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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: J Surg Res. 2020 Dec 17;260:488–498. doi: 10.1016/j.jss.2020.11.061

Sex based differences among experimental swine abdominal aortic aneurysms

J Michael Cullen 1, Alexander H Shannon 1, Guanyi Lu 2, Gang Su 2, Michael D Spinosa 1, William G Montgomery 1, Gorav Ailawadi 1, Morgan Salmon 1, Gilbert R Upchurch Jr 2
PMCID: PMC7946779  NIHMSID: NIHMS1650679  PMID: 33341252

Abstract

Background:

Female sex protects against abdominal aortic aneurysms (AAAs); however, the mechanisms behind these sex-based differences remain unknown. The purpose of this study was to explore the role of sex and sex hormones in AAA formation among swine.

Materials and Methods:

Using a previous validated model, infrarenal AAA were surgically created in uncastrated male (n=8), female (n=5), and castrated male (n=4) swine. Aortic dilation was measured on post-operative day 28 during the terminal procedure and compared to initial aortic diameter measured during the index procedure. Tissue was analyzed for immunohistochemistry, cytokine array, gelatin zymography, serum 17β–Estradiol, and testosterone assay.

Results:

Uncastrated males had significantly larger maximal aortic dilation compared to castrated males (113.5%±11.4% vs 38.1%±4.5%, p=.0012). Females had significantly higher mean aortic dilation compared to castrated males (96.2%±7.5% vs 38.1%±4.5%, p=.0004). Aortic diameters between females and uncastrated males were not significantly different on day 28. Female swine had significantly higher concentrations of 17β-estradiol compared with uncastrated males (1590±873.3 ng/mL vs 95.2±2.3 ng/mL, p=.047), with no significant difference between females and castrated males. Uncastrated male AAA demonstrated significantly more elastin degradation compared with female and castrated males (p=0.01 and <.01, respectively). No differences existed for T-cells or smooth muscle cells between groups. Multiple proinflammatory cytokines were elevated within uncastrated male aortic walls compared to females and castrated males.

Conclusions:

Sex hormones, specifically 17β–Estradiol and testosterone, influence experimental swine AAA formation as demonstrated by increased aneurysm size, collagen turnover, and elastolysis in uncastrated males in processes reflective of human disease.

Keywords: Aneurysm, aorta, abdominal aortic aneurysm, hormones, estrogen

INTRODUCTION

The 21st century has brought significant changes to the management and treatment of abdominal aortic aneurysms (AAAs). Non-invasive screening and endovascular repair have contributed to improved outcomes, and basic science research has furthered our understanding of the disease biology.1 Despite these revolutions, AAAs remain a significant public health burden representing the 15th leading cause of death in the United States with a strong sex component, as men are four times more likely to have an AAA compared to women.1–4 However, no medical treatment currently exists to prevent or slow the growth rates of this deadly disease.

Epidemiologic studies show female sex to be protective against AAA formation.3 However, when women have an aneurysm, they tend to have higher rates of rupture and increased mortality after surgery.4 Our laboratory and others have attempted to determine the importance of sex on aneurysm formation and rupture; however, the influence of sex and related hormones on the nature and biology of AAAs remains poorly understood.4–7

In the present study, we sought to evaluate sex differences in AAA biology using a recently published experimental swine model.8 Aneurysms were surgically induced in female, castrated male, and uncastrated male swine. The aneurysms were studied for differences in size, histology, enzyme expression, and inflammatory mediators. It is hypothesized that female and castrated male swine would be protected from aneurysm development.

METHODS and MATERIALS

Animals

Experiments were conducted with the approval of the University of Virginia Institutional Animal Care and Use Committee (Protocol #3848). Domestic swine weighing 20–30 kilograms were used for the experiments. Three experimental groups (female (n=5), castrated male (n=4), uncastrated male (n=8)) underwent AAA induction surgery. The swine were fed usual chow and unlimited water; the animals were fasted the night before surgery. Two control groups (uncastrated male (n=5) and female (n=5)) did not undergo the aneurysm surgery but underwent aorta harvest. All experimental swine were fed β–aminopropionitrile (BAPN) with a weight-based dose of (0.15 g/kg) once daily mixed in whole milk plain yogurt for one week before surgery and daily postoperatively. Weights were obtained weekly.

