Castration of male mice prevents the progression of established angiotensin II-induced abdominal aortic aneurysms

Abstract

Objective

Male sex is a non-modifiable risk factor for abdominal aortic aneurysm (AAA) development. Similar to humans, male mice are more susceptible to angiotensin II (AngII)-induced AAAs than females. Previous studies demonstrated that castration of males markedly reduced the formation of AngII-induced AAAs. Progression of AAA size is associated with increased risk of aneurysm rupture. In this study, we hypothesized that castration of male mice would reduce the progression of established AngII-induced AAAs.

Methods

Male apolipoprotein E (ApoE)-/- mice were infused with AngII for 1 month to induce AAA formation. Aortic diameters were measured by ultrasound and mice were stratified into 2 groups that were either sham-operated or castrated. AngII infusions were continued for a further 2 months. Ultrasound was used to quantify lumen diameters, and excised aortas were processed for quantification of AAA size, volume, and tissue characteristics.

Results

Sham-operated mice exhibited progressive dilation of suprarenal aortic lumen diameters during continued AngII infusion. Castration significantly decreased aortic lumen diameters (study endpoint: 1.88 ± 0.05 mm vs 1.63 ± 0.04 mm; P<.05; sham-operated [n = 15] vs castration [n = 17], respectively). However, maximal external AAA diameters were not significantly different between sham-operated and castrated mice. The vascular volume/lumen volume ratio of excised AAAs imaged by ultrasound was significantly increased by castration (sham-operated, 4.8 ± 0.9; castration, 9.5 ± 2.0 %; n = 11/group; P<.05). Moreover, compared to thin walled AAAs of sham-operated mice, aneurysm sections from castrated mice exhibited increased smooth muscle -actin and collagen.

Conclusions

Removal of endogenous male hormones by castration selectively reduces aortic lumen expansion while not altering the external AAA dimensions.

Introduction

The natural history of AAA progression is gradual expansion of aortic diameters obtained by ultrasound with increased risk of rupture as AAA size increases. Ruptured AAAs are a leading cause of death in western countries and result in approximately 15,000 deaths per year in the United States.¹ Based on current clinical practice guidelines, the only effective therapeutic option to prevent AAA rupture is open repair or endovascular surgery if AAA size (by ultrasound) exceeds 5-5.5 cm.^2;3 To date, no pharmaceutical drugs have been proven to prevent the progression of AAA size and/or prevent ruptures of small AAAs.

The renin angiotensin system (RAS) has been demonstrated to be important in AAA pathogenesis in experimental models, and there is increasing evidence that it is a contributor to human AAA pathology^4;5. Chronic infusion of angiotensin II (AngII), the primary peptide of the RAS, induces AAA formation in hypercholesterolemic male mice⁶. Several studies demonstrate that male sex is one of the strongest non-modifiable risk factors for human AAAs.^7-9 Similar to humans, male mice exhibit a 4-fold higher prevalence of AngII-induced AAAs compared to age-matched females.¹⁰ Testosterone was demonstrated to be a primary mediator of sex differences in AngII-induced AAAs, as castration of male mice reduced AAA incidence to the level of age-matched females while administration of dihydrotestosterone restored AAA incidence in castrated males.¹¹

While several studies have addressed mechanisms contributing to the formation of AngII-induced AAAs, few studies have focused on mechanisms contributing to the progression of AAA pathology. Recent studies demonstrated that prolonged infusion of AngII to male apolipoprotein E (ApoE) deficient mice resulted in progressive aortic lumen dilation associated with increased macrophage accumulation in regions of medial disruption.¹² These results suggest that interventions introduced after an AAA is formed from AngII infusion could be used to develop novel therapeutic targets that may blunt AAA progression and/or rupture. While one study demonstrated that introduction of a JNK inhibitor caused regression of established AngII-induced AAAs¹³, administration of doxycycline had no effect on the progression of established AngII-induced AAAs.¹⁴ Unfortunately, few studies have demonstrated effective modes of preventing AAA progression in experimental models.

