pathophysiological role of neutrophils in acute myocardial ... · pathophysiological role of...

© Schattauer 2013 Thrombosis and Haemostasis 110.3/2013

501Review Article

Pathophysiological role of neutrophils in acute myocardial infarctionFederico Carbone1; Alessio Nencioni1; François Mach2; Nicolas Vuilleumier3,4; Fabrizio Montecucco1,2

1Department of Internal Medicine, University of Genoa, Genoa, Italy; 2Division of Cardiology, Foundation for Medical Researches, Faculty of Medicine, Department of Internal Medicine, Geneva University Hospital, Geneva, Switzerland; 3Division of Laboratory Medicine, Department of Genetics and Laboratory Medicine, Geneva University Hospitals, Switzerland; 4Department of Human Protein Science, Geneva Faculty of Medicine, Geneva, Switzerland

SummaryThe pathogenesis of acute myocardial infarction is known to be me-diated by systemic, intraplaque and myocardial inflammatory pro-cesses. Among different immune cell subsets, compelling evidence now indicates a pivotal role for neutrophils in acute coronary syn-dromes. Neutrophils infiltrate coronary plaques and the infarcted myocardium and mediate tissue damage by releasing matrix-degrad-ing enzymes and reactive oxygen species. In addition, neutrophils are also involved in post-infarction adverse cardiac remodelling and neointima formation after angioplasty. The promising results obtained in preclinical models with pharmacological approaches interfering

with neutrophil recruitment or function have confirmed the patho-physiological relevance of these immune cells in acute coronary syn-dromes and prompted further studies of these therapeutic interven-tions. This narrative review will provide an update on the role of neut-rophils in acute myocardial infarction and on the pharmacological means that were devised to prevent neutrophil-mediated tissue dam-age and to reduce post-ischaemic outcomes.

KeywordsAtherosclerosis, neutrophils, inflammation, acute myocardial infarc-tion

Correspondence to: Fabrizio MontecuccoCardiology Division, Department of MedicineGeneva University Hospital, Foundation for Medical Researches64 Avenue Roseraie, 1211 Geneva, SwitzerlandTel.: +41 223827238, Fax: +41 223827245E-mail: [email protected]

Financial support:This research was funded by EU FP7, Grant number 201668, AtheroRemo to Dr. F. Mach. This work was also supported by the Swiss National Science Foundation Grants to Dr. F. Mach (#310030–118245), Dr N. Vuilleumier (#310030–140736), and to Dr. Montecucco (#32003B-134963/1) and by the Italian Ministry of Health (GR-2008–1135635 to Dr. A. Nencioni).

Received: March 12, 2013 Accepted after major revision: May 4, 2013 Prepublished online: June 6, 2013

doi:10.1160/TH13-03-0211Thromb Haemost 2013; 110: 501–514

Introduction

Atherosclerotic plaque rupture promotes atherothrombosis, vessel occlusion and consequent ischaemic organ damage. This patho-physiological sequence is the primary cause of acute cardiovascular events (such as myocardial infarction [MI] and stroke). Both sys-temic and intraplaque atherosclerotic inflammation represent main risk factors, closely related to each other in determining the plaque composition and related vulnerability (1). In fact, culprit lesions are typically not flow-limiting stenoses, but rather inflamed lipid-loaded lesions (2). Although the clinical assessment of coronary athero-sclerosis severity is based on vascular lumen occlusion degree deter-mination (3), retrospective analyses as well as prospective observa-tions suggested that in 60–70% of the patients with acute coronary syndromes (ACS), the culprit site exhibits a diameter narrowing below 70% and frequently below 50% (2). Macrophages are the most abundant cell subtype within atherosclerotic plaques. However, other leukocytes are also well represented and have important roles in de-termining local inflammation and in promoting plaque rupture (4). Among these, polymorphonuclear neutrophils (PMNs) are now under the spotlight for their role in acute cardiovascular events and,

in particular, during the very early and late stages of atherogenesis. Previous lack of research attention to neutrophils was potentially de-pendent on their rare detection, short life span and high tissue turn-over (5). As of now, most of the knowledge on the role of PMNs with-in atherosclerotic plaques has derived from studies on experimental mouse models, human atherosclerotic plaque biopsies and epidemi-ological data. The aim of this narrative review is to highlight the step of coronary atherogenesis and post-infarction cardiac remodelling which PMNs partake in. In addition, we will also focus on pharma-cological intervention strategies targeting PMNs in acute MI as po-tential sources of future therapeutic perspectives.

