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Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis
Cardiovascular Pathology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Cardiovascular Pathology
Original Article
Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis Marja-Leena Kortelainen ⁎, Katja Porvari Institute of Diagnostics, Department of Forensic Medicine, P.O. Box 5000, 90014 University of Oulu, Finland
a r t i c l e
i n f o
Article history: Received 18 November 2013 Received in revised form 13 March 2014 Accepted 13 March 2014 Available online xxxx Keywords: Atherogenesis Arteriosclerosis Adventitia Inflammation Macrophages Lymphocytes
a b s t r a c t Background: Atherosclerosis is considered a chronic inflammatory disease of the entire arterial wall, including the adventitia. Advanced coronary lesions with lipid cores are associated with adventitial inflammation, but the early inflammatory process in human coronary adventitia is largely unknown. We hypothesized that adventitial inflammatory cell infiltration accompanies the early stages of atherogenesis in human coronary arteries, and it is synchronous with the inflammatory process in the intima. Methods: Coronary artery samples were obtained from 111 forensic autopsy cases aged from 7 to 25 years. Adventitial and intimal macrophages, T lymphocytes and B lymphocytes, and intimal microvessels were detected by immunohistochemical methods and quantified by computerized image analysis. Body height, weight, waist circumference, and the size of mesenteric and omental fat depots were measured. Results: Adventitial densities of macrophages and T lymphocytes were significantly higher in arteries showing intimal xanthomas than in cases with only scattered intimal macrophages. The xanthoma group also had significantly higher body mass index and larger visceral fat depots. Highest densities of all adventitial cell types were seen in intermediate lesions and fibroatheromas. There were significant positive correlations between intimal and adventitial densities of T cells and B cells in the groups with or without intimal xanthomas, but the positive correlation between intimal and adventitial macrophages was significant only in the group without xanthomas. Conclusions: Adventitial immune-inflammatory cell accumulation accompanies the early stages of coronary atherogenesis in young individuals, and lymphocyte accumulation seems to be synchronous in the intima and adventitia. Macrophage accumulation is also synchronous before xanthomas are seen. © 2014 Elsevier Inc. All rights reserved.
1. Introduction At present, atherosclerosis is considered a chronic inflammatory disease of the entire arterial wall, including the adventitia [1]. Traditional risk factors for atherosclerosis are linked to the disease process by inflammatory mechanisms [2] Monocyte-macrophages and lymphocytes are recruited into the intima from the arterial lumen, where they interact with one another and with the cells of the arterial wall [3–7], but these cells are thought to reach the intima also from the adventitial side [8]. Due to the lack of any functional border between coronary adventitia and epicardial fat, adipokines produced by this adipose tissue depot may contribute locally to coronary atherosclerosis [9]. Intimal thickening promotes growth of neovessels, through which adventitial inflammatory cells are able to reach the intima [10]. It has been assumed that monocyte-macrophages could enter a plaque through adventitia also in the early stages of the atherosclerotic process [11].
Funding: University of Oulu, Finland. ⁎ Corresponding author. Tel.: +358 40 1547501; fax: +358 8 5315037. E-mail address: Marja-Leena.Kortelainen@oulu.fi (M.-L. Kortelainen).
Epicardial fat shares the same origin with intraabdominal visceral fat [9], which has an important role in systemic inflammation [12]. These fat depots have also the same kind of inflammatory features associated with central type of obesity [13]. Local effects of epicardial fat may, however, differ from those of intraabdominal fat. Epicardial adipose tissue has a double role as pro- and anti-inflammatory organ [14], and it plays a role also in the regulation of vascular tone [15]. Perivascular adipose tissue may also have some protective effect against atherosclerosis through its thermogenic potential resembling that of brown adipose tissue [16]. Macrophages are known to appear in the normal arterial intima as isolated cells [17], and macrophages and T lymphocytes are always found in arterial intimal regions subjected to major hemodynamic stress [18]. Adventitial inflammatory cells are also known to be present in the aortas of hypercholesterolemic experimental animals already in the early developmental phase of atherosclerosis and even prior to lesion development [19]. Early inflammatory–immunological lesions have been reported also in the human aortas of the Pathobiological Determinants of Atherosclerosis in Youth study material [18]. Studies on human coronary adventitial inflammation have been focused on advanced disease. Plaques with well-developed lipid cores are associated with prominent adventitial infiltrates of
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Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001
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M-L. Kortelainen, K. Porvari / Cardiovascular Pathology xxx (2014) xxx–xxx
macrophages, lymphocytes, and mast cells [20–22]. Coronary fatty streaks from cases with fatal lesions have not been observed to be in relation to large amounts of adventitial inflammatory cells [21]. To our knowledge, systematic observations on coronary adventitial inflammatory status of young healthy individuals showing merely subclinical stages of atherogenesis have not been reported. The purpose of the present study was to find out the level of adventitial inflammatory cell infiltration associated with early coronary lesions appearing in children and young adults and compare this to macroscopically normal arteries, in which microscopic features of fatty streaks were not found either. We hypothesized that there is increased adventitial immune-inflammatory cell infiltration in the early phase of atherogenesis in human coronary arteries, and this is synchronous with the inflammatory process in the intima. The study was based on a series of 111 medicolegal autopsies on victims of nonnatural death who were otherwise healthy.