While it is clear that testosterone contributes to AAA formation in male mice, it is undefined whether male sex hormones contribute to previously observed progressive aortic lumen dilation and vascular remodeling of established AngII-induced AAAs.¹² In this study, we hypothesized that male sex hormones contribute to the progression of established AngII-induced AAAs. In male ApoE-/- mice with an established AAA from AngII infusion, we performed orchiectomy to remove effects of endogenous androgen and quantified AAA progression. Results demonstrate that male sex hormones markedly influence vascular wall remodeling and the progression of AAAs.

Methods

Animals

Male ApoE-/- mice were purchased from The Jackson Laboratory (2 months of age; n = 60; Bar Harbor, ME). All mice were maintained in a pathogen-free environment. Water and normal laboratory diet were available ad libitum. At 2 months of age, all mice were infused with AngII (1,000 ng/kg/min) for 1 month to induce AAA formation. On day 25 of AngII infusion, abdominal aortas of all mice were scanned by noninvasive high-frequency ultrasound to quantify aortic lumen diameter as an index of AAA formation. An AAA was defined as a >30% increase in lumen diameter compared to baseline (day 0 of infusion). Mice (n = 46) exhibiting an AAA were assigned to either sham-operated or castration groups (n = 23/group). Large and small AAAs, reflected by abdominal aortic lumen diameters, were evenly assigned to both sham-operated and castration groups to control for differences in AAA size as a mediator contributing to AAA progression. After castration surgery, mice in each group continued to be infused with AngII for an additional 2 months. Over 3 months of AngII infusion, aortic rupture occurred in n = 3 sham-operated mice and n = 1 castrated mice, decreasing the experimental number to n = 20 sham-operated, n = 22 castrated mice. We infused paraformaldehyde at physiological pressure (≈120 mmHg) in anesthetized mice (n = 12 mice/group) at study endpoint for histological analysis of AAAs. We chose mice with suprarenal aortic lumen diameters that were similar to the mean for each group for whole body perfusion fixation. Of the remaining mice, we froze aortas (n = 4 mice/group) for gene expression analysis, and placed one AAA/group into isopentane. This resulted in n = 15 sham-operated and n = 17 castrated mice for quantification of maximal external diameters of cleaned AAAs. Abdominal aortic lumen diameters were monitored by ultrasound on days 42, 56, 70 and 84 of AngII infusion in mice from each group (n = 23 mice/group with the exception of those exhibiting ruptures during chronic AngII infusion).

AngII Infusion

Alzet (Durect Corp; Cupertino, CA) osmotic pumps (model 1004) were filled with AngII (infusion rate of 1000 ng/kg/min) as described previously.^6;15 Pumps were implanted subcutaneously on the right flank via an incision in the scapular region. AngII infusions were continued each month by replacement of new osmotic minipumps.

Orchiectomy

Male ApoE-/- mice were anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.). Both testes were moved to the scrotal sacs by gently applying pressure to the abdomen. An 8-10 mm incision through the skin was made along the midline of the scrotal sac. Another incision was made into the midline wall between the testes sacs under the covering membranes. The testis, the vas deferens and epididymal fat pad were pulled carefully out through the incision. The blood vessels supplying the testis were clamped with a hemostat and the testis was dissected away. The vas deferens and the fat pad were cauterized and placed back into the scrotal sac. This procedure was repeated for the other testis. The incision through the skin was closed using wound clips. For sham-operated controls, mice were anesthetized, incisions were made and sex organs were localized.

Ultrasound Imaging

Abdominal aortas of mice (n = 60 mice at study onset) were visualized with high-frequency ultrasound (Vevo 660, VisualSonics, Toronto, Canada) as described previously.^12;16 Briefly, mice were anesthetized with isoflurane and restrained in a supine position for ultrasonography. Short axis scans of aortas were performed on abdominal aortas from the level of the left renal arterial branch moving vertically to the suprarenal region. Cine loops of 300 frames were acquired throughout the suprarenal region of abdominal aortas and maximal luminal diameters in the suprarenal region of abdominal aortas were measured on images at maximal dilation. To reconstruct three-dimensional (3D) images of AAAs, cleaned aortas from n = 11 mice/group, where whole body fixation preserved aortic structure, were submerged in saline solution. Ex vivo three-dimensional scan (3D mode) combined with two-dimensional imaging (B mode) was performed on abdominal aortas to obtain sequential 2D cross section images of AAAs. Vevo® software was used to construct the serial scans into 3D images and quantify total and lumen volumes of AAAs. Vascular volumes of AAAs were calculated by subtracting lumen volumes from external dimensions of the aorta. To compare AAA volumes between sham and castrated mice, we used volume measurements from 2 mm length of reconstructed 3D AAA segments with the most dilated lumens at the center to minimize variations caused by the heterogeneity of AAA pathologies.

