2. 40 C. Villard et al. / Maturitas 96 (2017) 39ā44
observed when the recipient was female was lost if the recipient
instead was male [15].
Studies on the potential effect of gender on aneurysm devel-
opment in humans are scarce. The observed negative association
between AAA occurrence and long-term hormone therapy and the
lower menopausal age in women with larger AAA support the the-
ory of a protective effect of female sex hormones [16,17]. On the
other hand, lower levels of testosterone are associated with AAA
occurrence in elderly men [18].
Studies on gender aspects on aneurysm formation primarily
focus on an effect of endogenous as well as exogenous sex hor-
mones and little is known about the expression proļ¬le of sex
hormone receptors in the aneurysm wall [12,15,18,19]. The aim of
this study was to examine the expression proļ¬le of the sex hormone
receptors: ERā£, ERā¤, progesterone receptor (PR) and androgen
receptor (AR) in the aneurysmal walls of men and women and com-
pare with the expression proļ¬le in non-aneurysmal aortic walls.
The study focused on the medial layer of the aortic vessel wall as it
is most affected by degradation in aneurysm development [20].
2. Material and method
2.1. Study population and tissue handling
All women treated electively for AAA with open repair (OR)
at Karolinska University Hospital, Stockholm Sweden, November
2008āJune 2014, were included (n = 16). Male patients treated
during the same time period (n = 16), were chosen to match the
age and aneurysm diameter of the participating women. Mycotic
aneurysms were not included. The patients were treated with OR,
as they were considered unsuitable for endovascular aneurysm
repair (EVAR). Biopsies of the ventral, infrarenal aneurysm wall,
at the maximum diameter, were obtained during the surgical pro-
cedure. Only thrombus covered aneurysm walls were used since
non-thrombus covered aneurysm walls could not be obtained from
all participants, which is probably due to the low occurrence
of thrombus free walls in such large aneurysms. Patient char-
acteristics were obtained from hospital charts. Body mass index
(BMI) was deļ¬ned as the ratio of weight/square of the height
[21]. Body surface area (BSA) was predicted using DuBois formula:
(weight0.425xheight0.725) Ć 0.007184 [22]. Aortic size index (ASI)
was deļ¬ned as the ratio of aneurysm diameter/BSA [23]. All patients
had signed an informed consent prior to the surgical procedure.
The control group consisted of 6 organ donors, male and female.
The aortic diameters of the organ donors were not measured, but
deļ¬ned non-aneurysmal by the transplant surgeon. During the
organ donation biopsies of the infrarenal aorta were obtained. The
aortic biopsies from organ donors and patients with AAA were han-
dled according to the same protocol and stored during transport
in RNAlater and formalin. The aortic walls, both aneurysmal and
non-aneurysmal, were divided into initimal, medial and advential
layers. The biopsies from organ donors could not be transported
freshly frozen, which limited the protein expression analysis. All
donors, or their close relatives, had signed an informed consent
regarding donation of the tissue for research purposes. Patient
characteristics were obtained from a form ļ¬lled out by the organ
coordinator in charge and based on information from hospital
charts and from the patientsā relatives. The study was approved
by the local Ethics Committee.
2.2. Immunohistochemical analysis
5 ā®m sections of aortic walls were deparafļ¬nised in Tissue-
Clear (Sakura) and rehydrated in ethanol. The sections were boiled
under high pressure in DIVA-buffer (BioCare Medical) and Back-
ground Sniper Solution (BioCare Medical) was used for background
blocking. Antibodies ERā¤ (Thermo), PR (Dako) and AR (Dako) were
diluted in DaVinci Green Solution (BioCare Medical) for 60 min
in room temperature, followed by short incubations with Probe
and āPolymer Kit speciļ¬c for mouse/rat (BioCare Medical). Vulcan
Fast Red Solution (BioCare Medical) was used for detection and
counterstaining was performed with Mayerās hematoxillin (Vec-
tor Laboratories). Double staining with ERā¤ and smooth muscle
ā£-actin (Abcam) were performed in a similar manner but with a
double staining probe and polymer Kit (BioCare Medical) and Warp
Red Solution together with Vine Green (Biocare Medical) for detec-
tion. Double stainings with AR and smooth muscle ā£-actin and PR
and smooth muscle ā£-actin were unfortunately not possible and
therefore the stainings were performed on consecutive sections.
