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Dietary phytoestrogens inhibit experimental aneurysm formation in male mice

Published:December 05, 2013DOI:https://doi.org/10.1016/j.jss.2013.11.1108

      Abstract

      Background

      The purpose of these experiments was to test the hypothesis that dietary phytoestrogens would diminish experimental aortic aneurysm formation.

      Materials and methods

      Six-wk-old C57BL/6 mice were divided into groups, fed either a diet with minimal phytoestrogen content or a regular commercial rodent diet with high phytoestrogen content for 2 wk. At the age of 8 wk, aortic aneurysms were induced by infusing the isolated infrarenal abdominal aorta with 0.4% elastase for 5 min. Mice were recovered and the diameter of the infused aorta was measured at postoperative days 3, 7, and 14. Abdominal aorta samples were collected for histology, cytokine array, and gelatin zymography after aortic diameter measurement. Blood samples were also collected to determine serum phytoestrogens and estradiol levels. Multiple-group comparisons were done using an analysis of variance with post hoc Tukey tests.

      Results

      Compared with mice on a minimal phytoestrogen diet, mice on a regular rodent diet had higher levels of serum phytoestrogens (male, 1138 ± 846 ng/dL; female, 310 ± 295 ng/dL). These serum phytoestrogen levels were also much higher than their own endogenous estradiol levels (109-fold higher for males and 35.5-fold higher for females). Although aortic diameters of female mice were unaffected by the phytoestrogen concentration in the diets, male mice on the regular rodent diet (M+ group) developed smaller aortic aneurysms than male mice on the minimal phytoestrogen diet (M− group) on postoperative day 14 (M+ 54.8 ± 8.8% versus M− 109.3 ± 37.6%; P < 0.001). During aneurysm development (postoperative days 3 and 7), there were fewer neutrophils, macrophages, and lymphocytes in the aorta from the M+ group than from the M− group. Concentrations of multiple proinflammatory cytokines (matrix metalloproteinases [MMPs]; interleukin 1β [IL-1β]; IL-6; IL-17; IL-23; monocyte chemoattractant protein-1; regulated on activation, normal T cell expressed and secreted; interferon γ; and tumor necrosis factor α) from aortas of the M+ group were also lower than those from the aortas of the M− group. Zymography also demonstrated that the M+ group had lower levels of aortic MMP-9s than the M− group on postoperative day 14 (P < 0.001 for pro-MMP-9, P < 0.001 for active MMP-9).

      Conclusions

      These results suggest that dietary phytoestrogens inhibit experimental aortic aneurysm formation in male mice via a reduction of the inflammatory response in the aorta wall. The protective effect of dietary phytoestrogens on aneurysm formation warrants further investigation.