Atherosclerosis and AAA Quantification

With the exception of n= 5 aortas/group where vessels were removed and either snap frozen in liquid nitrogen or placed in isopentane, aortas were removed and fixed with formalin overnight at room temperature. Extraneous tissue, including fat and connective tissues were removed from aortas. Atherosclerotic lesions were quantified en face on the luminal surface of aortic arches as described previously.^6;15;17 Briefly, atherosclerotic lesions were determined by measuring the area of the intimal surface covered by grossly discernible atherosclerotic plaque in mice where the total area of the aortic arch was within 10% standard error of the mean (n = 14 mice/group) using ImagePro Plus software (Media Cybernetics,Bethesda, MD). The extent of AngII-induced atherosclerosis was expressed as the percentage of total lesion area to the total aortic arch intimal area. Maximal diameters of suprarenal abdominal aortas were measured on cleaned aortas from n = 15 sham-operated and n = 17 castrated mice by ImagePro Plus software (Media Cybernetics, Bethesda, MD).

Frozen serial sections of AAAs were cut from formalin-fixed aneurysm samples using a cryostat (Leica Microsystems Inc., Buffalo Grove, IL) as described previously.¹² Sections from proximal to distal end of representative AAAs (n = 6/group) were stained with Gomori trichrome ((Poly Scientific, Bay Shore, NY) and immunostained for smooth muscle α-actin (5 □g/mL; Rabbit polyclonal ab5694; Abcam, Cambridge MA). To quantify various tissue/cell types in aneurysmal tissues, we used the HSL (hue, saturation, and lightness) mode in Image-Pro Plus software (Media Cybernetics, Rockville, MD) to process positive stained colors from AAA sections. Briefly, a unique set of Hue values was determined for any particular positive stain by comparing the color histograms between positive and negative stained areas. The quantification of any positive staining was subsequently calculated in a defined area of interest by using the staincolor specific Hue values.

Smooth Muscle Cells

Primary cultures of smooth muscle cells were harvested from the abdominal aortas of adult C57BL/6 male mice as described previously.¹⁷ Confluent cells were incubated in low serum media (0.1% vol/vol charcoal-stripped fetal bovine serum without phenol Red) with vehicle (0.3% DMSO) or testosterone (10 nM) for 12, 24 or 48 hours. Cells were harvested for RNA extraction and real time PCR quantification of gene expression as described previously¹⁷.

Statistical Analysis

Data are represented as mean ± SEM. To compare 2 groups, Student's t tests were performed for parametric data and Mann-Whitney Rank Sum Tests were utilized for nonparametric data. Two-way repeated measures ANOVA with Holm-Sidak post hoc analysis were performed to compare body weight and ultrasound data. P<0.05 was considered statistically significant. All statistical analyses were performed using SigmaStat (SPSS Inc).

Results

Castration Reduced the Progressive Lumen Dilation of Established AngII-induced AAAs in Male Mice

