Female Mice Exhibit Abdominal Aortic Aneurysm Protection in an Established Rupture Model

Abstract

Background:

Male gender is a well-established risk factor for AAA (abdominal aortic aneurysm) while estrogen is hypothesized to play a protective role. Although rupture rates are higher in women, these reasons remain unknown. In the present study, we sought to determine if female mice are protected from AAA rupture.

Materials and Methods:

7-week-old apolipoproteinE deficient (ApoE-/) male and female mice (n=25/group) were infused with Angiotensin II (2000 ng/kg/min) plus β-aminopriopionitrile(BAPN) in the drinking water for 28 days to test the effects of gender on AAA rupture.Separately, a second group of male ApoE−/− mice underwent AngII infusion + BAPN while being fed high-fat phytoestrogen free or a high-fat phytoestrogen diet to assess effects of phytoestrogens on rupture. In a third group, female mice either underwent oophorectomy or sham operation 4 weeks prior to infusion of AngII and BAPN to further test the effects of female hormones on AA rupture.. Surviving mice abdominal aorta were collected for histology, cytokine array, and gelatin zymography on postoperative day 28.

Results:

Female mice had decreased AAA rupture rates (16% vs. 46%, P=0.029). Female mice expressed fewer elastin breaks (p=0.0079) and decreased SMC degradation (p=0.0057). Multiple cytokines were also decreased in the female group. Gelatin zymography demonstrated significantly decreased pro MMP2 in female mice (p=0.001). Male mice fed a high dose phytoestrogen diet failed to decrease AAA rupture. Female mice undergoing oophorectomy did not have accelerated aortic rupture.

Conclusion:

These data are the first to attempt to tease out hormonal effects on AAA rupture and the possible role of gender in rupture.

Introduction

Abdominal aortic aneurysms (AAA) are a major health threat in the United States as they remain the 15^th leading cause of death, specifically affecting men 4:1 over women^1–4. Even more concerning, AAAs results in more than 15,000 surgical procedures annually. Abdominal Aortic Aneurysm (AAA) is a disease that involves several complex factors that can lead to rupture⁵. In adults 60–64 years old, AAA rupture in general has a 50–80% mortality rate. Even more concerning, there is currently no medical treatment strategy for slowing the growth rates of this deadly disease^1–4

Aortic aneurysms have been characterized initially by elevation of pro-inflammatory cytokines associated with elastin and collagen degradation, apoptosis of smooth muscles and influx of inflammatory cells.. Gender is a well-established risk factor associated with increased AAA incidence with a prevalence of 4:1 in males when compared to females. Estrogens have been shown by our groups and others to play a protective role in the development of AAAs^5–8. Phytoestrogens are plant-derived chemicals that are strikingly similar to estrogens both in structure and function and are contained in many common foods consumed on a daily basis. Therefore, there exist a number of potential benefits and risks of phytoestrogen exposure based on the amounts consumed^9–12. Major sources of phytoestrogens include soybeans, alfalfa, and flaxseed. Phytoestrogens are selective estrogen receptor (ER) modulators and have anti-inflammatory, antioxidant, and antiproliferative properties.^13–15 Animal experiments in other models of vascular diseases have demonstrated that phytoestrogens can reduce plasma cholesterol and attenuate atherosclerosis^16–18. However, little is known regarding the effects of phytoestrogens on aortic aneurysm formation. In a mouse experimental topical elastase AAA model from our laboratory, Lu at al. showed that male mice placed on a high phytoestrogen diet 2 weeks prior to surgery have decreased AAA size compared to male mice fed a low estrogen diet¹⁹. However, these studies failed to examine the role of rupture in male or female mice or whether phytoestrogens could play a role in inhibition of AAA rupture.