2.3. mRNA expression analysis
Frozen medial layers of aortic walls were homogenized with
tissue lyser (Qiagen). RNA was isolated with Qiazol (Qiagen), RLT
buffer (from Rneasy Mini kit, Qiagen) and Dnase1 (Rnase free Dnase
Set, Qiagen) according to a standardized protocol. RNA was quanti-
ļ¬ed by a Nanodrop (NanoDrop Products). RNA quality and integrity
were veriļ¬ed using the Agilent 2100 Bioanalyzer System (Agilent
Technologies). For quantiļ¬cation of gene expression, total RNA was
reversely transcribed to cDNA using High capacity RNA to cDNA
kit (Life technologies) the manufacturers protocol. Real time PCR
was performed on the Applied Biosystems 7000 Real-Time PCR Sys-
tem with TaqMan Assays-on-Demand Gene Expression Probes for
ERā£, ERā¤, PR and AR. Robust multiarray average normalization was
performed and gene expression data were log2-transformed. The
housekeeping gene Ribosomal Protein Large P0 (RPLP0) was used
for normalization.
2.4. Western blot analysis
The medial layers of the aneurysm samples were shred and
mixed with a lysis buffer containing 50 ā®l protease inhibitor and
30 ā®l 1 M Tris-HCl pH 8,0. The mixture of samples and lysis buffer
samples were then granulated with a Tissuelyzer according to
manufacturerās protocol and centrifuged for 5 min at 220 rpm. The
supernatants were sonicated for 5 min at high level followed by
centrifugation for 10 min at 12000 rpm. The protein content in the
supernatants was determined using Bradford protein assay. The
samples were diluted with lysis buffer before being loaded on a
4ā12% SDS gel (Novex NuPAGE 4ā12% Bis-Trisgel 15well, Invit-
rogen) in MOPS-SDS running buffer. Electrophoresis was run for
90 min at 120 V, in a cold room. The gel and membrane (Hybond
PVDF transfer membrane, GE Healthcare) were equilibrated in
transfer buffer before transfer by electroblotting for 90 min at
400 mA, in a cold room. For blocking, the membrane was suspended
in blocking buffer (3% bovine serum albumin/TTBS) for 60 min.
The membrane was incubated over night with ERā¤ (Thermo), AR
(Abcam) and GAPDH (Abcam) followed by the second antibody
(anti-Mouse and anti-Rabbit HRP, BioRab) for 45 min. Finally, the
developing solution from ECL Prime Western Blotting Detection
Reagent kit (GE Healthcare) and CCD camera (Fujiļ¬lm LAS-1000)
were used for chemiluminescent detection.
2.5. Statistical analysis
Statistical analysis was performed with SPSS 21.0. Independent
t-test was used for gender comparisons of normally distributed data
and Mann U test for not normally distributed data. Pearsonās chi-
square test and Fischerās exact test were used for normally and not
normally distributed categorical variables, respectively. AAA occur-
rence and AAA diameter were estimated by multivariate logistic
3. C. Villard et al. / Maturitas 96 (2017) 39ā44 41
Table 1
Patient characteristics.
Patients with AAA n = 32 Controlsn = 6 P-value
men women P-value
Age 69 Ā± 5 71 Ā± 7 0.327 72Ā± 5 0.448
BMIa
25.6 Ā± 3.2 25.8 Ā± 4.0 0.899 26.0 Ā± 5.1 0.992
BSAb
1.9 Ā± 0.2 1.7 Ā± 0.2 0.004 1.8 Ā± 0.3 0.998
Aneurysm diameter (cm) 5.8 (.7) 5.7 (.6) 0.867 ā ā
ASIc
3.1 (.6) 3.5 (.5) 0.008 ā ā
Smoking habits (%) .380 0.001
Current 5 (31) 3 (9) 1 (17)
Prior 11 (69) 12 (75) 1 (17)
Never 0 1 (6) 4 (17)
High blood pressured
11 (69) 10 (63) .500 4 (67) 0.672
Heart conditione
3 (19) 3 (19) .673 0 (0) 0.328
Lung diseasef
3 (19) 3 (19) .673 1 (17) 0.698
Diabetes mellitus 3 (19) 0 0.113 0 0.588
Values are presented as mean Ā± standard deviation for normally distributed data and median (range) for not normally distributed data. Categorical variables are presented
as number (percent).