Neutrophils in atherogenesis

In the last two decades, a large number of studies documented a direct correlation between a high number of circulating white blood cells (WBC) and an increased cardiovascular risk. The pa-thophysiological hypotheses (6) underlying this observation have been interestingly reviewed by Coller in 2005 (7). In subsequent analyses, a high neutrophil count and neutrophils/lymphocytes

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Thrombosis and Haemostasis 110.3/2013 © Schattauer 2013

502 Carbone et al. Neutrophils in acute myocardial infarction

(N/L) ratio have been proved as useful prognostic predictors in pa-tients with ACS (8) (▶ Table 1).

In addition, individual studies as well as multivariate analyses showed a direct correlation between neutrophil count and cardio-vascular risk factors, such as hypertension (34), metabolic syn-drome (21), type 2 diabetes mellitus (35), smoking (36), hyperlipi-daemia (37), obstructive sleep apnea (38) and end-stage renal dis-

ease (39). Finally, PMN-related biomarkers (such as systemic levels of MPO and elastase) were also shown associated with athero-sclerosis (▶ Table 2).

Low amounts of PMNs were initially found within human atherosclerotic plaques and this might explain why PMNs have been classically neglected in the pathophysiology of athero-sclerosis. Mehta et al. firstly showed in 1989 an increased neutro-

Table 1: Neutrophil count and cardiovascular risk.

Author (reference)

Dinerman J. L. et al. [9]

Bell D. et al. [10]

Mazzone A. et al. [11]

Takeshita S. et al. [12]

Leckie M. J. et al. [13]

Avanzas P. et al. [14]

Avanzas P. et al. [15]

Haumer M. et al. [16]

Grau A. J. et al.(CAPRIE study) [17]

Gillum R. F. et al.(NHANES I study) [18]

Horne B. D. et al. [19]

Nijm J. et al. [20]

Tsai J. C. et al. [21]

Zazula A. D. et al. [22]

Year

1990

1990

1993

1997

2004

2004

2004

2004

2004

2005

2005

2005

2007

2008

Study design (n)

Case-control(93 patients; 17 AMI, 29 UA, 25 SA and 22 healthy controls)

Case-control (97 patients; 32 AMI, 30 IHD and 35 healthy controls)

Case-control(29 patients; 15 UA and 14 SA)

Case-control (166 patients; 68 ACS, 83 CAD and 15 SA)

Case-control(53 patients; 9 UA, 18 SA and 26 healthy controls)

Prospective cohort study(55 AMI NSTEMI)

Case-control (150 patients; 121 CAD and 29 healthy controls)

Prospective observational(398 patients with peripheral artery disease)

Prospective observational(18558 patients with previous car-diovascular disease)

Prospective observational(5027 healthy controls)

Prospective observational(3227 patients angiographically en-rolled)

Case-control(105 patients; 20 UA, 45 SA and 45 healthy controls)

Prospective observational(1872 T2DM patients)

Prospective observational178 patients with chest pain

Target

Neutrophil count

Neutrophil count

Neutrophil count (CD11b)

Neutrophil count

Neutrophil count

Neutrophil count

Neutrophil count

Neutrophil count

Neutrophil count

Neutrophil count

N/L ratio

Neutrophil count

Neutrophil count; N/L ratio

N/L ratio (N/L) ratio

Outcome and Prediction

Positively correlates with CAD presence. AMI/UA vs. SA/control group (p<0.01)

Positively correlates with CAD presence. AMI (p<0.0001) and IHD (p<0.004) vs healthy con-trols

Positively correlates with coronary inflammation in coronary ACS.UA vs. SA group (p<0.01)

Positively correlate with CAD severity (p<0.05)

Positively correlates with CAD presence.UA (p=0.007) and SA (p<0.001) group vs. healthy controls

Positively correlates with coronary stenoses complex-ity (r=0.36; p=0.007)

Positively correlates with coronary stenoses complex-ity (r=0.28; p=0.002) and is independent stenosis complexity predictor (OR: 4.05; CI 1.9–10.4; p=0.038)

Positive predictor of major adverse cardiovascular events (multivariate analysis: HR 1.83; CI 1.29–3.00; p<0.017).

Positive predictor of recurrent ischaemic events (multi-variate analysis: RR 1.09; CI 1.06–1.13; p<0.001)

Indipendent death risk factor from all causes (RR 1.29; CI 1.14–1.47; p<0.01) and CVD death (RR 1.39; CI 1.15–1.67; p<0.001)

Positive predictor of death/AMI (multivariate analysis: HR 0.83; CI 0.68–1.03; p<0.001).