Rupture rates are suggested to be higher in women, but this may represent an advanced disease in the females due to a smaller baseline aortic diameter²⁰. A pre-established murine rupture model could help further evaluate the AAA differences between genders and determine if there are gender related impacts on aortic rupture, both in the ascending and abdominal aorta. We have recently developed two different models of abdominal aortic aneurysm rupture, one using the porcine pancreatic elastase model and the other using Angiotensin II infusion plus water supplemented with B-aminopropionitrile^{21, 22}. We wanted to determine whether female gender and/or phytoestrogen treatments might play a role in prevention of AAA rupture. We have previously seen in gender comparison studies that female mice were protected from forming AAA; therefore, we sought to determine whether female mice could also be protected from AAA rupture. In this study, it is hypothesized that female mice are protected against AAA rupture using a recently established murine AAA rupture model¹¹

Methods

Angiotensin II infusion

Experiment 1 (gender-based study):

7-week-old ApoE −/− male and female mice (Jackson Laboratory, Bar Harbor, ME) were housed and maintained at 70°F, 50% humidity, in 12-hour light-dark cycles per institutional animal protocols. Male and female mice were fed water and placed on a high fat diet (TD 09682, Harlan Teklad Inc, Indianapolis, IN) introduced one week prior to the day of surgery. 49 (n = 24 males and 25 females) 6-wk-old wild-type ApoE−/− (Jackson Laboratory, Bar Harbor, Maine) were divided into two groups based on gender. AngII was delivered via osmotic pump at 2,000 ng/kg/min. 0.2% β-aminopropionitrile (BAPN) (Sigma Aldrich) was dissolved in the mice’s drinking water 3 days before osmotic pump placement and both groups were continuously given BAPN until the end of the experiment²¹. If a mouse died before day 28, an autopsy was performed to determine the cause of death. After 28 days, surviving mice were euthanized under anesthesia by overdose and exsanguination.\

Experiment 2 (phytoestrogen Angiotensin II study):

Another group of male ApoE−/− mice (n=38) were fed either a high phytoestrogen or a phytoestrogen free diet. The mice were divided into two groups of 19 mice each based on¹⁹ dietary phytoestrogen exposure to determine the influence of phytoestrogen content on aortic aneurysm formation and rupture. Thus, two groups were evaluated: (1) male mice fed a diet with a high fat but minimal phytoestrogen diet (NO PE), (2) male mice fed a control high fat diet (PE). The isoflavone content, one of the major classes of phytoestrogens, ranged under 1 ppm for the minimal phytoestrogen diet (TD 09682 Teklad Global Western diet modified to contain no phytoestrogens), whereas the regular diet (TD 09682 Teklad High Fat Diet Mouse/Rat Diet, Madison, WI) had between 600 and 850 mg/kg. The other ingredients in the both diets were similar (see www.harlan.com). Both rodent diets were commercially available. Four weeks after mice were placed on the diets, AAAs were induced using Angiotensin II and BAPN as described in experiment 1 above. . If a mouse died before day 28, an autopsy was performed to determine the cause of death. After 28 days, surviving mice were euthanized under anesthesia by overdose and exsanguination.\

Experiment 3 (phytoestrogen Elastase study):

A group of male C57/B6 mice (n=20) were fed either a high phytoestrogen or a phytoestrogen free diet. The mice were divided into two groups of 10 mice each based on dietary phytoestrogen exposure to determine the influence of phytoestrogen content on aortic aneurysm formation and rupture. Thus, two groups were evaluated: (1) male mice fed a diet with a high fat but minimal phytoestrogen diet (NO PE), (2) male mice fed a control high fat diet (PE). The isoflavone content, one of the major classes of phytoestrogens, ranged under 1 ppm for the minimal phytoestrogen diet (TD 09682 Teklad Global Western diet modified to contain no phytoestrogens), whereas the regular diet (TD 09682 Teklad High Fat Diet Mouse/Rat Diet, Madison, WI) had between 600 and 850 mg/kg. The other ingredients in the both diets were similar (see www.harlan.com). Both rodent diets were commercially available. Four weeks after mice were placed on the diets, mice were placed on 0.2% β-aminopropionitrile (BAPN) (Sigma Aldrich) dissolved in the mice’s drinking water 3 days before AAA induction and both groups were continuously given BAPN until the end of the experiment²¹. AAAs were induced using the following protocol, infrarenal abdominal aorta was isolated and porcine pancreatic elastase (0.4 U/mL; Sigma, St. Louis, MO) was applied for 5 min. Elastase solution was evacuated and the mice were allowed to recover. Mice abdominal aortic diameters (n = 10/group) were measured immediately after application to ensure similar dilation. On postoperative day 14, the infrarenal abdominal aorta was dissected and the maximal aortic diameter was measured using video microscopy with NIS-Elements D.3.10 software attached to the microscope (Nikon SMX-800, Melville, NY). Aortic dilation was determined using the formula (maximal aortic diameter − internal control diameter)/maximal aortic diameter × 100%. The internal control diameter was the diameter of un-infused infrarenal aorta just above the infused section. A dilation of 50% or more was considered to be positive for AAA formation. All measurements were performed when the animal was alive. Aorta samples were harvested for protein analysis (n = 5/group) and histologic studies (n = 3/group) should there be dilation differences following phytoestrogen treatments.