a
BMI ā body mass index.
b
BSA ā body surface area = (weight425
Ć height725
) Ć 0.007184.
c
ASI ā aortic size index = aneurysm diameter/BSA.
d
High blood pressure ā antihypertensive medication.
e
Heart condition ā prior coronary bypass surgery, arterial ļ¬brillation and/or heart failure.
f
Lung disease ā chronic obstructive pulmonary disease and/or asthma.
and linear regression, respectively. The data was analysed using
a univariate model (sex, age, hypertension, heart condition, lung
disease, diabetes mellitus, smoking habits, BMI, ASI, ERā£, ERā¤, PR
and AR were included) and clinically relevant variables or those
signiļ¬cant from the univariate analysis were analysed using a mul-
tiregression model. Statistical signiļ¬cance was deļ¬ned as P < 0.05.
3. Results
3.1. Patient characteristics
Age, BMI as well as BSA were similar in patients with AAA and
controls (69 years vs. 70, P = 0.496, 25.7 vs. 26.0, P = 0.873 and 1.8 vs.
1.8, P = 0.987, respectively). Occurrence of comorbid conditions was
similar in patients with AAA and controls (Table 1)(Supplementary
Table I). Patients with AAA were prior and current smokers to a
greater extent than the control group, 25% vs. 17% and 72% vs. 17%,
P = 0.001.
Mean age of and aneurysm diameter in men and women with
AAA were similar (Table 1). Women with AAA had lower BSA
and higher ASI compared with men (Table 1). Smoking habits and
comorbid conditions were similar in men and women with AAA.
3.2. mRNA expressions of sex hormone receptors
Patients with AAA had higher mRNA expression levels of AR
and PR than controls (7.26 vs. 5.14, P = 0.001 and 8.73 vs. 6.21,
P = 0.003, respectively) (Table 2). mRNA expression of ERā¤ was
lower in patients with AAA compared with controls (9.15 vs. 12.29,
Table 2
mRNA expression levels of sex hormone receptors in patients with AAA and controls.
Patients with AAA
n = 32
Controls
n = 6
P-value
Estrogen receptor ā£ 5.92 Ā± 0.75 6.47 Ā± 0.30 0.090
Estrogen receptor ā¤ 9.15 Ā± 1.45 12.29 Ā± 1.06 < 0.001
Progesterone receptor 8.73 (1.68) 6.21 (1.42) 0.003
Androgen receptor 7.26 Ā± 1.33 5.14 Ā± 0.51 0.001
Values, normalized to RPLP0, are presented log2-transformed and as arbitrary units.
Values are presented as mean Ā± standard deviation for normally distributed data and
median (range) for not normally distributed data.
P < 0.001) (Table 2). There was no difference in expression levels of
ERā£ between aneurysms and control aortas (Table 2).
Men and women with AAA did not differ regarding mRNA
expression levels of sex hormone receptors (Supplementary Table
II). No correlations were found between aneurysm size and mRNA
expression levels of sex hormone receptors.
A multiple logistic regression was calculated to predict AAA
occurrence in the study population based on: sex, age, hyper-
tension, diabetes mellitus, heart condition, lung disease, smoking
habits, BMI, BSA, expression levels of ERā£, ERā¤, PR and AR. A sig-
niļ¬cant regression equation was found (F(1,36) = 25.336, P < 0.001),
with an R2 of 0.413 identifying the expression level of ERā¤ as a sig-
niļ¬cant predictor of AAA occurrence. A multiple linear regression
was calculated to predict AAA diameter in patients with AAA. A sig-
niļ¬cant regression equation was found (F (2,28) = 15.316, P < 0.001)
with an R2 of 0.488 identifying both ASI and heart condition as
signiļ¬cant predictors of AAA diameter.
