Yonago Acta medica 2001;44:25–35

Expression of MMP-2, MMP-9 and TIMP-1 in the Wall of Abdominal Aortic
Aneurysms
Kengo Nishimura, Shigetsugu Ohgi and Eiji Nanba*
Second Department of Surgery, Faculty of Medicine, Tottori University and *Gene ResearchCenter, Tottori University, Yonago 683-0826 Japan
An impaired mechanism of regulatory feedback by matrix metalloproteinases (MMPs)
and tissue inhibitors of metalloproteinases (TIMPs) has been implicated in the development of abdominal aortic aneurysms (AAAs). This study examined the pathogenesis ofAAAs with respect to pathological characteristics and expressions of MMP-2, MMP-9
and TIMP-1. Their expressions were evaluated by immunohistochemistry, competitive
polymerase chain reaction (PCR) and Western blotting in a total of 23 consecutive
AAAs. The AAA specimens were obtained by surgery, while control specimens were obtained at autopsy. Specimens consisted of 6 patients with small-diameter AAAs (30–45mm), 17 with medium-large-diameter AAAs (> 45 mm) and 11 controls (17–25 mm).
Immunohistochemistry showed MMP-2- and TIMP-1-positive cells mainly in the intima,
and MMP-9-positive cells in the intima and adventitia. Competitive PCR showed asignificantly higher expression of MMP-2 messenger RNA (mRNA) in the small-diameter
AAAs, and higher expressions of MMP-9 mRNA in the small-diameter and mediumlarge-diameter AAAs than in the controls. The mRNA levels significantly correlated
between TIMP-1 and MMP-9, and between MMP-2 and MMP-9 in the AAAs, especiallyin the medium-large-diameter AAAs. Western blotting revealed the expression of MMPs
and TIMP-1 variably in all the specimens examined. These results indicated that MMP2 and MMP-9 might act cooperatively and play a crucial role in the development ofAAAs, and that TIMP-1 inhibits MMP-9 in the AAAs, especially in those mediumlarge-diameter AAAs.
Key words: abdominal aortic aneurysm; competitive polymerase chain reaction; immunohistochemistry; matrix metalloproteinase; tissue inhibitor of metalloproteinase

The pathologic hallmark of aortic aneurysm isconsidered to be the remodeling of the aortic
wall, involving fragmentation and decrease ofelastic fibers in the tunica media (Baxter et al.,
1992; Gandhi et al., 1994). True aneurysms
formed in Marfan’s syndrome (Chikumi et al.,2000) were found to be due to a mutation in the
fibrillin gene (Dietz et al., 1991; Tsipouras etal., 1992), indicating that aneurysms may becaused by genetic disorders affecting 2 or morecomponents of the aortic wall. Recent hypotheses have indicated that matrix metallo-

proteinases (MMPs) involved in the metabolism of elastin in the wall of aortic aneurysms
may be one of the etiologic factors in the development of abdominal aortic aneurysm(AAA) (Campa et al., 1987; Tilson et al., 1988;
Baxter et al., 1992; Freestone et al., 1995;
Newman et al., 1994; Thompson et al., 1995;
McMillan et al., 1995, 1995, 1997; Tamarina etal., 1997; Davis et al., 1998; Elmore et al., 1998).
However, the precise roles of monocytes, macrophages and vascular smooth muscle cells which
are considered to be involved in the early changes

Abbreviations: AAA, abdominal aortic aneurysm; cDNA, complementary DNA ESDNA, external standard
DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP, matrix metalloproteinase;mRNA,
messenger RNA; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SAB, streptavidin-biotinperoxidase; SCDNA, specimen cDNA;TIMP, tissue inhibitor of metalloproteinase25

K. Nishimura et al.

of vascular remodeling, have not been adequately elucidated. It is important to examine
the entire wall as a functioning organ using
molecular-biological techniques, to compare
the pathological changes of the individual
layers including the intima, tunica media andadventitia. AAAs might result from impairment of the mechanism of regulatory feedbackby MMPs and tissue inhibitors of metalloproteinases (TIMPs). A comparative analysisof anatomic changes and tissue metabolism inAAAs small in diameter without obvious structural destruction may provide a clue to the pathogenesis.
With this background, the present study
examined the metabolism of elastin in the human aortic wall during the process of expansionof aortic diameter with respect to the expressionof MMP-2, MMP-9 and TIMP-1.