Experiment 4 (oophorectomy study):

6-wk-old wild-type ApoE−/− female mice (n = 15) were divided into two groups based on whether the female received removal of her ovaries (oophorectomy) or a control, sham surgery. Female mice were allowed to recover for 4 weeks on high fat western diet. At 10 weeks of age, AAAs were induced using Angiotensin II and BAPN as described in experiment 1 above. If a mouse died before day 28, an autopsy was performed to determine the cause of death. After 28 days, surviving mice were euthanized under anesthesia by overdose and exsanguination.\

For all surviving mice at day 28, video micrometry measurements of the aortic wall diameter were performed in situ using a Q-Color3 Optical Camera (Olympus Corp., Center Valley, Pennsylvania) using QCapture Pro Software version 6.0 (QImaging Inc., Surrey, Canada). Kaplan-meier curves and log-rank (Mantel-Cox) tests tracked the percentage survival of mice over the 28-day period. Suprarenal abdominal aortic tissue was collected and stored at −80°C or embedded in paraffin for histology. Animal care and use were in accordance with the Guide for the Care and Use of Laboratory Animals. The animal protocol was approved by the University of Virginia Institutional Animal Care and Use Committee (#3848) in compliance with the Office of Laboratory Animal Welfare.

Histology

Aortic tissue was fixed in 4% buffered formaldehyde for 24 hours, transferred to 70% ethanol, and then embedded in paraffin. Day 28 harvested (n=5/group) cross-sections were prepared with Verhoeff-Van Gieson (VVG) for elastin, and immunohistochemical staining for anti-mouse SM α-actin for smooth muscle α-actin (1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, California). Images were acquired using AxioCam Software v 4.6 with a 10× objective. Histology was graded by 3 blinded reviewers. For elastin grading, 1 corresponded to no degradation, 2= mild degradation, 3 =moderate degradation, and 4= severe degradation. For smooth muscle cell (SMC) grading, 1 corresponded to no SMC loss, 2 = mild SMC loss, 3 = moderate SMC loss, and 4 = severe SMC loss.

Protein extraction and Cytokine Array

Protein was extracted from frozen aortic tissue using phosphate buffer saline (PBS) and protease inhibitors (Roche Diagnostics, Indianapolis, IN) as previously described²³. Cytokine array (R&D Systems, Minneapolis, MN) was completed according to manufacturer instructions, on mice harvested on day 28 (n=4 pooled aortas/group). Tissue was pooled for protein analysis and all samples were run in duplicate. Because aortic the samples were pooled, these results were not statistically compared.

Gelatin Zymography

Gelatin zymography was performed using abdominal aortic tissue samples (n=4–6/group) to measure pro and active MMP 2 and 9 activity. Precast zymography gels (10%, Invitrogen, Carlsbad, CA) were loaded with 3μg of aortic protein from each sample. Samples were diluted into 2x Tris-glycine SDS sample buffer and separated using electrophoreses under non-reducing conditions. The gels were renatured for 30 minutes in renaturing buffer (Invitrogen) and incubated for 1 day at 37°C while rocking in developing buffer (Invitrogen, Carlsbad, CA). The gels were then stained in Simply Blue Safe Stain (Invitrogen, Carlsbad, CA). Optical density levels were quantified using the Bio-Rad Image Lab Software version 4.0²⁴.

Statistical methods

GraphPad Prism 7 software (GraphPad Software, La Jolla, CA) was used for testing statistical analysis. AAA rupture results were analyzed using a Chi-squared test. Statistical analysis of aortic diameters was performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). Maximal aortic dilation (%) was calculated as [maximal aortic diameter – internal control diameter] ÷ internal control diameter * 100%. The internal control was a small segment of normal proximal abdominal aorta just distal to the renal arteries that was above the proximal extent of the AAA. Values are reported as mean ± standard error of the mean. Aortic dilation between groups was compared using a student’s t-test for 2 sample groups or ANOVA for 3 or more sample groups. To compare two groups in other samples, a Student’s t-test was used. Paired data were analyzed by paired student’s t test. Differences between 2 or more groups versus a control group were analyzed with One-way ANOVA plus Bonferroni correction for multiple comparisons. Non-parametric data were analyzed by Mann-Whitney U test.

