Key Words
Abdominal aortic aneurysm - Gender - Women - Elastin - MMP-9 - Progression - Rupture
Introduction
Abdominal aortic aneurysms (AAAs) in women differ in some important aspects from those
in men. The lower prevalence rate, higher rupture rate and potentially increased growth
rate in women with AAA suggest gender to be of importance for aneurysm development
and progression.[1]
[2]
[3].
The development of AAA is characterized by the loss of elastin in the medial layer
of the aortic wall together with an increased collagen turnover[4]
[5]. With the depletion of elastin and collagen the aorta loses its elasticity and tensile
strength[6]. Elastin, which is synthesized by vascular smooth muscle cells (VSMCs) in the medial
layer of the aortic wall, is composed to impart resilience for a lifetime[7]
[8].
The breakdown of elastin in AAA development is induced by proteolytic enzymes, produced
by infiltrating inflammatory cells and modified VSMCs[9]. The family of matrix metalloproteinases (MMPs) are the proteolytic enzymes most
associated with AAA development[10]. Especially MMP-2 and -9, with substantial elastolytic capacity, contribute to both
AAA development and growth[11]
[12]. The family of cathepsins is another group of matrix-degenerating enzymes inducing
elastolysis that has been implicated in the formation and growth of AAA[13].
The intraluminal thrombus (ILT), often prevalent in AAA, has been show to influence
the deterioration of the underlying aneurysm wall[14]. Beneath the ILT the fragmentation of elastic fibers is more pronounced, the vessel
wall is thinner, and there are more inflammatory cells and fewer VSMCs than in the
non-thrombus-covered wall[14]
[15]. The effect mediated by the ILT has been related to its thickness and size; a thin
ILT has been shown to be more biomechanically active and to more effectively promote
proteolysis in the underlying aneurysm wall, whereas a thicker ILT contributes to
the deterioration of the underlying aneurysm wall by inducing hypoxia[16]
[17].
Women have smaller aortas than men, rendering the relative enlargement in women with
AAA greater compared to that of men at any given diameter, but little is known of
how it relates to the deterioration of the aneurysm wall[18]
[19]. Aortic size index (ASI) is a measurement taking the relative dilatation into consideration[20]. Gender differences in vascular aging have been ascribed to the effect of sex hormones[21]. Differences between the sexes in the degradation of the arterial wall, induced
by senescence, have been illustrated by the maintenance of elasticity in the aorta
of women compared to that of men[22]. Thereto, estrogen has been shown to increase the deposition of elastin by VSMCs[23].
A difference in elastin and elastolytic proteins between men and women with AAA could
help explain the gender disparities in AAA progression. The aim of the study was to
compare elastin content and some of the elastolytic enzymes mostly associated with
AAA development, between men and women with equally large AAA.
Materials and Methods
All women treated electively for AAA with open aneurysm repair at Karolinska University
Hospital, November 2008 to December 2012, were included (n = 14). Male AAA patients within similar age and aneurysm diameter, treated during
the same time period, were also included (n = 23). The patients were treated with open repair (OR) either because they were deemed
unfit for endovascular aneurysm repair (EVAR) or because of their comparatively young
age. A biopsy of the ventral infrarenal aneurysm wall, preferably in midline, often
at the maximum diameter, was obtained during operation. Two biopsies were taken from
each patient, if possible: thrombus-covered and non-thrombus-covered aneurysm wall.
The thrombus was removed and stored separately.
Patient characteristics were obtained from hospital charts. Body surface area (BSA)
was calculated according to DuBois: body surface area ((weight0.425 × height0.725) × 0.007184))[24]. ASI was calculated: aneurysm diameter (cm)/BSA (m2)[20].
All patients had signed an informed consent prior to the surgical procedure. The study
was approved by the local Ethics Committee.
Immunohistochemistry
Sections (5 μm) of thrombus-covered and non-thrombus-covered aneurysm walls were deparaffinized
in Tissue-Clear™ (Sakura, Leiden, The Netherlands) and rehydrated in ethanol. Weigert-van
Gieson staining was then performed by immersing the sections in the various solutions:
Weigert's hematoxylin, Weigert's elastin, and Van Gieson solutions, all from Sigma-Aldrich
(St. Louis, Missouri, USA), according to a standardized protocol. Elastic fibers turned
dark purple, muscle turned yellow, nuclei turned brown, and connective tissue turned
red. The investigator was blinded for sex and AAA diameter when valuing the elastin
staining.
