International Journal of Cardiology 176 (2014) 1167–1169
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Characterisation of novel cytokines in human atherosclerotic plaque Qiong Xia a, Andranik Kahramanian a, Clare Arnott b, Shisan Bao a,1, Sanjay Patel b,c,d,⁎,1 a
Discipline of Pathology, D17, The University of Sydney, New South Wales 2006, Australia Sydney Medical School, A27, The University of Sydney, New South Wales 2006, Australia c Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, New South Wales 2050, Australia d Heart Research Institute, 7 Eliza Street, Newtown, New South Wales 2042, Australia b
a r t i c l e
i n f o
Article history: Received 19 July 2014 Accepted 27 July 2014 Available online 4 August 2014 Keywords: Atherosclerosis Inflammation Cytokines Carotid
Inflammation plays a critical role in all stages of atherosclerotic plaque development, particularly so in inducing an unstable plaque phenotype [1]. Cytokines modulate this inflammatory process, with emerging data showing that both pro- and anti-inflammatory cytokines are present in atherosclerotic plaques [2]. The cytokines IL-31, IL-32, IL-33 and IL-34 have recently been characterised with regard to various inflammatory conditions, with IL-31, -32 and -34 having proinflammatory effects whereas IL-33 has been shown to be potentially athero-protective both in vitro and in animal studies of experimental atherosclerosis [3–6]. Our recently published data described the distribution of these cytokines in post-mortem coronary samples [7]; however their expression in ex-vivo human carotid plaques, particularly their correlation with stable versus unstable plaque phenotypes and relationship with patient characteristics, has not yet been explored. Forty-five consecutive patients with haemodynamically significant carotid artery disease and in whom carotid endarterectomy was deemed appropriate were selected to participate in the study [8]. Patients were classified as symptomatic (n = 23) or asymptomatic (n = 22) according to the presence or absence of cerebrovascular symptoms, respectively. As previously described, the majority of plaques from asymptomatic subjects were Type V lesions, whilst plaques from symptomatic subjects were predominantly Type VI lesions, as defined by the American Heart Association Vascular Lesion Classification [9]. Cardiovascular risk factors, ⁎ Corresponding author at: Department of Cardiology, Royal Prince Alfred Hospital, Sydney 2050, Australia. Tel.: +61 2 95157615; fax: +61 2 95506262. E-mail address: [email protected] (S. Patel). 1 Equal final authors.
http://dx.doi.org/10.1016/j.ijcard.2014.07.252 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
lipid profile and medication use were recorded for each patient. Informed consent was obtained from each patient and the study was approved by the South Western Sydney Area Health Ethics Committee, NSW, Australia. Excised plaques were immediately fixed in formalin and transverse 5 mm paraffin blocks were prepared from each plaque, from which 5 μM serial sections were obtained. Sections were stained with haematoxylin–eosin and Movat's pentachrome, and further adjacent sections were used for immunohistochemistry (IL-31–IL-34). Each of the polyclonal rabbit anti-human primary antibodies (Abcam, Sydney, Australia) was followed by polymer HRP goat anti-rabbit immunoglobulin (Dako, Sydney, Australia). The positive staining was photographed and quantified using Image-Pro® Plus 7. Quantitative real-time PCR was performed on cDNA prepared from total RNA isolated from snap frozen carotid plaques (10 asymptomatic, 8 symptomatic). Gene expression of IL-31–IL-34 was quantified according to the manufacturer's instructions (Qiagen, Chadstone, Australia) using relevant primers. The statistical analyses were performed using GraphPad Prism® Version 5.0 by one way ANOVA. p-Value b 0.05 was considered significant. Asymptomatic and symptomatic groups were well matched in terms of baseline patient characteristics (Table 1). IL-31, -32, -33 and -34 were detected in plaques from both asymptomatic and symptomatic patients. Immunostaining for IL-31, -32 and -34 was significantly greater in plaques from symptomatic compared with asymptomatic subjects (2.4-, 3.5- and 2.3-fold higher, respectively, p b 0.05 for all). However, IL-33 