Therapeutic Effect of Rapamycin on Aortic Dissection in Mice

Abstract

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.

Keywords: aortic dissection; inflammation; mTOR; rapamycin; smooth muscle cells.

Figure 1

Effect of cell cycle inhibitors on aortic dissection (AD). (A,B) Effects of rapamycin and gefitinib on the expression of cyclin D3 (A) and macroscopic findings (B) in a murine model of AD. Scale bar: 5 mm. (C,D) Quantitative analysis of the effect of rapamycin on AD lesion length (C), p70 S6 kinase (S6K), activated (phosphorylated) S6K (pS6K), and cyclin D3 (D). The figures in parentheses indicate the numbers of independent observations. * p < 0.05, ** p < 0.01, *** p < 0.001. Among the multiple experimental groups, all comparisons between two groups were tested and for simplicity only those with statistically significant differences are indicated.

Figure 2

Effect of rapamycin in aortic tissue. Immunofluorescence staining of the aortic tissue for Ki67 and SMA (A) or CD45 (B) with nuclear 4’,6-diamidino-2-phenylindole (DAPI) staining, before (Pre) or after 3 days of BAPN + AngII challenge with vehicle (BA+DMSO) or rapamycin (BA+Rapa) treatment. Representative immunofluorescence images (upper panels) and scattergrams of imaging cytometry (bottom panels) are shown. The percentages of cell populations are indicated in the scattergrams of the quadrants as arbitrarily determined by the thresholds for the signal intensities of Ki67 (A,B), SMA (A) and CD45 (B). The thresholds are set constant for Pre, BA+DMSO, and BA+Rapa in a given double staining. Scale bars: 50 µm.

Figure 3

Gene expression network in AD. Organic plot of the Bayesian network analysis for gene expression. Each node represents a single gene that is color coded for induction (red) or suppression (green) by rapamycin in the presence of BAPN + AngII challenge. The genes are connected by red or blue edges that represent positive or negative correlations, respectively, between a pair of genes on both sides of the edge. The higher the correlations are, the closer the genes are plotted. Three clusters of the nodes represent the groups of genes making tightly coupled subnetworks (#1, #2, and #3) for their expressions.

Figure 4

Effect of rapamycin on cellular signaling in AD. Effect of rapamycin on protein expression in the AD model with or without 3 days of BAPN + AngII challenge. Mice were treated with vehicle (DMSO) or rapamycin without or during the BAPN + AngII challenge (BA). Representative images for Western blotting (A) and quantitative analysis of the selected proteins (B) are shown. The figures in parentheses indicate the numbers of independent observations. * p < 0.05, ** p < 0.01, *** p < 0.001. Among the multiple experimental groups, all comparisons between two groups were tested and for simplicity only those with statistically significant differences are indicated.

Figure 5

Effect of rapamycin on cellular signaling in SMCs. Effect of 3 days of BAPN + AngII (BA) and rapamycin on pStat3, pAkt1, and pAkt2 in aortic tissue (A) or in SMCs in culture (B, C). (A) Representative immunofluorescence images of triple staining for SMA (red), nuclear DAPI (blue) staining, and pStat3, pAkt1, or pAkt2 (green). Scale bar: 50 µm. (B) Representative images of Western blotting for the indicated proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal loading control. (C) Quantitative analysis of the indicated proteins. The figures in parentheses indicate the numbers of independent observations. * p < 0.05, ** p < 0.01, *** p < 0.001. Among the multiple experimental groups, all comparisons between two groups were tested and for simplicity only those with statistically significant differences are indicated.

Figure 6

Therapeutic effect of rapamycin on AD. (A) Representative macroscopic findings at the indicated periods of BAPN + AngII infusion (BA) with vehicle (DMSO) or rapamycin (Rapa). Scale bar: 5 mm. (B) Quantitative analysis of the AD lesion length in the indicated experimental groups. The figures in parentheses indicate the numbers of independent observations. * p < 0.05, ** p < 0.01, *** p < 0.001.