order to visualize the structure of the different layers of the vessels. Macrophages were detected immunohistochemically with CD68 antibody (Dako). T lymphocytes were detected with CD3 antibody (Dako); and B lymphocytes, with CD20 antibody (Dako). An antibody to von Willebrand factor (Dako) was used to detect intimal microvessels. The cases were divided into groups according to the macroscopic and microscopic appearance of the most advanced lesion observed in each case, based on the American Heart Association classification [26] but modified according to Virmani et al. [27]. Group 1 represented cases in which the arteries were macroscopically normal and microscopic examination did not reveal any groups of lipid-laden macrophages in the intima. Group 2 represented intimal xanthomas with macrophage foam cells seen in groups but no morphological evidence of extracellular lipid accumulation or lipid core. Group 3 represented all more advanced lesions including pathological intimal thickening (intermediate lesions) or more severe lesions.
2. Material and methods 2.1. Subjects The subjects were forensic autopsy cases examined at the Department of Forensic Medicine, University of Oulu, Finland. The material was collected from medicolegal autopsies from 1995 to 2003, and the procedure was approved by the ethical committee of the Medical Faculty, University of Oulu. The autopsy cases were 111 children and young adults (73 males and 38 females) aged from 7 to 25 years who died from external causes (34 traffic accidents; 26 other accidental deaths; 47 suicides by shooting, hanging, other selfinflicted injury, or intoxication; and 4 homicides). The subjects did not have any clinical diagnoses of inflammatory/immune diseases, and there were no signs of such diseases or pericardial effusions either at autopsy or in the microscopic examination. 2.2. Anthropometric measurements and visceral fat Height (cm), body weight (kg), and waist circumference (cm) were measured in all cases. The level for measuring the waist circumference was based on standard skeletal reference points [23]. The cadavers were lying naked in supine position on the autopsy table during the procedure. Body mass index (BMI, weight divided by height squared) and waist-to-height ratio (waist divided by height) were calculated from the measurements. Waist-to height-ratio was used instead of waist-to hip-ratio because the same values of this ratio can be used in both genders as indicators of abdominal obesity [24]. The greater omentum was excised free from intraabdominal tissues and the mesenteric fat from the gut, and these fat deposits were weighed fresh. 2.3. Coronary arteries After weighing the heart, the left and right coronary arteries were excised free from the surrounding tissues. Samples were taken from each main branch, left anterior descending artery (LAD), circumflex artery, and right coronary artery (RCA) from where macroscopically visible lesions were observed. There were no visible lesions in the left main artery (LM) in any of cases, and therefore, LM samples, even if taken, were not included in this study. In cases where no visible lesions existed in any of the main branches, samples were taken from the proximal part of each vessel. The samples were fixed in neutral formalin and embedded in paraffin. 2.4. Histology and immunohistochemistry The paraffin-embedded coronary artery samples were sectioned at 5 μm and stained by the Verhoeff–Masson trichrome method [25] in
2.5. Quantification of macrophages and lymphocytes and intimal microvessels The most advanced lesion from each subject was chosen for quantification of macrophages, T lymphocytes, and B lymphocytes. The CD68-, CD3-, and CD20-positive cells in the intima and in the adventitia, and intimal microvessels positive for von Willebrand factor were counted in five fields in each section at a magnification of ×400 in each sample. The fields were selected from the regions where the maximum densities of the cells/vessels were seen, and in cases with only scattered cells/vessels, five fields were randomly selected. The mean values of three sections were calculated and expressed as means/mm 2 [28]. 2.6. Statistical analysis All the analyses were performed using the Statistical Package for Social Sciences 19 software. Differences in interval variables between the groups were tested by Kruskal–Wallis test, and the difference between the dichotomial variable (gender) was tested by χ 2 test. The third group showing most advanced lesions was not included in pairwise comparisons between the groups because the total amount of cases in this group was considered too low for this. Bivariate correlations between adventitial and intimal densities of various cell types were analyzed by Spearman correlation. P values of .05 were considered significant. 3. Results Adventitial densities of both macrophages and T lymphocytes were significantly higher in the group showing intimal xanthomas than in the group with only scattered intimal macrophages (Table 1). There were only eight individuals with more advanced intimal lesions classified as pathological intimal thickening (six cases) or fibroatheromas with lipid core (two cases), all of whom were males. Highest densities of macrophages and T and B lymphocytes were seen in this group, and intimal microvessels in close proximity to macrophages were present only in four of these cases (Table 1). There were significant positive correlations between intimal and adventitial macrophage densities and T cell and B cell densities in the whole material, but when the groups with and without xanthomas were analyzed separately, the correlations between macrophage densities reached the level of significance only in the group without xanthomas (Table 2). The average age in the group with xanthomas was slightly higher than in the group without xanthomas, but not significantly so. There were significantly more males in the xanthoma group than in the group without these. BMI and the size of visceral fat depots were
Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001
M-L. Kortelainen, K. Porvari / Cardiovascular Pathology xxx (2014) xxx–xxx
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Table 1 Descriptive statistics of 111 autopsy cases Group 1: no lesions (n=52)
Male/female Age (y) Height (cm) Weight (kg) BMI (kg/m2) Waist-to-height ratio Omental and mesenterial fat (g) Intimal macrophages/mm2 Adventitial macrophages/mm2 Intimal T cells/mm2 Adventitial T cells/mm2 Intimal B cells/mm2 Adventitial B cells/mm2 Intimal microvessels/mm2 a b
Group 2: intimal xanthomas (n=51)
Group 3: pathological intimal thickening or fibroatheromas (n=8)
Mean±S.D. median
Mean±S.D. median
Mean±S.D. median
28/24 16±5 18 161±17 165 54±14 55 20.3±3.1 19.8 0.43±0.05 0.42 250±187 188 48±47 32 100±61 86 1.0±1.0 0 1.0±1.0 0 0.1±0.4 0 0.1±0.4 0 00
39/12 18±4 19 171±11 172 66±14 66 22.2±3.6 22.0 0.44±0.05 0.43 315±150 287 205±273 112 185±120 163 4.5±7.7 1.0 2.0±2.5 1.0 0.1±0.4 0 0.1±0.4 0 00
8/0 20±1 21 174±7 175 74±17 71 24.5±4.4 23.2 0.46±0.06 0.45 413±280 321 860±511 768 459±857 118 56±43 50 12.2±9.8 10.0 1.9±2.5 1.0 1.1±1.3 1.0 9.6±13.7 0
P P values values
.001 .013 .029 .000 .001 .148 .003 .000 .000 .000 .000 .000 .000 .000
a
b b b
b b b b
Refers to χ2 test. All other P values refer to Kruskal–Wallis test. Refers to pairwise comparisons between groups 1 and 2, Pb.05.
significantly higher in the group with xanthomas than without, but waist-to-height ratios were in the same range (Table 1). Macrophage densities were much higher than T lymphocyte densities both in the intima and in the adventitia in all groups. Adventitial and intimal densities of B cells were practically on the same very low level in both xanthoma and nonxanthoma groups, but in cases with more advanced lesions, the densities were a little higher. Maximal densities of adventitial macrophages were usually seen around small blood vessels and nerves both in normal arteries and in the vessels with intimal lesions. Adventitial T cells were generally not seen in groups of several cells, but when appearing in groups, these cells were also arranged around small blood vessels and nerves. Examples of macrophage and lymphocyte distribution are presented in Figs. 1, 2, and 3. 4. Discussion Increased density of adventitial macrophages and T lymphocytes in association with intimal xanthomas suggests that adventitial inflammatory process is involved in early subclinical stages of atherogenesis in human coronary arteries. Positive correlations between intimal and adventitial immune-inflammatory cells in the group without xanthomas may indicate early communication between adventitia and intima before development of visible lesions [1]. Adventitial densities of macrophages in association with intimal xanthomas did not follow the same pattern as lymphocyte densities which increased along with intimal densities. Since perivascular fat is a diet-regulated adipose depot [29], it may be possible that changes in diet-related risk factors or probably also in other risk factors may modulate adventitial macrophage status more rapidly than they are able to affect the intimal environment. The growth of intimal neovessels observed in the four cases with more advanced lesions in this material may have offered a route to inflammatory cells from the adventitia [1]. Macrophages itself are able to produce matrix-degrading enzymes, leading to medial barrier
disruption [1]. Based on the present observations, it is, however, not possible to make any conclusions about a possible outside-in trafficking of macrophages or lymphocytes in this developmental phase of atherogenesis. Lymphocyte accumulation in the intima and adventitia seems to be synchronized when fatty streaks are present, and in the group with more advanced lesions, adventitial lymphocyte densities were also in a parallel direction with intimal densities. In the aorta, adventitial immune response has been observed to become more intense along with progression of early atherosclerosis referring to structural organization of the adventitial infiltrates [1]. Lymphoid follicle-like structures were not seen in the present material, but groups of several adventitial T lymphocytes were occasionally seen both in association with fatty streaks and with more advanced lesions. Larger quantities of T cells have been observed around adventitial nerves associated with early atherosclerotic lesions in human aortas [18]. Aggregation on adventitial lymphocytes together with macrophages in the aortas of mice fed an atherogenic diet has been suggested to represent the beginning of structural organization