immunostaining was 1.9-fold greater in stable versus unstable plaques (p b 0.05) (Fig. 1). Similarly mRNA transcript levels of IL-31, -32 and -34 were significantly higher in unstable v stable plaques (1.5-, 1.8-, 1.9-fold, respectively, p b 0.05 for all) whereas for IL-33 there was a significant 2.1-fold increase in mRNA expression in stable versus unstable plaques (Fig. 2). When statin-treated and statin-naïve patients were compared, IL-33 immunostaining was higher in treated versus untreated (1.6-fold, p b 0.05), with these groups otherwise being well matched in terms of the presence or absence of symptoms or other cardiovascular risk factors. There was a non-significant increase in IL-33 mRNA levels in statin-treated compared with statin naïve patients. Cardiovascular risk factors were otherwise not significantly associated with the presence of any of these cytokines. IL-31, -32, and -34 are potent inflammatory cytokines, whose presence has been demonstrated in inflammatory diseases such as colitis and psoriasis. We have previously shown the presence of these
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Q. Xia et al. / International Journal of Cardiology 176 (2014) 1167–1169
Table 1 Clinical characteristics of study patients.
Age (years) Sex (male/female) Hypertension (%) Current or past smoking (%) Diabetes (%) Coronary artery disease (%) Total cholesterol (mmol/L) Statin use (%)
Symptomatic (n = 23) mean ± SD
Asymptomatic (n = 22) mean ± SD
69 ± 9 19/4 61 52 39 30 3.9 ± 0.7 75
76 ± 8 20/2 64 59 36 32 3.9 ± 1 80
cytokines in post-mortem coronary artery sections of culprit atheromatous plaque from premature and mature CAD cases. Here, we confirm the presence of these cytokines in ex vivo human carotid atheroma at both gene and protein level, and importantly demonstrate that the
expression of the inflammatory cytokines IL-31, -32 and -34 strongly correlates with an unstable plaque phenotype, implying their role in atherosclerosis-associated inflammation. Notably, IL-33 has been previously shown to have anti-inflammatory effects, with IL-33 treatment significantly reducing plaque size and inflammatory content in fat-fed mice [5]. In this current study, IL-33 was expressed in significantly greater amounts in stable plaques, suggesting that it may play an active role in maintaining a stable plaque phenotype. This is in contrast to our previous studies, where we demonstrated an over-abundance of the putative anti-inflammatory mediators, Foxp3 and IL-22 in unstable plaques, suggesting that these moieties are likely recruited to local areas of athero-inflammation but are ineffective [8,9]. Furthermore, we also demonstrated that IL-33 immunostaining was significantly and positively associated with statin use, suggesting that statins may exert their plaque stabilising effects, at least in part through IL-33. This observation is consistent with in vitro studies demonstrating that simvastatin up-regulates IL-33 expression in human endothelial cells [10].
Fig. 1. IL-31, IL-32, IL-33 and IL-34 immunostaining in carotid plaques from asymptomatic (A, C, E, G) vs symptomatic (B, D, F, H) patients. * represents p b 0.05.
Q. Xia et al. / International Journal of Cardiology 176 (2014) 1167–1169
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Fig. 2. IL-31, IL-32, IL-33 and IL-34 mRNA levels (relative units) in carotid plaques from asymptomatic (white bars) vs symptomatic (black bars) patients. * represents p b 0.05.
In summary, our data demonstrates, for the first time, a significant correlation between these novel inflammatory cytokines and plaque vulnerability, and also supports the notion that the anti-inflammatory cytokine IL-33 plays an active role in plaque stabilisation, possibly mediated through statin use. Further mechanistic studies are required to explore the role of IL-33 as a potential therapeutic target in atheroma stabilisation. Conflict of interest The authors report no relationships that could be construed as a conflict of interest. References [1] Libby P, Okamoto Y, Rocha VZ, Folco E. Inflammation in atherosclerosis: transition from theory to practice. Circ J 2010;74:213–20. [2] Patel S, Celermajer DS, Bao S. Atherosclerosis-underlying inflammatory mechanisms and clinical implications. Int J Biochem Cell Biol 2008;40:576–80.