3.3. Protein expressions of sex hormone receptors
Protein expression of ERā¤ in vascular smooth muscle cells
was lower in patients with AAA compared with controls, whereas
expression of AR was higher in patients with AAA compared with
controls, assessed by immunohistochemistry (Fig. 1). There was no
difference in the expression of PR between patients with AAA and
controls.
Protein expressions of ERā¤ and AR were similar in men and
women with AAA, assessed by western blot analysis (Fig. 2).
4. Discussion
The observed gender differences in aneurysm development
have been ascribed to effects of sex hormones on the vasculature,
yet to our knowledge this is the ļ¬rst study to investigate the expres-
sion proļ¬le of sex hormone receptors in human AAA. The results
show differences in the expression proļ¬le of sex hormone receptors
between aneurysmal and non-aneurysmal aortic walls, indepen-
dent of gender. The observed lower expression of ERā¤ and higher
expression of AR in aneurysms, could suggest that an increase of
AR and a decrease of ERā¤ are associated with the development of
AAA.
4. 42 C. Villard et al. / Maturitas 96 (2017) 39ā44
Fig. 1. Double staining of ERā¤ (red) and smooth muscle ā£-actin (green) of (a) unaffected aorta and (b) aneurysmatic aorta. Medial layer enlarged. (For interpretation of the
references to colour in this ļ¬gure legend, the reader is referred to the web version of this article.)
Staining on consecutive sections for AR (c) unaffected aorta, (e) aneurysmatic aorta and smooth muscle ā£-actin (d) unaffected aorta, (f) aneurysmatic aorta. Medial layer
enlarged.
The expression of sex hormones and the vascular effects that
they mediate are inļ¬uenced by sex, age and menopause, which
could partly explain the lack of differences between the sexes in this
study [24,25]. Thereto, for apparent reasons, tissue from aneurysms
can only be obtained when patients are subjected to OR, which limit
the analysis to an end-stage disease. Since AAA affects an elderly
population it is also for practical reasons impossible to obtain biop-
sies of younger AAA patients, i.e. premenopausal women. With the
aid of results from studies on animals, we can only speculate on the
hormonal changes occurring in the aneurysm wall as AAA evolves
in men and women.
Estrogen receptors play an intricate role in many physiological
processes in the vasculature by modulating vascular tone, inļ¬am-
matory response and smooth muscle cell proliferation [26,27]. ERā£
and ERā¤ are present in equal quantities in the aortic vascular
smooth muscle cells (VSMCs) of men, whereas ERā¤ is the primary
estrogen receptor in the aortic VSMCs of women [25]. The preven-
tive effect of exogenous estrogen on aneurysm development shown
in animal models is associated with an inhibition of the proteolytic
activity mediated by matrix metalloproteinases [12,15,19]. In this
study we found a lower expression of ERā¤, mRNA and protein,
in aneurysmal media compared with unaffected media. We found
Fig. 2. Western blot analysis and densitometry of ERā¤ in the thrombus covered aneurysm wall of 5 men and 5 women (1.47 vs. 1.12, P = 0.841). Loading control: GAPDH.
5. C. Villard et al. / Maturitas 96 (2017) 39ā44 43
no difference in the expression of ERā£ between aneurysmal and
non-aneurysmal tissue. It could suggest that a loss of a potentially
protective effect, mediated by ERā¤, is associated with aneurysm
development in women.
The higher prevalence of AAA in men, even after adjustment for
risk factor distribution, suggests a susceptibility to aneurysm for-
mation dependent on male sex [5]. In animal models, AR has been
shown to induce aneurysm formation in mice by modulating the
inļ¬ammatory response and inļ¬uencing the contractile response
of the vessel [28,29]. The ļ¬nding of a higher expression of AR in
AAA compared with unaffected aorta could suggest an association
between aneurysm formation and the expression of AR. Further
studies are required to investigate a potential causation.
PR is expressed in VSMCs in a greater extent in pre- and post-
menopausal women compared with men, without correlation to
circulating levels of progesterone [30]. Polymorphisms of ERā¤ and
PR have been associated with AAA [31]. The effect of progesterone
on the vasculature is contradictory. The adverse events observed
in the WHI- and HERS trials have partly been ascribed to a pro-
thrombotic effect of medroxyprogesterone [32]. On the other hand
progesterone inhibits VSMC proliferation and thereby mediates
an antiatherogenic effect in cultured VSMCs and animal models
[33,34]. We found higher mRNA expression levels of PR in aneurys-
matic aortic walls compared with the unaffected aortic walls but
could not conļ¬rm the difference in protein expression analysis.