of lymphoid tissue [1]. It remains to be studied in the future whether the present observations could represent the same kind of development in human coronary adventitia. Obesity in general – especially visceral obesity – is one of the known risk factors of atherogenesis. Intimal atherogenesis and corresponding adventitial inflammation were seen in those with higher BMI and more visceral fat. The thickness of the epicardial fat in normal-weight children and adolescents is lower than in the obese ones [30]. Coronary arterial samples in the present study contained variable amounts of epicardial fat cells, but the whole thickness of the perivascular fat was not measured at any level. However, in some cases with larger amount of fat, macrophages and lymphocytes were seen also among fat cells with longer distance from the adventitia. The study of Verhagen et al. [31] showed that local coronary perivascular adipose tissue quantity and macrophage infiltration were related to atherosclerotic plaque size and plaque composition in patients with coronary atherosclerosis. In the present study, analysis of the full
Table 2 Bivariate correlations (Spearman) between adventitial and intimal macrophages and lymphocytes
Adventitial macrophages/mm2–intimal macrophages/mm2 Adventitial T cells/mm2–intimal T cells/mm2 Adventitial B cells/mm2–intimal B cells/mm2
No lesions (n=52)
Intimal xanthomas (n=51)
Intimal xanthomas and more advanced lesions (n=59)
All cases (n=111)
r=0.716, P=.000 r=0.696, P=.000 r=1.000, P=.000
r=0.129, P=.365 r=0.682, P=.000 r=0.998, P=.000
r=0.437, P=.103 r=0.774, P=.000 r=0.996, P=.000
r=0.474, P=.000 r=0.763, P=.000 r=0.999, P=.000
Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001
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Fig. 3. LAD adventitia of a 22-year-old female with coronary fatty streaks observed at autopsy. CD3 antibody. B indicates blood vessel; N, nerve. The arrows indicate T lymphocytes. Original magnification × 400.
thickness of surrounding fat together with adventitia and histological examination of intraabdominal fat depots would have given a more profound picture of the role of the perivascular milieu and its relationship to adipose tissue distribution in early human coronary atherogenesis. The well-known limitation of this work is the selection bias common to all autopsy-based studies. In addition, there were no available data on ante mortem cholesterol levels, alcohol consumption, or duration of more or less regular smoking or lifetime passive exposure to tobacco smoke, although, in some cases, postmortem toxicological analysis revealed presence of nicotine in the blood suggesting recent antemortem exposure to tobacco smoke. Some cases were also tested positive for alcohol and/or other drugs, but these results do not allow any firm conclusions to be made on habitual consumption of these substances or on their significance in the adventitial inflammatory process. Fig. 1. LAD of a 15-year-old male with coronary intimal fatty streaks observed at autopsy. (A) Verhoeff–Masson trichrome staining. (B) CD68 antibody, intimal and adventitial macrophages. A indicates adventitia; I, intima; M, media. The arrows indicate macrophages. Original magnification × 100.
Fig. 2. RCA adventitia of a 13-year-old male with coronary fatty streaks observed at autopsy. CD68 antibody. B indicates blood vessel; N, nerve. The arrows indicate macrophages. Original magnification × 400.
5. Conclusions Lymphocyte and macrophage accumulation seems to be synchronous in the normal coronary intima and adventitia of children and young adults. Adventitial and intimal lymphocyte accumulation was also synchronous in coronary xanthomas, but no such tendency was seen in association with macrophage accumulation. Fatty streaks are potentially reversible lesions [32] and clinically insignificant, but if they proceed to pathological intimal thickening, the situation may become more complicated. Erosions with adjacent thrombosis may occur in these intermediate lesions, causing either total coronary occlusion or rapid plaque growth by organized thrombi [33], and predispose young individuals to sudden death even before further progression to classical rupture-prone atheromas [27]. This material included a few cases with these potentially dangerous lesions, which were accompanied with the most intensive adventitial macrophage and lymphocyte infiltration. Further studies with more autopsy cases showing this lesion type will probably reveal more on the adventitial inflammation in this stage of the atherosclerotic process. Lifestyle and therapeutic interventions targeting adventitia-associated perivascular fat have been suggested in order to prevent cardiovascular complications [34,35]. Future analyses covering the whole thickness of the epicardial fat, including different subsets of T cells and macrophage phenotypes, may produce a more detailed picture on the associations between early atherogenesis and adventitial and perivascular inflammation in children and young adults.
Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001
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Acknowledgments The authors are grateful to the autopsy technicians and to the laboratory staff of the Department of Forensic Medicine for the excellent technical assistance.