The experimental design for this study is illustrated in Figure 1A. Castration resulted in a significant decrease in body weight at termination (Table I; P<.05). Moreover, castration resulted in significant atrophy of the prostate, seminal vesicles, and vas deferens (Table 1; P<.05). Abdominal aortic lumen diameters at baseline and following 1 month of AngII infusion were not significantly different between mice assigned to different groups (baseline, 1.1 ± 0.1 vs 1.1 ± 0.1 mm; sham-operated versus castrated, respectively; n = 46 mice; 1 month, 1.6 ± 0.1 vs 1.6 ± 0.4 mm; n = 23 mice/group, sham-operated versus castrated, respectively), demonstrating that AAAs of different sizes were evenly assigned to sham-operated and castrated groups. In sham-operated mice, abdominal aortic lumen diameters increased progressively from month 1-3 of AngII infusion, with significant increases in lumen diameter at study endpoint compared to month 1 of AngII infusion (month 1 vs 3, 1.6 ± 0.1 vs 1.9 ± 0.1 mm; n = 20; P<.001; Figure 1B). In contrast, abdominal aortic lumen diameters did not increase in size with continued AngII infusion in castrated mice (month 1 vs 3, 1.6 ± 0.1 vs 1.6 ± 0.1 mm; n = 22; Figure 1B). At study endpoint, abdominal aortic lumen diameters were significantly decreased in castrated compared to sham-operated mice (Figure 1B; P<.05). In sham-operated mice, 3 out of 20 mice died from ruptured AAA, while 1 out of 22 castrated mice died of ruptured AAA (Figure 1C; p = 0.31).

Figure 1.

Castration reduces abdominal aortic lumen dilation in AngII-infused mice. A, Experimental design for quantifying effects of castration on the progression of established AngII-induced AAAs. B, Suprarenal aorta lumen dilation at various time points following AngII infusion in sham and castrated mice (n = 20 sham-operated; n = 22 castrated). Arrow indicates sham or castration surgery. C, Percent survival of mice in each group from day 28 to day 84 of AngII infusion. *, P<.05 compared to day 28 of AngII infusion. **, P<.05 compared to sham.

Table I.

Characteristics of sham-operated and castrated mice continuously infused with AngII.

	Sham-operated	Castrated
Body Weight (g)	27.7 ± 0.6	24.6 ± 0.3*
Seminal vesicle (g)	0.99 ± 0.25	0.06 ± 0.01*
Prostate (g)	0.049 ± 0.007	0.003 ± 0.002*
Vas deferens (g)	0.064 ± 0.008	0.028 ± 0.004*

Data are mean ± SEM from n = 7 (sham-operated) or n = 9 (castrated) mice

^*P<.05 compared to sham-operated.

Castration had no Effect on Maximal External Diameters of AngII-induced AAAs

At study endpoint, maximal external diameters of excised, cleaned AAAs were not significantly reduced by castration (sham-operated vs castration, 2.3 ± 0.2 vs 1.9 ± 0.2 mm; n = 15 sham-operated, n = 16 castrated; p = 0.137; Figure 2). Since castration resulted in a reduction in lumen diameter, but no change in external diameter of AAAs, we imaged AAAs by ex vivo ultrasound (Figure 3A). Three dimensional reconstruction of aortas from a representative mouse in each group (with similar maximal external AAA diameters) illustrate marked differences in vascular wall remodeling between castrated versus sham mice (n = 11 mice/group). We quantified total aortic volume, vascular volume/lumen volume ratio, and percent vascular wall volume to define differences between groups. While AAAs from sham-operated and castrated mice exhibited similar total AAA volumes (Figure 3B), the vascular volume/lumen volume ratio was significantly increased in AAAs from castrated versus sham-operated mice (Figure 3C; P<.05).

Figure 2.

Castration has no effect on AAA maximum external diameters from AngII-infused mice. A, Aortas from mice in each group who underwent whole body perfusion fixation (n = 12/group). B, Maximum external diameters of suprarenal abdominal aortas. Data are mean ± SEM from n = 15 sham-operated mice and n = 17 castrated mice.

Figure 3.

AAAs from castrated mice have increased vascular volume. A, 3-D image of a representative AAA from mice in each group illustrating changes in aortic remodeling. B, Total volume (mm³) of AAAs imaged by ultrasound (A) (n = 11 mice/group). C, Vascular volume/lumen volume ratio from AAAs imaged by ultrasound in mice from each group. *, P<.05 compared to sham.