      Keywords

      1. Introduction

      Abdominal aortic aneurysms (AAAs) are a gender-related disease with a prevalence of a male to female ratio of 4:1. Estrogens play a protective role in the development of AAAs [
      • Woodrum D.T.
      • Ford J.W.
      • Cho B.S.
      • et al.
      Differential effect of 17-beta-estradiol on smooth muscle cell and aortic explant MMP2.
      ,
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      • Tham D.M.
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      17β-Estradiol attenuates development of angiotensin II–induced aortic abdominal aneurysm in apolipoprotein E–deficient mice.
      ,
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      • Hannawa K.K.
      • Pearce C.G.
      • et al.
      Tamoxifen up-regulates catalase production, inhibits vessel wall neutrophil infiltration, and attenuates development of experimental abdominal aortic aneurysms.
      ]. Phytoestrogens are plant-derived chemicals that are strikingly similar to estrogens both in structure and function. Therefore, the potential benefits and risks of phytoestrogen exposure have already attracted much attention [
      • Cassidy A.
      Potential risks and benefits of phytoestrogen-rich diets.
      ,
      • van der Schouw Y.T.
      • de Kleijn M.J.
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      • Grobbee D.E.
      Phytoestrogens and cardiovascular disease risk.
      ,
      • Hall W.L.
      • Vafeiadou K.
      • Hallund J.
      • et al.
      Soy-isoflavone-enriched foods and inflammatory biomarkers of cardiovascular disease risk in postmenopausal women: interactions with genotype and equol production.
      ,
      • Anderson J.J.
      • Anthony M.
      • Messina M.
      • Garne S.C.
      Effects of phyto-oestrogens on tissues.
      ,
      • McCarty M.F.
      Isoflavones made simple—genistein's agonist activity for the beta-type estrogen receptor mediates their health benefits.
      ,
      • Jackman K.A.
      • Woodman O.L.
      • Sobey C.G.
      Isoflavones, equol and cardiovascular disease: pharmacological and therapeutic insights.
      ]. Major sources of phytoestrogens include soybeans, alfalfa, and flaxseed. Phytoestrogens are selective estrogen receptor (ER) modulators and have anti-inflammatory, antioxidant, and antiproliferative properties [
      • Ondricek A.J.
      • Kashyap A.K.
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      A comparative study of phytoestrogen action in mitigating apoptosis induced by oxidative stress.
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      • Tiulea C.
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      • Dehelean C.
      • Motoc A.
      A comparison regarding antiproliferative action between soy total extract and genistein.
      ,
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      • Pepper M.S.
      • Montesano R.
      • et al.
      Phytoestrogens and inhibition of angiogenesis.
      ,
      • Yu X.
      • Zhu J.
      • Mi M.
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      • Pan Q.
      • Wei M.
      Anti-angiogenic genistein inhibits VEGF-induced endothelial cell activation by decreasing PTK activity and MAPK activation.
      ,
      • Xu J.X.
      • Zhang Y.
      • Zhang X.Z.
      • Ma Y.Y.
      Anti-angiogenic effects of genistein on synovium in a rat model of type II collagen-induced arthritis.
      ]. Animal experiments have demonstrated that phytoestrogens can reduce plasma cholesterol and attenuate atherosclerosis [
      • Kirk E.A.
      • Sutherland P.
      • Wang S.A.
      • Chait A.
      • LeBoeuf R.C.
      Dietary isoflavones reduce plasma cholesterol and atherosclerosis in C57BL/6 mice but not LDL receptor-deficient mice.
      ,
      • Yamakoshi J.
      • Piskula M.K.
      • Izumi T.
      • et al.
      Isoflavone aglycone-rich extract without soy protein attenuates atherosclerosis development in cholesterol-fed rabbits.
      ,
      • Clarkson T.B.
      • Anthony M.S.
      • Morgan T.M.
      Inhibition of postmenopausal atherosclerosis progression: a comparison of the effects of conjugated equine estrogens and soy phytoestrogens.
      ]. However, little is known regarding the effects of phytoestrogens on aortic aneurysm formation. Therefore, we hypothesized that dietary supplementation with phytoestrogens might reduce the inflammation in the aortic wall and thus inhibit aneurysm formation in an experimental model.

      2. Materials and methods

      2.1 Experimental design

      Experiment 1 (gender-based study): 32 (n = 16 males and 16 females) 6-wk-old wild-type C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) were divided into four groups of eight mice based on dietary phytoestrogen exposure to determine the influence of phytoestrogen content on aortic aneurysm formation. Thus, four groups were evaluated: (1) male mice fed a diet with minimal phytoestrogen (M−), (2) male mice fed a regular diet (M+), (3) female mice fed a diet with minimal phytoestrogen (F−), and (4) female mice fed a regular diet (F+). The isoflavone content, one of the major classes of phytoestrogens, ranged from non-detectable to 20 mg/kg for the minimal phytoestrogen diet (2016 Teklad Global 16% Protein Rodent Diet, Madison, WI), whereas the regular diet (7012 Teklad LM485 Mouse/Rat Diet, Madison, WI) had between 300 and 500 mg/kg. The other ingredients in the both diets were similar (see www.harlan.com). Both rodent diets were commercially available and obtained on April 2011. Two weeks after mice were placed on the diets, AAAs were induced surgically [
      • Thompson R.W.
      • Curci J.A.
      • Ennis T.L.
      • Mao D.
      • Pagano M.B.
      • Pham C.T.
      Pathophysiology of abdominal aortic aneurysms: insights from the elastase-induced model in mice with different genetic backgrounds.
      ,
      • DiMusto P.D.
      • Lu G.
      • Ghosh A.
      • et al.
      Increased PAI-1 in females compared with males is protective for abdominal aortic aneurysm formation in a rodent model.
      ].
      Briefly, infrarenal abdominal aorta was isolated and infused in situ with porcine pancreatic elastase (0.4 U/mL; Sigma, St. Louis, MO) for 5 min at a pressure of 100 mm Hg. Elastase solution was evacuated and the mice were allowed to recover. Mice abdominal aortic diameters (n = 8/group) were measured immediately after infusion 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. Blood samples were collected immediately after the measurement of the aorta diameter. Aorta samples were harvested for protein analysis (n = 5/group) and histologic studies (n = 3/group). Serum samples were used to determine phytoestrogens and estradiol levels.
      Experiment 2 (time course study): 6-wk-old wild-type C57BL/6 male mice (n = 24) were divided into two groups based on diet. As mentioned previously, one group was fed with the minimal phytoestrogen 2016 diet, whereas the other group was maintained on the phytoestrogen-rich 7012 diet. At the age of 8 wk, all mice underwent elastase infusion. To evaluate AAA formation at earlier time points, six mice in each group were evaluated at postoperative days 3 and 7. After aortic diameters were measured (n = 6/group), samples were collected for histology (n = 3/group) and protein analysis (n = 3/group).
      All experiments were conducted in accordance with the standards approved by the Animal Care and Use Committee of University of Virginia (IACUC #3848).