Results

Female mice have decreased AAA rupture compared to male mice

Female gender has been suggested to be protective in AAA as males are 4:1 more likely than females to have a AAA; however, to date no studies have examined whether female mice have decreased rates of AAA rupture compared to males mainly because there have been no effective models to study aortic rupture in murine models. Therefore, we sought to examine the role of rupture in male versus female mice in our new AAA murine model of rupture. Male and female ApoE−/− mice were infused Angiotensin II plus 0.2% BAPN in the drinking water for 28 days. Survival rates were recorded and mice still survived at day 28 were harvested for histology and protein analysis. Female versus male survival rates were not different between male and female mice over the time period examined. Interestingly, female mice demonstrated decreased AAA rupture rates when compared to male mice (16% vs. 46%, p=0.029, Fig. 1). However, AAA sizes between male and female mice that survived to 28 days were not statistically different (Figure 1 B and D). In addition, there were no differences in the rate of ascending aortic rupture.

Figure 1. Female mice had significantly less AAA Ruptures than male mice.

A) Male and female were treated with 2000 ng/kg/min Angiotensin II+ BAPN for 28 days and survival was tracked. Male and female showed a non-significant difference in survival rate (n=24/group). B) Graphical Representation of the aortic dilation as compared with a control segment in male and female mice which survived to the end of the 28-day period (p=0.8407). C) Graphical representation of the number of total ruptures, AAA ruptures and AA rupture (ascending aneurysm) in female versus male mice over a 28-day period. (AAA rupture; 11 ruptures, 46% vs. 4 ruptures, 16%, P=0.032, n=24–25/group). D) Picture representation of AAAs from mice harvested at day 28 following Angiotensin II infusion.

Following harvest of male and female mice at 28 days, abdominal aortas were placed in 4% paraformaldehyde, embedded, sectioned and immune-stained for elastin breakage, SM α-actin, macrophages and neutrophils. Female mice expressed significantly fewer elastin breaks (Fig 2, p=0.0079) and increased levels of SMC staining as compared to male mice treated with Angiotensin II and BAPN for 28 days (Fig. 2, p=0.0057). Interestingly, there appeared to be no change in neutrophil staining but decreased macrophage staining. These immuno-histochemical results suggested further that female mice might exhibit protective characteristics compared to males. Following immune-histochemical analysis, we next sought to determine whether inflammatory cytokines were decreased in pooled groups of female versus male mice at 28 days post Angiotensin II infusion. Pooled aortic tissue from female versus male mice that survived to day 28 showed down regulated inflammatory cytokines IL-1β, CXCL1, MIP-2, CCL17, TREM-1, TNF-α, TNF-γ, IL-6, IL-17, RANTES, TIMP-1, MIP-1α, CXCL10, IL-27, IL-4, and M-CSF when compared to male mice via cytokine array (Fig. 3). Finally, gelatin zymography demonstrated significantly decreased Pro MMP2 activity in female mice compared to males (4,533,497 vs. 8,746,652 densitometry units, P=0.001, Fig. 4). Active MMP2 and both latent and active MMP9 were also measured by gelatin zymography but were not found to be statistically different between male and female mice (data not shown). Collectively, these data suggest that female gender could exert protective effects over AAA rupture.

Figure 2. Abdominal aortic wall in female mice who survived had less elastin breaks and increased SM actin staining compared with males.

A and B) Female and male ApoE−/− mice were treated with Angiotensin II for 28 days. Following harvest, a subset was analyzed for immuno-histochemical analysis using VVG, MAC2, and SM actin staining. VVG staining demonstrated decreased elastin breaks (p=0.0079, n=5/group), while SM actin staining was increased in the female group (p=0.0057, n=5/group).

Figure 3. Female mice have decreased inflammatory cytokine levels compared to male mice in their abdominal aorta. A-D).