Method for collection of aortic wall
After approval by the Ethics Committee of
Tottori University Faculty of Medicine, surgical specimens of the aortic wall were collectedfrom the patients after obtaining informed consent. During surgery, a rectangular strip of theanterior wall was resected from the site of maximal diameter to the aneurysmal-normal junction throughout the wall. At autopsy, a section
was obtained from the abdominal aorta with agrossly atheroma-free intima throughout thewall. Immediately after resection, part of eachspecimen was fixed in 10% neutral bufferedformalin for 48 h, and the rest was frozen inliquid nitrogen and stored at –80˚C.

Histopathologic analysis
Three-micron-thick sections were cut fromparaffin-embedded human AAA walls and abdominal aortas from autopsy materials. The
sections were stained with hematoxylin-eosin
staining and elastica van Gieson. For the localization of MMP-2, MMP-9 and TIMP-1, thestreptavidin-biotin-peroxidase complex (SAB)
method was used for immunohistochemical
staining. As the first antibody, purified mousemonoclonal anti-human MMP-2 antibody(diluted 1:200), purified mouse monoclonalanti-human MMP-9 antibody (diluted 1:400),
purified mouse monoclonal anti-human TIMP1 antibody (diluted 1:200) (Fuji Chemical Inc.,
Takaoka, Japan), and purified mouse monoclonal anti-human HAM56 antibody (DAKO,
Carpenteria, CA) were used. As the 2nd antibody and enzyme reagents, a Histofine SABPO kit (Nichirei, Tokyo, Japan) was used. Endogenous peroxidase activity was blocked withmethanol containing 0.3% hydrogen peroxide
at room temperature for 30 min. For the retrieval of the antigens, all slides were placed in 10
mmol/L citrate buffer (pH 6.0) and heated at
94˚C for 15 min in a microwave oven. Nonspecific staining was prevented by treatment
with phosphate-buffered saline (PBS) containing 2% fetal calf serum. The sections weretreated with diaminobezidine for color develop-

Materials and Methods

Specimens
The subjects in this study consisted of 23 consecutive patients with AAA who had undergone
an elective excision with graft replacement between November 1998 and December 1999.
Eleven specimens obtained at autopsy performed between February 1999 and March 2000
served as controls. Table 1 lists clinicopathological profiles of the AAA patients and the
autopsy cases.

Measurement of aortic diameter and staging of AAA
During surgery, the maximal diameter of theaorta distal to the renal artery was measured directly using the divider. The aortic diameter inthe control group was measured at autopsy. In
this study, an aorta with a diameter more than
30 mm was defined as an AAA. An aorta with adiameter between 30 mm and 45 mm was defined as a small-diameter AAA, and that with adiameter over 45 mm as a medium-largediameter AAA.

26

MMP-2, MMP-9 and TIMP-1 in AAAs

Table 1. Patient Characteristics
Variable

AAA

Small
AAA†

Number of patients
Total
23
6
Male
22
5
Female11Age(year)
Mean ± SD
72.4 ± 6.8
70.3 ± 3.7
Range
62–85
66–77
Diameter(mm)
Mean ± SD
53.3 ± 10.8
40.8 ± 5.6
Range
30–80
30–45
History of smoking
16 (69.6)
5 (83.3)
Chronic obstructive
pulmonary disease
8 (34.8)
1 (16.7)
Hypertension
14 (60.9)
4 (66.7)
Diabetes mellitus
5 (21.7)
2 (33.3)
Hyperlipidemia
6 (26.1)
2 (33.3)
Ischemic heart disease
10 (43.5)
1 (16.7)
Arteriosclerosis obliterans
7 (30.4)
2 (22.2)
Cerebrovbascular disease
3 (13.0)
1 (16.7)
Malignant tumor
5 (21.7)
3 (50.0)( ), %.
AAA, abdominal aorataic aneurysm; NS, not significant.
† Aneurysmal diameter, 30–45 mm.
‡ Aneurysmal diameter, > 45 mm.