Abdominal aortic aneurysm swine model

The AAA induction surgery procedure was performed as previously described.8 In short, after induction of general anesthesia the swine abdomen was prepped and draped in sterile fashion and a midline laparotomy performed. Abdominal viscera were displaced and the retroperitoneum incised. The aorta is dissected circumferentially from the renal arteries to the aortic trifurcation. Wire access to the aorta is obtained and the infrarenal aorta is sequentially balloon dilated, perfused with an elastase + collagenase solution, and topically treated with elastase. The abdomen is then irrigated and closed in layers.

Postoperative care and the harvest procedure followed previously published standardized protocol.8 All experimental animals were harvested on postoperative day 28.Ten milliliters of blood was collected from control animals prior to harvest and prior to AAA induction surgery for experimental swine. This was spun for 10 minutes and the plasma was extracted and stored at −80 Celsius. After euthanasia, the aorta was dissected from the trifurcation to the renal arteries and explanted. Samples of the mid-infrarenal aorta of 20 millimeters in size were flash-frozen with liquid nitrogen or fixed in formalin for histology. Size comparisons of the infrarenal treated aorta were performed at time point day 28 between the experimental groups. Histologic and cytokine analyses were performed on the infrarenal aortas for all animals. 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.

17β – Estradiol (E2) Assay

An E2 (Pig) ELISA assay (Abnova, Taipei City, Taiwan) was performed using the isolated plasma from swine blood samples for quantitative determination of 17β – Estradiol levels. The assay procedure and calculation of results was completed according to the manufacturer instructions.

Testosterone Assay

A Testosterone assay was performed using the isolated plasma from swine blood samples for quantitative determination of Testosterone level according to the manufacturer’s instructions (R and D Systems, Minneapolis, MN USA).

Histology

Aortic tissue was fixed in 4% buffered formaldehyde for 24 hours, transferred to 70% ethanol, and then embedded in paraffin. Cross-sectional samples were prepared using Verhoeff-van Gieson trichrome to stain for collagen and elastin. Two independent blinded reviewers scored elastin breaks using a previously published scale.9 Immunohistochemical staining was also performed for anti-mouse α smooth muscle actin (α-SMA) (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), macrophages (anti-rat Mac-2; 1:10,000, Cedarlane Laboratories, Burlington, ON, Canada), macrophage-specific (Galectin-3 ligand; 1:200, Abcam, and CD68 ligand; 1:200, Abcam), M1 macrophages (IL-1β ligand; 1:200, Abcam), M2 macrophages (Arginine type 1; 1:200; Abcam), and CD3 for T cells (1:500; Santa CruzBiotechnology). Visualization color development was completed using alkaline phosphatase (Dako, Glostrup, Denmark) for α-SMA, and diaminobenzidine for Mac-2, anti-neutrophil, and CD3. ·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 described13. Cytokine array was performed on aortic tissue to determine the concentrations of granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN- γ), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-1α, −1β, −1RA, −2, −4, −6, −8, −10, −12, and −18. Using the isolated protein homogenate, cytokine arrays (Millipore Sigma, Bellerica, MA) were completed according to the manufacturer instructions. Samples from the groups were run in duplicate, and the mean value used.

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 4.014.

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 the diameter of the infrarenal aorta at the induction operation just prior to treatment. 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

Male sex lends to increased AAA size

Uncastrated male swine had significantly larger percent increase in aortic diameter compared to castrated males (113.5% ± 11.4% vs 38.1% ± 4.5%, p=0.0012). Interestingly, female swine also had significantly higher percent increase aortic dilation compared to castrated males (96.2% ± 7.5% vs 38.1% ± 4.5%, p=0.0004; Figure 1). Figure 1B demonstrates an aneurysmal swine aorta. Of further interest, there was no significant difference between female and uncastrated males on Day 28 (p = .29) in the swine model. These results then lead us to speculate as to the mechanisms why castrated males demonstrated significant decreases in AAA formation compared with both intact females and males at day 28 in the swine model.