Protein was isolated from the abdominal aorta of male and female mice following harvest and protein cytokine arrays were conducted (n=4 aortas pooled sample/group). Females demonstrated decreased IL-1β (428,987 vs 119,494), CXCL1 (375,784 vs. 227,306), MIP-2 (325,049 vs. 98,572), CCL17 (269,933 vs. 90,714), TREM-1 (283,916 vs. 80,786), TNF-α (1,095,109 vs. 161,944), TNF-γ (780,659 vs. 143,500), IL-6 (129,648 vs. 62,173), IL-17(662,829 vs. 155,417), RANTES (234,489 vs. 114,393), TIMP-1 (2,575,212 vs. 1,301,407), MIP-1α (349,216 vs. 151,289), CXCL10 (315,555 vs. 91,915), IL-27 (378384 vs. 90,113), IL-4 (484,098 vs. 79,423), and M-CSF (454,038 vs. 91,664).

Figure 4. Female mice have decreased Pro MMP2 levels compared with male mice using Gelatin Zymography in the abdominal aorta at 28 days. A and B).

Female mice have decreased Pro MMP2 activity compared to males (8,746,652 vs. 4,533,497 densitometry units, p=0.001, n=4–6/group).

High-Phytoestrogen diet does not impact male rupture rates or survival rates in a AAA rupture model

Since we were able to demonstrate that female mice protect against AAA rupture versus male mice in a AAA rupture model, we next sought to determine if high-phytoestrogen diet could mimic these effects for male mice. We treated two groups of male mice, a phytoestrogen free Western diet group and a high-phytoestrogen Western diet group, with Angiotensin II plus BAPN in the drinking water for 28 days. Interestingly, although more male mice fed a high-phytoestrogen diet survived to harvest, there was no statistical difference in survival rates between male mice in either the phytoestrogen-free group or the high phytoestrogen group (p=0.223, Fig 5). The surviving male mice in both groups were harvested at 28 days following Angiotensin II infusion did not demonstrate a significant difference between percent aortic dilation between groups (Fig. 5 B). Male mice fed a high phytoestrogen diet also did not show protection against AAA rupture when compared to mice fed a phytoestrogen free diet but did demonstrate a decrease in ascending aneurysm rupture rates (66% versus 36%, p=0.041, n=24/group). To test the role of phytoestrogen in a second model of aortic aneurysm rupture, a control Western diet group and a phytoestrogen free Western diet group (n=10/group), underwent topical elastase application plus BAPN in the drinking water (0.2% BAPN) for 28 days. Aortas were harvested and aortic dilation was measured via video micrometry. Percent aortic dilation in phytoestrogen treated mice was found to not be satistically different from control diet treated mice. These data suggest that the addition of phytoestrogens, granted a weak estrogen, to the diet would not alone decrease AAA rupture in inflammatory models of AAA such as the Angiotensin II plus BAPN mouse model.

Figure 5. Phytoestrogen Diet does not affect survival in male mice using two models of rupture.

Male mice on phytoestrogen positive and negative diet were treated with 2000 ng/kg/min Angiotensin II+ BAPN (0.2% in drinking water) for 28 days and survival was tracked. A) Male mice on a diet containing supplemental phytoestrogens showed a non-significant difference in survival rate compared to mice not on a phytoestrogen diet (p=0.2166 n=24/group). B) Male mice that survived to 28 days following pump insertion were harvested and their aortic aneurysm was measured in reference to a control segment. Representative images from each group were also included. C) Graphical representation of the number of total ruptures, AAA ruptures and AA rupture (ascending aneurysm) in male control versus male phytoestrogen treated mice over a 28-day period. (AA rupture; 10 ruptures, 66% vs. 5 ruptures, 36%, p=0.041; n=24/group). D) Male mice on a diet containing supplemental phytoestrogens were subjected to topical elastase application + BAPN (0.2% in drinking water) for 28 days and then harvested and aortic dilation were assessed.

Oopherectomy does not accelerate aortic rupture in female mice.

In a final set of experiments, we sought to determine if estrogens derived from the female gonads could represent the possible cause of the decreased rates of AAA rupture in the female versus male experiments performed previously in this study. We treated two groups of female mice, a group of females that underwent oophorectomy 4 weeks prior to surgery and a control group that underwent sham surgery, with our Angiotensin plus BAPN rupture model. Female mice that underwent oophorectomy did not demonstrate increased survival or rupture rates in comparison to control female mice (p=0.66, Fig. 6). Female mice that survived through the course of the experiment were harvest and percent aortic dilation was measured and demonstrated no significant difference between female mice with and without gonads (Fig. 6B). Rupture rates, AAA rupture rates and ascending aneurysm rupture rates were quantified and demonstrated no significant differences between groups. These data suggest that estrogens provided by the gonads are not sufficient to alter AAA rupture rates within our model.