[3] Kasraie S, Niebuhr M, Werfel T. Interleukin (IL)-31 induces pro-inflammatory cytokines in human monocytes and macrophages following stimulation with staphylococcal exotoxins. Allergy 2010;65(6):712–21. [4] Heinhuis B, Popa CD, van Tits BL, et al. Towards a role of interleukin-32 in atherosclerosis. Cytokine 2013;64:433–40. [5] Miller AM, Xu D, Asquith DL, et al. IL-33 reduces the development of atherosclerosis. J Exp Med 2008;205:339–46. [6] Chang EJ, Lee SK, Song YS, et al. IL-34 is associated with obesity, chronic inflammation, and insulin resistance. J Clin Endocrinol Metab 2014;99:1263–71. [7] Fang BA, Dai A, Duflou J, Zhang X, Puranik R, Bao S. Age-related inflammatory mediators in coronary artery disease (II). Int J Cardiol 2013;168:4839–41. [8] Patel S, Chung SH, White G, Bao S, Celermajer DS. The “atheroprotective” mediators apolipoproteinA-I and Foxp3 are over-abundant in unstable carotid plaques. Int J Cardiol 2010;145:183–7. [9] Xia Q, Xiang X, Patel S, Puranik R, Xie Q, Bao S. Characterisation of IL-22 and interferon-gamma-inducible chemokines in human carotid plaque. Int J Cardiol 2012;154:187–9. [10] Hot A, Lavocat F, Lenief V, Miossec P. Simvastatin inhibits the pro-inflammatory and pro-thrombotic effects of IL-17 and TNF-α on endothelial cells. Ann Rheum Dis 2013;72:754–60.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Characterisation of novel cytokines in human atherosclerotic plaque Qiong Xia a, Andranik Kahramanian a, Clare Arnott b, Shisan Bao a,1, Sanjay Patel b,c,d,⁎,1 a
Discipline of Pathology, D17, The University of Sydney, New South Wales 2006, Australia Sydney Medical School, A27, The University of Sydney, New South Wales 2006, Australia c Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, New South Wales 2050, Australia d Heart Research Institute, 7 Eliza Street, Newtown, New South Wales 2042, Australia b
a r t i c l e
i n f o
Article history: Received 19 July 2014 Accepted 27 July 2014 Available online 4 August 2014 Keywords: Atherosclerosis Inflammation Cytokines Carotid
Inflammation plays a critical role in all stages of atherosclerotic plaque development, particularly so in inducing an unstable plaque phenotype [1]. Cytokines modulate this inflammatory process, with emerging data showing that both pro- and anti-inflammatory cytokines are present in atherosclerotic plaques [2]. The cytokines IL-31, IL-32, IL-33 and IL-34 have recently been characterised with regard to various inflammatory conditions, with IL-31, -32 and -34 having proinflammatory effects whereas IL-33 has been shown to be potentially athero-protective both in vitro and in animal studies of experimental atherosclerosis [3–6]. Our recently published data described the distribution of these cytokines in post-mortem coronary samples [7]; however their expression in ex-vivo human carotid plaques, particularly their correlation with stable versus unstable plaque phenotypes and relationship with patient characteristics, has not yet been explored. Forty-five consecutive patients with haemodynamically significant carotid artery disease and in whom carotid endarterectomy was deemed appropriate were selected to participate in the study [8]. Patients were classified as symptomatic (n = 23) or asymptomatic (n = 22) according to the presence or absence of cerebrovascular symptoms, respectively. As previously described, the majority of plaques from asymptomatic subjects were Type V lesions, whilst plaques from symptomatic subjects were predominantly Type VI lesions, as defined by the American Heart Association Vascular Lesion Classification [9]. Cardiovascular risk factors, ⁎ Corresponding author at: Department of Cardiology, Royal Prince Alfred Hospital, Sydney 2050, Australia. Tel.: +61 2 95157615; fax: +61 2 95506262. E-mail address: [email protected] (S. Patel). 1 Equal final authors.