Figure 1. Uncastrated males had significantly higher mean aortic dilation compared to castrated males.

Figure 1.

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A. Swine Aortic Diameter at Day 28 was measured in uncastrated males, females, and castrated males. (113.5% ± 11.4% vs 38.1% ± 4.5%, ** p = .0012). Female swine had significantly higher mean aortic dilation compared to castrated males (96.2% ± 7.5% vs 38.1% ± 4.5%, * p = .0004). There was no significant difference between female and uncastrated males. B. An aneurysmal male swine infrarenal aorta.

Decreased negative characteristics of AAA in castrated male swine following aneurysm induction

We first sought to determine whether estrogen levels could account for these differences. Figure 2A shows serum 17β – Estradiol (estradiol) concentrations between the groups on day 28. Female swine had significantly higher concentrations of estradiol compared with uncastrated males (1590 ± 873.3 ng/mL vs 95.2 ± 2.3 ng/mL, p=0.047, respectively); however, concentrations between females and castrated males did not significantly differ (Figure 2). The average estradiol concentration in uncastrated male serum was also significantly less than castrated males (95.2 ± 2.3 ng/mL vs. 123.3 ± 9.5 ng/mL, p < 0.01, respectively). We also sought to determine whether testosterone serum levels could account for the differences seen in the castrated male swine. Figure 2B demonstrates that castrated male swine did not have significantly lower levels of testosterone as compared to normal males (4.601 ± .7528 ng/mL vs 3.386 ± 1.8 ng/mL, p=0.48, respectively) and these results were between the testosterone levels seen in female swine (1.841 ± .5843 ng/mL vs 3.386 ± 1.8 ng/mL, p=.9048, respectively).

Figure 2. Serum Estradiol levels were significantly higher in females while Testosterone levels were higher in males following AAA.

Figure 2.

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A.17-Beta Estradiol serum concentrations among the three groups at Day 28. (1590 ± 873.3 ng/mL vs 95.2 ± 2.3 ng/mL, # p = .048). Significant differences in serum estradiol were seen between castrated and uncastrated males (123.3 ± 9.5 ng/mL vs 95.2 ± 2.3 ng/mL, ** p = .003). There was no significant difference between serum estradiol between males and castrated males (p = .18). B. Testosterone serum concentrations among the three groups at Day 28( 4.601 ± .7528 ng/mL vs 1.841 ± .5843 ng/mL, # p=.0451 ). Significant differences in serum testosterone levels were seen between females and uncastrated males. There was no significant difference between females and castrated males (1.841 ± .5843 ng/mL vs 3.386 ± 1.8 ng/mL, p=.9048).

Figure 3A displays elastin degradation between the groups on day 28 using Verheoff-van Gieson staining. Uncastrated male AAA demonstrated significantly more elastin degradation compared to female and castrated male swine (p=0.01 and < .01, respectively). However, female swine had significantly more elastin degradation compared with castrated males (p =0.02). Verhoeff-van Gieson staining revealed disruption of the collagen architecture within the aneurysm wall on day 28 (Figure 3B). Collagen content was significantly less in the female AAA than castrated males (p = .01). Statistical analysis did not show a significant difference between uncastrated male AAA collagen content and female or castrated males. Quantification of T lymphocytes and smooth muscle cells did not reveal statistical differences between the groups (Figure 3C and D, respectively). Figure 4 displays macrophage quantification among the different groups. While not significant, overall macrophage numbers trended lower in female swine (Figure 4A and B). Castrated male AAAs had significantly more pro-inflammatory M1 macrophages as measured by IL-1β staining than uncastrated male AAA (p< 0.01). Female AAAs also showed significantly higher levels of the M1 macrophage compared with uncastrated males (p < .01). Female and castrated male AAA did not show a statistical difference in M1 macrophage polarization. The pro-resolving M2 macrophage phenotype as measured by liver arginase was significantly more abundant in castrated male AAAs than uncastrated (p <0 .01).