Gene Expression Analysis
Snap-frozen thrombus- and non-thrombus-covered media were homogenized in Lysing Matrix
D tubes with Fastprep® (MP Biomedicals, Solon, Ohio, USA). RNA was isolated with TRIzol
(Life Technologies, Grand Island, New York, USA), RLT buffer (from RNeasy Mini kit,
Qiagen, Hilden, Germany) and DNase1 (RNase free DNase Set, Qiagen) according to the
manufacturer's protocol. RNA was quantified by a NanoDrop (NanoDrop Products, Wilmington,
Delaware, USA), and RNA quality and integrity were verified using the Agilent 2100
Bioanalyzer System (Agilent Technologies, Santa Clara, California, USA).
For quantification of gene expression, total RNA, was reverse-transcribed to cDNA
using Superscript II according to the manufacturer's protocol (Life Technologies).
Real-time polymerase chain reaction (PCR) was performed on the Applied Biosystems
(Foster City, California, USA) 7000 Real-Time PCR System with TaqMan Assays-on-Demand
Gene Expression Probes for elastin, MMP-9, MMP-2, and cathepsin K. 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.
Western Blot
Thrombus- and non-thrombus-covered medial layers were shred and put in tubes with
a lysis buffer containing 50 protease inhibitor and 30 1 mol/L Tris-HCl, pH 8.0. The
samples were then granulated with a Qiagen Tissuelyser according to the manufacturer's
protocol, followed by centrifugation for 5 minutes at 220 rpm. The supernatants were
sonicated for 5 minutes at high level followed by centrifugation for 10 minutes at
12,000 rpm. The protein content in the supernatants was measured using a Bradford
protein assay.
The protein was diluted with lysis buffer and then loaded on a 4-12% sodium dodecyl
sulfate (SDS) gel [Novex NuPAGE® 4-12% Bis-Tris gel, 15 well (Life Technologies)]
in MOPS-SDS running buffer. The electrophoresis was performed in a cold room for 90
minutes at 100 V. Before transfer by electroblotting in a cold room for 90 minutes
at 400 mA, the gel and membrane (Hybond Polyvinylidene Flouride transfer membrane,
GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) were equilibrated
in transfer buffer. For blocking, the membrane was suspended in blocking buffer (3%
bovine serum albumin/Tween Tris-buffered saline) for 60 minutes. The membrane was
incubated overnight with elastin (Abcam, Cambridge, United Kingdom), MMP-9 (Cell Signaling
Technology, Danvers, Massachusetts, USA), β-tubulin (Santa Cruz Biotechnology, Santa
Cruz, California, USA), and Glyceraldehyde 3-phosphate dehydrogenase (Abcam) followed
by the second antibody (anti-mouse and anti-rabbit HRP, Bio-Rad Laboratories, Hercules,
California, USA) for 45 minutes. For the final step of chemiluminescent detection,
the developing solution from ECL Prime Western Blotting Detection Reagent kit (GE
Healthcare) and film (Amersham Hyperfilm ECL, GE Healthcare) were used. Densitometry
was performed using ImageJ analysis software.
Statistics
The statistical analysis was performed with SPSS 21.0. The independent t test was used for gender comparisons of normally distributed data and the Mann-Whitney
U test for continuous, not normally distributed, data. Pearson's χ2 test and Fisher's exact test were used for parametric and nonparametric categorical
variables, respectively. Statistical significance was defined as P < 0.05.
Results
Body Surface Area and Aortic Size Index in Men and Women with AAA
Men and women with AAA were similar in age and had similar aneurysm diameter and body
mass index (BMI). Men with AAA had higher BSA than women with AAA, 1.9 versus 1.7,
P < 0.001. There was no difference in the absolute diameter relative to BSA (ASI),
i.e., AAA diameter (cm)/BSA (m2), between men and women ([Table 1]). Men and women with AAA were similar regarding smoking habits, occurrence of comorbid
conditions, relevant laboratory parameters, and medical treatments, with the exception
of beta blockers, which was more frequently prescribed to women ([Table 1]).
Table 1.