Medium-large
AAA‡

Control

P value

17
17
0

11
7
4

73.1 ± 7.8
62–85

66.1 ± 14.1
47–92

NS

57.5 ± 8.6
48–80
11 (64.7)

20.4 ± 2.4
17–25
7 (63.6)< 0.01

7 (41.1)
10 (58.8)
4 (23.5)
4 (23.5)
9 (52.9)
5 (29.4)
2 (11.8)
2 (11.8)

1 ( 9.1)
2 (18.2)
2 (18.2)
2 (18.2)0( 0 )
1 ( 9.1)
3 (27.3)
8 (72.7)

NS
NS
NS
NS
< 0.01NSNS< 0.05NS

WI) and 2 µL of 200-units/µL Moloney murine
leukemia virus reverse transcriptase (Gibco
BRL Products, Life Technologies, Rockville,
MD). Control reactions omitting the reverse
transcriptase (reverse transcriptase negative)
were set up for each RNA sample.
The primers of human glyceraldehyde-3phosphate dehydrogenase (GAPDH), MMP-2,
MMP-9 and TIMP-1 were designed based on
Tamarina’s report (Tamarina et al., 1997) byusing the Genetyx-Mac Macintosh software(Software Development Co., Ltd., Tokyo)(Table 2). We designed the external standard
DNA (ESDNA) for each studied gene thatlacked from 10 to 20 base pairs (bp) so that the
ESDNA differed in size from the specimen
cDNA (SCDNA) obtained by the previous
method (Ho et al., 1989). The identity of the
SCDNA and ESDNA was verified by DNA sequence analysis with ABI310 (Applied Biosystems, Tokyo).
The PCR reaction mixture (12 µL final volume) contained 0.64 µL of each cDNA, 0.5

ment, and counterstained with 3% methyl
green. The degree of tissue staining was judged
under a light microscope at 4 × 3.3 magnification for elastica van Gieson staining and at 20 ×
5 magnification for immunohistochemical
staining, and then the expression was analyzed.

Competitive polymerase chain reaction(PCR)
The frozen tissue samples were homogenized in
guanidium isothiocyanate using a Polytron
homogenizer (Kinematica, Luzern, Switzerland).
After homogenization, total RNA was isolated
by CsCl in ultracentrifugation at 100,000 g for
20 h. Total RNA was further treated with gribonuclease-free deoxyribonuclease I (Nippon
gene, Tokyo) and extracted with phenol/chloroform/isoamylalcohol (50:49:1) and precipitatedwith ethanol. Reverse transcription of 1.5-µg
total RNA was performed to synthesize complementary DNA (cDNA) using 2 µL of 500µg/mL Random Hexamer (Promega, Madison,27

K. Nishimura et al.

Table 2. Primers used for competitive PCR
Gene

PCR

Length (bp)

MMP-2

SP

398

Primer5’=CAGGCTCTTCTCCTTTCACAAC
3’=AAGCCACGGCTTGGTTTTCCTC
ESP
379
5’=ACTCCAGACCCCTGGCTTTT
3’=CCAGGGGTCTGGAGTTGTCCCACTGCCCTGTGCCA
MMP-9
SP
172
5’=TGGGCTACGTGACCTATGACAT
3’=GCCCAGCCCACCTCCACTCCTC
ESP
162
5’=AAGGAGCCAGTTTGCCGGAT
3’=GCAAACTGGCTCCTTGGTCCCAGTGGGGATTTACA
TIMP-1SP279
5’=GGGGCTTCACCAAGACCTACAC
3’=AAGAAAGATGGGAGTGGGAACA
ESP
267
5’=TCTGAAAAGGGCTTCCAGTC
3’=GAAGCCCTTTTCAGACTGGTCCGTCCACAAGCAAT
GAPDH
SP
216
5’=TGCCTCCTGCACCACCAACTGC
3’=ATGACCTTGCCCACAGCCTT
ESP
205
5’=TGGAAGGACTCATGACCACA
3’=TCATGAGTCCTTCCATTGTCATGGATGACCTTGGC
bp, base pair; ESP, external standard primer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP,
matrix metalloproteinase; PCR, polymerase chain reaction; SP, specimen primer; TIMP, tissue inhibitor of
metalloproteinase.