Positively correlates with CAD severityIncreasing neutrophil count in UA (p<0.05) and SA (p<0.05) vs control group and in UA vs SA group (p<0.01)

Correlates with systolic BP (r=0.002, p=0.003) and low HDL-cholesterol (r=−0.003, p=0.004).N/L ratio positive correlates with CAD risk (multiple linear regression: OR 237.458; CI 50.242–1147.02; p=0.001)

Is predictor of ACS diagnosis in chest pain (p<0.0001)



503Carbone et al. Neutrophils in acute myocardial infarction

phil elastase activity in patients with ACS (40). Dinerman (9) and Bell (10) in 1990 and others later confirmed these results by flow cytometry. This difficulty in detecting PMNs in human and mouse atherosclerotic plaques can be attributed to their biological fea-tures, but also to the methods that are commonly used to detect them. Firstly, within inflamed tissues, PMNs rapidly undergo apoptosis and are cleared by phagocytes (57). On the other hand, activated human PMNs also might exhibit marked phenotypic changes by acquiring antigen-presenting cell features or shedding their surface membrane receptors (58). These plastic and dynamic properties might modify the expression of neutrophil specific markers. In addition, for many years, sensitive and specific detec-tion methods for PMNs also lacked. Anti-mouse Ly6G antibodies

allowed to identify PMNs in atherosclerosis mouse models (from early stages to rupture-prone lesions) (59, 60). Similar results were obtained by combining the staining for MPO and elastase (61) or formyl peptide receptor (FPR2) and p22phox (62). Apoe-/-Lys-megfp/egfp mice allow tracking PMNs in late atherosclerotic plaques. Using this model, Rotzius et al. noticed a PMN-endothelium inter-action and PMN accumulation in shoulder region of mouse atherosclerotic plaques, thus suggesting a significant PMN recruit-ment from the vessel lumen to the arterial wall (63). These results from human and mouse studies suggest that neovessels, which have a more fragile in lipid core, might be a preferred target for PMN homing.

Author (reference)

Papa A. et al. [23]

Karabinos I. et al. [24]

Poludasu S. et al. [25]

Setianto B. Y. et al. [26]

Muhmmed Suliman M. A. et al. [27]

Husser O. et al. [28]

Park B.-J. et al. [29]

O’ Hartaigh B. et al [30]

Arbel A. et al. [31]

Kaya N. et al. [32]

Park J. J. et al. [33]

Year

2008

2009

2009

2010

2010

2010

2011

2012

2012

2012

2012

Study design (n)

Prospective observational (422 CAD patients)

Prospective observational (160 NSTEMI patients)

Prospective observational (327 lower N/L ratio, 45 higher N/L ratio)

Case-control(79 patients; 54 AMI, 25 UA)

Prospective observational(300 ACS patients)

Prospective observational (267 rep-erfused STEMI patients)

Cross-sectional(849 healthy controls)

Prospective(3316 patient undergoing coronary angiography)

Prospective observational(3005 patient undergoing coronary angiography)

Case-control(394 repeating angiography; 196 progressive disease and 198 non-progressive disease)

Prospective observational(325 patients with STEMI treated with PTCA)

Target

N/L ratio

Neutrophil count

N/L ratio

neutrophil count

N/L ratio

Neutrophil count

N/L ratio

Neutrophil count

N/L ratio

N/L ratio

N/L ratio


Positive predictor of cardiac death (multivariate analy-sis: HR 8.13; CI 1.42–46.57; p<0.02))

Positively correlate with major in-hospital events (p=0.02), independently predict major in-hospital events (OR: 7.74; CI: 2.79–21.47; p<0.001) and is an independent in-hospital prognostic factor (OR: 6.52; CI: 1.56–27.22; p=0.01)

Positively correlates with mortality rate (p<0.001) and is indipendent long-term mortality predictor (covari-ates analysis: HR 2.1; CI 1.1–4; p=0.02

Positively correlates with CAD severity.AMI vs UA group (p<0.001).

Positively correlates with all-cause in-hospital mortal-ity (2 test: p<0.003)

Positively correlate with larger infarction (p=0.0001), independently predict larger infarction (OR 1.14; CI 1.04–1.26; p=0.008) and major adverse cardiac events (HR 1.2; CI 1.1–1.4; p=0.003).

