ORIGINAL ARTICLE - ADULT CARDIAC

Interactive CardioVascular and Thoracic Surgery 16 (2013) 1115

doi:10.1093/icvts/ivs421 Advance Access publication 9 October 2012

Characteristics of aortic wall extracellular matrix in patients

with acute myocardial infarction: tissue microarray detection
of collagen I, collagen III and elastin levels
Chee Hoe Konga,b,c,, Xiao Yun Lina,b,, Chin Cheng Wooc, Hung Chew Wongd, Chuen Neng Leea,b,c,
A. Mark Richardsb,e and Vitaly A. Sorokina,b,*
a
b
c
d
e

Department of Cardiac, Thoracic and Vascular Surgery, National University Heart Centre, Singapore
National University Health System, Singapore
Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Biostatistics Unit, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Cardiovascular Research Institute, National University Heart Centre, Singapore

Received 15 May 2012; received in revised form 20 August 2012; accepted 31 August 2012

Abstract
OBJECTIVES: Extracellular matrix (ECM) remodelling of the vessel wall is hypothesized to be an important step in atherosclerosis.
Changes of the ECM are associated with the gradual progression of an atherosclerotic lesion from a lipid streak to complicated unstable
plaque, leading to a complete vessel occlusion and eventually myocardial infarction (MI). Understanding of this process is critical in the
treatment and prevention of ischaemic heart disease (IHD).
METHODS: We investigated the histopathological characteristics of aortic wall ECM in IHD patients. Collagen I, collagen III and elastin
were assessed immunohistochemically in patients with acute MI and those with stable angina, using aortic punch tissues obtained from
coronary artery bypass graft surgery. Fluorescence tissue images were analysed using the tissue microarray technique.
RESULTS: The results showed that collagen III expression was found to be signicantly lower in the acute MI group (P < 0.001). As a
result of this change, the patients with MI also revealed a signicant reduction in the collagen III/collagen I ratio. The elastin/collagen
III ratio was signicantly higher in the MI group (P < 0.001).
CONCLUSIONS: Our study provided evidence of a decrease in collagen III content in patients with MI, which could possibly explain
the mechanism of plaque vulnerability and weakening of the plaque cap. A reduction in collagen III content, particularly away from the
atherosclerotic lesions, might be explained by the systemic vascular changes in patients with MI, and inammation and immune
responses could be potential causes of these systemic transformations. The biochemical mechanisms and factors regulating collagen III
production might be potential markers to predict possible cardiovascular events.
Keywords: Extracellular matrix Atherosclerosis Myocardial infarction Ischaemic heart disease Immunohistochemistry Tissue
microarray

INTRODUCTION
The vessel wall, comprising endothelial cells, vascular smooth
muscle cells and extracellular matrix (ECM), is very sensitive to
diverse stimuli, including mechanical forces and neurohumoral
factors. The vascular smooth muscle cells will sense any changes
that may lead to the modication and remodelling of ECM in
the vessel wall.
Collagen types I and III and elastin are the predominant constituents of the cardiovascular ECM. These components are
synthesized and regulated by the vascular cells. They are
arranged into an interlocking mesh to provide the structural and

Both authors contributed equally to this manuscript.

mechanical properties for vessel functions. Remodelling involves

the dynamic interaction between the vessel wall cells and the
ECM through a wide range of intracellular signalling pathways,
leading to the phenotypic adaptation of the ECM. Therefore, the
ECM remodelling is hypothesized to be an important step in the
pathogenesis of vascular diseases, including atherosclerosis [1].
The coronary arteries are one of the most common vessels
affected by the atherosclerotic process. The transformation of a
normal coronary artery to a completely occluded vessel is a
process developed over years via a series of complex changes.
The gradual progression from lipid streaks to complicated unstable plaque is strongly associated with the remodelling of the
ECM, but the molecular mechanism is not well-explained [2].
Understanding the transformation process of a stable to an

The Author 2012. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

ORIGINAL ARTICLE

* Corresponding author. Department of Cardiac, Thoracic and Vascular Surgery, National University Hospital, 1E Kent Ridge Road, NUHS Tower Block, Level 9,
Singapore 119228, Singapore. Tel: +65-67726507; fax: +65-67766475; e-mail: vitaly_sorokin@nuhs.edu.sg (V.A. Sorokin).