      2.2 Mass spectrometry

      Mass spectrometry was performed on serum samples to identify and quantify four common phytoestrogens in serum. Four standards were used in the test (Sigma, St Louis, MO; purity: daidzein ≥97%, genistein ≥98%, equol ≥99%, and coumestrol ≥95%). The test used a liquid chromatography–mass spectrometry system, which consisted of a Thermo Electron TSQ Quantum Access MAX mass spectrometer system with a Protana nanospray ion source (Thermo Scientific Inc, San Jose, CA) interfaced to a self-packed 8 cm × 75 μm (inner diameter) Phenomenex Jupiter 10 μm C18 reversed-phase capillary column (Phenomenex Inc, Torrance, CA). Samples were prepared as published [
      • Morandi S.
      • Locati D.
      • Ferrario F.
      • Chiesa G.
      • Arnoldi A.
      A simple method for the characterization and quantification of soy isoflavone metabolites in the serum of MMTV-Neu mice using high-performance liquid chromatography/electrospray ionization mass spectrometry with multiple reaction monitoring.
      ]. Dry samples were resuspended using 5% ethanol to a volume equal to 25% of the plasma volume processed. Samples were vortexed and spun at 14K rpm. A 5-μL aliquot of each extract was injected and four compounds were eluted from the column by an acetonitrile of 0.1 mol per liter acetic acid gradient at a flow rate of 0.5 μL per min over 0.5 h. Nanospray ion source was operated at −3 kV (negative ions). Data were analyzed using the peak area multiple reaction monitoring for each compound in each sample and comparing it with areas obtained for standard curves generated at the start and end of the sample set. The limit of detection and limit of quantification were 0.5 and 5 fmol per mL, respectively.

      2.3 Radioimmunoassay

      To determine circulating estradiol concentration in the serum, a 200-μL aliquot of each serum sample was used for radioimmunoassay with a commercial kit from Siemens Healthcare Diagnostic Inc (Tarrytown, NY). TKE21 kit was used to determine the serum estradiol levels according to the manufacturer's instructions at the Ligand Assay and Analysis Core Facility at the University of Virginia Center for Research in Reproduction.

      2.4 Histology and immunohistochemical stainings

      Aortic specimens were fixed in 4% buffered formaldehyde for 24 h, transferred to 70% ethanol, and subsequently embedded in paraffin. Aortic cross-sections were stained with Verhoeff-Van Gieson and picrosirius red for elastin and collagen, respectively. Immunohistochemical stainings for neutrophils, migratory macrophages, CD3+ T cells, and smooth muscle actin-α were performed as published [
      • Lau C.L.
      • Zhao Y.
      • Kron I.L.
      • et al.
      The role of adenosine A2A receptor signaling in bronchiolitis obliterans.
      ,
      • Zhao Y.
      • LaPar D.J.
      • Steidle J.
      • et al.
      Adenosine signaling via the adenosine 2B receptor is involved in bronchiolitis obliterans development.
      ,
      • Zhao Y.
      • Xiao A.
      • diPierro C.G.
      • et al.
      An extensive invasive intracranial human glioblastoma xenograft model: role of high level matrix metalloproteinase 9.
      ]. For grading, integrated optical density value of positive staining areas of each section was randomly selected and measured. Four randomly selected 20× fields per aneurysm section were assessed for the density of staining by blinded observers.

      2.5 Angiogenesis and cytokine arrays

      Protein from aortic samples was isolated and pooled protein (30 μg) from each group was used in proteome profiler mouse cytokine array and proteome profiler mouse angiogenesis array according to the manufacturer's instruction (R&D Systems, Minneapolis, MN; cat# ARY 006, ARY 015). All samples were run in duplicate and the mean value was used to represent each cytokine level.

      2.6 Zymography

      Using aortic protein isolated from the samples, gelatin zymography was performed to determine the amounts of matrix metalloproteinase (MMP)-2 and MMP-9. Precast zymography gels (Invitrogen, Grand Island, NY) were loaded with 3 μg of tissue protein from each aortic sample diluted into 2× Tris–glycine sodium dodecyl sulfate sample buffer and electrophoretically separated under nonreducing conditions. The gels were renatured for 30 min in renaturing buffer (Invitrogen) and incubated in the developing buffer (Invitrogen) for 24 h at 37°C rocker. Then, the gels were stained in Simply Blue Safe Stain (Invitrogen). Pro and active forms of MMP-9 and MMP-2 appeared as a clear band against the blue background. Quantification was determined according to the optical density using Bio-Rad Image Lab Software version 4.0 (Bio-Rad Laboratories, Inc, Hercules, CA).

