Testosterone Metabolite 6β-Hydroxytestosterone Contributes to Angiotensin II-Induced Abdominal Aortic Aneurysms in Apoe-/- Male Mice

Abstract

Background Sex is a prominent risk factor for abdominal aortic aneurysms (AAAs), and angiotensin II (Ang II) induces AAA formation to a greater degree in male than in female mice. We previously reported that cytochrome P450 1B1 contributes to the development of hypertension, as well as AAAs, in male mice. We also found that a cytochrome P450 1B1-generated metabolite of testosterone, 6β-hydroxytestosterone (6β-OHT), contributes to Ang II-induced hypertension and associated cardiovascular and renal pathogenesis in male mice. The current study was conducted to determine the contribution of 6β-OHT to Ang II-induced AAA development in Apoe^-/- male mice. Methods and Results Intact or castrated Apoe^-/-/Cyp1b1^+/+ and Apoe^-/-/Cyp1b1^-/- male mice were infused with Ang II or its vehicle for 28 days, and administered 6β-OHT every third day for the duration of the experiment. Abdominal aortas were then evaluated for development of AAAs. We observed a significant increase in the incidence and severity of AAAs in intact Ang II-infused Apoe^-/-/Cyp1b1^+/+ mice, compared with vehicle-treated mice, which were minimized in castrated Apoe^-/-/Cyp1b1^+/+ and intact Apoe^-/-/Cyp1b1^-/- mice infused with Ang II. Treatment with 6β-OHT significantly restored the incidence and severity of AAAs in Ang II-infused castrated Apoe^-/-/Cyp1b1^+/+ and intact Apoe^-/-/Cyp1b1^-/- mice. However, administration of testosterone failed to increase AAA incidence and severity in Ang II-infused intact Apoe^-/-/Cyp1b1^-/- mice. Conclusions Our results indicate that the testosterone-cytochrome P450 1B1-generated metabolite 6β-OHT contributes to Ang II-induced AAA development in Apoe^-/- male mice.

Keywords: 6β‐hydroxytestosterone; CYP1B1; abdominal aortic aneurysm; angiotensin II; castration.

Figure 1. 6β‐OHT treatment restores Ang II‐induced AAA incidence and severity in castrated Apoe^–/– mice.

Representative images of aorta (A); maximal aortic diameter (B: mean [left]; maximum–minimum [right]); incidence of AAAs (C) and severity of AAAs (D) in intact or castrated Apoe^–/–/Cyp1b1^+/+ mice infused with Ang II or its vehicle (saline) for 28 days and administered 6β‐OHT or its vehicle DMSO; n=5 for Vehicle group, n=12 for Ang II group, n=5 for 6β‐OHT group, n=10 for Ang II+6β‐OHT group, n=12 for Cas+Ang II group, n=14 for Cas+Ang II+6β‐OHT group. Data are expressed as mean±SEM and analyzed using 1‐way ANOVA, with Tukey's multiple comparisons test (for aortic diameter) and Fisher exact test (for AAA incidence); *P<0.05; **P<0.01. AAA indicates abdominal aortic aneurysm; Ang II, angiotensin II; Cas, castrated; Cyp1b1, cytochrome P450 1b1; DMSO, dimethyl sulfoxide; and 6β‐OHT, 6β‐hydroxytestosterone.

Figure 2. 6β‐OHT treatment restores Ang II‐induced AAA incidence and severity in Apoe^–/–/Cyp1b1^–/– mice.

Representative images of aorta (A); maximal aortic diameter (B: mean [left]; maximum‐minimum [right]); incidence of AAAs (C) and severity of AAAs (D) in Apoe^–/–/Cyp1b1^–/– mice infused with Ang II or its vehicle (saline) for 28 days and administered 6β‐OHT or testosterone or their vehicle DMSO; n=10 for Vehicle group, n=9 for Ang II group, n=5 for 6β‐OHT group, n=16 for Ang II+6β‐OHT group, n=4 for Testosterone group, n=10 for Ang II+Testosterone group. Data are expressed as mean±SEM and analyzed using 1‐way ANOVA, with Tukey's multiple comparisons test (for aortic diameter) and Fisher exact test (for AAA incidence); *P<0.05; **P<0.01; ****P<0.0001. AAA indicates abdominal aortic aneurysm; Ang II, angiotensin II; Cyp1b1, cytochrome P450 1b1; DMSO, dimethyl sulfoxide; and 6β‐OHT, 6β‐hydroxytestosterone.