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unstable plaque, leading to complete vessel occlusion and possible myocardial infarction (MI), is critical in preventing and
treating complications of ischaemic heart disease (IHD).
Although an unstable coronary plaque is a local complication of
atherosclerosis, available research information indicates that
transformation to unstable plaque might be related to systemic
processes, affecting the entire vascular system [3]. This concept
of unstable patient has been proven by the presence of systemic biomarkers correlated with coronary events [4]. One of the
major obstacles in the process of understanding unstable plaque
is that no experimental model has been established and most information about the coronary artery comes from autopsy.
In our study, we used fresh tissues of the aortic punch
obtained from coronary artery bypass graft (CABG) surgery. Our
aim was to examine the histopathological characteristics of the
aortic wall in patients with IHD. In particular, we investigated the
immunohistochemical expression of major ECM components:
collagen I, collagen III and elastin in patients with acute MI and
patients with stable angina.

MATERIALS AND METHODS

Patient groups
Our study cohort comprised 50 patients. Forty patients underwent coronary artery bypass surgery at National University
Hospital, Singapore, between 2009 and 2011. The surgical
patients were divided into two groups. The MI group included
19 patients who were operated within 5 days post-MI, and the
non-MI group consisted of 21 patients with stable angina
according to accepted criteria. The non-MI group was kept free
of patients who had MI within 3 months. To compare an IHD
aorta with a normal aorta, we designed a control group consisting of autopsy material from cadavers without any medical
history or evidence of atherosclerotic disease (control group
n = 10). All surgical patients underwent routine examination, including lipid prole, Troponin I, full blood count and C-reactive
protein. Patients in the MI group had non-ST elevated myocardial reaction with Troponin I levels signicantly higher compared
with the non-MI group (Table 1). Statistical analyses of demographical data, risk factors and the lipid prole between the two
groups were compared, and we did not nd any signicant difference in the demographical data (Table 1). Also, as expected,
the MI group had signicantly elevated inammatory markers
and raised cardiac enzymes.
CABG surgery was performed with standard full normothermic
cardiopulmonary bypass and antegrade cold (4C) cardioplegia.
Cardiopulmonary bypass time and aortic cross-clamp time were
not signicantly different between the two groups (Table 1).
During CABG surgery, the aorta was punched out in order to
create proximal anastomosis. Tissues from the aortic punches
were preserved on dry ice immediately after excision and were
collected in the operation theatre within 5 min by the
Cardio-vascular Tissue Bank research team. For the integrity of
ECM, time was not critical; however, we kept the time as short
as possible to preserve the tissue for potential genomic and
proteomic experiments in the future. In the laboratory, one of
the aortic punch tissues was blocked with formalin and embedded in parafn, paying attention to orientate the tissue in the
standard way. The rest of the collected tissue was preserved in
80C. Tissue collection was approved by the Institution Review

Table 1: Demographical data of myocardial infarction

(MI) and non-MI groups
MI

Non-MI

P-value

Gender male (%)

17 (89.5%)
16 (76.2%)
0.27
Diabetes mellitus
12 (63.2%)
12 (57.1%)
0.698
Age mean (SD)
57.42 (11.28)
62.10 (8.64)
0.147
Smoking
10 (52.6%)
10 (47.6%)
0.752
Preoperative
12 (63.2%)
18 (85.7%)
0.1
anti-hyperlipidaemic
medication
Cholesterol (mmol/l) mean
4.71 (1.11)
4.51 (1.79)
0.716
(SD)
LDL-C (mmol/l) mean (SD)
2.81 (0.86)
2.95 (1.65)
0.77
Triglycerides (mmol/l)
2.14 (2.05)
1.35 (0.50)
0.188
mean (SD)
WBC (10.9/l) mean (SD)
10.31 (3.05)
7.07 (1.79)
<0.001
CKMB (g/l) mean (SD)
73.14 (73.08)
1.16 (0.23)
0.04
Troponin I (g/l) mean
23.92 (23.60) 0.0162 (0.0064) 0.014
(SD)
CRP (mg/l) mean (SD)
92.5 (63.49) NA
NA
CPB time mean (SD)
132.78 (44.51) 129.25 (40.66)
0.8
ACC time mean (SD)
72.33 (35.68)
82.00 (32.90)
0.39
In the MI group, WBC, Troponin and CKMB levels were significantly
higher (P > 0.05).
ACC: aortic cross clamp; LDL: low density lipoprotein; CKMB: creatine
kinase MB fraction; CRP: Creactive protein; WBC: white blood cell.

