Maturitas 96 (2017) 3944

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Maturitas
journal homepage: www.elsevier.com/locate/maturitas

Differential expression of sex hormone receptors in abdominal aortic

aneurysms
Christina Villard a,b, , Per Eriksson c , Malin Kronqvist a , Mariette Lengquist a , Carl Jorns d ,
Johan Hartman e , Joy Roy a,b , Rebecka Hultgren a,b
a
Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
b
Department of Vascular Surgery, Karolinska University Hospital, Stockholm, Sweden
c
Atherosclerosis Research Unit, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden
d
Department of Transplant Surgery, CLINTEC, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
e
Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: Objective: Male sex is a signicant risk factor for abdominal aortic aneurysm (AAA). Female sex hormones
Received 15 June 2016 have been reported to prevent aneurysm formation in animal models. The study aims to describe the
Received in revised form 30 October 2016 expression prole of sex hormone receptors in the aneurysm wall of men and women with AAA and
Accepted 8 November 2016
compare with unaffected controls.
Methods: Aneurysm wall biopsies were obtained during elective open repair of AAA in men and women
Keywords:
(n = 16 + 16). Aortic vessel wall from controls were obtained at organ donation (n = 6). Western blot-,
Abdominal aortic aneurysm
mRNA expression- and immunohistochemical analyses were performed to assess the expression prole
Gender
Sex hormones
of the sex hormone receptors androgen receptor (AR), progesterone receptor (PR), estrogen receptor
Estrogen receptor (ER) and (ER).
Androgen receptor Results: The mRNA- and protein expression levels of AR were higher in AAA compared with control aorta
(7.26 vs. 5.14, P = 0.001). mRNA- and protein expression levels of ER were lower in AAA compared with
control aorta (9.15 vs. 12.29, P < 0.001). mRNA expression levels of PR were higher in AAA compared with
control aorta (8.73 vs. 6.21, P = 0.003), but could not be conrmed on protein level. The expression prole
of sex hormone receptors in men and women with AAA was similar.
Conclusion: Expression of sex hormone receptors differs in the aneurysmal aorta compared with unaf-
fected aorta in men and women. A higher expression of AR and a lower expression of ER suggest that
sex hormone activity could be associated with aneurysm development.
2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction directly and indirectly, by the estrogen receptor (ER) and

Male gender is an important risk factor for abdominal aortic
aneurysm (AAA) [1]. The prevalence in elderly men is approxi-
mately 2% compared with 0.5% in aging women [2,3]. Albeit the
prevalence of AAA in women is lower than in men, the risk of rup-
ture is higher, suggesting gender to be of importance for both the
development and the progression of AAA [4,5].
The lower prevalence of AAA and later onset of disease in
women resemble the gender differences observed in the presen-
tation of cardiovascular disease (CVD), which have been ascribed
to changes in the levels of female sex hormones [1,68]. The pro-
tective effects of estrogens on the vasculature are mediated, both
Corresponding author at.: Department of Vascular Surgery A2:01, Karolinska
University Hospital, 171 76 Stockholm, Sweden.
E-mail address: christina.villard@ki.se (C. Villard).
(ER) [9]. However, the antiatherogenic effects of estrogens were
seriously challenged, as hormone replacement therapy was found
to be associated with prothrombotic events in the Women Health
Initiative (WHI) trial and the Heart and Estrogen/Progestin Replace-
ment Study (HERS) [10,11].
An effect of sex hormones on aneurysm formation has
been demonstrated in animal models. In both angiotensin-II-
and elastase-induced aneurysms, estradiol attenuated aneurysm
formation by inhibiting the proteolytic activity [12,13]. In a
rodent model, orchidectomy and treatment with estradiol pre-
vented aneurysm enlargement, whereas no effect on aneurysm
growth was observed in females given testosterone or undergoing
oophorectomy [14]. An effect of gender on aneurysm formation
was again illustrated when rat aorta was transplanted from dif-
ferent sexed rats. The observed resistance to aneurysm formation

