β-Aminopropionitrile Induces Distinct Pathologies in the Ascending and Descending Thoracic Aortic Regions of Mice

Abstract

Background: β-aminopropionitrile (BAPN) is a pharmacological inhibitor of LOX (lysyl oxidase) and LOXLs (LOX-like proteins). Administration of BAPN promotes aortopathies, although there is a paucity of data on experimental conditions to generate pathology. The objective of this study was to define experimental parameters and determine whether equivalent or variable aortopathies were generated throughout the aortic tree during BAPN administration in mice.

Methods: BAPN was administered in drinking water for a period ranging from 1 to 12 weeks. The impacts of BAPN were first assessed with regard to BAPN dose, and mouse strain, age, and sex. BAPN-induced aortic pathological characterization was conducted using histology and immunostaining. To investigate the mechanistic basis of regional heterogeneity, the ascending and descending thoracic aortas were harvested after 1 week of BAPN administration before the appearance of overt pathology.

Results: BAPN-induced aortic rupture predominantly occurred or originated in the descending thoracic aorta in young C57BL/6J or N mice. No apparent differences were found between male and female mice. For mice surviving 12 weeks of BAPN administration, profound dilatation was consistently observed in the ascending region, while there were more heterogeneous changes in the descending thoracic region. Pathological features were distinct between the ascending and descending thoracic regions. Aortic pathology in the ascending region was characterized by luminal dilatation and elastic fiber disruption throughout the media. The descending thoracic region frequently had dissections with false lumen formation, collagen deposition, and remodeling of the wall surrounding the false lumen. Cells surrounding the false lumen were predominantly positive for α-SMA (α-smooth muscle actin). One week of BAPN administration compromised contractile properties in both regions equivalently, and RNA sequencing did not show obvious differences between the 2 aortic regions in smooth muscle cell markers, cell proliferation markers, and extracellular components.

Conclusions: BAPN-induced pathologies show distinct, heterogeneous features within and between ascending and descending aortic regions in mice.

Keywords: aorta; aorta, thoracic; aortic aneurysm; aortic dissection; protein-lysine 6-oxidase.

Figure 1.. Survival curves of BAPN-induced aortic rupture.

Survival curves of (A) male C57BL/6J mice administered BAPN 0.1%, 0.3%, or 0.5% wt/vol (N=10/concentration) for 12 weeks, (B) five mouse strains administered BAPN 0.5% (N=19–21/mouse strain, male), (C) male C57BL/6J mice started BAPN 0.5% at 3 (N=38), 4 (N=15), or 26 (N=15) weeks of age, and (D) male (N=24) and female (N=23) C57BL/6J mice administered BAPN 0.5%. P values were determined by Log-Rank analysis. P=0.008 for BAPN 0.1% vs 0.3% and P<0.001 for BAPN 0.1% vs 0.5% (A); P values for (B) are detailed in Table S1; P=0.003 for 4- and 26-week comparison, and P<0.001 for 3- vs 4-week comparison and 3- vs 26-week comparison (C). BAPN administration was started at 3 weeks of age in (A) and (B), and 4-weeks of age in (D).

Figure 2.. Locations of BAPN-induced aortic rupture in young C57BL/6J mice.

BAPN (0.5% wt/vol) was administered in drinking water for 12 weeks. Necropsy was performed for mice that died during BAPN administration within 12 weeks. (A) Examples of ex vivo images showing ascending or descending aortic rupture. (B) Death rate attributed to aortic rupture in the ascending versus descending aortic regions of male and female mice. Necropsy was performed on 78 male mice and 21 female mice. Incidence based on aortic rupture location was analyzed by Chi-square in male mice (P<0.001), and by Fisher-exact test in female mice (P<0.001).

Figure 3.. BAPN induced dilatation of the ascending and descending thoracic regions in young mice.

Representative ultrasound images and luminal diameters of ascending aortas in young (4-week-old) C57BL/6J mice administered vehicle (drinking water only) versus BAPN (A and B) and in young (4-week-old) versus mature (26-week-old) mice administered BAPN (C and D). BAPN versus vehicle after 12 weeks of BAPN administration in male and female mice: P<0.001 in both sexes, and the difference in changes over time between male and female mice of BAPN administration: P>0.99. (B). Young versus Mature mice within males and females: P<0.001 and P=0.015, respectively, and the difference in changes over time between young male and female mice: P=0.16. (D). In situ measurements of maximal diameters of ascending/arch regions (E) and descending thoracic regions (F) in 4-week-old C57BL/6J mice administered vehicle versus BAPN for 12 weeks. In situ measurements of maximal diameters of ascending/arch regions (G) and descending thoracic regions (H) in 4-week-old (Young) versus 26-week-old (Mature) C57BL/6J mice administered BAPN for 12 weeks. P=0.56 between male and female mice administered BAPN (E), P=0.096 between male and female mice administered BAPN (F), P=0.93 between male and female young mice administered BAPN (G), and P=0.086 between male and female young mice administered BAPN (H). P values in B and D were determined using the mixed effects model with post hoc contrast tests. Data in E-H were analyzed using two-way ANOVA with post hoc contrast tests within each sex.

Figure 4.. Temporal characteristics of BAPN-induced thoracic aortopathies in young male C57BL/6J mice.

BAPN was administered in drinking water for 1, 2, 3, or 4 weeks in 3–4-week-old male C57BL/6J mice. (A) Examples of in situ images of thoracic aortas. (B) Hematoxylin-eosin (H&E) and Verhoff staining images of ascending aortic dissection and descending thoracic dissection. Scale bar is 200 μm.

Figure 5.. Chronic administration of BAPN led to profound vascular remodeling following dissection in the descending thoracic region.

Representative images of hematoxylin-eosin (H&E), Verhoeff iron hematoxylin, Movat’s pentachrome, α-SMA (smooth muscle actin), and CD68 staining in ascending and descending aortas of mice with BAPN administration for 12 weeks. The approximate location of the tissue section from the ascending and descending region is indicated by the blue line. * indicates false lumen. Scale bar is 200 μm.

Figure 6.. Transcriptomic alteration by BAPN administration in the ascending and descending thoracic aortas.

(A) Principle component analysis for unfiltered transcriptomes of ascending and proximal descending thoracic aortas harvested from 4-week-old male C57BL/6J mice administered either vehicle or BAPN for 7 days. (B) Heatmap depicting z-scored normalized read counts of differentially expressed genes (DEGs) from the interaction analysis between sexes and administration in two-way ANOVA. (C) Bar plots for DEGs related to proliferation, smooth muscle cell contraction, the extracellular maturation (ECM). N=5 biological replicates per group. (D) Western blot and (E) gelatin zymography and their quantifications. N=4–5/group and P values were calculated by Kruskal-Wallis test (pSMAD2) or two-way ANOVA followed by Holm-Sidak method (pERK and MMP2).