Figure 3. Castration or Cyp1b1 gene disruption attenuates Ang II‐induced histopathological features of AAAs in Apoe^–/– mice, which is restored by 6β‐OHT administration.

Representative images of H&E staining (A), VVG staining of elastin fibers (B), α‐actin expression (brown) (C; arrows indicate attenuated α‐actin expression in medial layers adjacent to the aneurysmal lesion), and Masson's trichrome staining of collagen (collagen: blue; smooth muscle: red) (D) in abdominal aortas of Apoe^–/–/Cyp1b1^+/+ and Apoe^–/–/Cyp1b1^–/– mice. Scale bars represent 200 µm (upper panel), 20 µm (lower panel) for H&E, elastin and α‐actin, and 50 µm for collagen. Quantitation of elastin degradation grade (E), smooth muscle α‐actin (SMA) intensity (F) and aortic collagen (G) in Apoe^–/–/Cyp1b1^+/+ and Apoe^–/–/Cyp1b1^–/– mice. n=3 to 4 in each group. Data are expressed as mean±SEM and analyzed using 1‐way ANOVA with Tukey's multiple comparison test; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. AAA indicates abdominal aortic aneurysm; Ang II, angiotensin II; Cas, castrated; Cyp1b1, cytochrome P450 1b1; H&E, hematoxylin and eosin; 6β‐OHT, 6β‐hydroxytestosterone; and VVG, Verhoeff–Van Gieson.

Figure 4. Castration or Cyp1b1 gene disruption prevents inflammatory cell infiltration in Ang II‐induced AAAs in Apoe^–/– mice, which is restored by 6β‐OHT administration.

Representative images of infiltration of CD68⁺ macrophages (red) and nuclear staining with 4′,6‐diamidino‐2‐phenylindole (blue) and quantitative analysis of total number of CD68⁺ macrophages per high‐power field (hpf) in abdominal aortas of (A) Apoe^–/–/Cyp1b1^+/+ and (B) Apoe^–/–/Cyp1b1^–/– mice. Arrows indicate CD68⁺ macrophages. Scale bars represent 20 µm. n=3 in each group. Data are expressed as mean±SEM and analyzed using 1‐way ANOVA with Tukey's multiple comparison test; *P<0.05; **P<0.01. Ang II indicates angiotensin II; Cas, castrated; Cyp1b1, cytochrome P450 1b1; and 6β‐OHT, 6β‐hydroxytestosterone.

Figure 5. Castration or Cyp1b1 gene disruption attenuates Ang II‐induced increase in systolic blood pressure in Apoe^–/– mice, which is restored by 6β‐OHT administration.

Systolic blood pressure during Ang II infusion in Apoe^–/–/Cyp1b1^+/+ (A) and Apoe^–/–/Cyp1b1^–/– (B) mice; (A) ****P<0.0001, Vehicle vs Ang II; ^# P<0.05 Ang II vs Cas+Ang II; ^† P<0.05; ^†† P<0.01, Cas+Ang II vs Cas+Ang II+6β‐OHT; (B) *P<0.05, Vehicle vs Ang II; ^†††† P<0.0001 Ang II vs Ang II+6β‐OHT. For Apoe^–/–/Cyp1b1^+/+ mice: n=4 for Vehicle group, n=3 for Ang II group, n=5 for Cas+Ang II group, n=5 for Cas+Ang II+6β‐OHT group; for Apoe^–/–/Cyp1b1^–/– mice: n=4 for Vehicle group, n=5 for Ang II group, n=8 for Ang II+6β‐OHT group. Data are expressed as mean±SEM and analyzed using repeated‐measures 2‐way ANOVA, with Tukey's multiple comparisons test. Ang II indicates angiotensin II; Cas, castrated; Cyp1b1, cytochrome P450 1b1; and 6β‐OHT, 6β‐hydroxytestosterone.