Figure 3. Immunohistochemistry of swine abdominal aortic aneurysms at Day 28.

Figure 3.

Figure 3.

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Elastin fragmentation was significantly increased in uncastrated males compared to both females and castrated males (A). Collagen was significantly altered in castrated males compared to females (B). CD3+ T-cells and Smooth muscle cells demonstrated no significant difference between the three groups (C and D, respectively). * p < .05, Females vs Castrated Males, # p = .01, Uncastrated males vs Females, ** p < .0001, Uncastrated Males vs Castrated Males.

Figure 4. M1 and M2 macrophage polarization in swine infrarenal aorta as measured by densitometry units at Day 28.

Figure 4.

Figure 4.

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Galectin-3/Mac2 levels trended higher but were non-significant in both male swine groups (uncastrated males vs females, p=0.10; castrated male versus females, p=0.246) (A). CD68 levels trended toward being higher but were not significant in both male swine groups (uncastrated males vs females, p=0.22; castrated males vs females, p=0.166) (B). M1 macrophages were significantly higher in both Females and castrated males compared to uncastrated males (C). Castrated males demonstrated significantly higher levels of M2 macrophages than uncastrated males (D). ** p < .007, Castrated males vs uncastrated males, # p = .0016, Females vs Uncastrated males (L=lumen).

Figure 5 displays pro-MMP2, active-MMP2, pro-MMP9 and active-MMP9 among the three groups compared to both male and female control aorta on day 28. Female AAA demonstrated significantly higher levels of pro-MMP2 and active-MMP2 compared to female control aorta (both p <0.03, Figure 5A and 5B, respectively). However, there was no significant difference in pro-MMP2 and active-MMP2 between female control swine tissue and uncastrated or castrated male swine tissue. Significant differences for active MMP9 were seen between female control aorta and female AAA as well as female control aorta and castrated males (both p <0.03, Figure 5C). Additionally, male control aorta significantly differed from castrated males AAA in active MMP9 (p=0.02, Figure 5C). Finally, MMP9P was significantly increased in uncastrated males (p=0.04), females (p < 0.03), and castrated males (p <0.03) compared to female controls (Figure 5D). These data suggest there are complicated mechanisms at work with regard to MMPs in the swine models and male gonad removal might not be the only mechanism in swine AAA.

Figure 5. Matrix metalloproteinases (MMP) 2A, 2P, 9A, 9P in day 28 swine abdominal aortic aneurysms (AAA) vs day 28 male and female control swine aorta.

Figure 5.

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MMP2A and 2P were significantly higher in female AAAs than female control aorta (A and B, respectively). MMP9A was significantly higher in females and castrated males AAAs compared to female control aorta and higher in castrated males AAAs compared to male control aorta (C). MMP9P was significantly higher in uncastrated males, females, and castrated males AAAs compared to female control aorta (D). # p < .03, Female Control Aorta vs Females AAA; ## p = .02, Male Control Aorta vs Castrated Males AAA; * p = .04, Female Control Aorta vs Uncastrated Males AAA; ** p < .03, Female Control Aorta vs Castrated Males AAA.

Cytokine array on day 28 is demonstrated in Figure 6 for the three groups. Interferon gamma (INF-G) was significantly increased in uncastrated males compared to females and castrated males (both p <0.05). Additionally, proinflammatory cytokine tumor necrosis factor alpha (TNF-a) was significantly higher in uncastrated male swine compared to castrated males (p <0.05). There were no significant differences between interleukin (IL) 1 alpha (IL-1a), IL-1 receptor antagonist (IL-1RA), IL-2, IL-6, IL-8, IL-10, and IL-18.

Figure 6. Swine abdominal aortic aneurysm (AAA) cytokine levels among the three groups at Day 28.

