K. Nishimura et al.

noted in 2 of the small AAAs. A higher expression of MMP-2 was also noted in 1 of the 3aneurysms of the control group. On the otherhand, MMP-9 showed lower or almost no expressions in all the specimens examined.

16MMP-2/GAPDH

141210Discussion

86This study analyzed the relationship between
the development of AAAs and the expression ofMMP-2, MMP-9 and TIMP-1 in the aorticwalls. The AAAs were subclassified into 2
categories due to the diameter of AAAs; small
type being 45 mm or less, and medium-large
type being greater than 45 mm. This classification is based on a previous observation, which
confirmed that AAAs with diameters greaterthan 45 mm showed a higher frequency of aneurysmal rupture (Kanaoka et al., 1999). Maeda
et al. (1996) described 3 stages of AAA progression; development, growth and rupture.
They corrected the diameter of AAAs followedby body height as a congenital factor and by age
as an acquired factor, and found that small andmedium-large AAAs corresponded well to thestages of growth and rupture, respectively.
The present study confirmed the highest
expression of MMP-2 mRNA as well as protein
levels among the 3 molecules. The expression
was significantly higher in the small-diameter
AAAs than in the controls, suggesting the involvement of MMP-2 in the growth of AAAs.
This is partially consistent with the report byFreestone et al. (1995), who found that theproduction of MMP-2 protein was higher in theAAAs of 40 to 55 mm in diameter. Moreover, it
is well known that MMP-2 is expressed only insmall amounts in obstructive arteriosclerotic
lesions (Freestone et al., 1995; Davis et al.,1998). Thus, it is conceivable that MMP-2
might play a crucial role in the development andgrowth, i.e., an early stage of AAAs.

4201525354555

657585 (mm)657585 (mm)657585 (mm)Diameter

MMP-2
0.16

MMP-9/GAPDH

0.14
0.12
0.10
0.08
0.06
0.04
0.02
01525354555

Diameter

MMP-9

TIMP-1/GAPDH

2.9
2.4
1.9
1.4
0.9
0.4
01525354555

Diameter

TIMP-1
Fig. 4. Scatter diagrams show the expressions of matrix metalloproteinase (MMP)2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 mRNA in smallabdominal aortic aneurysms (AAAs) [ n = 6] and medium-large AAAs [n = 14] andin control specimens [n = 10]. The RNA amount is presented as a percentage toglyceraldehyde-3-phosphate dehydrogenase (GAPDH).

32

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

Table 3. mRNA expressions of MMP-2, MMP-9 and TIMP-1 in the small and medium-largeAAAs and controls
MMP-2

MMP-9TIMP-1

Controls[10]0.02 ± 0.03
0.001 ± 0.002**
0.01 ± 0.03**
Small AAAs
[ 6]
3.17 ± 6.04
0.03 ± 0.03
0.58 ± 0.98
Medium-large AAAs
[14]
0.10 ± 0.20
0.02 ± 0.05
0.29 ± 0.47
[ ], number of specimens
AAA, abnormal arotic aneurysm; MMP, matrix metalloproteinase; mRNA, messenger RNA; TIMP, tissue
inhibitor of metalloproteinase.
*P < 0.05; **P < 0.01.

Both small and medium-large AAAs showed higher expressions of MMP-9 than controls.
When the medium-large AAAs were divided
into 2 subgroups by size, the mean expression
ratio of MMP-9 mRNA was higher in themedium-type AAAs than in the large-typeAAAs, although the difference was not significant. Similar results were reported by
McMillan et al. (1997), who divided AAAs into
3 groups; small (less than 49 mm), medium

Table 4. Correlation of MMP-2, MMP-9 and TIMP in the small, medium-large and total AAAs
and controls
Small AAA

Medium-large AAA

AAAControls

0.15

0.15

0.13

0.13

MMP-9/GAPDH

MMP-9/GAPDH

MMP-2 and MMP-9–0.20 [6]0.62 [15]*0.46 [21]*0.06 [10]MMP-2 and TIMP-1
0.26 [6]0.42 [14]0.30 [20]0.39 [10]MMP-9 and TIMP-1
0.77 [6]0.75 [15]**0.70 [21]**0.52 [11][ ], number of specimens
AAA, abnormal arotic aneurysm; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase.
Spearman’s correlation coefficient by rank (*P < 0.05, **P < 0.01).