Positively correlates in multivariate analysis with arter-ial stiffness (OR 2.12; CI 1.18–3.83; p=0.012) and cor-onary calcium score (OR 2.19; CI 1.02–4.70; p=0.045)

Positive predictor of CVD mortality (multivariate analysis: HR 1.93; CI 1.39–2.67; p<0.001).

Positive predictor of CAD presence and severity (OR 2.45; CI 1.76–3–42; p<0.001).Also positively correlated.N/L ratio correlate with CVD events both in crude HR 2.3 (CI 1.72–3.16; p<0.001) than after regression analysis: HR 1.55 (CI 1.09–2.2; p=0.01

Positively correlates with progressive disease (multi-variate analysis: RR 2.267; CI 1.068–4.815; p=0.003

Positive predictor of mortality (HR 3.12; CI 1.14–8.55; p<0.05)

Table 1: Continued




Neutrophils in the pathophysiology of plaque ruptureA potential relationship between the risk of plaque rupture and its composition and structure has been recently reported (64). Spe-cific histological features characterise the so-called “vulnerable plaque” (65). The feature that is most highly predictive of plaque rupture is the necrotic lipid core, a mixture of necrotic cellular de-bris, macrophages, foam cells, growth factors, cytokines and extra-cellular lipid pools composed of free cholesterol, cholesterol cryst-

als, and cholesterol esters. A large, eccentric lipid core unbalances circumferential plaque stress to the shoulder lesion, where nearly 60% of plaque ruptures tend to occur (66). A necrotic lipid core also contributes to inflammation, thrombosis, proteolytic plaque breakdown, and physical stress at the fibrous cap level (67). The fi-brous cap is a connective tissue layer which covers the necrotic lipid core and separates it from the arterial lumen. If necrotic lipid core miss there is no fibrous cap and the plaque cannot rupture. Structural components of the fibrous cap components include ma-trix molecules such as collagen, elastin, and proteoglycans that are

Table 2: Neutrophil products and cardiovascular risk.

Author (reference)

Mehta J. et al. [40]

Dinerman J. L. et al. [9]

Bell D. et al. [10]

De Servi S. et al. [41]

Amaro A. et al. [42]

Biasucci L. M. et al. [43]

Koşar F. et al. [44]

Smith F. B. et al. [45]

Zhang R. et al. [46]

Garlichs C. D. et al. [47]

Buffon A. et al. [48]

Saìnchez de Miguel L. et al. [49]

Smith C. et al. [50]

Year

1989

1990

1990

1995

1995

1996

1998

2000

2001

2003

2002

2002

2006

Study design (n)

Case-control(57 patients; 17 AMI or UA, 20 SA and 20 healthy controls)


Case-control(97 patients; 32 AMI, 30 IHD and 35 healthy controls)

Case-control(60 patients; 38UA and 22 SA)

Case-control(42 patients; 19 CAD and 23 not CAD)

Case-control(86 patients; 16 AMI, 30 UA, 40SA)

Case-control(185 patients; 135 CAD, 50 not-CAD)

Prospective observational(207 patients with SA)

Case-control(333 patients; 158 CAD, 175 CAD)


Case-control (52 patients; 33UA, 13 SA and 6 healthy controls)

Case-control(69 patients; 31 AMI, 18 UA and 20 healthy controls)

Case-control(100 patients; 40 UA, 40 SA, 20 healthy controls)

Target

HNE

HNE

HNE

Neutrophil adhesion molecule CD11/CD18b

HNE

MPO

HNE

HNE

MPO

PMNs apoptosis

MPO

eNOSiNOS

NAP-2


Plasma levels (B peptide) positively correlate with CAD severity.AMI/UA group vs SA (p<0.01) and healthy controls (p<0.001)

Plasma levels (B peptide) positively correlate with CAD severity.AMI (p<0.02) and UA (p<0.006) vs healthy controls

Plasma levels (B peptide) positively correlate with CAD severityAMI (p<0.0001) and IHD (p<0.004) vs control group

Plasma levels positively correlate with CAD severity UA vs SA (p<0.0001)

Plasma levels positively correlate with CAD presence (p<0.05)

Intracellular MPO was reduced in AMI e UA vs SA (p<0.01)

Plasma levels positively correlate with CAD presence (p<0.001) and plaque complexity (p<0.001)

Plasma levels positively correlate with cardiovascular events risk during follow-up(RR: 1.74; CI 1.04–2.95; p<0.005)

Plasma levels positively correlate with CAD presence (p<0.001).MPO is an independent factors CAD (multivariate analysis: OR 20.4; CI 8.9–47.2, (p<0.001)