Figure 6.

Figure 6.

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Levels of interferon gamma (IFN-γ), interleukin (IL) 1 alpha (IL-1a), IL-1 receptor antagonist (IL-1RA), IL-2, IL-6, IL-8, IL-10, IL-18, and tumor necrosis factor alpha (TNF-α). # p < .05 Females vs Uncastrated Males. ** p < .05 Castrated Males vs Uncastrated Males.

DISCUSSION

The present study reports sex-based differences in experimental AAA biology using a large animal model. Hynecek and colleagues first described the swine model11, and our laboratory recently modified this protocol in an attempt to produce a more robust and reproducible AAA phenotype. Recently published, this swine model exhibits significant aneurysm formation and progression while also mimicking the ultrastructural remodeling, inflammatory milieu, proteinase expression, and effector cell infiltration that reflect the hallmarks of chronic human AAA biology.8 We sought to expand on this prior work by investigating experimental AAA from a sex and related hormone perspective.

We found uncastrated swine had larger AAAs compared with females and castrated males, while female AAAs were significantly larger than castrated males. For circulating estradiol concentrations, the opposite was true with uncastrated males having the lowest concentrations compared to females and castrated males. The above findings are in accordance with human data published in the Women Health Initiative cohort study showing long-term estrogen replacement therapy was protective against AAA, and parallel experimental animal data that estradiol protects male rats from aneurysm formation.12,13 In an Angiotensin-II murine model, castration/orchiectomies were performed resulting in reduced aneurysm growth and minimal elastolysis.14,15 Likewise, estradiol is known to influence the extracellular matrix by increasing elastin deposition and decreasing collagen deposition.16–18 In the present study, significantly less elastin degradation was observed in the wall of female swine AAAs compared with uncastrated males, and female AAAs showed significantly less collagen deposition compared with castrated males. Furthermore, testosterone has been shown to increase the collagen to elastin ratio, mediating the opposite effect of estradiol.19 Overall, these data also suggest that removal of male gonads leads to decreased aneurysm formation in the swine model; however, it is certain that estradiol and testosterone concentrations are not the sole drivers of elastin degradation and collagen turnover in this model, nor are they in human disease. Further studies of this large animal model will certainly be necessary to shed light on other forces involved in AAA pathogenesis and its relation to human disease.

The pro-inflammatory M1 macrophage and the pro-resolving M2 macrophage were significantly more abundant in castrated male AAA compared with the uncastrated group. This finding contrasts with data published by Henriques et al that showed Angiotensin-II-induced aneurysms in castrated mice had less macrophage infiltration.15 Female swine AAA also showed significantly more M1 polarization than uncastrated swine. The finding that pro-inflammatory macrophages are more abundant in the walls of castrated swine AAA, which have significantly smaller aneurysm size than uncastrated swine, is discordant from experimental rodent AAA data published by our lab.6,13 Macrophages are believed to play an important role in MMP production, perhaps via estrogen-dependent mechanisms as suggested in some animal studies.20,21 The concomitant presence of both the pro- and anti-inflammatory macrophage phenotypes highlight the complexity of the inflammatory milieu and indicate immune deregulation may play a role in this experimental model. Furthermore, sex and sex hormones are likely one of several factors driving aneurysm formation and inflammation, and these factors may act at different timepoints. It is possible that the increased polarization of both M1 and M2 macrophages in the castrated males who also had overall smaller aneurysms were at a different stage of aneurysm development, and reflective of sex, hormones, or macrophages playing a more central force on development at that specific phase.

Statistically significant differences were not observed in MMP2 or MMP9 expression between experimental swine AAA in the present study. Differences were seen with normal swine aorta controls; pro-MMP9P and active MMP-9 expression was significantly higher in female and castrated swine AAA compared with female control aorta. A recent study of human AAAs found increased expression of MMP9 in females with non-thrombus-covered aneurysm wall compared with non-thrombus-covered male AAA wall.22 This human data supports the hypothesis that different elastolytic processes exist between the sexes. The data from the present study supports MMP activation and inactivation are not the sole drive of aneurysm formation. Further, there may be differences at various timepoints, not just at day 28. The present study did not venture to assess the possible role intraluminal thrombus (ILT) has on experimental swine AAAs. However, ILTs were present in some swine AAAs at harvest.