AAAs from Castrated Male Mice Exhibited Thickened Walls with Increased Smooth Muscle -Actin and Collagen Content

Since the wall/lumen ratio of AAAs from castrated mice was increased compared to sham-operated mice, we examined tissue morphology in AAA sections from mice in each group. Tissue sections from proximal to distal regions of AAAs from sham-operated mice exhibited pronounced lumen dilation with very thin walls (Figure 4, A-D). Smooth muscle -actin immunostaining in dilated regions of AAAs was more pronounced in AAA sections from castrated compared to sham-operated mice (Figure 4E; P<0.05). Similarly, collagen immunostaining was more pronounced in AAA sections from castrated versus sham-operated mice (Figure 5).

Figure 4.

AAA sections from castrated mice have increased smooth muscle α-actin immunostaining. AAA sections from the proximal (A,B) to distal (C,D) end of AAA tissue sections from sham (A,C) and castrated (B,D) mice were immunostained for smooth muscle α-actin. E, Quantification of α-actin immunostaining in AAA sections from each group exhibiting the largest luminal dilation. Data are from n = 6 AAAs/group. *, P<0.05 compared to sham.

Figure 5.

AAA sections from castrated mice have increased ground substance and collagen content. AAA sections from the proximal (top) to distal (bottom) end of AAA tissue sections from mice in each group were stained using Gomori trichrome. Data are from n = 6 AAAs/group. Blue/green, collagen.

Castration had no Effect on Atherosclerosis in Male Mice Chronically Infused with AngII

In addition to inducing AAAs, infusion of AngII also promotes atherosclerosis in the aortic arch of hypercholesterolemic mice.⁶ Castration had no significant effect on the percent lesion surface area in the aortic arch of AngII-infused mice (n = 14 mice/group; p = 0.69; Figure 6).

Figure 6.

Castration has no effect on atherosclerosis in mice infused chronically with AngII. Lesion surface area in the aortic arch was quantified by en face methods. Circles indicate individual mice, with diamonds mean ± SEM. Data are mean ± SEM from n = 14 mice/group.

Testosterone Reduced mRNA Abundance of Transforming Growth Factor (TGF) and Plasminogen Activator Inhibitor-1 (PAI-1) in Abdominal Aortic Smooth Muscle Cells

To define mechanisms for effects of castration to increase vascular wall volumes in progressing AAAs, we quantified effects of testosterone on mRNA abundance of two factors implicated in fibrosis, TGF¹⁸ and PAI-1¹⁹, in abdominal aortic smooth muscle cells from male C57BL/6 mice. Testosterone resulted in a significant reduction in mRNA abundance of TGF and PAI-1 in abdominal aortic smooth muscle cells (Figure 7; P<.05).

Figure 7.

Testosterone reduces mRNA abundance of TGF-β1 and PAI-1 in abdominal aortic smooth muscle cells. Cells were incubated with vehicle or testosterone for 12, 24 or 48 hours prior to quantification of mRNA abundance. Data are n = 3 experiments. *, P<.05 compared to vehicle.

Discussion

This study demonstrated that castration of male mice prevented progressive lumen dilation of established AngII-induced AAAs in male mice. However, despite an attenuation of lumen dilation, the size of excised AAAs was not influenced by castration. As a result, AAAs from castrated mice exhibiting smaller lumen diameters had thickened vascular walls with more pronounced -smooth muscle actin and collagen immunostaining. Effects of castration may result from regulation of genes (TGF, PAI-1) associated with fibrosis and protection against AAA formation and/or progression, as testosterone significantly reduced mRNA abundance of TGF and PAI-1 in abdominal aortic smooth muscle cells. These results demonstrate that testosterone contributes to both the formation and progression of AngII-induced AAAs.

In contrast to effects of castration to blunt progressive increases in aortic lumen diameters of AAAs, castration of male mice had no significant effect on the level of atherosclerosis in mice chronically infused with AngII. These results are in agreement with previous data demonstrating that male sex hormones markedly influence AngII-induced AAAs.¹⁰ We previously demonstrated that castration had opposing effects on AngII-induced atherosclerosis and AAAs, with increased atherosclerosis and marked reductions in AAA formation in castrated hypercholesterolemic male mice exhibiting low sera testosterone concentrations.¹⁰ Similar to previous findings, results from this study suggest that mechanisms for chronic progression of AngII-induced AAAs and atherosclerotic lesions are distinct.