http://dx.doi.org/10.1016/j.ijcard.2014.07.252 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
lipid profile and medication use were recorded for each patient. Informed consent was obtained from each patient and the study was approved by the South Western Sydney Area Health Ethics Committee, NSW, Australia. Excised plaques were immediately fixed in formalin and transverse 5 mm paraffin blocks were prepared from each plaque, from which 5 μM serial sections were obtained. Sections were stained with haematoxylin–eosin and Movat's pentachrome, and further adjacent sections were used for immunohistochemistry (IL-31–IL-34). Each of the polyclonal rabbit anti-human primary antibodies (Abcam, Sydney, Australia) was followed by polymer HRP goat anti-rabbit immunoglobulin (Dako, Sydney, Australia). The positive staining was photographed and quantified using Image-Pro® Plus 7. Quantitative real-time PCR was performed on cDNA prepared from total RNA isolated from snap frozen carotid plaques (10 asymptomatic, 8 symptomatic). Gene expression of IL-31–IL-34 was quantified according to the manufacturer's instructions (Qiagen, Chadstone, Australia) using relevant primers. The statistical analyses were performed using GraphPad Prism® Version 5.0 by one way ANOVA. p-Value b 0.05 was considered significant. Asymptomatic and symptomatic groups were well matched in terms of baseline patient characteristics (Table 1). IL-31, -32, -33 and -34 were detected in plaques from both asymptomatic and symptomatic patients. Immunostaining for IL-31, -32 and -34 was significantly greater in plaques from symptomatic compared with asymptomatic subjects (2.4-, 3.5- and 2.3-fold higher, respectively, p b 0.05 for all). However, IL-33 immunostaining was 1.9-fold greater in stable versus unstable plaques (p b 0.05) (Fig. 1). Similarly mRNA transcript levels of IL-31, -32 and -34 were significantly higher in unstable v stable plaques (1.5-, 1.8-, 1.9-fold, respectively, p b 0.05 for all) whereas for IL-33 there was a significant 2.1-fold increase in mRNA expression in stable versus unstable plaques (Fig. 2). When statin-treated and statin-naïve patients were compared, IL-33 immunostaining was higher in treated versus untreated (1.6-fold, p b 0.05), with these groups otherwise being well matched in terms of the presence or absence of symptoms or other cardiovascular risk factors. There was a non-significant increase in IL-33 mRNA levels in statin-treated compared with statin naïve patients. Cardiovascular risk factors were otherwise not significantly associated with the presence of any of these cytokines. IL-31, -32, and -34 are potent inflammatory cytokines, whose presence has been demonstrated in inflammatory diseases such as colitis and psoriasis. We have previously shown the presence of these
1168
Q. Xia et al. / International Journal of Cardiology 176 (2014) 1167–1169
Table 1 Clinical characteristics of study patients.