Patients' Characteristics
|
Men with AAA (N = 23)
|
Women with AAA (N = 14)
|
P-value
|
|
Age
|
71 ± 5
|
72 ± 7
|
0.595
|
|
AAA diameter
|
6.3 ± 0.9
|
6.0 ± 0.6
|
0.263
|
|
BMI
|
26.0 (5.2)
|
25.0 (3.0)
|
0.752
|
|
BSA
|
1.9 (0.2)
|
1.7 (0.2)
|
<0.001
|
|
ASI
|
3.2 ± 0.5
|
3.5 ± 0.3
|
0.107
|
|
Risk factors and comorbid conditions
|
|
|
|
|
Smoking
|
|
|
0.172
|
|
Current
|
12 (52)
|
4 (29)
|
|
|
Prior
|
10 (44)
|
9 (64)
|
|
|
Hypertension
|
11 (55)
|
8 (57)
|
0.901
|
|
Diabetes mellitus
|
3 (15)
|
0 (0)
|
0.261
|
|
Renal insufficiency
|
1 (5)
|
0 (0)
|
1.000
|
|
Cardiac disease[a]
|
3 (15)
|
4 (29)
|
0.410
|
|
Connective tissue disorder
|
1 (5)
|
1 (8)
|
1.000
|
|
Medical treatment
|
|
|
|
|
Beta blockers
|
4 (21)
|
10 (72)
|
0.004
|
|
Antiplatelet
|
15 (75)
|
7 (50)
|
0.163
|
|
Statins
|
12 (60)
|
8 (57)
|
0.868
|
|
Laboratory values
|
|
|
|
|
Albumin[b]
|
36 ± 4
|
38 ± 3
|
0.128
|
|
Creatinine[b]
|
86 ± 19
|
82 ± 24
|
0.614
|
Values are presented as mean ± standard deviation for normally distributed data, median
(IR) for not normally distributed data, and frequencies (%) for categorical variables.
Significance calculated by independent t test, Mann Whitney U test, Pearson's chi-square test, and Fisher's exact test. BMI = body mass index;
BSA = body surface area (weight0.425 × height 0.725) × 0.007184); ASI = aortic size index {aneurysm diameter (cm)/BSA (m2)}.
a Cardiac disease defined as cardiac insufficiency, coronary artery disease, and atrial
fibrillation.
b Laboratory values obtained 1 or 2 days prior to the elective surgery.
The Non-Thrombus-Covered Aneurysm Wall in Men and Women
Western blot analysis and immunohistochemistry showed that protein expression of elastin
in the non-thrombus-covered aneurysm wall was lower in women than in men ([Fig. 1]) There was no difference in mRNA expression of elastin between men and women ([Table 2]). Women had a higher mRNA expression of MMP-9 than men ([Table 2]). Similar results were observed in Western blot analysis ([Fig. 1]). There were no differences in MMP-2 and cathepsin K between men and women in mRNA
expression analysis ([Table 2]). Prior and current smokers as well as smoking and non-smoking men and women were
similar in the mRNA expressions of elastin, MMP-9 and cathepsin K.
Figure 1. A. Weigert-van Gieson's staining of elastin in the non-thrombus-covered aneurysm
wall of two men. B. Weigert-van Gieson's staining of elastin in the non-thrombus-covered
aneurysm wall of two women. C. Western blot and densitometry of elastin in the non-thrombus-covered
aneurysm wall of three women and two men (0.81 versus 2.30, P = 0.200). D. Western blot and densitometry of MMP-9 in the non-thrombus-covered aneurysm
wall of three women and two men (0.65 versus 0.22, P = 0.200).
Table 2.
Gene Expression Analysis of Elastin, MMP-2, MMP-9, and Cathepsin K in the Non-Thrombus-Covered
Aneurysmal Wall of Men and Women
|
Non-Thrombus-Covered Wall
|
P-value
|
|
Men with AAA
|
Women with AAA
|
|
Elastin
|
4.55 ± 3.21
|
5.01 ± 3.31
|
0.722
|
|
MMP-2
|
1.52 ± 1.22
|
1.74 ± 1.51
|
0.744
|
|
MMP-9
|
−0.83 ± 1.08
|
0.90 ± 2.59
|
0.041
|
|
Cathepsin K
|
1.68 ± 1.06
|
2.63 ± 1.01
|
0.063
|
Values are presented as mean ± standard deviation for normally distributed data. Significance
calculated by independent t test. Values are presented log2-transformed and as arbitrary units.
The Thrombus-Covered Aneurysm Wall in Men and Women
There was no difference in mRNA expression or protein expression of elastin, MMP-2,
MMP-9, and cathepsin K in the thrombus covered aneurysm wall between men and women
([Table 3]). There were no differences in mRNA expressions of elastin, MMP-2 and -9, and cathepsin
K between prior and current smokers or between smoking and nonsmoking men and women.