      2.7 Statistical analysis

      The overall difference among groups, together with gender and diet factor, was determined using a two-way analysis of variance. Post hoc Tukey test was used for multiple comparisons between groups (PRISM 5; GraphPad Software, La Jolla, CA). Data are presented as the mean ± standard deviation. P value <0.05 was considered statistically significant.

      3. Results

      3.1 Reduced AAA diameter in male mice on the phytoestrogen-rich diet

      In minimal phytoestrogen diet groups, aortic dilation was significantly greater in male mice compared with females (M− 109.3 ± 37.6% versus F− 52.9 ± 11.5%; P < 0.001); in phytoestrogen-rich diet groups, this difference was negated. The aortic dilation of male mice was similar to that of female mice (M+ 54.8 ± 8.8% versus F+ 67.1 ± 17.4%; P = 0.68). Comparing the two male groups on different diets, mice on the phytoestrogen-rich diet had significantly smaller aortic dilation than male mice on the minimal phytoestrogen diet (M+ 54.8 ± 8.8% versus M− 109.3 ± 37.6%; P < 0.001). M+ group also had decreased incidence of AAA (75%) compared with the M− group (100%). There were no statistical differences between two female groups either in aortic dilation or in AAA incidence (Fig. 1A and B). Aortic dilation increased over time after elastase perfusion. Aortic dilations were similar in male mice fed the minimal phytoestrogen diet or phytoestrogen-rich diet at days 3 and 7. Between days 7 and 14, dilation progressed in male mice fed the minimal phytoestrogen diet, whereas further dilation was halted in male mice fed the phytoestrogen-rich diet (Fig. 2).
      Figure thumbnail gr1
      Fig. 1(A) Comparison of infrarenal aorta diameter. On postoperative day 14, the dilation of male mice on the minimal phytoestrogen diet (M−) was more obvious than the other groups (M+, F−, and F+). (B) Comparison of infrarenal abdominal aortic dilation on postoperative day 14. N = 8 mice per group. Male mice on the minimal phytoestrogen diet (M−) had larger dilation than those of other groups.P value less than 0.05 is considered significant.
      Figure thumbnail gr2
      Fig. 2Comparison of infrarenal abdominal aorta dilation of male mice on different diets. Male mice on the phytoestrogen-rich diet (M+) failed to dilate during the second week compared with male mice on the minimal phytoestrogen diet (M−). D0-pre means day 0 before infusion. D0-post means day 0 after infusion. D3, D7, and D14 mean days 3, 7, and 14 after infusion, respectively.

      3.2 Significant higher level of serum phytoestrogens in mice on the phytoestrogen-rich diet

      At postoperative day 14, phytoestrogen levels were non-detectable in both groups of mice on the minimal phytoestrogen diet (M− and F−), whereas the other two groups on the phytoestrogen-rich diet had much higher levels of total phytoestrogen in their sera (M+, 1138 ± 846 ng/dL; F+, 310 ± 295 ng/dL). The predominant phytoestrogen was equol (M+, 87.3%; F+, 65.5%). The total serum phytoestrogen levels of M+ and F+ groups were significantly higher than their own endogenous estradiol levels, respectively (M+, 109-fold; F+, 35.5-fold) (Table 1).
      Table 1Serum phytoestrogens and endogenous estrogen.
      GroupGenderDietCoumestrolDaidzeinGenisteinEquolTotal phytoestrogenEstradiol
      F−Female7012 TekladNDNDNDNDND10.11 ± 1.16
      M−Male7012 TekladNDNDNDNDND11.74 ± 0.77
      F+Female2016 TekladND63 ± 9944 ± 45203 ± 152310 ± 2958.74 ± 1.44
      M+Male2016 TekladND85 ± 6660 ± 40993 ± 7941138 ± 84610.40 ± 1.04
      ND = non-detectable.
      All units were expressed as nanograms per deciliter. Eight mice per group. In mice on the phytoestrogen-rich diet (2016 Teklad), the total serum phytoestrogen levels were much higher than their endogenous estradiol levels (109-fold in males and 35.5-fold in females).