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Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001
Contents lists available at ScienceDirect
Cardiovascular Pathology
Original Article
Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis Marja-Leena Kortelainen ⁎, Katja Porvari Institute of Diagnostics, Department of Forensic Medicine, P.O. Box 5000, 90014 University of Oulu, Finland
a r t i c l e
i n f o
Article history: Received 18 November 2013 Received in revised form 13 March 2014 Accepted 13 March 2014 Available online xxxx Keywords: Atherogenesis Arteriosclerosis Adventitia Inflammation Macrophages Lymphocytes
a b s t r a c t Background: Atherosclerosis is considered a chronic inflammatory disease of the entire arterial wall, including the adventitia. Advanced coronary lesions with lipid cores are associated with adventitial inflammation, but the early inflammatory process in human coronary adventitia is largely unknown. We hypothesized that adventitial inflammatory cell infiltration accompanies the early stages of atherogenesis in human coronary arteries, and it is synchronous with the inflammatory process in the intima. Methods: Coronary artery samples were obtained from 111 forensic autopsy cases aged from 7 to 25 years. Adventitial and intimal macrophages, T lymphocytes and B lymphocytes, and intimal microvessels were detected by immunohistochemical methods and quantified by computerized image analysis. Body height, weight, waist circumference, and the size of mesenteric and omental fat depots were measured. Results: Adventitial densities of macrophages and T lymphocytes were significantly higher in arteries showing intimal xanthomas than in cases with only scattered intimal macrophages. The xanthoma group also had significantly higher body mass index and larger visceral fat depots. Highest densities of all adventitial cell types were seen in intermediate lesions and fibroatheromas. There were significant positive correlations between intimal and adventitial densities of T cells and B cells in the groups with or without intimal xanthomas, but the positive correlation between intimal and adventitial macrophages was significant only in the group without xanthomas. Conclusions: Adventitial immune-inflammatory cell accumulation accompanies the early stages of coronary atherogenesis in young individuals, and lymphocyte accumulation seems to be synchronous in the intima and adventitia. Macrophage accumulation is also synchronous before xanthomas are seen. © 2014 Elsevier Inc. All rights reserved.
1. Introduction At present, atherosclerosis is considered a chronic inflammatory disease of the entire arterial wall, including the adventitia [1]. Traditional risk factors for atherosclerosis are linked to the disease process by inflammatory mechanisms [2] Monocyte-macrophages and lymphocytes are recruited into the intima from the arterial lumen, where they interact with one another and with the cells of the arterial wall [3–7], but these cells are thought to reach the intima also from the adventitial side [8]. Due to the lack of any functional border between coronary adventitia and epicardial fat, adipokines produced by this adipose tissue depot may contribute locally to coronary atherosclerosis [9]. Intimal thickening promotes growth of neovessels, through which adventitial inflammatory cells are able to reach the intima [10]. It has been assumed that monocyte-macrophages could enter a plaque through adventitia also in the early stages of the atherosclerotic process [11].
Funding: University of Oulu, Finland. ⁎ Corresponding author. Tel.: +358 40 1547501; fax: +358 8 5315037. E-mail address: Marja-Leena.Kortelainen@oulu.fi (M.-L. Kortelainen).
Epicardial fat shares the same origin with intraabdominal visceral fat [9], which has an important role in systemic inflammation [12]. These fat depots have also the same kind of inflammatory features associated with central type of obesity [13]. Local effects of epicardial fat may, however, differ from those of intraabdominal fat. Epicardial adipose tissue has a double role as pro- and anti-inflammatory organ [14], and it plays a role also in the regulation of vascular tone [15]. Perivascular adipose tissue may also have some protective effect against atherosclerosis through its thermogenic potential resembling that of brown adipose tissue [16]. Macrophages are known to appear in the normal arterial intima as isolated cells [17], and macrophages and T lymphocytes are always found in arterial intimal regions subjected to major hemodynamic stress [18]. Adventitial inflammatory cells are also known to be present in the aortas of hypercholesterolemic experimental animals already in the early developmental phase of atherosclerosis and even prior to lesion development [19]. Early inflammatory–immunological lesions have been reported also in the human aortas of the Pathobiological Determinants of Atherosclerosis in Youth study material [18]. Studies on human coronary adventitial inflammation have been focused on advanced disease. Plaques with well-developed lipid cores are associated with prominent adventitial infiltrates of
1054-8807/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.carpath.2014.03.001
Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001
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M-L. Kortelainen, K. Porvari / Cardiovascular Pathology xxx (2014) xxx–xxx
macrophages, lymphocytes, and mast cells [20–22]. Coronary fatty streaks from cases with fatal lesions have not been observed to be in relation to large amounts of adventitial inflammatory cells [21]. To our knowledge, systematic observations on coronary adventitial inflammatory status of young healthy individuals showing merely subclinical stages of atherogenesis have not been reported. The purpose of the present study was to find out the level of adventitial inflammatory cell infiltration associated with early coronary lesions appearing in children and young adults and compare this to macroscopically normal arteries, in which microscopic features of fatty streaks were not found either. We hypothesized that there is increased adventitial immune-inflammatory cell infiltration in the early phase of atherogenesis in human coronary arteries, and this is synchronous with the inflammatory process in the intima. The study was based on a series of 111 medicolegal autopsies on victims of nonnatural death who were otherwise healthy.
