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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


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

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 ground Sniper Solution (BioCare Medical) was used for background
instead was male [15]. blocking. Antibodies ER (Thermo), PR (Dako) and AR (Dako) were
Studies on the potential effect of gender on aneurysm devel- diluted in DaVinci Green Solution (BioCare Medical) for 60 min
opment in humans are scarce. The observed negative association in room temperature, followed by short incubations with Probe
between AAA occurrence and long-term hormone therapy and the and Polymer Kit specic for mouse/rat (BioCare Medical). Vulcan
lower menopausal age in women with larger AAA support the the- Fast Red Solution (BioCare Medical) was used for detection and
ory of a protective effect of female sex hormones [16,17]. On the counterstaining was performed with Mayers hematoxillin (Vec-
other hand, lower levels of testosterone are associated with AAA tor Laboratories). Double staining with ER and smooth muscle
occurrence in elderly men [18]. -actin (Abcam) were performed in a similar manner but with a
Studies on gender aspects on aneurysm formation primarily double staining probe and polymer Kit (BioCare Medical) and Warp
focus on an effect of endogenous as well as exogenous sex hor- Red Solution together with Vine Green (Biocare Medical) for detec-
mones and little is known about the expression prole of sex tion. Double stainings with AR and smooth muscle -actin and PR
hormone receptors in the aneurysm wall [12,15,18,19]. The aim of and smooth muscle -actin were unfortunately not possible and
this study was to examine the expression prole of the sex hormone therefore the stainings were performed on consecutive sections.
receptors: ER, ER, progesterone receptor (PR) and androgen
receptor (AR) in the aneurysmal walls of men and women and com- 2.3. mRNA expression analysis
pare with the expression prole in non-aneurysmal aortic walls.
The study focused on the medial layer of the aortic vessel wall as it Frozen medial layers of aortic walls were homogenized with
is most affected by degradation in aneurysm development [20]. 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-
2. Material and method
ed by a Nanodrop (NanoDrop Products). RNA quality and integrity
were veried using the Agilent 2100 Bioanalyzer System (Agilent
2.1. Study population and tissue handling
Technologies). For quantication of gene expression, total RNA was
reversely transcribed to cDNA using High capacity RNA to cDNA
All women treated electively for AAA with open repair (OR)
kit (Life technologies) the manufacturers protocol. Real time PCR
at Karolinska University Hospital, Stockholm Sweden, November
was performed on the Applied Biosystems 7000 Real-Time PCR Sys-
2008June 2014, were included (n = 16). Male patients treated
tem with TaqMan Assays-on-Demand Gene Expression Probes for
during the same time period (n = 16), were chosen to match the
ER, ER, PR and AR. Robust multiarray average normalization was
age and aneurysm diameter of the participating women. Mycotic
performed and gene expression data were log2-transformed. The
aneurysms were not included. The patients were treated with OR,
housekeeping gene Ribosomal Protein Large P0 (RPLP0) was used
as they were considered unsuitable for endovascular aneurysm
for normalization.
repair (EVAR). Biopsies of the ventral, infrarenal aneurysm wall,
at the maximum diameter, were obtained during the surgical pro-
2.4. Western blot analysis
cedure. Only thrombus covered aneurysm walls were used since
non-thrombus covered aneurysm walls could not be obtained from
The medial layers of the aneurysm samples were shred and
all participants, which is probably due to the low occurrence
mixed with a lysis buffer containing 50 l protease inhibitor and
of thrombus free walls in such large aneurysms. Patient char-
30 l 1 M Tris-HCl pH 8,0. The mixture of samples and lysis buffer
acteristics were obtained from hospital charts. Body mass index
samples were then granulated with a Tissuelyzer according to
(BMI) was dened as the ratio of weight/square of the height
manufacturers protocol and centrifuged for 5 min at 220 rpm. The
[21]. Body surface area (BSA) was predicted using DuBois formula:
supernatants were sonicated for 5 min at high level followed by
(weight0.425 xheight0.725 ) 0.007184 [22]. Aortic size index (ASI)
centrifugation for 10 min at 12000 rpm. The protein content in the
was dened as the ratio of aneurysm diameter/BSA [23]. All patients
supernatants was determined using Bradford protein assay. The
had signed an informed consent prior to the surgical procedure.
samples were diluted with lysis buffer before being loaded on a
The control group consisted of 6 organ donors, male and female.
412% SDS gel (Novex NuPAGE 412% Bis-Trisgel 15well, Invit-
The aortic diameters of the organ donors were not measured, but
rogen) in MOPS-SDS running buffer. Electrophoresis was run for
dened non-aneurysmal by the transplant surgeon. During the
90 min at 120 V, in a cold room. The gel and membrane (Hybond
organ donation biopsies of the infrarenal aorta were obtained. The
PVDF transfer membrane, GE Healthcare) were equilibrated in
aortic biopsies from organ donors and patients with AAA were han-
transfer buffer before transfer by electroblotting for 90 min at
dled according to the same protocol and stored during transport
400 mA, in a cold room. For blocking, the membrane was suspended
in RNAlater and formalin. The aortic walls, both aneurysmal and
in blocking buffer (3% bovine serum albumin/TTBS) for 60 min.
non-aneurysmal, were divided into initimal, medial and advential
The membrane was incubated over night with ER (Thermo), AR
layers. The biopsies from organ donors could not be transported
(Abcam) and GAPDH (Abcam) followed by the second antibody
freshly frozen, which limited the protein expression analysis. All
(anti-Mouse and anti-Rabbit HRP, BioRab) for 45 min. Finally, the
donors, or their close relatives, had signed an informed consent
developing solution from ECL Prime Western Blotting Detection
regarding donation of the tissue for research purposes. Patient
Reagent kit (GE Healthcare) and CCD camera (Fujilm LAS-1000)
characteristics were obtained from a form lled out by the organ
were used for chemiluminescent detection.
coordinator in charge and based on information from hospital
charts and from the patients relatives. The study was approved
2.5. Statistical analysis
by the local Ethics Committee.