Uncastrated swine AAA tissue revealed on average higher concentrations of IL-8 and IL-10 than female AAA tissue, although this did not achieve statistical significance. IL-10 is a powerful regulatory cytokine and human studies have shown it to be depleted in the serum of patients with known AAA.23 IFN-γ concentrations were significantly higher in the wall of uncastrated male AAA than both females and castrated males, as well as significantly more compared with female and male controls. Uncastrated male swine AAA had greater IL-2 expression compared with castrated male and female swine control aorta. These data likely reflect the inflammatory insult of aneurysm induction surgery owing to statistical differences existing predominantly between control and experimental aorta and not between experimental groups. Cytokine data in the present study is limited to just one time-point (Day 28), and additional time-points would possibly reveal temporal associations of expression and help with interpretation and generalizability. The inflammatory response and immune system’s contribution to AAA pathophysiology is poorly understood. Attempts at identifying biomarkers for AAAs have proved to be difficult as many of the inflammatory cytokines important to AAA formation also overlap with other cardiovascular diseases.24,25 Certain inflammatory mediators are consistently increased in AAA cytokine profiles, but attempts to pharmacologically block these mediators and their respective pathways have also proved ineffective in human disease.9,26–30

Several limitations are apparent in the present study. Uncertainty remains as to which of the multiple interventions employed during the surgical procedure is most critical for AAA formation and progression. While we improved the perfusion aspect of the procedure with practice, it remains variable from animal to animal. An important limitation is that estradiol was quantified without knowledge of the gilt’s pubertal status. Additionally, we did not include an ovarectomized female group and testosterone levels were not measured before and/or after castration to ensure these had decreased prior to surgery. It is possible that sex and sex hormones affect the aneurysm at different timepoints, and that some of the animals featured in these studies were at different stages of AAA development. Future experiments will address these limitations. Despite these limitations, this model could allow for experimentation with targeted drug therapies and endovascular device development.

Conclusions:

In conclusion, these data suggest that while gonad removal in the male swine leads to decreased aneurysm formation, as well as female sex. It does not appear to fully explain nor provide complete aneurysm ablation. herefore suggesting that sex might only be one component of AAA formation in humans as well. Sex-based differences in experimental swine AAA models appear to reflect the human AAA condition and could perhaps represent great pre-clinical large animal models for the testing of possible medical treatment therapies for AAA.

ACKNOWLEDGEMENTS

The authors would like to thank Anthony Herring and Cindy Dodson for their dedication to this project and contributing their expertise in the perioperative and postoperative care of the study animals.

Sources of Funding

This work was supported by American Heart Association Scientist Development Grant 14SDG18730000 (M.S.), R01 HL126668 (G.A.), RO1 HL081629 (G.R.U.), and R01 HL132395 (G.R.U.) grants. This project was supported by Award Number T32HL007849 from the National Heart, Lung, and Blood Institute (NHLBI) (J. Cullen, PI: Irving L. Kron, MD), and by the Thoracic Surgery Foundation for Research and Education (TSFRE) Research Grant (PI: G. Ailawadi). The content is solely the responsibility of the authors and does not necessarily represent the views of the NHLBI or the TSFRE.

Non-standard Abbreviation and Acronyms:

AAA

Abdominal aortic aneurysm

ACTA2

smooth muscle α-actin

BAPN

B-aminopropionitrile

ECM

extracellular matrix

MMP2

Matrix metallo-proteinase 2

MMP9

Matrix metallo-proteinase 9

MYH11

smooth muscle cell myosin heavy chain

SM22α

transgelin

VSMC

vascular smooth muscle cell

Footnotes

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Disclosures

None.

Conflicts of Interest: None

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