      3.3 Decreased inflammatory cell infiltration in the early stages of AAA formation in male mice on phytoestrogen-rich diet

      Compared with the M− group, male aortas from mice fed the phytoestrogen-rich diet (M+) had fewer lymphocytes on postoperative day 7 (P = 0.02). The phytoestrogen-rich diet group also had fewer neutrophils on postoperative day 7 and fewer macrophages on postoperative day 3 (Fig. 3A and B). By postoperative day 14, aorta samples of the M− group demonstrated obvious elastin fragmentation and disorganization and increased collagen deposition in the media and adventitia of aorta, whereas the elastin and collagen contents in the M+ group were relatively intact, similar to the female groups (Fig. 4A and B). Male groups still contained more inflammatory cells in the aorta compared with the female groups on postoperative day 14. However, no diet-related differences in inflammatory cell staining were found at this time point (Fig. 4C–E). There were no differences in smooth muscle actin-α staining among groups.
      Figure thumbnail gr3a
      Fig. 3(A) Representative immunohistochemistry samples of postoperative days 3 and 7 demonstrating macrophage, neutrophil, and lymphocyte infiltration in aorta. Macrophage and neutrophil were red, CD3+ T cell was brown in their respective staining. On postoperative days 3 and 7, there were more inflammatory cells in the M− group than in the M+ group (marked by arrows). (B) Quantification of immunohistochemistry evaluating neutrophil, macrophage, and T cell in aorta samples of postoperative days 3 and 7. N = 3 mice per group. M− group had more T cells in aorta than the M+ group. D3 and D7 mean postoperative days 3 and 7, respectively.
      Figure thumbnail gr3b
      Fig. 3(A) Representative immunohistochemistry samples of postoperative days 3 and 7 demonstrating macrophage, neutrophil, and lymphocyte infiltration in aorta. Macrophage and neutrophil were red, CD3+ T cell was brown in their respective staining. On postoperative days 3 and 7, there were more inflammatory cells in the M− group than in the M+ group (marked by arrows). (B) Quantification of immunohistochemistry evaluating neutrophil, macrophage, and T cell in aorta samples of postoperative days 3 and 7. N = 3 mice per group. M− group had more T cells in aorta than the M+ group. D3 and D7 mean postoperative days 3 and 7, respectively.
      Figure thumbnail gr4
      Fig. 4Representative samples of postoperative day 14, demonstrating elastin fragmentation (Van Gieson) and collagen disruption (picrosirius red) in the M− group (A and B). Quantification of immunohistochemistry evaluating neutrophils, macrophages, T cells, and smooth muscle cells (C–F). N = 3 mice per group. P value less than 0.05 is considered significant.

      3.4 Lower inflammatory cytokine levels in the aorta of male mice on the phytoestrogen-rich diet

      Several dozen cytokines of interest and MMPs were analyzed in the present study. According to the arrays' result, the following MMPs and cytokines were found more closely related to aneurysm formation in this animal model: MMP-9; tissue inhibitor of metalloproteinase 1 (TIMP-1); interleukin 1β (IL-1β); IL-6; IL-17; IL-23; monocyte chemoattractant protein-1; regulated on activation, normal T cell expressed and secreted; tumor necrosis factor α; and interferon γ. The array analysis indicated that most of these MMPs and inflammatory cytokines were upregulated in the M− group at postoperative day 3, 7, or 14, whereas the M+ group had relatively lower levels of the previously mentioned proteins in their aortas (Table 2, Figs. 5 and 6).
      Table 2Upregulated cytokine levels in the M− group at different time points.
      CytokineDay 3Day 7Day 14
      TNF-α0.452.580.07
      INF-γ0.295.630.17
      IL-1β1.811.480.91
      IL-60.091.704.15
      IL-170.820.560.00
      IL-230.404.360.89
      MCP-10.392.360.46
      RANTES0.932.990.27
      TIMP-10.130.660.18
      MMP-9 (pro and active)7.97
      INF-γ = interferon γ; M− = male mice on minimal phytoestrogen diet; M+ = male mice on phytoestrogen-rich diet; MCP-1 = monocyte chemoattractant protein-1; RANTES = regulated on activation, normal T cell expressed and secreted; TNF-α = tumor necrosis factor α.
      Cytokine levels are presented in the fold of increase compared with the M+ group.
      Figure thumbnail gr5
      Fig. 5Cytokine array of mouse aorta samples of postoperative days 3 and 7. Three pooled samples per group. Multiple inflammatory cytokines were upregulated in the M− group compared with the M+ group on the same experiment end point. D3 and D7 mean postoperative days 3 and 7, respectively. M+ means male mice on the phytoestrogen-rich diet; M− means male mice on the minimal phytoestrogen diet. INF-γ = interferon γ; MCP-1 = monocyte chemoattractant protein-1; RANTES = regulated on activation, normal T cell expressed and secreted; TNF-α = tumor necrosis factor α.
      Figure thumbnail gr6
      Fig. 6Cytokine array of postoperative day 14 mouse aorta samples. Five pooled samples per group. IL-6, IL-1β, IL-23, monocyte chemoattractant protein-1, and MMP-9 were upregulated in male mice on the minimal phytoestrogen diet (M−) compared with the M+ group. There were also gender differences in the levels of multiple inflammatory cytokines. INF-γ = interferon γ; MCP-1 = monocyte chemoattractant protein-1; RANTES = regulated on activation, normal T cell expressed and secreted; TNF-α = tumor necrosis factor α.

