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 significantly lower in the acute MI group (P < 0.001). As a result of this change, the patients with MI also revealed a significant reduction in the collagen III/collagen I ratio. The elastin/collagen III ratio was significantly 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 inflammation 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 modification 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 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 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 profile, Troponin I, full blood count and C-reactive protein. Patients in the MI group had non-ST elevated myocardial reaction with Troponin I levels significantly higher compared with the non-MI group (Table 1). Statistical analyses of demographical data, risk factors and the lipid profile between the two groups were compared, and we did not find any significant difference in the demographical data (Table 1). Also, as expected, the MI group had significantly elevated inflammatory markers and raised cardiac enzymes.
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 anti-hyperlipidaemic medication | 12 (63.2%) | 18 (85.7%) | 0.1 |
| Cholesterol (mmol/l) mean (SD) | 4.71 (1.11) | 4.51 (1.79) | 0.716 |
| LDL-C (mmol/l) mean (SD) | 2.81 (0.86) | 2.95 (1.65) | 0.77 |
| Triglycerides (mmol/l) mean (SD) | 2.14 (2.05) | 1.35 (0.50) | 0.188 |
| WBC (×10.9/l) mean (SD) | 10.31 (3.05) | 7.07 (1.79) | <0.001 |
| CKMB (µg/l) mean (SD) | 73.14 (73.08) | 1.16 (0.23) | 0.04 |
| Troponin I (µg/l) mean (SD) | 23.92 (23.60) | 0.0162 (0.0064) | 0.014 |
| 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.
CABG surgery was performed with standard full normothermic cardiopulmonary bypass and antegrade cold (4°C) cardioplegia. Cardiopulmonary bypass time and aortic cross-clamp time were not significantly 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 paraffin, paying attention to orientate the tissue in the standard way. The rest of the collected tissue was preserved in −80°C. Tissue collection was approved by the Institution Review 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 paraffin-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 immunofluorescence
The sections were deparaffinized 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-specific 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 4°C. 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 finally counterstained for nuclei with Vectashield Hard Set mounting medium with 4,6 diamidino 2 phenylindole.
Image analysis and grading system for quantification
Fluorescence images were scanned sequentially and fluorescence images of each channel were analysed separately using Ariol software (Genetix). Fluorescence cross talk between fluorophores was tested and found to be negligible. For each stain, a mask was generated for all pixels with a fluorescence level above background. The area and mean intensity of this region were recorded.
The natural auto fluorescence in the green spectra was used to establish the total area of tissue sampled. The fluorescence score for each tissue section was calculated as follows:
 |
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 Kruskal–Wallis test was used to evaluate the differences in collagen I total fluorescence, collagen III total fluorescence, elastin total fluorescence, 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 Mann–Whitney U-test with the Bonferroni correction technique was used. The statistical significance was set at a 5% level.
RESULTS
The fluorescence images of the triple-stained tissue microarray sections were compared among the three groups using fluorescence score. The summarized data of the fluorescence score analysis is presented in Table 2. The analysis showed that the amount of elastin staining showed no differences among the MI, 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).
Table 2:
Scanned data of fluorescence score specimens from myocardial infarction (MI), non-MI and control groups
| Median | MI (n = 19) | Non-MI (n = 21) | Control (n = 10) |
P-value |
||
|---|---|---|---|---|---|---|
| MI vs non-MI | MI vs control | Non-MI vs control | ||||
| Collagen I total fluorescence (×1 000 000) median (minimum–maximum) | 2.43 (0.10–9.22) | 2.01 (0.002–13.64) | 2.81 (0.77–5.21) | 1 | 1 | 1 |
| Collagen III total fluorescence (×10 000) median (minimum–maximum) | 0.03 (0.0004–0.89) | 18.04 (0.51–352.47) | 7.45 (0.04–74.85) | <0.001 | <0.001 | 0.417 |
| Elastin total fluorescence ×1 000 000) median (minimum–maximum) | 1.27 (0.07–3.42) | 0.72 (0.001–5.59) | 1.32 (0.23–2.35) | 1 | 1 | 0.762 |
| Collagen III/collagen I median (minimum–maximum) | 0.0002 (0.00002–0.004) | 0.12 (0.001–171.88) | 0.03 (0.0002–0.20) | <0.001 | <0.001 | 0.297 |
| Elastin/collagen III median (minimum–maximum) | 2246.66 (53.55–20708.49) | 5.48 (0.003–130.54) | 13.71 (1.80–3853.87) | <0.001 | 0.001 | 0.324 |
However, a statistically significant difference was observed in collagen III expression. In the non-MI group, the median amount of collagen III total fluorescence was not statistically significantly different from that of the control group (P = 0.417). On the other hand, the median result for the MI group was significantly lower than both the non-MI and control groups (P < 0.001) (Fig. 1).
Figure 1:
Comparison of fluorescence scores between the myocardial infarction (MI), non-MI and control groups. A significant 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.
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 significantly lower compared with that of the non-MI and control groups (P < 0.001) (Fig. 2). The ratio of elastin/collagen III was significantly higher in the MI group compared with that of the non-MI (P < 0.001) and control groups (P = 0.001) (Fig. 3).
Figure 2:
Comparison between the collagen III/collagen I fluorescence score ratios of the myocardial infarction (MI), non-MI and control groups. Due to the significantly lower collagen III score in the MI group, the ratio of collagen III/collagen I was also significantly 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 fluorescence score ratios of the myocardial infarction (MI), non-MI and control groups. Due to significantly lower collagen III score in the MI group, the ratio of elastin/collagen III was significantly 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 patient's vascular system susceptibility is supported by systemic inflammatory 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 inflammatory 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 fibrillar aggregation, whereby collagen I is assembled into mainly thicker fibres and collagen III stays as thin fibres. 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 fibrous caps.
Inflammation may have caused a systemic modification of the ECM contents via the release of inflammatory cytokines such as interleukin-1 and tumour necrosis factor-α. 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 inflammation 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 fibrous caps due to the decrease in collagen III. Since collagen III helps to regulate the fibrillogenesis of collagen I, the loss of collagen III causes type I fibrils to be inconsistent. The abnormal fibril packing causes a decrease in the amount of mature collagen fibres, which directly affects the mechanical properties of the arterial walls and destabilizes the fibrous caps.
Inflammatory 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 fibroblast 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 proteases—more specifically, matrix metalloproteinases—in the shoulder regions of the plaque, causing degradation and thinning of the cap via the induction of interleukin-1 and tumour necrosis factor-α. The matrix metalloproteinases-1, being the most abundant interstitial collagenase, has greater affinities 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 findings, in patients with acute MI, the collagen III content was significantly reduced, while amounts of collagen I and elastin were not considerably different.
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 inflammation 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.
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 fitches but the result might be related to the death/autopsy period and thus may not reflect 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 fitches and could be reflected in the ascending aorta as well.
Funding
Funding was provided by Clinical Scientist Unit, Yong Loo Lin School of Medicine, National University Hospital Health System, Singapore.
Conflict of interest: none declared.
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).
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