Board of the hospital. The autopsy materials were obtained from

the NICHD Brain and Tissue Bank for Developmental Disorders
at the University of Maryland, Baltimore, MD, USA.

Tissue processing
To analyse tissues in a standardized way, a tissue microarray was
designed. The tissue microarray was created with specimens
from all the patients in the study, using current techniques in the
Histopathology Facility of Institute of Molecular and Cell Biology,
Singapore. Small disks of tissues were harvested from the
parafn-embedded histological specimens of the aortic punch.
All tissue disks were placed on the array, stained and analysed
simultaneously. The slides were triple-stained for collagen I, collagen III and elastin antigens.

Histology and immunouorescence

The sections were deparafnized in xylene and rehydrated
through descending percentages of ethanol to water. We used
0.4 mg/ml Proteinase K to expose the epitopes for 5 min, and
non-specic binding was blocked with 10% goat serum in
Tris-buffered saline-Tween 20 for 60 min. The sections were then
incubated with primary antibodies: collagen I (Abcam ab34710)
(dilution 1:250), collagen III (Abcam ab6310) (dilution 1:100) or
elastin (Abcam ab52115) (dilution 1:100) overnight at 4C. This was
followed by incubation with secondary antibodies: anti-rabbit Alexa
594 (Molecular Probes A11037), anti-mouse Alexa 488 (Molecular
Probes A11029) and anti-guinea pig Alexa 546 (Molecular Probes
A11074) in dilution 1:1000 for 30 min in the dark. The sections
were nally counterstained for nuclei with Vectashield Hard Set
mounting medium with 4,6 diamidino 2 phenylindole.

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Table 2: Scanned data of fluorescence score specimens from myocardial infarction (MI), non-MI and control groups

Collagen I total fluorescence

(1 000 000) median (minimum
maximum)
Collagen III total fluorescence
(10 000) median (minimum
maximum)
Elastin total fluorescence 1 000 000)
median (minimummaximum)
Collagen III/collagen I median
(minimummaximum)
Elastin/collagen III median
(minimummaximum)

MI (n = 19)

2.43 (0.109.22)

Non-MI (n = 21)

Control (n = 10)

P-value
MI vs
non-MI

MI vs
control

Non-MI vs
control

2.01 (0.00213.64)

2.81 (0.775.21)

18.04 (0.51352.47)

7.45 (0.0474.85)

0.72 (0.0015.59)

1.32 (0.232.35)

0.0002 (0.000020.004)

0.12 (0.001171.88)

0.03 (0.00020.20)

<0.001

<0.001

0.297

2246.66 (53.5520708.49)

5.48 (0.003130.54)

13.71 (1.803853.87)

<0.001

0.001

0.324

0.03 (0.00040.89)

1.27 (0.073.42)

<0.001

<0.001

0.417

0.762

ORIGINAL ARTICLE

Median

Image analysis and grading system for

quantication
Fluorescence images were scanned sequentially and uorescence images of each channel were analysed separately using
Ariol software (Genetix). Fluorescence cross talk between uorophores was tested and found to be negligible. For each stain, a
mask was generated for all pixels with a uorescence level above
background. The area and mean intensity of this region were
recorded.
The natural auto uorescence in the green spectra was used
to establish the total area of tissue sampled. The uorescence
score for each tissue section was calculated as follows:
Total fluorescence

Mean fluorescence  Threshold level  Fluorescent area

Total tissue area

Statistical analysis
All the statistical analyses were performed using IBM SPSS
Statistics version 19 (SPSS Inc., Chicago, IL, USA). The demographic data were presented using descriptive statistics. The
KruskalWallis test was used to evaluate the differences in collagen I total uorescence, collagen III total uorescence, elastin
total uorescence, ratio of collagen III and collagen I and the
ratio of elastin and collagen III among the MI, non-MI and
control groups, respectively. For multiple pair-wise comparisons,
the MannWhitney U-test with the Bonferroni correction technique was used. The statistical signicance was set at a 5% level.