      3.5 Less MMP-9s in the aorta of male mice on the phytoestrogen-rich diet

      MMPs are known to be critical in blood vessel matrix degradation and AAA formation. Therefore, gelatin zymography was used to further evaluate MMP activities. On postoperative day 14, the male group fed the phytoestrogen-rich diet (M+) had much lower MMP-9s levels in their aortas than the male group fed the minimal phytoestrogen diet (M−) (P < 0.001 for pro-MMP-9, P < 0.001 for active MMP-9). Within the two groups of mice fed the minimal phytoestrogen diet (F− versus M−), the female group also had less MMP-9s compared with the male group (P = 0.001 for pro-MMP-9, P < 0.001 for active MMP-9) (Fig. 7). MMP-2s had similar trends at postoperative day 14 as MMP-9s, but the differences were not significant. The present study found no differences in MMPs levels among all groups on postoperative days 3 and 7 (data not shown).
      Figure thumbnail gr7
      Fig. 7Gelatin zymography of postoperative day 14 samples (A) demonstrating a significant higher level of both pro and active forms of MMP-9s in male mice on the minimal phytoestrogen diet (B and C). There were similar trends for pro and active forms of MMP-2s (D and E).

      4. Discussion

      In the present study, we tested four phytoestrogens that are most commonly found in commercial rodent diets. Our study showed that mice on the regular commercial rodent diet had much higher levels of serum phytoestrogens than mice on the minimal phytoestrogen diet. The phytoestrogens in our study were mainly in the form of equol (87.3% in males, 65.5% in females). Equol is the metabolite of daidzein with a longer half-life and greater biological activity. We also tested endogenous serum estradiol levels to compare it with phytoestrogen levels in the blood. Our results showed that the total phytoestrogen levels in mice on the regular commercial rodent diet were alarmingly higher than their own endogenous estradiol levels (109-fold for males, 35.5-fold for females). Phytoestrogens come mainly from soybean in the diet. Phytoestrogen concentration in soybean varies from harvest to harvest, so does the phytoestrogen content in each batch of the rodent diet. Unfortunately, not all investigators doing experimental AAA studies are aware of the influence of phytoestrogens on AAA formation. Our study demonstrated that it is critical to use the minimal phytoestrogen diet in experimental AAA investigations.
      Gender-related differences in aneurysm formation have been well documented in both human and animal studies. Male gender is a well-known risk factor for AAA development [
      • Lederle F.A.
      • Johnson G.R.
      • Wilson S.E.
      Abdominal aortic aneurysm in women.
      ,
      • Katz D.J.
      • Stanley J.C.
      • Zelenock G.B.
      Gender differences in abdominal aortic aneurysm prevalence, treatment, and outcome.
      ,
      • Bengtsson H.
      • Sonesson B.
      • Bergqvist D.
      Incidence and prevalence of abdominal aortic aneurysms, estimated by necropsy studies and population screening by ultrasound.
      ]. Experiments with rodent AAA models have demonstrated that male animals develop a positive AAA phenotype compared with female animals [
      • DiMusto P.D.
      • Lu G.
      • Ghosh A.
      • et al.
      Increased PAI-1 in females compared with males is protective for abdominal aortic aneurysm formation in a rodent model.
      ,
      • DiMusto P.D.
      • Lu G.
      • Ghosh A.
      • et al.
      Increased JNK in males compared with females in a rodent model of abdominal aortic aneurysm.
      ,
      • Ailawadi G.
      • Eliason J.L.
      • Roelofs K.J.
      • et al.
      Gender differences in experimental aortic aneurysm formation.
      ]. In the present study, gender-related differences in AAA phenotype between the two groups of mice on the minimal phytoestrogen diet (M− versus F− group) were clearly demonstrated (109.3 ± 37.6% versus 52.9 ± 11.5%; P < 0.001). However, gender-related differences were obviated with phytoestrogen-rich diets, as both male and female mice had similar aortic dilation. Both male and female mice on the phytoestrogen-rich diet had decreased aortic dilation in comparison with male mice on the minimal phytoestrogen diet. Male mice on the phytoestrogen-rich diet (M+ group) had a much lower aortic dilation than male mice on the minimal phytoestrogen diet (M− group) (54.8 ± 8.8% versus 109.3 ± 37.6%; P < 0.001), whereas the aortic dilation of both female groups was low and similar. These results suggested that the phytoestrogen-rich diet blunted the aortic dilation that is normally seen in the male mice in the gender-related AAA study. Because female mice are protected against AAA formation, it is suspected that phytoestrogens would not have a further impact on AAA protection.