The potential role of PR in the formation of AAA requires further
investigation in human and animal models.
The effects mediated by the sex hormone receptors are depen-
dent on their ligands, the sex hormones. Menopause in women
results in lower levels of circulating estrogens, whereas aging men
have increasing levels, due to continued production in the testis and
peripheral aromatization [35,36]. The levels of progesterone are
higher in women compared with men at all ages, while the opposite
is observed for testosterone. Both hormones decrease slightly with
increasing age in men and women [37ā39]. A feedback mechanism
could theoretically affect the expression proļ¬le of sex hormone
receptors in the aorta, as has been observed for other vascular
disorders [25,40]. Theoretically, the susceptibility to aneurysm for-
mation in men is related to effects mediated by AR. In women, an
aneurysm evolves as the aortic wall loses the protective effect of
being female or due to āmale adaptationā.
Women have proportionally smaller aortas than men and there-
fore in AAA development the relative aneurysm enlargement in
womenās AAA exceeds that of menās at any given diameter [41].
Aortic size index (ASI) is a measurement, which takes the relative
enlargement into account, and has been associated with the risk of
aneurysm rupture in women [23]. In this ASI was higher in women
than in men.
There are limitations to this study such as the limited sample
size, however it is similar to other studies within the ļ¬eld [42,43].
The study illustrates a correlation between the expression levels of
sex hormone receptors and AAA; it is not designed to show cau-
sation. Still, as it is the ļ¬rst of its kind in humans, the data does
provide important information. The lack of appropriate aortic wall
tissue from controls limited the protein expression analysis. The
two splice variants of the ERā¤; ERā¤1 and ERā¤cx, could not be
analysed separately with the used probes and antibodies. These
variants have been shown to have antagonising roles in breast can-
cer but are not widely studied in arteries [44]. Blood samples could
not be obtained from the controls and consequently an analysis
of circulating levels of sex hormones could not be performed. As
in all studies, especially those with a small sample size, both beta
errors (due to small cohorts) and alpha errors (due to multiplicity
of comparisons) should be considered.
In conclusion, the expression proļ¬le of sex hormone recep-
tors differs in the aneurysmal aorta compared to unaffected aorta.
A higher expression of AR and a lower expression of ERā¤ sug-
gest that sex hormone activity could be associated with aneurysm
development. Further studies are required to determine the clin-
ical implications for patients suffering from sex hormone related
alterations and risk of aneurysm development.
Conļ¬ict of interest
The authors declare that they have no conļ¬ict of interest.
Contributors
CV was responsible for study design, data collection from
controls, laboratory analysis, statistical analysis, and writing the
manuscript.
PE was responsible for study design, collection of patient sam-
ples, ļ¬nancial support, and critical review of the manuscript.
MK was responsible for laboratory analysis.
ML was responsible for laboratory analysis.
CJ was responsible for data collection from controls, laboratory
analysis, and critical review of the manuscript.
JH was responsible for study design, laboratory analysis, and
critical review of manuscript.
JR was responsible for study design, collection of patient sam-
ples, ļ¬nancial support, and critical review of the manuscript.
RH was responsible for study design, collection of patient and
control aortic wall samples, ļ¬nancial support, critical review of the
manuscript, and had overall responsibility.
Funding
This study was supported by the Swedish Heart-Lung Founda-
tion (Hultgren) and by the regional agreement on medical training
and clinical research (Villard) between Stockholm County Council
and the Karolinska Institutet.
Ethical approval
The study was approved by the local Ethics Commit-
tee (application numbers 2011/1863-3, 2009/4:2, 2009/9-31/4,
2013/615-31/4).
Provenance and peer review
This article has undergone peer review.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.maturitas.2016.
11.005.
References
[1] H.J.H.A. Pleumeekers, E. van der Does, H. van Urk, A. Hofman, P.T. de Jong, D.E.