We demonstrated previously that castration strikingly reduced the formation of AngII-induced AAAs in male mice while exogenous androgen administration restored AAA formation in castrated males, indicating an important role of androgen in processes leading to the formation of AngII-induced AAAs.^10;11 In this study, we demonstrate that castration significantly reduced the progressive lumen dilation of established AngII-induced AAAs, suggesting that androgen also plays a role in the progression of established AAAs in male mice. While it is unequivocal that male sex is a prominent risk factor for AAA development in humans^7-9;20, it is unclear whether male gender influences growth rates of established AAAs. Results from the United Kingdom Small Aneurysm Trial demonstrated no association of gender with AAA growth.³ Rather, despite a lower incidence of AAAs in females, the growth rate and proportional dilations of AAAs were greater in females compared to males.^21-23 A recent study directly compared biomechanical and clinical factors in AAAs from males versus females, and concluded that increased stiffness, lower energy to propagate a dissection, and lower elastin content of the intima-media were associated with higher risks for AAA growth in males.²⁴ While castration is not an amenable therapy to treat AAAs in humans, these results suggest that androgen receptor blockade may be of benefit to blunt progressive lumen dilation of advancing AAAs, or alternatively that use of anabolic steroids may be contraindicated in males of advancing age with a family history of AAAs.

Despite an ability of castration to significantly reduce aortic lumen dilation of progressing AAAs, external AAA diameters were only modestly decreased by castration. A reduction in AAA lumen diameter in the face of a modest but yet insignificant decrease in external diameter suggests that castration also influenced aortic remodeling associated with prolonged AngII infusion. Indeed, quantification of vascular volume/lumen volume ratios demonstrated that AAAs from castrated male mice had increased vascular volumes. Moreover, in contrast to thin-walled AAAs from sham-operated mice, AAAs from castrated male mice exhibited more pronounced smooth muscle and collagen content, suggesting a stronger vascular wall. While it is not clear if these changes are favorable to protect against AAA ruptures, castrated male mice did exhibit a modest, but insignificant reduction in death from aortic rupture.

Results from this study suggest that testosterone, the primary male sex hormone influenced by castration, contributes to both the formation and progression of AngII-induced AAAs. Our results from studies examining effects of testosterone in abdominal aortic smooth muscle cells suggest that TGF and/or PAI-1, proteins involved in the regulation of fibrosis and implicated in AAA formation and/or rupture²⁵, may be targets of testosterone to influence aortic remodeling and AAA progression. Administration of a neutralizing antibody to TGF in AngII-infused male mice promoted AAA formation and rupture²⁵, suggesting that loss of testosterone in castrated mice may increase TGF expression and protect against AAA progression. Since testosterone suppressed TGF mRNA abundance in aortic smooth muscle cells and TGF is a regulator of smooth muscle cell differentiation²⁶, castration-induced regulation of TGF may have contributed to a more muscularized aneurysmal wall. Similarly, regulation of PAI-1 gene expression arising from castration-induced reductions in testosterone may have contributed to increased matrix deposition and protection against AAA progression.^27;28

In summary, results from this study demonstrate that removal of male sex hormones blunted progressive lumen dilation of AngII-induced AAAs. AAAs from castrated male mice exhibited an increase in vascular volume associated with more pronounced smooth muscle -actin and collagen immunostaining. These results suggest that male sex hormones contribute to the progression of experimental AAAs.

Clinical Relevance.

There are no therapeutics that slow the progression of AAAs, and as the size of AAAs increases so does the risk of rupture and mortality. Male sex is a non-modifiable risk factor for AAA development, but it is unclear if male sex hormones have a similar impact on AAA progression. Using an established mouse model of AngII-induced AAAs, results demonstrate that removal of male sex hormones reduced progressive lumen dilation while not altering external AAA dimensions. Therapies that limit androgen action may provide benefit against AAA progression. Alternatively, supplemental testosterone may be contraindicated in males diagnosed with an AAA.