Fig. 3. Zymogram of three atherosclerotic coronary artery segments. Equal amounts of protein were used. The arterial segment in lane 1
shows less MMP-2 compared with the segments in lanes 2 and 3. Active MMP-2 is most prevalent in lane 2.
on incubation was followed by staining the gel with Coommassie blue stain and additional destaining of the
gel so that clear white bands could be seen against a blue background. The white bands were quantified
using a densitometric method on a Geldoc 1000 system Bio-Rad using the Molecular Analist software. The
white bands for activeMMP-2 Fig. 3 and MMP-9 were identified on the basis of their molecular size and
by taking into account the recombinant MMP-2 and MMPs-9 proteins activated and non-activated on zy-
mography gel.
2
.
4
. Analysis The sections immunostained on the presence of
macrophages, MMP-1, MMP-2 and MMP-9 were ana- lyzed quantitatively by light microscopy using Sis-anal-
ysis 2.1 software. The sections were carefully studied and color thresholds were set and adjusted until the
computerized detection met the visual interpretation. The degree of staining was expressed in mm
2
. For all stains, the plaque, media and adventitia were analyzed
separately. For macrophages and MMP-1 the cap shoulder of the plaque and the core of the plaque were
analyzed separately. In addition, the type of staining co-localization with macrophages, diffuse staining was
noted.
For each immuno staining, cross sections were cate- gorized into three groups based on the values obtained
after computerized quantitative analysis: A, absentmi- nor staining 0 – 1000 mm
2
; B, moderate staining 1000 – 10 000 mm
2
; and C, extensive staining \ 10 000 mm
2
.
2
.
5
. Statistical analysis All values are presented as mean 9 S.D. A P-value of
B 0.05 was considered significant. Student’s t-test was
used to compare morphometric measurements and zy- mographic determined activity among groups. A x
2
-test was used to compare the area of immuno-stain among
groups.
3. Results
From 36 coronary arteries, 45 and 51 segments were selected with a vessel area that were found to be \ 10
smaller and larger compared with the adjacent seg- ments, respectively two to four segmentsartery. Mor-
phometric measurements of the cross-sections that demonstrated expansive or constrictive remodeling are
depicted in Table 1. Lumen area, plaque area, IEL-area and EEL-area were all larger in the expansively remod-
eled cross-sections compared with the cross-sections classified in the constrictive remodeling group.
taining 1 BSA and 1 normal human serum for 1 h. Next, the sections were rinsed in PBS three times for 5
min and incubated for one hour with 2 mgml strep- tavidine-horse radish peroxidase Southern Biotechnol-
ogy Associates.
To visualize the horse radish peroxidase, the sections were treated for 7 min with a sodium acetate buffer pH
5.0 containing amino-ethyl-carbazole AEC substrate or with a Tris – HCl buffer pH 7.5 containing DAB
imidazole substrate. In addition, the sections were counterstained with heamatoxylin, to visualize all nuclei
in the sections. For each staining, extra stainings with an omission of the primary antibody that served as
negative controls were taken into account. When using the monoclonal antibodies for detection of MMP-2 and
CD68, additional stainings were performed using an isotype matched control antibody in the same concen-
tration and served as negative controls.
2
.
3
. Zymography For a subgroup of cross-sections stained for MMP-2
and MMP-9 of which adjacent tissue was available, zymography was performed n = 35 for segments with
constrictive remodeling and n = 37 for segments with expansive remodeling. The zymography procedure was
performed using the next 10 – 15 adjacent 10 mm sec- tions. The sections were dissolved in 100 ml PBS con-
taining 0.1 Tween and 1 SDS. Non dissolved tissue remnants were removed by centrifuging the samples for
5 min at 13 000 rpm. In addition, the amount of protein in each sample was determined by using a standard
Lowry protein assay kit Bio-Rad and measuring at 655 nm with a automated ELISA-plate reader Bio-
Rad.
