Vascular and inflammatory cells can modulate the structure and composition of the extracellular matrix
by producing enzymes involved in its degradation. In vulnerable plaques matrix metalloproteinases MMPs,
that are secreted by macrophages, digest the matrix components within the fibrous cap. In cross-sections of
atherosclerotic arteries revealing extreme expansive re- modeling, like aneurysms, increased MMP activity
within the arterial wall and a higher density of macrophages may be observed within the arterial wall
[14]. Mainly the gelatinases MMP-2 and MMP-9 are highly expressed and more active in enlarged aneurys-
matic arteries.
It remains to be investigated whether the increased prevalence of inflammatory cells in expansively remod-
eled segments compared with constrictively remodeled segments is also evident for the atherosclerotic coronary
artery. In addition, it is unknown if such higher preva- lence of macrophages in enlarged segments is also
associated with an enhanced release of matrix metallo- proteases that are thought to play a pivotal role in both
matrix degradation and plaque destabilization [15 – 18] and atherosclerotic remodeling [14,19].The aim of the
present study was to investigate the association between the
mode of
remodeling and
the presence
of macrophages and MMP-1, MMP-2 and MMP-9 in
coronary atherosclerotic cross-sections. In addition, zy- mography was performed to study the gelatinase activ-
ity in atherosclerotic cross-sections that show expansive and constrictive remodeling.
2. Methods
Fourteen hearts were obtained within 24 h post- mortem eight men and six women, 71 9 8 years from
patients who did not die of cardiovascular disease. The right coronary artery RCA, n = 13, left anterior de-
scending coronary artery LAD, n = 12 and left cir- cumflex
LCX, n = 11
were dissected
from the
epicardium over a length of 5 – 10 cm from their origin. Six arteries were not used for analysis due to severe
calcifications or extensive cutting artifact. The dissected coronary arteries were frozen in liquid nitrogen and
stored at − 80°C.
2
.
1
. Morphometric analysis The frozen coronary arteries were cut into segments
of 2.5 mm that were subsequently numbered from proximal to distal. From each segment the side of the
proximal cutting face was marked with east-Indian ink. Odd numbered segments were stained by Lawson
elastin stain. Even numbered segments were stored for additional immunohistological staining. Morphometric
measurements were performed on both the proximal and sital site from each odd-numbered segment. If
selected, the adjacent cutting face of the even-numbered segment was used for additional staining. For example,
if the distal part of segment 1 was selected, then the proximal part of segment 2 was used for additional
staining [20].
The microscopic images of the cross-sections stained with Lawson elastic tissue stain were recorded on VHS
videotape with a 3CCD video camera for further image analysis. A ruler was used for distance calibration.
From each cross-section we measured the following parameters: lumen area, area encompassed by the inter-
nal elastic lamina IEL-area mm
2
, IEL-area circum- ference mm, area encompassed by the external elastic
lamina EEL-area mm
2
and EEL-area circumference mm.
Plaque area mm
2
was calculated by subtracting the lumen area from the IEL-area. The media area was
calculated by subtracting the EEL-area from the IEL- area. The area encompassed by the EEL-area was
recalculated as if the vessel was circular corrected EEL-area. Subsequently, the corrected IEL-area and
corrected lumen area were calculated assuming a circu- lar EEL-area. The corrected IEL-area and lumen area
were calculated by subtracting the media area from the corrected EEL-area and the plaque area from the cor-
rected IEL-area, respectively. Throughout the text, the lumen area and IEL-area will be referred as being the
corrected lumen areas and corrected IEL-areas.
2
.
2
. Selection and immunohistochemical staining Prior to histological staining a selection was made
based on arterial geometry. All measurements obtained from the arterial segments were plotted as demon-
strated in Fig. 1. Two groups of cross-sections were selected for staining that met the following criteria: A,
IEL-area \ 10 larger compared with adjacent proxi- mally and distally located cross-sections within 5 mm
expansive remodeling; B, IEL-area \ 10 smaller compared with adjacent proximally and distally located
cross-sections within 5 mm constrictive remodeling. No major side branches should originate between the
cross-section under investigation and the adjacent cross-sections.
From the selected segments 8 mm thick cross-sections were cut using a Microm HM 500 OM cryomicrotome.
From all selected arterial locations n = 84 cross-sec- tions were stained for macrophages with a monoclonal
antibody anti-CD68 Dakopatts and an acid phos- phatase staining. MMP-1 was located using a poly-
clonal antibody Biogenesis. A monoclonal antibody directed against MMP-2 Oncogene and a polyclonal
antibody against MMP-9 Biogenesis were used on a subgroup of 68 and 61 cross-sections, respectively. Ex-
amples for the different stainings are depicted in Fig. 2.
In short, 8 mm frozen sections were cut and stored at −
80°C until use. The sections were dried at room temperature for 1 h and fixed for 10 min in fresh
aceton, containing 0.03 H
2
O
2
to block endogenous peroxidase. The sections were dried, rinsed in PBS and
incubated with normal horse serum 10 in PBS for 30 min. Additional incubation with the correct primary
monoclonal antibody was performed overnight at 4°C in PBS containing 0.1 BSA. After the overnight incu-
bation the sections were rinsed in PBS three times for 5 min and incubated with 2.5 mgml biotynilated horse
anti-mouse polyclonal antibody Vector in PBS con-
Fig. 1. Cross-sectional changes along a post mortem obtained coronary artery segment. The arterial segment is represented as if all lesions were completely concentric. The interrupted line represents the luminal border, the solid line the internal elastic lamina. Arrows indicate the selected
cross-sections which revealed an IEL-area 10 larger or smaller than the adjacent cross-sections representative for local expansive and constrictive remodeling and shrinkage, respectively.
Fig. 2. Examples of the different immunohistochemical stainings. Left upper panel: MMP-9 in the cap of a small atherosclerotic plaque. The plaque near the arrow is illustrated magnified in the middle upper panel. Right upper panel is positive MMP-1 staining in the shoulder of an
atherosclerotic plaque. CD-68 positive staining is depicted in the bottom panels. The left panel shows CD-68 positive cells macrophages in the cap of the plaque. The right panel illustrates CD-68 positive cells in the adventitia ADV of a coronary artery segment.
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