      Statistic analysis showed that the interaction of gender and diet factor accounted for 31.33% of the total aortic dilation variance (F = 19.56, P = 0.0001). This interaction indicates that the male and female mice had different responses to the same diet factor. The gender factor in this experiment accounted for approximately 12.97% of the total variance in aortic dilation (F = 8.094, P = 0.0082). The effect is considered very significant. The diet factor in the experiment accounted for about 10.83% of the total variance (F = 6.760, P = 0.0147) and the effect is considered significant.
      When evaluating aneurysm formation over time, our results showed that the diets did not make a significant difference in aortic dilation during the early stages of AAA (postoperative days 3 and 7). Male mice on the minimal phytoestrogen diet gained much of their aorta dilation during the second week (P = 0.012), which is one of the characteristics of the elastase-induced model [
      • Thompson R.W.
      • Curci J.A.
      • Ennis T.L.
      • Mao D.
      • Pagano M.B.
      • Pham C.T.
      Pathophysiology of abdominal aortic aneurysms: insights from the elastase-induced model in mice with different genetic backgrounds.
      ]. On the contrary, the aortic diameter of male mice on the phytoestrogen-rich diet remained stable over the second half of the experimental model (P = 0.999), indicating the importance of diet. Diet factor in the time course experiment accounted for approximately 4.409% of all the variance (F = 22.21, P < 0.0001). However, the previously mentioned results do not mean that dietary phytoestrogens affect only the later stage of aneurysm formation. Aorta dilation in the early stages of this model reflects mainly the mechanical pressure and elastase digestion on the blood vessel, whereas the aortic wall degradation in the later stages of the AAA model is caused by inflammation, which mimics the human aneurysm formation. The logical implication of this observation was that dietary phytoestrogens might reduce aneurysm formation through anti-inflammation properties.
      In the elastase-induced AAA model, the inflammation in the aorta wall usually peaks around 5–7 d after surgery, then elastin and collagen disruption and remodeling of the aorta occurs. Our histology demonstrated that there was more inflammatory cell infiltration in the aortas of male mice on the minimal phytoestrogen diet on postoperative day 7 compared with male mice fed a diet rich in phytoestrogens. On postoperative day 14, the histology showed increased elastin fragmentation and collagen deposition in the animals on the minimal phytoestrogen diet. In contrast, male mice on the phytoestrogen-rich diet experienced less inflammation during the early stage of this model, indicating the anti-inflammatory effects of phytoestrogen. Also in this model, aortic dilation usually peaks around postoperative day 14, then, the aorta begins to heal. In the present study, there were still more inflammatory cells left in male samples than in female samples on postoperative day 14. Endogenous estrogen is believed to account for this gender-related difference. We are not sure why there were no diet-related differences at this time point, but hypothesized that the anti-inflammatory effects of phytoestrogen may be detected more easily at the peak of inflammation. By day 14, the acute injury is basically resolving, so gender-related differences at this particular point might have more influence on the recovery process, not in the formation of AAA.
      To explore the mechanisms behind diet-related phenotypic differences, multiple MMPs and cytokines in aortic samples were examined. Although the function of each of these proinflammatory molecules in aneurysm formation has been studied extensively [
      • DiMusto P.D.
      • Lu G.
      • Ghosh A.
      • et al.
      Increased JNK in males compared with females in a rodent model of abdominal aortic aneurysm.
      ,
      • Ailawadi G.
      • Eliason J.L.
      • Roelofs K.J.
      • et al.
      Gender differences in experimental aortic aneurysm formation.
      ,
      • Sharma A.K.
      • Lu G.
      • Jester A.
      • et al.
      Experimental abdominal aortic aneurysm formation is mediated by IL-17 and attenuated by mesenchymal stem cell treatment.
      ,
      • Johnston W.F.
      • Salmon M.
      • Su G.
      • et al.
      Genetic and pharmacologic disruption of interleukin-1β signaling inhibits experimental aortic aneurysm formation.
      ,
      • Ailawadi G.
      • Knipp B.S.
      • Lu G.
      • et al.
      A nonintrinsic regional basis for increased infrarenal aortic MMP-9 expression and activity.
      ,
      • Moehle C.W.
      • Bhamidipati C.M.
      • Alexander M.R.
      • et al.
      Bone marrow-derived MCP1 required for experimental aortic aneurysm formation and smooth muscle phenotypic modulation.
      ,
      • Ehrlichman L.K.
      • Ford J.W.
      • Roelofs K.J.
      • et al.
      Gender-dependent differential phosphorylation in the ERK signaling pathway is associated with increased MMP2 activity in rat aortic smooth muscle cells.
      ,
      • Sinha I.
      • Cho B.S.
      • Roelofs K.J.
      • Stanley J.C.
      • Henke P.K.
      • Upchurch Jr., G.R.
      Female gender attenuates cytokine and chemokine expression and leukocyte recruitment in experimental rodent abdominal aortic aneurysms.
