A validated mouse model capable of recapitulating the protective effects of female sex hormones on ascending aortic aneurysms and dissections (AADs)

Figures

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  FIGURE 1. The combined challenge of BAPN and AngII induces acute aortic rupture and AAD formation in adult normolipidemic mice. (a) Weighted log rank survival analysis. Reduction of AngII from 1,000 ng kg−1 min−1 (n = 10) to 500 ng kg−1 min−1 (n = 10) postponed aortic rupture (p = .029). (b) Dilation of the AADs evaluated with ultrasound imaging. Reduction of AngII dosage to 500 ng did not slow down aortic dilation (p = .897, two‐way repeated‐measures ANOVA). (c) Gross specimens collected on d28. Segments below the level of the diaphragm were cropped off. Blue color in the saline control group resulted from the Evans blue administered prior to gross evaluation. Ruler scale: 1.0 mm. Note the similar enlargement of the ascending aorta and the aortic arch between groups subjected to low and high AngII dosages

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  FIGURE 2. BAPN and AngII act synergistically to promote acute intimal/medial tears in the ascending aorta. Specimens were collected 3 days after AngII‐infusion. (a) En face view of the luminal surface of ATAs challenged with the indicated substances. Areas with extravasation of Evans Blue appeared in blue (black arrows). Note the extravasation of Evans Blue in the ATA challenged with BAPN only and a long intimal/medial tear (white arrow) in the ATA challenged with BAPN + AngII. Scaler bars: 1.0 mm. (b) Fractional area positive for Evans Blue staining (n = 3 per treatment). Data were analyzed using one‐way ANOVA. (c) Microscopic evaluation of cross‐sections of the ATAs. Left panel: bright field microscopy. An intimal/medial tear (black arrow) was located at the center of the area where elastic fibers were stained in blue. Right panel: fluorescence microscopy. Red: elastic fibers stained by Evans blue (Evans blue emits red fluorescence); green: autofluorescence. Note the co‐localization of Evans Blue extravasation with breaks of the intimal layer and elastic fibers (white arrow). Scale bars: 100 µm

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  FIGURE 3. AADs induced by BAPN + AngII continue to expand in the absence of AngII. (a) Dilation of the AADs (n = 7–12) before and after discontinuation of AngII‐infusion (‐AngII). Osmotic pumps loaded with AngII or saline control were removed on d28 (black arrow). Note that only the AADs of the group initially challenged with both BAPN and AngII expanded persistently after cessation of AngII‐infusion. *p = .429, AngII versus BAPN; **p = .017, d56 versus d28 for the BAPN + AngII group. Data were analyzed using two‐way repeated‐measures ANOVA. (b) Dilation of the established AADs in the absence of AngII‐stimulation. Data were expressed as percent increase of the AAD diameter in the last 4 weeks and analyzed using one‐way ANOVA. (c) Gross specimens collected on d56. Aortic segments below the level of diaphragm were cropped off. Ruler scale: 1.0 mm. Note the severe dilation of ATAs and presence of intramural hematoma in the descending aorta of the BAPN + AngII group

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  FIGURE 4. AADs induced by BAPN + AngII advance the aneurysmal degeneration through a sequence of pathologies. All specimens were stained with Movat's staining except those in E (Prussian blue staining) and F (immunofluorescence staining for α‐actin). (a and b) intimal/medial tears (a, arrows) and intramural hematoma (b, asterisks) presented by AADs at d3. (c and d) Full thickness intimal/medial tears (c) and penetrating aortic ulcers (PAUs) presented by AADs at d14. (e and f) Resolved hematoma (e, arrows) and widening of interlaminar space by hyperplastic SMCs (f) detected on d14 AADs. (g and h) PAUs (g) and medial diminishment (h, arrows) exhibited by d28 AADs. Areas with and without medial diminishment are shown with images placed in the mid and right, respectively, in the panel H. Scale bars: 100 µm

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  FIGURE 5. The challenge with BAPN + AngII induces the massive influx of macrophage, neutrophil, and B‐cell to AADs. (a) Immunofluorescence staining of markers specific for macrophage (CD68), B‐cell (CD19), neutrophil (Ly6b.2), and T‐cell (CD3). Specimens were collected 3 days after AngII‐fusion (d3). (b and c) Immunofluorescence labeling of the indicated inflammatory infiltrates in AADs collected on d14 (b) and d28 (c). (d) A representative image for negative controls in which the primary antibodies were replaced with normal rabbit IgG and rat isotype IgG2a. White dash lines depict the border between medial and adventitial layers. Adv., adventitia; AutoF, autofluorescence; M, media. Scale bars: 50 µm

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  FIGURE 6. The BAPN + AngII model is capable of recapitulating the protective effect of female sex on type A aortic dissections. (a) Survival of mice in the first 2 weeks of AAD induction. M: male mice; F: female mice. (b) Number of mice either dead from aortic rupture or alive by d28. (c) Dilation of the AADs evaluated by ultrasound imaging. Data were analyzed using two‐way repeated‐measures ANOVA. (d) Gross specimens collected on d28. Note the difference in the width of the AADs between male and female mice. Ruler scale: 1.0 mm. (e) Medial degeneration evaluated with Movat's staining (n = 4–5). Representative images of male and female AADs are provided in the left panel, with the degeneration score assigned to these AADs plotted in the right panel. Data were analyzed using unpaired t test. Scare bars: 100 µm

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  FIGURE 7. β‐estradiol (E2) attenuates the dilation of the AADs induced in OVXed female mice. (a) Survival of mice in 4 weeks. Note that aortic rupture in OVXed, E2‐treated mice did not occur until d14. (b) Dilation of the AADs estimated with ultrasound imaging. Data were analyzed using two‐way repeated‐measures ANOVA. (c) Changes in the AAD diameter over 4 weeks of AngII‐infusion. Data were analyzed using unpaired t test. (d) Representative images of AADs subjected to the indicated treatments. Scale bars: 100 µm. Note the difference in adventitial thickness between those AADs. (e) Adventitial thickening in the AADs treated with the indicated reagents (n = 8–9). Data were acquired analyzed using unpaired t test. Histologic data presented in E and F were obtained from AADs collected 28 days after AngII‐infusion