Figure 6. Oophorectomy does not affect survival in female mice using a model of rupture.

Female mice were oophorectomized or a control and then placed on Phytoestrogen free Western diet(Harlan Teklad). 4 weeks following oophorectomy, female mice were infused with an osmotic pump with 2000 ng/kg/min Angiotensin II for 28 days and BAPN (.2% in drinking water) and survival was tracked. A) Female mice with oophorectomy on a phytoestrogen negative diet showed a non-significant difference in survival rate compared to mice not on a phytoestrogen diet (p=0.66; n=15/group). B) Female mice that survived to 28 days following pump insertion were harvested and their aortic aneurysm was measured in reference to a control segment. Representative images from each group were also included. C) Graphical representation of the number of total ruptures, AAA ruptures and AA rupture (ascending aneurysm) in female control versus female oophorectomized mice over a 28-day period. (n=15/group).

Discussion

Using a new murine AAA rupture model that we had previously established²¹, we documented that female mice have a lower AAA rupture rate than male mice. Although the human disease suggests that females have increased AAA rupture compared to males in humans, studies such as Ailawadi et al. state that females could have protective traits against AAA growth and their relative smaller baseline aortic size could be what cause an increase amount of ruptures in women with advanced stage AAA disease⁶. In this study, differences between female and male murine AAA sizes were not significantly different at baseline or at termination of the study, which could suggest a protective nature of female physiology against AAA rupture if female and male aortic sizes were similar. However, future studies are warranted to further delineate the mechanisms of the difference between AAA rupture rates between males and females in the model.

In addition, supplemental phytoestrogen, which has been shown to inbibit AAA growth in a topical elastase AAA model failed to inhibit AAA rupture in this AngII/BAPN model and in a topical elastase plus BAPN model. Initially, we hypothesized that supplement phytoestrogens would be inhibitory in our murine models since we had previously seen inhibition in our topical elastase mouse model and we were surprised with the results of our phytoestrogen studies. Observations in our laboratory suggest that the present model is much more vigorous that the elastase model with a significant percentage of rupture. Oral phytoestrogens, typically derived from soy products, have both pro- and anti-estrogen properties but in general are considered weak estrogens, and thus their ability to inhibit rupture in this model was a surprise but not totally unexpected. Future experiments with chemical derivations of phtyoestrogens, such as genisten, where we can increase the dosage might provide better future experiments to determine the role of exogenous phytoestrogen derivatives in aneurysm rupture.

Finally, as most humans females that rupture their AAA are post-menopausal, we sought to determine the impact of oophorectomy and removal of female estrogen production from the ovaries in aortic rupture in this model. Female mice undergoing oophorectomy did not have accelerated aortic rupture perhaps due to continued circulating estrogen produced by fat, adrenal glands or brain. Our work using aromatase knockout mice, which is the enzyme responsible for the conversion of testosterone to estrogen, showed increased AAA growth in females who were both aromatase −/− and who underwent oophorectomy. These data are related to our oophorectomy experiments since gonad removal also removes aromatase conversion of testosterone to estrogen that occurs in the ovaries and suggests that estrogens are complex in aneurysm formation. Teasing out the exact mechanism the gonadal hormones play in aortic rupture will be the subject of further investigations in our laboratory.