      ,
      • Sinha I.
      • Hannawa K.K.
      • Eliason J.L.
      • et al.
      Early MT-1 MMP expression following elastase exposure is associated with increased cleaved MMP-2 activity in experimental rodent aortic aneurysms.
      ,
      • Colonnello J.S.
      • Hance K.A.
      • Shames M.L.
      • et al.
      Transient exposure to elastase induces mouse aortic wall smooth muscle cell production of MCP-1 and RANTES during development of experimental aortic aneurysm.
      ,
      • Eagleton M.J.
      • Peterson D.A.
      • Sullivan V.V.
      • et al.
      Nitric oxide inhibition increases aortic wall matrix metalloproteinase-9 expression.
      ], little is known regarding how these inflammatory biomarkers in aorta are regulated in the presence of dietary phytoestrogens. In our study, each of these inflammatory cytokines in male mice on the minimal phytoestrogen diet (M−) was upregulated at different time points. These findings demonstrate that phytoestrogens impact AAA formation process through not one, but multiple cytokine pathways.
      Of all the MMPs and cytokines evaluated in the present study, the most interesting ones were MMP-9/TIMP-1 pair. First, the results demonstrated that the rodent diets in the present study have a significant impact on MMPs levels in the aorta. For pro-MMP-9, diet factors accounted for approximately 22.55% of the total variance (F = 9.90, P = 0.0071), which was considered very significant. The diet factor for active MMP-9 was about 30.35% of the total variance (F = 19.89, P = 0.0005, extremely significant). The diet factor of pro-MMP-2 and active MMP-2 was 24.6% (F = 5.66, P = 0.0321, significant), and 17.73% (F = 3.75, P = 0.0734, not significant), respectively. Second, on postoperative day 14, male mice on the minimal phytoestrogen diet (M−) had significantly higher levels of MMP-9 activity, but the increase of TIMP-1 was not in proportion to the increase in MMP-9. This change in an MMP/TIMP-1 ratio was consistent with the aortic remodeling time frame and the positive aneurysm phenotype of the M− group. Considering MMPs' critical role in matrix degradation, the high level of MMP-9 activity in the second week may play an important role in the aorta dilation in the M− group.
      The anti-inflammatory mechanisms of phytoestrogens are complicated. It could be ER-dependent or ER-independent, or both. Phytoestrogens have the ability to bind both ER-α and ER-β, but with a preference for ER-β. These exogenous phytoestrogens may compete with endogenous estrogens on the same ERs. Although phytoestrogens' affinities for ERs are weak, they can be present in large amounts in the blood. Depending on their concentrations, they can elicit estrogenic or antiestrogenic effects [
      • Johnston I.
      • Williamson G.
      Phytochemical functional foods.
      ,
      • Ososki A.L.
      • Kennelly E.J.
      Phytoestrogens: a review of the present state of research.
      ,
      • Brown N.M.
      • Setchell K.D.
      Animal models impacted by phytoestrogens in commercial chow: implications for pathways influenced by hormones.
      ]. The ER-independent anti-inflammatory effect probably relies on phytoestrogen's antioxidant, antiproliferative, and antiangiogenic properties. Although the present study demonstrated that phytoestrogens reduced the inflammation in the aorta of male mice, the results did not indicate if this anti-inflammatory effect was ER-dependent or ER-independent. Experiments with ER knockout animals would be helpful to detail the mechanisms. Because MMPs play such an important role in AAA formation, using MMP-9 and TIMP-1 knockout mice in future experiments could also help us understand the relationship between phytoestrogen and MMPs. Other AAA animal models (e.g., angiotensin II model) would be useful to investigate the different mechanisms at play in a model with advanced inflammation.

      5. Conclusion

      The present study showed that dietary phytoestrogens inhibit male experimental aneurysm formation by reducing the inflammatory response in the aorta of male mice. Further studies should reveal more details of the mechanisms involved. Because the effect of phytoestrogen may be tissue specific, their selectivity may provide the beneficial effect for AAA prevention without the side effects of traditional hormone therapy. Further studies should examine the potential of dietary phytoestrogens for AAA risk reduction in humans.

      Acknowledgment

      This work was supported by NIH RO1 HL081629 (G.R.U).
      The authors would like to thank Erin D. Jeffery of W.M. Keck Biomedical Mass Spectrometry Laboratory of University of Virginia for her skillful technical assistance with serum phytoestrogen tests. The authors would also like to thank the University of Virginia Center for Research in Reproduction Ligand and Analysis Core, supported by the Eunice Kennedy Shriver NICHD/NIH (SCCPIR) Grant U54-HD28934, for conducting the serum sex hormone assays.

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