percentage to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

units Taq polymerase (Gene Amp Taq Gold,
Perkin Elmer, Branchburg, NJ), 12 pmol of forward primer, 6 pmol of reverse primer conjugated with Cy5 (Amersham Pharmacia Biotech,
Buckinghamshire, England) and 6 pmol of reverse primer. For each sample, 4 or 5 equal
aliquots were prepared with a dilution series of
the ESDNA, and the PCR mixture was spiked
into these aliquots. Amplification was performed in the thermal cycler (Labosystems Japan
Co., Tokyo) for 35 cycles: denaturation at 95˚C
for 1 min; annealing for 1 min at 55˚C forGAPDH, MMP-9 and TIMP-1 and at 60˚C for
MMP-2; and extension at 72˚C for 1 min.
As the reverse primer was fluorescently
labeled with Cy5, PCR products were detectedwith an ALF red DNA Sequencer (Amersham
Pharmacia Biotech). After competitive PCR
was completed, the products corresponding to
the SCDNA and the ESDNA were electrophoresed on a 6% polyacrylamide and 6 mol/Lurea gel on the Sequencer. The peaks weredetected and analyzed with the Allele Links
software (Amersham Pharmacia Biotech). The
specific SCDNA template in each starting
cDNA sample was equimolar to ESDNA whenthat ESDNA area/SCDNA area was equal to 1
(Fig. 1). The RNA amount is presented as aWestern blotting
Frozen specimens of human AAA walls or
autopsied abdominal aortic walls were homogenized in a homogenizer, and the homogenate
was dissolved in lysis buffer containing 0.1%
nonylphenoxy polyethoxy ethanol (NP-40).
The solution was centrifuged in an ultracentrifuge at 15,000 revolutions per minute for 10 min,and the supernatant was used as a test sample.
The concentration of protein was measured
with a Protein Assay Kit (Bio-Rad, Hercules,
CA). Test samples of the same protein content(375 µg) were electrophoresed on a 5% sodium
dodecyl-sulfate-polyacrylamide gel at a constant voltage of 150 V, and the proteins weretransferred onto a nitrocellulose membrane at
80 V for 150 min. Markers (Bio-Rad Labs.,
Richmond, CA) were dissolved in a Coomassie
brilliant blue solution and used for electrophoresis. After blocking nonspecific reaction
with 10% skim milk in PBS, the membrane wasreacted at 4˚C overnight with one of the following first antibodies: purified mouse monoclonalanti-human MMP-2 antibody (diluted 1:50),
28

MMP-2, MMP-9 and TIMP-1 in AAAs

A

B
ESDNA (b)log(b/a)

SCDNA (a)2.5
2.0

Y = 3.376 + 0.409X
r 2 = 0.997

1.5
1.0
0.5
0.0
–0.5
–1.0
–1.5
–12 –11 –10 –9 –8

–7

–6

–5 –4

χ

–3 log(5E )Fig. 1. A: Polymerase chain reaction (PCR) products were detected with an ALF red DNA Sequencer(Amersham Pharmacia Biotech), as the reverse primer was fluorescently labeled with Cy5. The Peaks weredetected and analyzed with the Allele Links software (Amersham Pharmacia Biotech). B: The specificspecimen cDNA (SCDNA) template in each starting cDNA sample was equimolar to the external standard
DNA (ESDNA) when that ESDNA area/SCDNA area was equal to 1.

purified mouse monoclonal anti-human MMP9 (diluted 1:50) and purified mouse monoclonalanti-human TIMP-1 (diluted 1:50) (Fuji Chemical Inc., Takaoka). Subsequently, the membrane was reacted with purified goat polyclonal
peroxidase-conjugated anti-mouse immunoglobulin G antibody (diluted 1:1,000) as the 2nd
antibody at room temperature for 1 h. The
membrane was washed with PBS, and treatedwith enhanced chemiluminescence Western
blotting solution. The enhanced chemiluminescence of protein bands was detected by exposing X-ray film to the membrane, and the expression of MMP-2, MMP-9 and TIMP-1 proteins
was evaluated.

Results

Staging of AAA
The 23 patients with AAA (≥ 30 mm) consistedof 22 men and 1 woman. The mean age (± SD)
was 72.4 ± 6.8 years, and the mean diameter (±SD) of aneurysms was 53.3 ± 10.8 mm. Of
them, 6 patients (5 males and 1 female) had
small AAAs (30–45 mm): their mean age was70.3 ± 3.7 years and the mean diameter of aneurysms, 40.8 ± 5.6 mm (small-diameter AAA
group). The remaining 17 patients (all male)
had medium or large AAAs (> 45 mm): theirmean age was 73.1 ± 7.8 years and the mean
diameter of aneurysms, 57.5 ± 8.6 mm (mediumlarge-diameter AAA group).
The control group of 11 autopsy cases consisted of 7 males and 4 females aged 66.1 ± 14.1
years: the mean aortic diameter was 20.4 ± 2.4
mm. There were no significant differencesamong the 3 groups in clinicopathological profiles, except for aortic diameter, history of ischemic heart disease and malignant disease(Table 1).