Despite typical limitations of animal models compared to human disease, the Ang II/BAPN rupture model is an established model that has biology consistent with human AAA ruptures²¹. Previous studies from our lab using the murine elastase model coupled with BAPN have demonstrated similar patterns of increasing aortic diameter and rupture²². Separately, we have previously published studies looking at the Angiotensin II mouse model and BAPN administration²¹. In these studies, we examined increasing doses of Angiotensin II or inclusion of BAPN to demonstrate that it is the combination of both a higher level of Angiotensin II and BAPN that induces the chronic murine model with increased rupture rates. Human AAA exhibits aortic wall destruction through degradation of elastin and SMCs aortic rupture similar to the results seen in our models. MMP-2 and MMP-9 are elevated in human aneurysmal aortic tissue while in our model we only saw differences between latent MMP2 between males and females^25–27. Cytokines IL-1B, IL-6, IL-8, TNF-a, and IFN-g are all increased in human AAA disease. In our study of murine AAA disease in a rupture model that more closely resembles an advanced stage of AAA disease, female mice had fewer elastin breaks and SMC degradation, decreased levels of latent MMP2, and possibly many down-regulated pro-inflammatory cytokine levels that are correlated with AAA protection as shown in pervious findings in our lab²⁴. Future experiments by our lab will be performed to further assess the roles of MMPs and pro-inflammatory cytokines in males versus females using our AAA rupture model coupled with mouse genetic knock-out studies. Genetic Knock-out studies in males versus females in the Angiotensin II rupture model would provide conclusive evidence of the possible roles of these factors in AAA rupture. It is recognized that this present model is likely too vigorous to examine subtle changes in aortic rupture risk. Ongoing experiments altering the amount of BAPN or AngII are being performed in order to attain a 50% rupture rate in males and females at 28 days. Despite the suggested limitation, these results show promising surrogate outcomes that are hypothesized to help us study how to possibly inhibit aortic rupture.

Our laboratory’s previous work has shown that sex hormones can play a role in AAA protection. Male rats treated with estrogen or receive an orchiectomy have decreased AAA size and females treated with testosterone did not show any change in AAA size⁷. Interestingly, deletion of aromatase in females mice led to increased AAA formation in females but no change in male mice.²⁸ Related to estrogens are phytoestrogens, which are plant derived chemicals that act and look like estrogen, male mice fed a phytoestrogen diet were shown to have significantly less AAA size compared to non-phytoestrogen fed male mice in an elastase perfusion model¹⁹. Although our study showed that a phytoestrogen diet does not change rupture rates or survival rates in male mice using this model, the data suggest that gender hormones are not only associated with AAA size, but also can affect AAA rupture rate. It can be hypothesized that phytoestrogens delivered intraperitoneally rather than through the diet could have a bigger impact such as administration of genisten, the chemical derivative of phytoestrogens. To further elucidate gender differences of AAA rupture, future studies could measure the differences between AAA rupture rates in male mice with supplemented amounts of female hormones directly injected into mice either in a min-osmotic pump or through a sustained release tablet under the skin. This could help us understand and target possible pathways protecting against AAA rupture.

Conclusion

Using a recently created murine rupture AAA model, we studied the role of gender differences in a more advanced stage of AAA disease. In this study, we demonstrated that female mice have fewer AAA rupture deaths than males. Female mice that survived to day 28 also had fewer elastin breaks and less SMC degradation versus male mice. Neither phytoestrogen treatment of male mice or oophorectomy of female mice demonstrated significant increases in survival nor did they affect AAA rupture rates. These results suggest that gender differences could play a role in events that lead to aortic rupture but suggest that detailed future studies are necessary to further delineate the exact roles of gender in AAA rupture.

Table 1.

Male versus Female Survival and Rupture Statistics

Survival and Rupture Statistics
	Male		Female
Number of Survivors at Harvest	5	20.83%	5	20.83%
Total Number of Ruptures	15	62.5%	16	66.67%
AAA Ruptures	11	45.83%	4	16.67%
AA Ruptures	10	41.67%	13	54.16%

Table 2.

Male control diet versus Phytoestrogen diet mice Survival and Rupture Statistics

Survival and Rupture Statistics
	Male No PE		Male PE
Number of Survivors at Harvest	5	20.83%	5	20.83%
Total Number of Ruptures	15	62.5%	14	58.33%
AAA Ruptures	11	45.83%	9	37.5%
AA Ruptures	10	41.67%	5	20.83%

Table 3.

Female Controls versus Oophorectomy mice Survival and Rupture Statistics

Survival and Rupture Statistics
	Female Control		Female Oophorectomy
Number of Survivors at Harvest	3	20.0%	4	26.67%
Total Number of Ruptures	12	80.0%	11	73.3%
AAA Ruptures	7	46.67%	6	40.0%
AA Ruptures	7	46.67%	5	33.33%

Non-standard Abbreviation and Acronyms:

AAA

Abdominal aortic aneurysm

ACTA2

smooth muscle α-actin

Ang II

Angiotensin II

ApoE

apolipoprotein E

BAPN

B-aminopropionitrile

ECM

extracellular matrix

MYH11

smooth muscle cell myosin heavy chain

SM22α

transgelin

VSMC

vascular smooth muscle cell