Statistical analysis was performed with SPSS 21.0. Independent
2.2. Immunohistochemical analysis t-test was used for gender comparisons of normally distributed data
and Mann U test for not normally distributed data. Pearsons chi-
5 m sections of aortic walls were deparafnised in Tissue- square test and Fischers exact test were used for normally and not
Clear (Sakura) and rehydrated in ethanol. The sections were boiled normally distributed categorical variables, respectively. AAA occur-
under high pressure in DIVA-buffer (BioCare Medical) and Back- 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 P < 0.001) (Table 2). There was no difference in expression levels of
a univariate model (sex, age, hypertension, heart condition, lung ER between aneurysms and control aortas (Table 2).
disease, diabetes mellitus, smoking habits, BMI, ASI, ER, ER, PR Men and women with AAA did not differ regarding mRNA
and AR were included) and clinically relevant variables or those expression levels of sex hormone receptors (Supplementary Table
signicant from the univariate analysis were analysed using a mul- II). No correlations were found between aneurysm size and mRNA
tiregression model. Statistical signicance was dened as P < 0.05. 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-
3. Results
tension, diabetes mellitus, heart condition, lung disease, smoking
habits, BMI, BSA, expression levels of ER, ER, PR and AR. A sig-
3.1. Patient characteristics
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-
Age, BMI as well as BSA were similar in patients with AAA and
nicant predictor of AAA occurrence. A multiple linear regression
controls (69 years vs. 70, P = 0.496, 25.7 vs. 26.0, P = 0.873 and 1.8 vs.
was calculated to predict AAA diameter in patients with AAA. A sig-
1.8, P = 0.987, respectively). Occurrence of comorbid conditions was
nicant regression equation was found (F (2,28) = 15.316, P < 0.001)
similar in patients with AAA and controls (Table 1)(Supplementary
with an R2 of 0.488 identifying both ASI and heart condition as
Table I). Patients with AAA were prior and current smokers to a
signicant predictors of AAA diameter.
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 3.3. Protein expressions of sex hormone receptors
AAA were similar (Table 1). Women with AAA had lower BSA
and higher ASI compared with men (Table 1). Smoking habits and Protein expression of ER in vascular smooth muscle cells
comorbid conditions were similar in men and women with AAA. was lower in patients with AAA compared with controls, whereas
expression of AR was higher in patients with AAA compared with
3.2. mRNA expressions of sex hormone receptors controls, assessed by immunohistochemistry (Fig. 1). There was no
difference in the expression of PR between patients with AAA and
Patients with AAA had higher mRNA expression levels of AR controls.
and PR than controls (7.26 vs. 5.14, P = 0.001 and 8.73 vs. 6.21, Protein expressions of ER and AR were similar in men and
P = 0.003, respectively) (Table 2). mRNA expression of ER was women with AAA, assessed by western blot analysis (Fig. 2).
lower in patients with AAA compared with controls (9.15 vs. 12.29,
4. Discussion
Table 2
mRNA expression levels of sex hormone receptors in patients with AAA and controls. The observed gender differences in aneurysm development
have been ascribed to effects of sex hormones on the vasculature,
Patients with AAA Controls P-value
n = 32 n=6 yet to our knowledge this is the rst study to investigate the expres-
sion prole of sex hormone receptors in human AAA. The results
Estrogen receptor 5.92 0.75 6.47 0.30 0.090
Estrogen receptor 9.15 1.45 12.29 1.06 < 0.001
show differences in the expression prole of sex hormone receptors
Progesterone receptor 8.73 (1.68) 6.21 (1.42) 0.003 between aneurysmal and non-aneurysmal aortic walls, indepen-
Androgen receptor 7.26 1.33 5.14 0.51 0.001 dent of gender. The observed lower expression of ER and higher
Values, normalized to RPLP0, are presented log2-transformed and as arbitrary units. expression of AR in aneurysms, could suggest that an increase of
Values are presented as mean standard deviation for normally distributed data and AR and a decrease of ER are associated with the development of
median (range) for not normally distributed data. 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 Estrogen receptors play an intricate role in many physiological
they mediate are inuenced by sex, age and menopause, which processes in the vasculature by modulating vascular tone, inam-
could partly explain the lack of differences between the sexes in this matory response and smooth muscle cell proliferation [26,27]. ER
study [24,25]. Thereto, for apparent reasons, tissue from aneurysms and ER are present in equal quantities in the aortic vascular
can only be obtained when patients are subjected to OR, which limit smooth muscle cells (VSMCs) of men, whereas ER is the primary
the analysis to an end-stage disease. Since AAA affects an elderly estrogen receptor in the aortic VSMCs of women [25]. The preven-
population it is also for practical reasons impossible to obtain biop- tive effect of exogenous estrogen on aneurysm development shown
sies of younger AAA patients, i.e. premenopausal women. With the in animal models is associated with an inhibition of the proteolytic
aid of results from studies on animals, we can only speculate on the activity mediated by matrix metalloproteinases [12,15,19]. In this
hormonal changes occurring in the aneurysm wall as AAA evolves study we found a lower expression of ER, mRNA and protein,
in men and women. 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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