order to visualize the structure of the different layers of the vessels. Macrophages were detected immunohistochemically with CD68 antibody (Dako). T lymphocytes were detected with CD3 antibody (Dako); and B lymphocytes, with CD20 antibody (Dako). An antibody to von Willebrand factor (Dako) was used to detect intimal microvessels. The cases were divided into groups according to the macroscopic and microscopic appearance of the most advanced lesion observed in each case, based on the American Heart Association classification [26] but modified according to Virmani et al. [27]. Group 1 represented cases in which the arteries were macroscopically normal and microscopic examination did not reveal any groups of lipid-laden macrophages in the intima. Group 2 represented intimal xanthomas with macrophage foam cells seen in groups but no morphological evidence of extracellular lipid accumulation or lipid core. Group 3 represented all more advanced lesions including pathological intimal thickening (intermediate lesions) or more severe lesions.
2. Material and methods 2.1. Subjects The subjects were forensic autopsy cases examined at the Department of Forensic Medicine, University of Oulu, Finland. The material was collected from medicolegal autopsies from 1995 to 2003, and the procedure was approved by the ethical committee of the Medical Faculty, University of Oulu. The autopsy cases were 111 children and young adults (73 males and 38 females) aged from 7 to 25 years who died from external causes (34 traffic accidents; 26 other accidental deaths; 47 suicides by shooting, hanging, other selfinflicted injury, or intoxication; and 4 homicides). The subjects did not have any clinical diagnoses of inflammatory/immune diseases, and there were no signs of such diseases or pericardial effusions either at autopsy or in the microscopic examination. 2.2. Anthropometric measurements and visceral fat Height (cm), body weight (kg), and waist circumference (cm) were measured in all cases. The level for measuring the waist circumference was based on standard skeletal reference points [23]. The cadavers were lying naked in supine position on the autopsy table during the procedure. Body mass index (BMI, weight divided by height squared) and waist-to-height ratio (waist divided by height) were calculated from the measurements. Waist-to height-ratio was used instead of waist-to hip-ratio because the same values of this ratio can be used in both genders as indicators of abdominal obesity [24]. The greater omentum was excised free from intraabdominal tissues and the mesenteric fat from the gut, and these fat deposits were weighed fresh. 2.3. Coronary arteries After weighing the heart, the left and right coronary arteries were excised free from the surrounding tissues. Samples were taken from each main branch, left anterior descending artery (LAD), circumflex artery, and right coronary artery (RCA) from where macroscopically visible lesions were observed. There were no visible lesions in the left main artery (LM) in any of cases, and therefore, LM samples, even if taken, were not included in this study. In cases where no visible lesions existed in any of the main branches, samples were taken from the proximal part of each vessel. The samples were fixed in neutral formalin and embedded in paraffin. 2.4. Histology and immunohistochemistry The paraffin-embedded coronary artery samples were sectioned at 5 μm and stained by the Verhoeff–Masson trichrome method [25] in
2.5. Quantification of macrophages and lymphocytes and intimal microvessels The most advanced lesion from each subject was chosen for quantification of macrophages, T lymphocytes, and B lymphocytes. The CD68-, CD3-, and CD20-positive cells in the intima and in the adventitia, and intimal microvessels positive for von Willebrand factor were counted in five fields in each section at a magnification of ×400 in each sample. The fields were selected from the regions where the maximum densities of the cells/vessels were seen, and in cases with only scattered cells/vessels, five fields were randomly selected. The mean values of three sections were calculated and expressed as means/mm 2 [28]. 2.6. Statistical analysis All the analyses were performed using the Statistical Package for Social Sciences 19 software. Differences in interval variables between the groups were tested by Kruskal–Wallis test, and the difference between the dichotomial variable (gender) was tested by χ 2 test. The third group showing most advanced lesions was not included in pairwise comparisons between the groups because the total amount of cases in this group was considered too low for this. Bivariate correlations between adventitial and intimal densities of various cell types were analyzed by Spearman correlation. P values of .05 were considered significant. 3. Results Adventitial densities of both macrophages and T lymphocytes were significantly higher in the group showing intimal xanthomas than in the group with only scattered intimal macrophages (Table 1). There were only eight individuals with more advanced intimal lesions classified as pathological intimal thickening (six cases) or fibroatheromas with lipid core (two cases), all of whom were males. Highest densities of macrophages and T and B lymphocytes were seen in this group, and intimal microvessels in close proximity to macrophages were present only in four of these cases (Table 1). There were significant positive correlations between intimal and adventitial macrophage densities and T cell and B cell densities in the whole material, but when the groups with and without xanthomas were analyzed separately, the correlations between macrophage densities reached the level of significance only in the group without xanthomas (Table 2). The average age in the group with xanthomas was slightly higher than in the group without xanthomas, but not significantly so. There were significantly more males in the xanthoma group than in the group without these. BMI and the size of visceral fat depots were
Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001
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Table 1 Descriptive statistics of 111 autopsy cases Group 1: no lesions (n=52)
Male/female Age (y) Height (cm) Weight (kg) BMI (kg/m2) Waist-to-height ratio Omental and mesenterial fat (g) Intimal macrophages/mm2 Adventitial macrophages/mm2 Intimal T cells/mm2 Adventitial T cells/mm2 Intimal B cells/mm2 Adventitial B cells/mm2 Intimal microvessels/mm2 a b
Group 2: intimal xanthomas (n=51)
Group 3: pathological intimal thickening or fibroatheromas (n=8)
Mean±S.D. median
Mean±S.D. median
Mean±S.D. median
28/24 16±5 18 161±17 165 54±14 55 20.3±3.1 19.8 0.43±0.05 0.42 250±187 188 48±47 32 100±61 86 1.0±1.0 0 1.0±1.0 0 0.1±0.4 0 0.1±0.4 0 00
39/12 18±4 19 171±11 172 66±14 66 22.2±3.6 22.0 0.44±0.05 0.43 315±150 287 205±273 112 185±120 163 4.5±7.7 1.0 2.0±2.5 1.0 0.1±0.4 0 0.1±0.4 0 00
8/0 20±1 21 174±7 175 74±17 71 24.5±4.4 23.2 0.46±0.06 0.45 413±280 321 860±511 768 459±857 118 56±43 50 12.2±9.8 10.0 1.9±2.5 1.0 1.1±1.3 1.0 9.6±13.7 0
P P values values
.001 .013 .029 .000 .001 .148 .003 .000 .000 .000 .000 .000 .000 .000
a
b b b
b b b b
Refers to χ2 test. All other P values refer to Kruskal–Wallis test. Refers to pairwise comparisons between groups 1 and 2, Pb.05.
significantly higher in the group with xanthomas than without, but waist-to-height ratios were in the same range (Table 1). Macrophage densities were much higher than T lymphocyte densities both in the intima and in the adventitia in all groups. Adventitial and intimal densities of B cells were practically on the same very low level in both xanthoma and nonxanthoma groups, but in cases with more advanced lesions, the densities were a little higher. Maximal densities of adventitial macrophages were usually seen around small blood vessels and nerves both in normal arteries and in the vessels with intimal lesions. Adventitial T cells were generally not seen in groups of several cells, but when appearing in groups, these cells were also arranged around small blood vessels and nerves. Examples of macrophage and lymphocyte distribution are presented in Figs. 1, 2, and 3. 4. Discussion Increased density of adventitial macrophages and T lymphocytes in association with intimal xanthomas suggests that adventitial inflammatory process is involved in early subclinical stages of atherogenesis in human coronary arteries. Positive correlations between intimal and adventitial immune-inflammatory cells in the group without xanthomas may indicate early communication between adventitia and intima before development of visible lesions [1]. Adventitial densities of macrophages in association with intimal xanthomas did not follow the same pattern as lymphocyte densities which increased along with intimal densities. Since perivascular fat is a diet-regulated adipose depot [29], it may be possible that changes in diet-related risk factors or probably also in other risk factors may modulate adventitial macrophage status more rapidly than they are able to affect the intimal environment. The growth of intimal neovessels observed in the four cases with more advanced lesions in this material may have offered a route to inflammatory cells from the adventitia [1]. Macrophages itself are able to produce matrix-degrading enzymes, leading to medial barrier
disruption [1]. Based on the present observations, it is, however, not possible to make any conclusions about a possible outside-in trafficking of macrophages or lymphocytes in this developmental phase of atherogenesis. Lymphocyte accumulation in the intima and adventitia seems to be synchronized when fatty streaks are present, and in the group with more advanced lesions, adventitial lymphocyte densities were also in a parallel direction with intimal densities. In the aorta, adventitial immune response has been observed to become more intense along with progression of early atherosclerosis referring to structural organization of the adventitial infiltrates [1]. Lymphoid follicle-like structures were not seen in the present material, but groups of several adventitial T lymphocytes were occasionally seen both in association with fatty streaks and with more advanced lesions. Larger quantities of T cells have been observed around adventitial nerves associated with early atherosclerotic lesions in human aortas [18]. Aggregation on adventitial lymphocytes together with macrophages in the aortas of mice fed an atherogenic diet has been suggested to represent the beginning of structural organization of lymphoid tissue [1]. It remains to be studied in the future whether the present observations could represent the same kind of development in human coronary adventitia. Obesity in