The “atheroprotective” mediators apolipoproteinA-I and Foxp3 are over-abundant in unstable carotid plaques

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Abstract

Objective

Inflammation is important in plaque vulnerability but the role of atheroprotective mediators in unstable plaques is not defined. The apolipoproteinA-I (apoA-I) component of HDL, and CD4+/CD25+ regulatory T cells (with their major transcription factor, Foxp3), have been implicated in the suppression of vascular inflammation. Our aim was to characterise the presence of these novel “protective” markers (apoA-I and Foxp3) in carotid plaques from symptomatic and asymptomatic subjects.

Methods and results

Plaques from 57 patients (25 symptomatic, 32 asymptomatic) were stained immunohistochemically for macrophages (CD68), T cells (CD3), monocyte chemotactic protein-1 (MCP-1), matrix metalloproteinase-2 (MMP-2), myeloperoxidase (MPO), apoA-I and Foxp3. Twelve randomly selected plaques (6 asymptomatic, 6 symptomatic) were immunostained for interleukin-10 (IL-10) and interleukin-17 (IL-17). Staining was quantified using Image-Pro Plus software.

Significantly greater areas of positive immunostaining for CD68, CD3, MCP-1, MMP-2, IL-17 and MPO were found in plaques from symptomatic patients compared with asymptomatic patients (p < 0.05 for all). Furthermore, significantly greater areas of positive immunostaining for apoA-I, Foxp3 and IL-10 were found in symptomatic versus asymptomatic plaques (p < 0.05 for all). The presence of apoA-I was correlated significantly and co-localised with CD3, CD68, MCP-1, MMP-2 and MPO immunostaining (R = 0.70, 0.63, 0.52, 0.55 and 0.79, respectively; p < 0.01 for all). Foxp3 immunostaining also correlated significantly with CD3 (R = 0.42), CD68 (R = 0.47), MCP-1 (R = 0.55) and MMP-2 (R = 0.44) immunostaining (p < 0.05 for all).

Conclusions

ApoA-I and Foxp3 are over-abundant in plaques from symptomatic subjects and co-localise with key inflammatory mediators. These data suggest ineffective/insufficient protection against atherosclerosis-mediated inflammation by these “atheroprotective” moieties.

Introduction

Inflammatory processes play an important role in several stages of atherosclerosis, including increasing the propensity of plaques to rupture [1], [2], [3]. Atherosclerotic plaques from symptomatic patients tend to exhibit significant infiltration by macrophages, T cells and pro-inflammatory cytokines and chemokines. These inflammatory cells release matrix-degrading enzymes and thrombogenic substances that may result in plaque disruption, local thrombosis and subsequent clinical events, such as acute coronary and cerebrovascular syndromes [4], [5]. Conversely, stable plaques are characterised by significantly fewer inflammatory cells [6].

Although the association between pro-inflammatory cells and mediators and unstable plaques has been well established, the local role of putative anti-inflammatory moieties has not been well characterised. Two key anti-inflammatory markers studied extensively to date include apolipoproteinA-I (apoA-I), the major protein constituent of High Density Lipoproteins (HDL) and T-regulatory (Treg) cells. Population studies have shown a consistent and significant negative correlation between plasma HDL and cardiovascular events [7]. Recently it has been shown in vitro and in vivo, that apoA-I alone or as a component of HDL, can reduce vascular inflammation [8], [9]. However its presence and role in human atherosclerotic plaque is much less well defined. Furthermore, it is well established that inflammatory T cells, characterised by the expression of CD3 antigen, are found in significantly greater numbers in unstable plaques compared with stable plaques, and play a pivotal role in local inflammation [1], [10]. On the other hand, a subset of T cells, Treg, has been shown in animal studies to have protective effects against immune-mediated vascular inflammation [11]. However, the role of Treg cells and their major transcriptional protein, Foxp3, has not been well defined in human atherosclerotic plaque.

Therefore, we aimed to study the amounts and distribution of apoA-I and Foxp3 in human carotid plaque, because of their increasing profile in the literature as potent anti-inflammatory mediators. Moreover, we aimed to correlate their presence with patient characteristics, chiefly stable versus unstable disease.

Section snippets

Patients

Fifty seven consecutively seen, consenting patients with haemodynamically significant carotid artery disease undergoing endartectomy, were evaluated. Indications for surgery were based on NASCET [12] and ACAS [13] criteria for symptomatic and asymptomatic subjects respectively. The carotid stenoses were diagnosed and classified by colour duplex ultrasound according to consensus criteria [14]. Patients were classified as symptomatic according to the presence or absence of cerebrovascular

Results

Baseline patient characteristics did not differ significantly between study groups (Table 1).

Significantly greater areas of fibrous tissue were found in plaques from asymptomatic subjects compared with symptomatic subjects (35.2 ± 10.7% asymptomatic, 17.1 ± 6.7% symptomatic, p < 0.05). Immunohistochemical analysis revealed significantly greater areas of positive staining for macrophages (CD68 positive cells) (2.7 ± 0.5% asymptomatic, 9.2 ± 1.5% symptomatic, p < 0.001), the inflammatory chemokine, MCP-1 (1.3

Discussion

Inflammation is involved in several key stages of plaque development, and especially so, in the process of plaque destabilisation, leading to symptoms of “unstable” cardiovascular disease [3], [15]. In the current study, our immunohistochemical analysis of human atherosclerotic lesions demonstrated the presence of two novel potentially anti-inflammatory moieties, apoA-I, the major protein constituent of HDL and Foxp3, the major transcription protein of Treg cells. Furthermore, significantly

Acknowledgement

The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [32].

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    Dr Patel is supported by a postgraduate scholarship from the National Heart Foundation of Australia. Professor Celermajer is supported by Grant 222722 from the National Health and Medical Research Council of Australia.

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