisons in GSE116434 [21], GSE147078 [8], and GSE138558, as depicted in Supplemental Figure S3. The changes in gene expressions were considered significant when the fold changes were more than 4 (induced) or less than 0.25 (suppressed), and p values were less than 0.01. Induced genes and suppressed genes are indicated by red and blue shading, respectively.
Supplemental Figure S7. The heat map representation of hierarchical clustering of the 1221 ADrelated genes extracted from datasets GSE116434 [21], GSE147078 [8], and GSE138558. The sample names are color coded in red for GSE116434, blue for GSE147078, and green for GSE138558. These data were used for the gene network analysis in Figure 3.
Supplemental Figure S9. Representative immunofluorescence staining images corresponding to pStat3 staining in Figure 5A. Separate images for DAPI, pStat3, and SMA staining are shown in gray scale. Merged images are also shown in pseudo color: blue, green, and red for DAPI, pStat3, and SMA, respectively. Scale bar: 50 m.
Supplemental Figure S10. Representative immunofluorescence staining images corresponding to pAkt1 and pAkt2 staining in Figure 5A. Separate images for DAPI, pAkt1 or pAkt2, and SMA staining are shown in gray scale. Merged images are also shown in pseudo color: blue for DAPI, green for pAkt1 or pAkt2, and red for SMA. Scale bar: 50 m.