general – especially visceral obesity – is one of the known risk factors of atherogenesis. Intimal atherogenesis and corresponding adventitial inflammation were seen in those with higher BMI and more visceral fat. The thickness of the epicardial fat in normal-weight children and adolescents is lower than in the obese ones [30]. Coronary arterial samples in the present study contained variable amounts of epicardial fat cells, but the whole thickness of the perivascular fat was not measured at any level. However, in some cases with larger amount of fat, macrophages and lymphocytes were seen also among fat cells with longer distance from the adventitia. The study of Verhagen et al. [31] showed that local coronary perivascular adipose tissue quantity and macrophage infiltration were related to atherosclerotic plaque size and plaque composition in patients with coronary atherosclerosis. In the present study, analysis of the full
Table 2 Bivariate correlations (Spearman) between adventitial and intimal macrophages and lymphocytes
Adventitial macrophages/mm2–intimal macrophages/mm2 Adventitial T cells/mm2–intimal T cells/mm2 Adventitial B cells/mm2–intimal B cells/mm2
No lesions (n=52)
Intimal xanthomas (n=51)
Intimal xanthomas and more advanced lesions (n=59)
All cases (n=111)
r=0.716, P=.000 r=0.696, P=.000 r=1.000, P=.000
r=0.129, P=.365 r=0.682, P=.000 r=0.998, P=.000
r=0.437, P=.103 r=0.774, P=.000 r=0.996, P=.000
r=0.474, P=.000 r=0.763, P=.000 r=0.999, P=.000
Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001
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Fig. 3. LAD adventitia of a 22-year-old female with coronary fatty streaks observed at autopsy. CD3 antibody. B indicates blood vessel; N, nerve. The arrows indicate T lymphocytes. Original magnification × 400.
thickness of surrounding fat together with adventitia and histological examination of intraabdominal fat depots would have given a more profound picture of the role of the perivascular milieu and its relationship to adipose tissue distribution in early human coronary atherogenesis. The well-known limitation of this work is the selection bias common to all autopsy-based studies. In addition, there were no available data on ante mortem cholesterol levels, alcohol consumption, or duration of more or less regular smoking or lifetime passive exposure to tobacco smoke, although, in some cases, postmortem toxicological analysis revealed presence of nicotine in the blood suggesting recent antemortem exposure to tobacco smoke. Some cases were also tested positive for alcohol and/or other drugs, but these results do not allow any firm conclusions to be made on habitual consumption of these substances or on their significance in the adventitial inflammatory process. Fig. 1. LAD of a 15-year-old male with coronary intimal fatty streaks observed at autopsy. (A) Verhoeff–Masson trichrome staining. (B) CD68 antibody, intimal and adventitial macrophages. A indicates adventitia; I, intima; M, media. The arrows indicate macrophages. Original magnification × 100.
Fig. 2. RCA adventitia of a 13-year-old male with coronary fatty streaks observed at autopsy. CD68 antibody. B indicates blood vessel; N, nerve. The arrows indicate macrophages. Original magnification × 400.
5. Conclusions Lymphocyte and macrophage accumulation seems to be synchronous in the normal coronary intima and adventitia of children and young adults. Adventitial and intimal lymphocyte accumulation was also synchronous in coronary xanthomas, but no such tendency was seen in association with macrophage accumulation. Fatty streaks are potentially reversible lesions [32] and clinically insignificant, but if they proceed to pathological intimal thickening, the situation may become more complicated. Erosions with adjacent thrombosis may occur in these intermediate lesions, causing either total coronary occlusion or rapid plaque growth by organized thrombi [33], and predispose young individuals to sudden death even before further progression to classical rupture-prone atheromas [27]. This material included a few cases with these potentially dangerous lesions, which were accompanied with the most intensive adventitial macrophage and lymphocyte infiltration. Further studies with more autopsy cases showing this lesion type will probably reveal more on the adventitial inflammation in this stage of the atherosclerotic process. Lifestyle and therapeutic interventions targeting adventitia-associated perivascular fat have been suggested in order to prevent cardiovascular complications [34,35]. Future analyses covering the whole thickness of the epicardial fat, including different subsets of T cells and macrophage phenotypes, may produce a more detailed picture on the associations between early atherogenesis and adventitial and perivascular inflammation in children and young adults.
Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001
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Acknowledgments The authors are grateful to the autopsy technicians and to the laboratory staff of the Department of Forensic Medicine for the excellent technical assistance.
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Please cite this article as: Kortelainen M-L, Porvari K, Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis, Cardiovasc Pathol (2014), http://dx.doi.org/10.1016/j.carpath.2014.03.001