http://dx.doi.org/10.1016/j.maturitas.2016.11.005
0378-5122/ 2016 Elsevier Ireland Ltd. All rights reserved.
40 C. Villard et al. / Maturitas 96 (2017) 3944
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 prole of sex
hormone receptors in the aneurysm wall [12,15,18,19]. The aim of
this study was to examine the expression prole of the sex hormone
receptors: ER, ER, progesterone receptor (PR) and androgen
receptor (AR) in the aneurysmal walls of men and women and com-
pare with the expression prole 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
2008June 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 dened as the ratio of weight/square of the height
[21]. Body surface area (BSA) was predicted using DuBois formula:
(weight0.425 xheight0.725 ) 0.007184 [22]. Aortic size index (ASI)
was dened 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
dened 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 lled out by the organ
coordinator in charge and based on information from hospital
charts and from the patients relatives. The study was approved
by the local Ethics Committee.
2.2. Immunohistochemical analysis
5 m sections of aortic walls were deparafnised 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 ER (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 Polymer Kit specic for mouse/rat (BioCare Medical). Vulcan
Fast Red Solution (BioCare Medical) was used for detection and
counterstaining was performed with Mayers hematoxillin (Vec-
tor Laboratories). Double staining with ER and smooth muscle
-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 -actin and PR
and smooth muscle -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-
ed by a Nanodrop (NanoDrop Products). RNA quality and integrity
were veried using the Agilent 2100 Bioanalyzer System (Agilent
Technologies). For quantication 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
ER, ER, 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 l protease inhibitor and
30 l 1 M Tris-HCl pH 8,0. The mixture of samples and lysis buffer
samples were then granulated with a Tissuelyzer according to
manufacturers 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
412% SDS gel (Novex NuPAGE 412% 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 ER (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 (Fujilm 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. Pearsons chi-
square test and Fischers exact test were used for normally and not
normally distributed categorical variables, respectively. AAA occur-
rence and AAA diameter were estimated by multivariate logistic
 C. Villard et al. / Maturitas 96 (2017) 3944 41

Table 1
Patient characteristics.

Patients with AAA n = 32 Controlsn = 6 P-value

men women P-value

Age 69 5 71 7 0.327 72 5 0.448

BMIa 25.6 3.2 25.8 4.0 0.899 26.0 5.1 0.992
BSAb 1.9 0.2 1.7 0.2 0.004 1.8 0.3 0.998
Aneurysm diameter (cm) 5.8 (.7) 5.7 (.6) 0.867
ASIc 3.1 (.6) 3.5 (.5) 0.008
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 standard deviation for normally distributed data and median (range) for not normally distributed data. Categorical variables are presented
as number (percent).
a
BMI body mass index.
b
BSA body surface area = (weight425 height725 ) 0.007184.
c
ASI aortic size index = aneurysm diameter/BSA.
d
High blood pressure antihypertensive medication.
e
Heart condition prior coronary bypass surgery, arterial brillation and/or heart failure.
f
Lung disease 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, ER, ER, PR
and AR were included) and clinically relevant variables or those
signicant from the univariate analysis were analysed using a mul-
tiregression model. Statistical signicance was dened 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 ER 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 Controls
n = 32 n=6
Estrogen receptor 5.92 0.75 6.47 0.30
Estrogen receptor 9.15 1.45 12.29 1.06
Progesterone receptor 8.73 (1.68) 6.21 (1.42)
Androgen receptor 7.26 1.33 5.14 0.51
Values, normalized to RPLP0, are presented log2-transformed and as arbitrary units.
Values are presented as mean 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
ER 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 ER, ER, PR and AR. A sig-
nicant regression equation was found (F(1,36) = 25.336, P < 0.001),
with an R2 of 0.413 identifying the expression level of ER as a sig-
nicant predictor of AAA occurrence. A multiple linear regression
was calculated to predict AAA diameter in patients with AAA. A sig-
nicant 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
signicant predictors of AAA diameter.
3.3. Protein expressions of sex hormone receptors
Protein expression of ER 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 ER 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,
P-value
yet to our knowledge this is the rst study to investigate the expres-
sion prole of sex hormone receptors in human AAA. The results
0.090
< 0.001
show differences in the expression prole of sex hormone receptors
0.003 between aneurysmal and non-aneurysmal aortic walls, indepen-
0.001 dent of gender. The observed lower expression of ER and higher
expression of AR in aneurysms, could suggest that an increase of
AR and a decrease of ER are associated with the development of
AAA.
42 C. Villard et al. / Maturitas 96 (2017) 3944

Fig. 1. Double staining of ER (red) and smooth muscle -actin (green) of (a) unaffected aorta and (b) aneurysmatic aorta. Medial layer enlarged. (For interpretation of the
references to colour in this 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 -actin (d) unaffected aorta, (f) aneurysmatic aorta. Medial layer
enlarged.