After equalizing the protein levels for all samples by dilution with ddH
2
O to 0.5 mgml, 30 ml of each sample was loaded on a standard 8 SDS-PAGE gel
containing 1 gelatin. The samples were run at 30 mA through the stacking gel and at 60 mA through the
running gel. After running, the gel was rinsed twice in a 2.5 Triton solution for 15 min each and then incu-
bated on at 37°C with a Tris – HCl buffer 50 mM, pH 7.4 containing 0.05 Brij and 10 mM CaCl
2
.The
Table 1 Measurements of geometric variables obtained in cross-sections that
showed constrictive remodeling and expansive remodeling compared with the adjacent cross-sections within the arterial segment
a
Expansion Constriction
Number of cross-sections 45
51 5.8 9 3.4
Lumen area mm
2
3.5 9 2.6 13.7 9 5.9
8.0 9 4.5 IEL-area mm
2
7.9 9 5.0 Plaque area mm
2
4.5 9 3.3 17.1 9 7.3
10.4 9 5.4 EEL-area mm
2 a
Values are mean 9 S.D. PB0.05.
Table 2 Computerized measured values for macrophage, MMP-1, MMP-2
and MMP-9 staining
a
Expansion Constriction
Macrophages, plaque mm
2
56835 87974
3811849856 Luminalmedial border
3160925226 57948
45352 Macrophages, adventitia mm
2
MMP-1, plaque mm
2
59432 52275
MMP-1, adventitia mm
2
10578 11343
MMP-2, plaque mm
2
148093 91985
54605 MMP-2, adventitia mm
2
55010 MMP-9, plaque mm
2
256861 149787
20328 MMP-9, adventitia mm
2
15800
a
The degree of staining was not normally distributed because of a substantial number of cross-sections with extreme staining for the
different assays. The measured values may differ among macrophages and the different MMPs because of different threshold values for the
different stains.
The area of staining for macrophages in the plaque tended to be more prevalent in expansively remodeled
cross-sections compared with the cross-sections in the constrictive remodeling group. This difference, how-
ever, was not significant P = 0.08 Fig. 4. The com- puterized measured areas of immunostaining on the
presence of macrophages were 56.835 versus 87.974 mm
2
for the constricted and expansively remodeled cross-sec- tions, respectively. These values for macrophage stain-
ing within the total plaque were representative for the observed values within the cap and shoulder region of
the plaque as well as at the medial border of the plaque Table 2. Overall, the media lacked macrophage stain-
ing in the cross-sections under study. The degree of macrophage stain did not differ for the adventitia
among groups. In the adventitia the extent of macrophage stain was found to be moderate in 4296
Fig. 4. Percentage of cross-sections with minor 0 – 1000 mm
2
, moderate 1000 – 10 000 mm
2
and heavy \ 10 000 mm
2
staining for macrophages, MMP-1, MMP-2 and MMP-9 in the cap and shoulder CS, only depicted for macrophages, the total plaque PL, the media ME and the
adventitia ADV in the groups of cross-sections with the larger IEL-areas + versus the cross-sections with the smaller IEL-areas − .
cross-sections and heavy in 4596 cross-sections Fig. 4.
No difference in MMP-1 staining was observed be- tween the cross-sections that showed expansive and
constrictive remodeling in the different layers of the arterial wall total plaque, cap and shoulder of the
plaque and adventitia Fig. 4. In contrast to the macrophages, the extent of staining for MMP-1 was
more prevalent in the plaque than in the adventitia Table 2.Both MMP-2 and MMP-9 immunostaining
were more prevalent in plaques of the expansively remodeled segments versus constrictively remodeled
cross-sections Fig. 4. This enhanced staining in the expansively enlarged segments was evident in the cap
and shoulder region as well as at the medial border of the plaque. MMP-9 mostly co-localized with present
macrophages. MMP-2 showed more diffuse staining throughout the atherosclerotic plaque not specifically
co-localizing with macrophages. The differences in staining among groups for MMP-2 and MMP-9 were
not observed in either the media or the adventitia of the atherosclerotic cross-sections. MMP-2 and MMP-9
staining in the adventitia was much less prevalent when compared with the plaque Table 2.
The plaque area was larger in the segments with expansive remodeling, compared with segments that
showed constrictive remodeling Table 1. The plaque area may, therefore, be considered a confounder for the
interpretation of the differences in staining among groups. Zymography, in which equal amount of
proteins had been loaded, revealed more active MMP-2 in the segments with expansive compared with segments
with constrictive remodeling 340 9 319 vs. 199 9 180 adjusted pixel countsmm
2
, respectively P = 0.018 whereas the inactive MMP-2 did not differ 3030 9
1290 vs. 3080 9 1339, respectively. MMP-9 did not differ for expansively and constrictively remodeled
cross-sections 2884 9 1988 vs. 2597 9 1674, respec- tively.
4. Discussion