Grobbee, Aneurysms of the abdominal aorta in older adults. The Rotterdam
Study, Am. J. Epidemiol. 142 (December (12)) (1995) 1291ā1299.
[2] M. Zarrouk, J. Holst, M. Malina, B. Lindblad, C. Wann-Hansson, M. Rosvall,
et al., The importance of socioeconomic factors for compliance and outcome
at screening for abdominal aortic aneurysm in 65-year-old men, J. Vasc. Surg.
58 (1) (2013) 50ā55.
[3] S. Svensjo, M. Bjorck, A. Wanhainen, Current prevalence of abdominal aortic
aneurysm in 70-year-old women, Br. J. Surg. 100 (3) (2013) 367ā372.
[4] M.J. Sweeting, S.G. Thompson, L.C. Brown, J.T. Powell, collaborators R,
Meta-analysis of individual patient data to examine factors affecting growth
and rupture of small abdominal aortic aneurysms, Br. J. Surg. 99 (5) (2012)
655ā665.
6. 44 C. Villard et al. / Maturitas 96 (2017) 39ā44
[5] K.C. Kent, R.M. Zwolak, N.N. Egorova, T.S. Riles, A. Manganaro, A.J. Moskowitz,
et al., Analysis of risk factors for abdominal aortic aneurysm in a cohort of
more than 3 million individuals, J. Vasc. Surg. 52 (3) (2010) 539ā548.
[6] R.A. Scott, S.G. Bridgewater, H.A. Ashton, Randomized clinical trial of screening
for abdominal aortic aneurysm in women, Br. J. Surg. 89 (3) (2002) 283ā285.
[7] D.J. Lerner, W.B. Kannel, Patterns of coronary heart disease morbidity and
mortality in the sexes: a 26-year follow-up of the Framingham population,
Am. Heart J. 111 (2) (1986) 383ā390.
[8] P. Anagnostis, J.C. Stevenson, D. Crook, D.G. Johnston, I.F. Godsland, Effects of
menopause, gender and age on lipids and high-density lipoprotein
cholesterol subfractions, Maturitas 81 (1) (2015) 62ā68.
[9] M.E. Mendelsohn, R.H. Karas, Molecular and cellular basis of cardiovascular
gender differences, Science 308 (5728) (2005) 1583ā1587.
[10] J.E. Rossouw, G.L. Anderson, R.L. Prentice, A.Z. LaCroix, C. Kooperberg, M.L.
Stefanick, et al., Risks and beneļ¬ts of estrogen plus progestin in healthy
postmenopausal women: principal results from the womenās health initiative
randomized controlled trial, JAMA 288 (3) (2002) 321ā333.
[11] S. Hulley, D. Grady, T. Bush, C. Furberg, D. Herrington, B. Riggs, et al.,
Randomized trial of estrogen plus progestin for secondary prevention of
coronary heart disease in postmenopausal women. Heart and
Estrogen/progestin Replacement Study (HERS) Research Group, JAMA 280 (7)
(1998) 605ā613.
[12] X.F. Wu, J. Zhang, S. Paskauskas, S.J. Xin, Z.Q. Duan, The role of estrogen in the
formation of experimental abdominal aortic aneurysm, Am. J. Surg. 197 (1)
(2009) 49ā54.
[13] B. Martin-McNulty, D.M. Tham, V. da Cunha, J.J. Ho, D.W. Wilson, J.C. Rutledge,
et al., 17 Beta-estradiol attenuates development of angiotensin II-induced
aortic abdominal aneurysm in apolipoprotein E-deļ¬cient mice, Arterioscler.
Thromb. Vasc. Biol. 23 (9) (2003) 1627ā1632.
[14] B.S. Cho, D.T. Woodrum, K.J. Roelofs, J.C. Stanley, P.K. Henke, G.R. Upchurch Jr.,
Differential regulation of aortic growth in male and female rodents is
associated with AAA development, J. Surg. Res. 155 (2) (2009) 330ā338.
[15] G. Ailawadi, J.L. Eliason, K.J. Roelofs, I. Sinha, K.K. Hannawa, E.P. Kaldjian, et al.,
Gender differences in experimental aortic aneurysm formation, Arterioscler.