The expression of sex hormones and the vascular effects that
they mediate are inuenced 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, inam-
matory response and smooth muscle cell proliferation [26,27]. ER
and ER are present in equal quantities in the aortic vascular
smooth muscle cells (VSMCs) of men, whereas ER 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 ER, mRNA and protein,
in aneurysmal media compared with unaffected media. We found

Fig. 2. Western blot analysis and densitometry of ER in the thrombus covered aneurysm wall of 5 men and 5 women (1.47 vs. 1.12, P = 0.841). Loading control: GAPDH.
 C. Villard et al. / Maturitas 96 (2017) 3944 43

no difference in the expression of ER between aneurysmal and A higher expression of AR and a lower expression of ER sug-
non-aneurysmal tissue. It could suggest that a loss of a potentially gest that sex hormone activity could be associated with aneurysm
protective effect, mediated by ER, is associated with aneurysm development. Further studies are required to determine the clin-
development in women. ical implications for patients suffering from sex hormone related
The higher prevalence of AAA in men, even after adjustment for alterations and risk of aneurysm development.
risk factor distribution, suggests a susceptibility to aneurysm for-
mation dependent on male sex [5]. In animal models, AR has been Conict of interest
shown to induce aneurysm formation in mice by modulating the
inammatory response and inuencing the contractile response The authors declare that they have no conict of interest.
of the vessel [28,29]. The nding of a higher expression of AR in
AAA compared with unaffected aorta could suggest an association
Contributors
between aneurysm formation and the expression of AR. Further
studies are required to investigate a potential causation.
CV was responsible for study design, data collection from
PR is expressed in VSMCs in a greater extent in pre- and post-
controls, laboratory analysis, statistical analysis, and writing the
menopausal women compared with men, without correlation to
manuscript.
circulating levels of progesterone [30]. Polymorphisms of ER and
PE was responsible for study design, collection of patient sam-
PR have been associated with AAA [31]. The effect of progesterone
ples, nancial support, and critical review of the manuscript.
on the vasculature is contradictory. The adverse events observed
MK was responsible for laboratory analysis.
in the WHI- and HERS trials have partly been ascribed to a pro-
ML was responsible for laboratory analysis.
thrombotic effect of medroxyprogesterone [32]. On the other hand
CJ was responsible for data collection from controls, laboratory
progesterone inhibits VSMC proliferation and thereby mediates
analysis, and critical review of the manuscript.
an antiatherogenic effect in cultured VSMCs and animal models
JH was responsible for study design, laboratory analysis, and
[33,34]. We found higher mRNA expression levels of PR in aneurys-
critical review of manuscript.
matic aortic walls compared with the unaffected aortic walls but
JR was responsible for study design, collection of patient sam-
could not conrm the difference in protein expression analysis.
ples, nancial support, and critical review of the manuscript.
The potential role of PR in the formation of AAA requires further
RH was responsible for study design, collection of patient and
investigation in human and animal models.
control aortic wall samples, nancial support, critical review of the
The effects mediated by the sex hormone receptors are depen-
manuscript, and had overall responsibility.
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 Funding
peripheral aromatization [35,36]. The levels of progesterone are
higher in women compared with men at all ages, while the opposite This study was supported by the Swedish Heart-Lung Founda-
is observed for testosterone. Both hormones decrease slightly with tion (Hultgren) and by the regional agreement on medical training
increasing age in men and women [3739]. A feedback mechanism and clinical research (Villard) between Stockholm County Council
could theoretically affect the expression prole of sex hormone and the Karolinska Institutet.
receptors in the aorta, as has been observed for other vascular
disorders [25,40]. Theoretically, the susceptibility to aneurysm for- Ethical approval
mation in men is related to effects mediated by AR. In women, an
aneurysm evolves as the aortic wall loses the protective effect of The study was approved by the local Ethics Commit-
being female or due to male adaptation. tee (application numbers 2011/1863-3, 2009/4:2, 2009/9-31/4,
Women have proportionally smaller aortas than men and there- 2013/615-31/4).
fore in AAA development the relative aneurysm enlargement in
womens AAA exceeds that of mens at any given diameter [41].
Provenance and peer review
Aortic size index (ASI) is a measurement, which takes the relative
enlargement into account, and has been associated with the risk of
This article has undergone peer review.
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 Appendix A. Supplementary data
size, however it is similar to other studies within the eld [42,43].
The study illustrates a correlation between the expression levels of Supplementary data associated with this article can be found, in
sex hormone receptors and AAA; it is not designed to show cau- the online version, at http://dx.doi.org/10.1016/j.maturitas.2016.
sation. Still, as it is the rst of its kind in humans, the data does 11.005.
provide important information. The lack of appropriate aortic wall
tissue from controls limited the protein expression analysis. The References
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