0.11
0.09
0.07
0.05
0.03

0.11
0.09
0.07
0.05
0.03
0.01

0.01

00
0

0.45

0.95

1.45

1.95

2.45

0

0.4

0.8

1.2

TIMP-1/GAPDH

TIMP-1/GAPDHAAAMedium-large AAA
MMP-9 and TIMP-1

Fig. 5. Correlations between matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase(TIMP)-1. There is a significant correlation between TIMP-1 and MMP-9 in AAAs [n = 21, r = 0.70, P <0.01] (left), especially in medium-large AAAs [n = 15, r = 0.75, P < 0.01] (right). The RNA amount ispresented as a percentage to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

33

K. Nishimura et al.

1234

56

7

8

9MMP-2

68 kDaMMP-997 kDa
29 kDaTIMP-1Fig. 6. Western blot analysis for the expression of matrix metalloproteinase (MMP)-2, MMP-9 and tissue
inhibitor of metalloproteinase (TIMP)-1 in amall-diameter abdominal aortic aneurysms (AAAs) (lanes 1–3),
medium-large-diameter AAAs (lanes 4–6) and in control specimens (lanes 7–9): n = 3 per group.

(50–69 mm) and large (more than 70 mm).
They found a significantly higher expression in
the medium type than in the small or large type.
In other words, the MMP-9 expression obviously decreased in the large AAAs. Moreover, immunohistochemistry confirmed a diffuse distribution of MMP-9-positive cells in the entire
aneurysmal wall, in contrast to mainly intimal
localization of MMP-2- and TIMP-1-positivecells. In spite of the expression ratio of MMP-9
being lower than that of MMP-2, the results
indicated that MMP-9 might influence the disorder of elastin metabolism and participate in
the growth stage of AAAs, as suggested by
McMillan et al. (1997).
It is of interest that the MMP-2 and MMP-9
expressions well correlated in the mediumlarge AAAs, but not in the small AAAs. The
simultaneous lower expressions of MMP-2 andMMP-9 could imply that the gene expression
might be regulated by the aneurysmal stroma or
the cell-stromal interaction in the remodeled
aneurysmal wall.
TIMP-1 has been shown to inhibit the activity of MMPs and to prevent the degeneration ofelastic fibers, as well as development and rupture of AAAs (Birkedal-Hansen et al., 1993;
Allaire et al., 1998). In the present study, theTIMP-1 expression significantly correlatedwith MMP-9 in the medium-large AAAs, suggesting that TIMP-1 might inhibit MMP-9 in areactive or protective manner. In fact, there wasno significant correlation between the expressions of TIMP-1 and MMP-9 in the smallAAAs and control specimens. Although there

was no statistical correlation between the expressions of TIMP-1 and MMP-2, there was atendency for the TIMP-1 expression to increase
along with the MMP-2 expression.
In conclusion, this study demonstrated that
MMP-2 and MMP-9 play crucial roles in thedevelopment or growth of AAAs, and that
TIMP-1 inhibits their proteinase activity. The
precise roles of other MMP and TIMP family
molecules, such as TIMP-2, await further clarification.

Acknowledgments: We are grateful to Professor
Hisao Ito, First Department of Pathology and Professor Tadashi Terada, Second Department of Pathology, Faculty of Medicine, Tottori University, fortheir helpful advice and suggestions. We are also
grateful to Associate Professor Hitoshi Ohshiro, Department of Public Health, Faculty of Medicine,Tottori University, for his helpful statistical advice.
Finally, we would like to thank Ms. Etsuko Ueta, Mr.
Toshiyuki Yamamoto and Ms. Ami Inata, Ms. Kaori
Adachi and Ms. Makiko Tamura, Gene ResearchCenter, Tottori University, for their technical help.

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Received December 18, 2000; accepted December 22, 2000
Corresponding author: Dr. Kengo Nishimura

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