Statistical analysis
Using the StatView (Abacus Concepts, Inc.,
Berkeley, CA) software, the difference between
2 groups was tested by the Mann-Whitney U
test. Differences among multiple groups were
tested by the Kruskal-Wallis test, and thegroups with significant differences were testedby the Tukey test (nonparametric test). The
correlation between 2 groups was tested with
Spearman’s correlation coefficient by rank. A
value of P < 0.05 was considered significant.
29

K. Nishimura et al.

abFig. 2. Tissue sections taken from a small-diameter
abdominal aortic aneurysm (AAA) (a), a mediumlarge AAA (b) and an autopsy case (c) were stainedwith elastica van Gieson. The degree of the staining
was judged under a light microscope at an original
magnification of 4 × 3.3.

Histopathologic findings
Medial elastic fibers in AAA walls weremarkedly fragmented or lost in the aortic wallof the aneurysms examined. The walls of
medium-large-diameter AAAs showed marked
thickening of the adventitia compared with thewalls of small-diameter AAAs (Figs. 2a and b).
In contrast, medial elastic fibers were preservedin the aortic walls of the autopsy cases (Fig. 2c).
Immunohistochemistry revealed that intracytoplasmic cells positive for MMP-2, MMP-9
and TIMP-1 were noted in macrophages andlymphocytes as well as vascular smooth muscle
cells. MMP-2-positive cells were present in theintimal plaques and thickened intimas in the
AAA walls (Fig. 3a). MMP-9-positive cells
were present in the outer layer of the tunica
media, adventitia and intimal plaques in the
AAA walls (Fig. 3b). TIMP-1-positive cells
were noted in the intimal plaques (Fig. 3c).
HAM56-positive cells were seen in similar locations to MMP-9-positive cells in the AAA
walls (Fig. 3d). A few cells positive for MMP2, MMP-9 and TIMP-1 were also demonstrated
in the control aortic walls.

cCompetitive PCR
Of the 34 specimens, messenger RNA (mRNA)
could be obtained from 10 controls, 6 smallAAAs and 14 medium-large AAAs. Figure 4
shows the expression ratios of MMP-2, MMP-9
and TIMP-1 to the internal control GAPDH.
Table 3 summarizes the mean expression ratiosof MMP-2, MMP-9 and TIMP-1 in the 3groups. The MMP-2 expression ratio was thehighest in the small AAAs, the value being
significantly higher than that of the 10 controls(P < 0.05). The MMP-9 expression ratio washigher in the small AAAs and the medium-large
30

MMP-2, MMP-9 and TIMP-1 in AAAs

a

c

b

d

Fig. 3. AAA tissue shows a positive immunoreaction for matrix metalloproteinase (MMP)-2 (a), MMP-9 (b),tissue inhibitor of metalloproteinase (TIMP)-1 (c) and HAM56 (d). The degree of immunohistochemical
staining was judged under a light microscope at an original magnification of 20 × 5.

AAAs than in the control group, P values being
less than 0.01 and 0.05, respectively. On theother hand, there was no significant difference
among the 3 groups on the TIMP-1 expression
ratio, although the expression ratio tended to behigher in the small AAAs. Overall, the highest
expression was noted in MMP-2, followed by
TIMP-1 and MMP-9, in the order given.
The medium-large AAAs were divided into
2 subgroups by diameter; medium type < 65mm and large type ≥ 65 mm. The mean expression ratio of MMP-9 was 0.026 ± 0.051 in themedium-type AAA [n = 11] and 0.006 ± 0.009
in the large-type AAA [n = 3], the value being
higher in the former than in the latter without
significant differences. The mean expression
ratios of MMP-2 and TIMP-1 were 0.045 ±0.073 and 0.334 ± 0.521 in the medium type and0.281 ± 0.419 and 0.128 ± 0.190 in the large
type, respectively. There was no statisticaldifference.

Table 4 shows the results of Spearman’scoefficient by rank to examine the relationshipof the expressions among MMP-2, MMP-9 and
TIMP-1. The expression levels of MMP-9 and
TIMP-1 signficantly correlated in all the AAAs
and the medium-large AAAs (P < 0.01, Fig. 5),
but not in the small AAAs and controls.
Moreover, the expression levels of MMP-2 and
MMP-9 also significantly correlated in a similar
fashion (P < 0.05, Table 4). No significant
correlation of expression was noted between
MMP-2 and TIMP-1.

Western blotting
Protein could be obtained from a total of 9
specimens, 3 each from the 3 groups. MMP-2,
MMP-9 and TIMP-1 were variably expressedas shown in Fig. 6. Of these, an obviously
higher expression of MMP-2 and TIMP-1 was
31