Table 3.
Gene Expression Analysis of Elastin, MMP-2, MMP-9, and Cathepsin K in the Thrombus-Covered
Aneurysmal Wall of Men and Women
|
Thrombus-Covered Wall
|
P-value
|
|
Men with AAA
|
Women with AAA
|
|
Elastin
|
−6.03 (1.69)
|
−6.03 (1.89)
|
0.830
|
|
MMP-2
|
−2.13 ± 0.60
|
−2.41 ± 0.78
|
0.282
|
|
MMP-9
|
−2.27 ± 1.08
|
−2.20 ± 1.35
|
0.875
|
|
Cathepsin K
|
−2.40 (1.58)
|
−2.82 (1.89)
|
0.940
|
Values are presented as mean ± standard deviation for normally distributed data and
median (interquartile range) for not normally distributed data. Significance calculated
by independent t test and Mann Whitney U test. Values are presented log2-transformed and as arbitrary units.
Discussion
The key findings of this study, less elastin in the non-thrombus-covered aneurysm
wall of women compared to that of men with a simultaneous greater expression of MMP-9,
imply that the elastolytic process in the aneurysm wall differs between the sexes.
Elastin breakdown has been shown to be of importance especially in the initial growth
of an AAA, whereas collagen degradation has been associated with the later stages
of aneurysm enlargement and rupture[6]. Less protein expression of elastin in the non-thrombus-covered aneurysm wall of
women than in men, together with the opposite relation in protein and mRNA expression
of MMP-9, suggests a more elastolytic process in that part of the aneurysm wall of
women. The lack of difference in elastin content and proteolytic enzymes in the thrombus-covered
aneurysm wall between the sexes, found in this study, suggest that the elastolysis
in that part of the aneurysm wall is similar in men and women. The impact of the limited
expression of elastin in the non-thrombus-covered aneurysm wall of women remains to
be investigated but could contribute to explaining gender differences in AAA pathogenesis.
A weaker part of the aneurysm wall, as a result of greater elastolysis, in women could
be of importance for the growth and rupture rate of women's AAAs. The difference in
protein expression of elastin between men and women without a simultaneous difference
in the mRNA expression could be explained by the limited turnover rate and synthesis
of elastin in adults[8]. The similar age and AAA diameter as well as smoking habits of the participating
men and women in this study rule them out as possible confounding factors. Tong et
al.[19] recently presented results of a higher growth rate in men than in women with AAA
and less elastin in men, in the thrombus-covered aneurysm wall. A possible explanation
for the contradicting results might be differences in age, AAA diameter, and smoking
habits between the participants of the two studies.
MMP-2 has been implicated in the initial AAA development, and higher levels have been
observed beneath the ILT[14]. We found no difference in mRNA expression of MMP-2 in men and women either beneath
the ILT or in the non-thrombus-covered aneurysm wall. The lack of difference in mRNA
expression of MMP-2 and cathepsin K between men and women suggest that the expression
of MMP-2 and cathepsin K is of less importance for gender differences in AAA progression.
Women have smaller BSA and suprarenal aortic diameter than men, making the relative
aneurysm enlargement in women exceed that of men[18]. ASI is a measure—until recently, more commonly used when categorizing thoracic
aneurysms—that takes the relative enlargement into account[25]. Lo et al.[20] recently showed that ASI is an important determinant for AAA progression in women.
In this study women were found to have significantly lower BSA than men, but the ASI
did not significantly differ between the sexes. We do not rule out that a weaker aneurysm
wall, due to a greater enlargement, in women could help explain gender differences
in aneurysm development. We cannot, however, conclude that the differences in biomechanical
properties found in this study are attributed to such an effect.
There are limitations to this study, of which the small sample size is one. However,
the number of patients included in this study is similar to those in other studies
within the field of research[12]
[15]. Another limitation to the study is the potential influence of other enzymes involved
in the elastolytic process not addressed in this study; for instance, tissue inhibitor
of MMP (TIMP). More potentially influential genes could have been found with the use
of microarray analysis, for instance. Finally, the biopsies of the aneurysm wall were
all obtained from the anterior wall, and consequently the generalizability of the
results to the rest of the aneurysm wall cannot be assured.
In conclusion, less elastin in the non-thrombus-covered aortic wall of women with
AAA than in men and the simultaneous higher level of MMP-9 suggest differences in
the elastolytic process of AAA in men and women.