Thromb. Vasc. Biol. 24 (11) (2004) 2116ā2122.
[16] F.A. Lederle, G.R. Johnson, S.E. Wilson, Abdominal aortic aneurysm in women,
J. Vasc. Surg. 34 (1) (2001) 122ā126.
[17] C. Villard, J. Swedenborg, P. Eriksson, R. Hultgren, Reproductive history in
women with abdominal aortic aneurysms, J. Vasc. Surg. 54 (2) (2011)
341ā345 (e2).
[18] B.B. Yeap, Z. Hyde, P.E. Norman, S.A. Chubb, J. Golledge, Associations of total
testosterone, sex hormone-binding globulin, calculated free testosterone, and
luteinizing hormone with prevalence of abdominal aortic aneurysm in older
men, J. Clin. Endocrinol. Metab. 95 (3) (2010) 1123ā1130.
[19] A. Laser, A. Ghosh, K. Roelofs, O. Sadiq, B. McEvoy, P. DiMusto, et al., Increased
estrogen receptor alpha in experimental aortic aneurysms in females
compared with males, J. Surg. Res. 186 (1) (2014) 467ā474.
[20] N. Sakalihasan, R. Limet, O.D. Defawe, Abdominal aortic aneurysm, Lancet 365
(9470) (2005) 1577ā1589.
[21] G.A. Bray, Overweight is risking fate. Deļ¬nition, classiļ¬cation, prevalence, and
risks, Ann. N. Y. Acad. Sci. 499 (1987) 14ā28.
[22] Y. Wang, J. Moss, R. Thisted, Predictors of body surface area, J. Clin. Anesth. 4
(1) (1992) 4ā10.
[23] R.C. Lo, B. Lu, M.T. Fokkema, M. Conrad, V.I. Patel, M. Fillinger, et al., Relative
importance of aneurysm diameter and body size for predicting abdominal
aortic aneurysm rupture in men and women, J. Vasc. Surg. (2013).
[24] T. Muka, K.G. Vargas, L. Jaspers, K.X. Wen, K. Dhana, A. Vitezova, et al.,
Estrogen receptor beta actions in the female cardiovascular system: a
systematic review of animal and human studies, Maturitas 86 (2016) 28ā43.
[25] Y.K. Hodges, L. Tung, X.D. Yan, J.D. Graham, K.B. Horwitz, L.D. Horwitz,
Estrogen receptors alpha and beta: prevalence of estrogen receptor beta
mRNA in human vascular smooth muscle and transcriptional effects,
Circulation 101 (15) (2000) 1792ā1798.
[26] E. Murphy, Estrogen signaling and cardiovascular disease, Circ. Res. 109 (6)
(2011) 687ā696.
[27] H. Sumino, S. Ichikawa, S. Kasama, H. Kumakura, Y. Takayama, T. Sakamaki,
et al., Effect of transdermal hormone replacement therapy on carotid artery
wall thickness and levels of vascular inļ¬ammatory markers in
postmenopausal women, Hypertens. Res. 28 (7) (2005) 579ā584.
[28] C.K. Huang, J. Luo, K.P. Lai, R. Wang, H. Pang, E. Chang, et al., Androgen
receptor promotes abdominal aortic aneurysm development via modulating
inļ¬ammatory interleukin-1alpha and transforming growth factor-beta1
expression, Hypertension (2015).
[29] T. Henriques, X. Zhang, F.B. Yiannikouris, A. Daugherty, L.A. Cassis, Androgen
increases AT1a receptor expression in abdominal aortas to promote
angiotensin II-induced AAAs in apolipoprotein E-deļ¬cient mice, Arterioscler.
Thromb. Vasc. Biol. 28 (7) (2008) 1251ā1256.
[30] Y. Nakamura, T. Suzuki, T. Inoue, C. Tazawa, K. Ono, T. Moriya, et al.,
Progesterone receptor subtypes in vascular smooth muscle cells of human
aorta, Endocr. J. 52 (2) (2005) 245ā252.
[31] F. Massart, F. Marini, A. Menegato, F. Del Monte, M. Nuti, F. Butitta, et al.,
Allelic genes involved in artery compliance and susceptibility to sporadic
abdominal aortic aneurysm, J. Steroid Biochem. Mol. Biol. 92 (5) (2004)
413ā418.
