Female Mice With an XY Sex Chromosome Complement Develop Severe Angiotensin II-Induced Abdominal Aortic Aneurysms

Abstract

Background: Abdominal aortic aneurysms (AAAs) are a deadly pathology with strong sexual dimorphism. Similar to humans, female mice exhibit far lower incidences of angiotensin II-induced AAAs than males. In addition to sex hormones, the X and Y sex chromosomes, and their unique complements of genes, may contribute to sexually dimorphic AAA pathology. Here, we defined the effect of female (XX) versus male (XY) sex chromosome complement on angiotensin II-induced AAA formation and rupture in phenotypically female mice.

Methods: Female low-density lipoprotein receptor (Ldlr) deficient mice with an XX or XY sex chromosome complement were infused with angiotensin II for 28 days to induce AAAs. Abdominal aortic lumen diameters were quantified by ultrasound, whereas AAA diameters were quantified at study end point. DNA microarrays were performed on abdominal aortas. To mimic males, female mice were administered a single dose of testosterone as neonates or as adults before angiotensin II infusions.

Results: Female Ldlr^-/- deficient mice with an XX and XY sex chromosome complement had similar sex organ weights and low serum testosterone concentrations. Abdominal aortas from female XY mice selectively expressed Y chromosome genes, whereas genes known to escape X inactivation were higher in XX females. The majority of aortic gene differences in XY versus XX females fell within inflammatory pathways. AAA incidences doubled and aneurysms ruptured in XY females. AAAs from XY females exhibited inflammation, and plasma interleukin-1β concentrations were increased in XY females. Moreover, aortas from XY females had augmented matrix metalloproteinase activity and increased oxidative stress. Last, testosterone exposure applied chronically, or as a single bolus at postnatal day 1, markedly worsened AAA outcomes in XY in comparison with XX adult females.

Conclusions: An XY sex chromosome complement in phenotypic females profoundly influenced aortic gene expression profiles and promoted AAA severity. When XY females were exposed to testosterone, aneurysm rupture rates were striking. Mechanisms for augmented AAA severity in XY females include increased inflammation, augmented matrix metalloproteineases, and oxidative stress. Our results demonstrate that genes on the sex chromosomes regulate aortic vascular biology and contribute to sexual dimorphism of AAAs. Sex chromosome genes may serve as novel targets for sex-specific AAA therapeutics.

Keywords: angiotensins; aortic aneurysm, abdominal; sex chromosomes; testosterone.

Figure 1

Sex chromosome complement influences abdominal aortic gene expression patterns. (A) Left, total number of probe sets on arrays filtered to retain transcripts with reliable signal intensity (FDR; False Discovery Rate). Right, number of genes whose expression level was significantly altered by main effect of genotype (XX, XY), surgery (ovariectomy, OVX, sham surgery) and interaction between genotype and surgery. (B), Volcano plot illustrating fold change in gene expression (x-axis) and statistical significance (y-axis). Genes labelled in blue exhibited significant increase in XY compared to XX abdominal aortas; genes labelled in red exhibited significant increase in XX aortas. (C), RT-PCR of selected abdominal aortic genes illustrated in (B). A,B: Filtered array data analyzed by 2-way ANOVA. C, 2-way ANOVA; data are mean ± SEM from n = 5 mice/surgery group/genotype. *, P<0.05 compared to XX within surgical group. **, P<0.05 compared to sham within genotype.

Figure 2

An XY sex chromosome complement markedly promotes formation and severity of AngII-induced AAAs in female mice. (A), Internal abdominal aortic lumen diameters over time of AngII infusions from mice of each genotype (OVX groups; n = 10–17 mice/genotype at study onset) that exhibited a 50% increase in lumen diameter over baseline, and that survived the 28 day infusion protocol. (B), Maximal external AAA diameters after 28 days of AngII infusion mice with an AAA (from A). Symbols represent individual mice with mean ± SEM as horizontal lines. (C), AAA incidence (which includes aneurysm rupture). The number of mice with an AAA/total number of mice in each group is illustrated above each bar of the histogram. (D), % ruptured AAAs. (E) Aortas from mice of each genotype. Data were analyzed by (A) two-way repeated measures ANOVA, (B) unpaired t-test (C) and (D) Fisher’s exact test. *, P<0.05 compared to XX within surgical group.

Figure 3

IL1β expression in abdominal aortas (A), bone marrow-derived macrophages (BMM, B) and plasma (C) of XX and XY Ldlr^−/− females. For A, IL1β mRNA abundance was quantified by RT-PCR in abdominal aortas from XX and XY Ldlr^−/− ovariectomized females. For B, cells were harvested from bone marrow of XX and XY Ldlr^−/− females and differentiated in vitro to macrophages. IL1β concentrations were quantified in media of BMM cells of each genotype incubated with LPS. For C, plasma IL1β concentrations were quantified from XX and XY Ldlr^−/− females infused with AngII for 28 days. Data were analyzed by Mann-Whitney test. Circles represent data from individual mice while mean ± SEM are illustrated by horizontal lines. *, P<0.05 compared to XX.

Figure 4

Aortas from XY females exhibited augmented MMP2 activity (A,B) and dihydroethidium (DHE) fluorescence (C,D). A, Zymograms of aortic extracts from XX and XY Ldlr^−/− females infused with AngII for 1 day. B, MMP2 (64 kDa) activity quantified by densitometric scans of zymograms (n = 4/genotype). C, Representative images of DHE stained aortic explants from XX and XY Ldlr^−/− females incubated with vehicle (V) or AngII+testosterone (10 μM and 10 nM, respectively; T=testosterone). D, Quantification of DHE staining in aortic explant sections from XX and XY females. *, P<0.05 compared to XX (B, or within treatment group in D). **, P<0.05 compared to vehicle.

Figure 5

Severe AAA rupture in XY females administered dihydrotestosterone (DHT). Adult female XX (n=18) and XY mice (n=11) were administered DHT (0.16 mg/d) for 2 weeks prior to and throughout AngII infusions. (A) % AAA rupture in XX and XY females. (B) Maximal AAA diameters at study endpoint. Symbols represent individual mice with horizontal lines representing mean ± SEM. Data analyzed by (A) Fisher’s exact test (B) Unpaired t-test. *, P<0.05 compared to XX+DHT.

Figure 6

Administration of testosterone (400 μg) to 1 day old neonatal female XY mice results in severe AAA rupture when adult XY females are infused with AngII. Females (n=6 XX, n=12 XY) were administered a single dose of testosterone within 24 hours of birth, and then infused with AngII at 2 months of age. Top, % AAA rupture. Bottom, representative aortas from mice of each group. Data were analyzed using Fisher’s exact test. *, P<0.05 compared to XX.