Age (years) Sex (male/female) Hypertension (%) Current or past smoking (%) Diabetes (%) Coronary artery disease (%) Total cholesterol (mmol/L) Statin use (%)
Symptomatic (n = 23) mean ± SD
Asymptomatic (n = 22) mean ± SD
69 ± 9 19/4 61 52 39 30 3.9 ± 0.7 75
76 ± 8 20/2 64 59 36 32 3.9 ± 1 80
cytokines in post-mortem coronary artery sections of culprit atheromatous plaque from premature and mature CAD cases. Here, we confirm the presence of these cytokines in ex vivo human carotid atheroma at both gene and protein level, and importantly demonstrate that the
expression of the inflammatory cytokines IL-31, -32 and -34 strongly correlates with an unstable plaque phenotype, implying their role in atherosclerosis-associated inflammation. Notably, IL-33 has been previously shown to have anti-inflammatory effects, with IL-33 treatment significantly reducing plaque size and inflammatory content in fat-fed mice [5]. In this current study, IL-33 was expressed in significantly greater amounts in stable plaques, suggesting that it may play an active role in maintaining a stable plaque phenotype. This is in contrast to our previous studies, where we demonstrated an over-abundance of the putative anti-inflammatory mediators, Foxp3 and IL-22 in unstable plaques, suggesting that these moieties are likely recruited to local areas of athero-inflammation but are ineffective [8,9]. Furthermore, we also demonstrated that IL-33 immunostaining was significantly and positively associated with statin use, suggesting that statins may exert their plaque stabilising effects, at least in part through IL-33. This observation is consistent with in vitro studies demonstrating that simvastatin up-regulates IL-33 expression in human endothelial cells [10].
Fig. 1. IL-31, IL-32, IL-33 and IL-34 immunostaining in carotid plaques from asymptomatic (A, C, E, G) vs symptomatic (B, D, F, H) patients. * represents p b 0.05.
Q. Xia et al. / International Journal of Cardiology 176 (2014) 1167–1169
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Fig. 2. IL-31, IL-32, IL-33 and IL-34 mRNA levels (relative units) in carotid plaques from asymptomatic (white bars) vs symptomatic (black bars) patients. * represents p b 0.05.
In summary, our data demonstrates, for the first time, a significant correlation between these novel inflammatory cytokines and plaque vulnerability, and also supports the notion that the anti-inflammatory cytokine IL-33 plays an active role in plaque stabilisation, possibly mediated through statin use. Further mechanistic studies are required to explore the role of IL-33 as a potential therapeutic target in atheroma stabilisation. Conflict of interest The authors report no relationships that could be construed as a conflict of interest. References [1] Libby P, Okamoto Y, Rocha VZ, Folco E. Inflammation in atherosclerosis: transition from theory to practice. Circ J 2010;74:213–20. [2] Patel S, Celermajer DS, Bao S. Atherosclerosis-underlying inflammatory mechanisms and clinical implications. Int J Biochem Cell Biol 2008;40:576–80.
[3] Kasraie S, Niebuhr M, Werfel T. Interleukin (IL)-31 induces pro-inflammatory cytokines in human monocytes and macrophages following stimulation with staphylococcal exotoxins. Allergy 2010;65(6):712–21. [4] Heinhuis B, Popa CD, van Tits BL, et al. Towards a role of interleukin-32 in atherosclerosis. Cytokine 2013;64:433–40. [5] Miller AM, Xu D, Asquith DL, et al. IL-33 reduces the development of atherosclerosis. J Exp Med 2008;205:339–46. [6] Chang EJ, Lee SK, Song YS, et al. IL-34 is associated with obesity, chronic inflammation, and insulin resistance. J Clin Endocrinol Metab 2014;99:1263–71. [7] Fang BA, Dai A, Duflou J, Zhang X, Puranik R, Bao S. Age-related inflammatory mediators in coronary artery disease (II). Int J Cardiol 2013;168:4839–41. [8] Patel S, Chung SH, White G, Bao S, Celermajer DS. The “atheroprotective” mediators apolipoproteinA-I and Foxp3 are over-abundant in unstable carotid plaques. Int J Cardiol 2010;145:183–7. [9] Xia Q, Xiang X, Patel S, Puranik R, Xie Q, Bao S. Characterisation of IL-22 and interferon-gamma-inducible chemokines in human carotid plaque. Int J Cardiol 2012;154:187–9. [10] Hot A, Lavocat F, Lenief V, Miossec P. Simvastatin inhibits the pro-inflammatory and pro-thrombotic effects of IL-17 and TNF-α on endothelial cells. Ann Rheum Dis 2013;72:754–60.