[32] M.L. Stefanick, Estrogens and progestins: background and history, trends in
use, and guidelines and regimens approved by the US Food and Drug
Administration, Am. J. Med. 118 (Suppl. 12B) (2005) 64ā73.
[33] A.K. Morey, A. Pedram, M. Razandi, B.A. Prins, R.M. Hu, E. Biesiada, et al.,
Estrogen and progesterone inhibit vascular smooth muscle proliferation,
Endocrinology 138 (8) (1997) 3330ā3339.
[34] R.H. Karas, M. van Eickels, J.P. Lydon, S. Roddy, M. Kwoun, M. Aronovitz, et al.,
A complex role for the progesterone receptor in the response to vascular
injury, J. Clin. Invest. 108 (4) (2001) 611ā618.
[35] A. Bjornerem, B. Straume, M. Midtby, V. Fonnebo, J. Sundsfjord, J. Svartberg,
et al., Endogenous sex hormones in relation to age, sex, lifestyle factors, and
chronic diseases in a general population: the Tromso Study, J. Clin.
Endocrinol. Metab. 89 (12) (2004) 6039ā6047.
[36] G. Rannevik, S. Jeppsson, O. Johnell, B. Bjerre, Y. Laurell-Borulf, L. Svanberg, A
longitudinal study of the perimenopausal transition: altered proļ¬les of
steroid and pituitary hormones, SHBG and bone mineral density, Maturitas 61
(1ā2) (2008) 67ā77.
[37] A.R. Genazzani, F. Petraglia, F. Bernardi, E. Casarosa, C. Salvestroni, A. Tonetti,
et al., Circulating levels of allopregnanolone in humans: gender, age, and
endocrine inļ¬uences, J. Clin. Endocrinol. Metab. 83 (6) (1998) 2099ā2103.
[38] T. Thomas, B. Burguera, L.J. Melton 3rd, E.J. Atkinson, W.M. OāFallon, B.L. Riggs,
et al., Relationship of serum leptin levels with body composition and sex
steroid and insulin levels in men and women, Metabolism 49 (10) (2000)
1278ā1284.
[39] J.E. Morley, F.E. Kaiser, H.M. Perry 3rd, P. Patrick, P.M. Morley, P.M. Stauber,
et al., Longitudinal changes in testosterone, luteinizing hormone, and
follicle-stimulating hormone in healthy older men, Metabolism 46 (4) (1997)
410ā413.
[40] R.C. Christian, P.Y. Liu, S. Harrington, M. Ruan, V.M. Miller, L.A. Fitzpatrick,
Intimal estrogen receptor (ER)beta, but not ERalpha expression, is correlated
with coronary calciļ¬cation and atherosclerosis in pre- and postmenopausal
women, J. Clin. Endocrinol. Metab. 91 (7) (2006) 2713ā2720.
[41] T.L. Forbes, D.K. Lawlor, G. DeRose, K.A. Harris, Gender differences in relative
dilatation of abdominal aortic aneurysms, Ann. Vasc. Surg. 20 (5) (2006)
564ā568.
[42] J.H. Lindeman, B.A. Ashcroft, J.W. Beenakker, M. van Es, N.B. Koekkoek, F.A.
Prins, et al., Distinct defects in collagen microarchitecture underlie
vessel-wall failure in advanced abdominal aneurysms and aneurysms in
Marfan syndrome, Proc. Natl. Acad. Sci. U. S. A. 107 (2) (2010) 862ā865.
[43] M. Carmo, L. Colombo, A. Bruno, F.R. Corsi, L. Roncoroni, M.S. Cuttin, et al.,
Alteration of elastin, collagen and their cross-links in abdominal aortic
aneurysms, Eur. J. Vasc. Endovasc. Surg. 23 (6) (2002) 543ā549.
[44] G. Rosin, J. de Boniface, G.M. Karthik, J. Frisell, J. Bergh, J. Hartman, Oestrogen
receptors beta1 and betacx have divergent roles in breast cancer survival and
lymph node metastasis, Br. J. Cancer 111 (5) (2014) 918ā926.