RESULTS
The uorescence images of the triple-stained tissue microarray
sections were compared among the three groups using uorescence score. The summarized data of the uorescence score
analysis is presented in Table 2. The analysis showed that the
amount of elastin staining showed no differences among the MI,

Figure 1: Comparison of uorescence scores between the myocardial infarction (MI), non-MI and control groups. A signicant difference was observed
in collagen III staining between the MI and non-MI groups (P < 0.001), and
between the MI and control groups (P < 0.001). The value of 3524729.723
from the non-MI group has been excluded from the graph for better graph
presentation.

non-MI and control groups (P = 0.586). The same trend was

noticed for collagen I, wherein all groups equally expressed collagen I staining (P = 0.895).
However, a statistically signicant difference was observed in
collagen III expression. In the non-MI group, the median
amount of collagen III total uorescence was not statistically signicantly different from that of the control group (P = 0.417). On
the other hand, the median result for the MI group was signicantly lower than both the non-MI and control groups
(P < 0.001) (Fig. 1).
In view of the lower collagen III level in the MI group, the
result of collagen III/collagen I ratio, as well as elastin/collagen III
ratio, were affected too. In the MI group, the ratio of collagen
III/collagen I was found to be signicantly lower compared with
that of the non-MI and control groups (P < 0.001) (Fig. 2). The
ratio of elastin/collagen III was signicantly higher in the MI
group compared with that of the non-MI (P < 0.001) and control
groups (P = 0.001) (Fig. 3).

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C.H. Kong et al. / Interactive CardioVascular and Thoracic Surgery

Figure 2: Comparison between the collagen III/collagen I uorescence score

ratios of the myocardial infarction (MI), non-MI and control groups. Due to the
signicantly lower collagen III score in the MI group, the ratio of collagen III/
collagen I was also signicantly lower in the MI group compared with that of
the non-MI and control groups (P < 0.001). The value of 171.88 from the
non-MI group has been excluded from the graph for better graph presentation.

Figure 3: Comparison between the elastin/collagen III uorescence score

ratios of the myocardial infarction (MI), non-MI and control groups. Due to
signicantly lower collagen III score in the MI group, the ratio of elastin/
collagen III was signicantly higher in the MI groups compared with that of
the non-MI and control groups (P < 0.001 and P = 0.001, respectively).

DISCUSSION
Atherosclerosis is a systemic process affecting the arterial wall
and is present in all age groups. The main pathological feature of
atherosclerosis is atherosclerotic plaque with widespread distribution in the vascular system. Any plaque can potentially
become unstable and vulnerable, leading to further complications. The systemic features of atherosclerosis lead to the shift in
the concept from vulnerable plaque to vulnerable artery or even
an entire vulnerable patient [5]. The idea of the entire patients
vascular system susceptibility is supported by systemic inammatory biomarkers and hypercoagulation in the bloodstream.
Pathologically, plaque rupture is believed to be the leading

cause of acute coronary syndromes, which, in turn, are the

biggest causes of morbidity and mortality in coronary artery
disease. Even though the mechanism of plaque rupture is poorly
understood, some published data reveal that the rupture might
be due to the immune system and the inammatory process in
the arterial wall [6, 7].
Collagen type I and type III are the predominant vessel wall
ECM, maintaining the arterial wall stability. The total collagen I
content and collagen III content are 85 and 11%, respectively [8].
Type I has been thought to contribute to vascular stiffness by
providing tensile strength, while type III contributes to the extensibility of the vessel. The maturation of collagen I involves collagen III brillar aggregation, whereby collagen I is assembled into
mainly thicker bres and collagen III stays as thin bres. This is
why collagen I also has a longer turnover time compared with
the relatively dynamic collagen III. In the atherosclerotic plaques,
collagen I, III and elastin are present together, where they
sustain the integrity of the brous caps.
Inammation may have caused a systemic modication of the
ECM contents via the release of inammatory cytokines such as
interleukin-1 and tumour necrosis factor-. As a response to the
cytokines, endothelial cells and macrophages are activated, with
smooth muscle cells migrating to the media layer of the vessel
wall. When smooth muscle cells migrate to the atherosclerotic
plaque, they divide and synthesize ECM due to the immune response of auto-antigens, contributing to the accumulation of the
lipid streaks [9].
Recent studies had shown that systemic inammation might
play a part in weakening the plaque caps [6, 7], leading to the
events of acute MI. The possible interaction between the atherosclerotic plaques cap and the immune pathway could have
caused the instability of the brous caps due to the decrease in
collagen III. Since collagen III helps to regulate the brillogenesis
of collagen I, the loss of collagen III causes type I brils to be inconsistent. The abnormal bril packing causes a decrease in the
amount of mature collagen bres, which directly affects
the mechanical properties of the arterial walls and destabilizes
the brous caps.
Inammatory cytokines work synergistically with the growth
factors to modulate cellular behaviour via an autocrine feedback
mechanism. Interestingly, collagen III seemed to decrease in
production in the presence of the basic broblast growth factor
secreted from smooth muscle cells, while collagen I level was in
abundance [10]. Macrophages, together with smooth muscle
cells, release a broad range of proteasesmore specically,
matrix metalloproteinasesin the shoulder regions of the
plaque, causing degradation and thinning of the cap via the induction of interleukin-1 and tumour necrosis factor-. The
matrix metalloproteinases-1, being the most abundant interstitial
collagenase, has greater afnities for collagens I and III [11, 12]. It
had been demonstrated that localized increment of matrix
metalloproteinases-1 could contribute to plaque instability [13].
Tissue inhibitors of metalloproteinases-1 that, in turn, suppress
and control matrix metalloproteinases-1 production, is reported
to be reduced at the site of the lesion. This process increases the
activity of the matrix metalloproteinases-1, which, in turn, might
increase the turnover of collagens and collagen III in particular.
Our study provided evidence of structural systemic changes in
the aortic wall of patients with acute MI. As illustrated in our
ndings, in patients with acute MI, the collagen III content was
signicantly reduced, while amounts of collagen I and elastin
were not considerably different.

C.H. Kong et al. / Interactive CardioVascular and Thoracic Surgery

STUDY LIMITATIONS
This work provides information on collagen content in the
ascending aorta. The coronary artery wall is the ideal tissue for
this experiment. However, the human coronary artery wall is
available for research in limited situations only. Post-mortem
material can be used to evaluate gross histological tches but
the result might be related to the death/autopsy period and thus
may not reect the real situation.
This limitation leads us to the idea of using ascending aortic
tissue, which is easily available in the daily practice of cardiac
surgeons as an indicator of the condition of the vessel walls. In
this study, we did not see histological changes in the coronary
artery in the group of patients with acute coronary syndrome.
Accepting the unavailability of the coronary artery in this particular project, we aim to emphasize the hypothesis that ECM
changes in the acute coronary syndrome might have systemic
tches and could be reected in the ascending aorta as well.

ACKNOWLEDGEMENTS
The authors are grateful to all surgeons and nurses for their kind
assistance with specimen collection. Also, we would like to
express our appreciation to the Histopathology Facility of
Institute of Molecular and Cell Biology, Singapore (headed by
Keith Rogers), for microarray design and immunohistochemistry
work. We also would like to acknowledge the continuous help
and advice from the National University Hospital Tissue

Repository of National University Health System, Singapore

(headed by Eng Chon Boon).

FUNDING
Funding was provided by Clinical Scientist Unit, Yong Loo Lin
School of Medicine, National University Hospital Health System,
Singapore.
Conict of interest: none declared.

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ORIGINAL ARTICLE

A decrease in collagen III content could possibly explain the

mechanism of plaque vulnerability and weakening of the plaque
cap. Of more importance, having a disproportionate content of
collagen III away from culprit atherosclerotic plaque supports
the concept of systemic vascular changes in vulnerable patients.
Although the mechanism of the selective inhibition of collagen
III production is poorly understood, the immune system and inammation could be the causes of fragile plaques and greater
propensity to rupture.
With an increasing emphasis on the prophylaxis of primary
and secondary cardiovascular events, biomarkers related to the
progression of IHD have great potential in the management of
atherosclerotic patients. Investigations of the collagen III biochemical pathway and metabolism could be considered as a potential source of biomarkers to predict cardiovascular events.

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