Serum of ACS patients inhibits PMNs.AMI/UA vs SA/healthy controls (p<0.001)

Intracellular MPO inversely correlate with CAD sever-ity. UA vs SA and healthy (p<0.05 for all comparisons)

In circulating PMN higher iNOS (p<0.05) and lower eNOS (p<0.05) levels in AMI/UA vs healthy controls

Plasma levels positively correlate with CAD severity. UA vs SA (p<0.05) and healthy controls (p<0.001).




derived from vascular smooth muscle cells (VSMCs). Fibrous cap thickness is one of the most well-known markers of plaque vulner-ability. Plaque rupture only occurs when the fibrous cap is ex-tremely thin (68). Additional distinctive features of the vulnerable plaque are represented by: outward remodelling, inflammatory cell infiltration within the fibrous cap and increased plaque neo-vascularisation (69). Through their activation, adhesion and infil-tration, neutrophils might influence all of these parameters of atherosclerotic plaque vulnerability and their multiple roles are ac-tually under investigation in both humans and animal models (5). The presence of neutrophils was clearly documented in human vulnerable atherosclerotic plaques by Naruko et al. (70) and by Ionita et al. (71). In addition, Leclercq et al. also showed a positive correlation between PMN markers and shoulder haemorrhages as well as in the necrotic core in human atherosclerotic plaque (72). On the other hand, the study by Zernecke et al. recently showed that neutrophil intraplaque infiltration might be critical also dur-ing early atherogenesis (59). In particular, the chronic blockade of chemotactic pathway involving the chemokine CXCL12 and its re-ceptor transmembrane CXCR4 was associated with protection from the infiltration of neutrophils within mouse atherosclerotic plaques, indicating these cells might be crucial players not only in atheroma dirsuption and complication, but also in its early formation (59).

Neutrophil-derived components in plaque vulnerability

PMNs are activated by micro environmental inflammatory signals, inducing mediators that are contained in their own intracellular granules and vesicles (▶ Figure 1) (73). Neutrophil gelatinases have been shown to play a critical role in mouse atherogenesis and atherothrombosis, as deduced from experiments in knockout ani-mals (74-76). Gelatinases mainly degrade type IV and denatured collagens (gelatins) but also many matrix substrates: collagen types (I, V, VII, X, and XI), elastin, fibronectin, laminin, aggrecan, vitro-nectin, brevican, neurocan and decorin (77). In addition, gelati-nases have been shown to cleave several immune modulatory mol-ecules, such as growth factors (stromal cell-derived factor), cyto-kines (transforming growth factor [TGF]-β, tumour necrosis fac-tor [TNF]-α, interleukin [IL]-1β), chemokines (IL-8), and en-dothelin (ET)-1. Another important property is represented by the gelatinase ability to cleave and control their own and other MMP activities (78). PMN granules have also been shown to contain and release some collagenases (i.e. MMP-1, -8, and -13), which were recently described to increase atherogenesis and extracellular ma-trix degradation in atherosclerotic mice (79). Consistent with this study, MMP-8 has been shown to potentially increase plaque vul-nerability, cleaving type I and III collagen and activating angioten-sin (AT)-I conversion to AT-II. Moreover, MMP8 knockout mice exhibit reduced endothelial cell activation as compared to controls

Author (reference)

Mocatta T. J. et al. [51]

Roman R. M. et al. [52]

Naruko T. et al. [53]

Cojocaru I. M. et al. [54]

Setianto B. Y. et al. [26]

Kafkas N. et al. [55]

Salonen I. et al. [56]

Year

2007

2010

2010

2010

2010

2012

2012

Study design (n)

Prospective case-control study(668 patients; 512 AMI, 156 healthy controls)

Prospective case-control study(308 patients; 130 ACS, 178 SA)

Case-control(467 patients; 236 UA, 146 SA, 85 healthy controls)

Case-control(150 patients; 78 IS; 72 healthy controls)

Case-control(79 patients; 54 AMI, 25 UA)

Case-control (140 patients; 25 STEMI, 40 NSTEMI, 35 UA, 40 SA and 20 healthy controls)

Prospective(53 patients with ischaemic heart disease)

Target

MPO

MPO

MPO

MPO

sCD40L level

NGAL

MPO


Plasma levels positively correlate with ACS (p<0.001) and mortality (OR 1.8; CI 1–3; p=0.034)

Plasma levels positively correlate with CRP (r=0.39; p<0.001) and CAD severity (p<0.05) in ACS (not in SA). MPO have prognostic value (OR 3.8; CI 1.2–12; p<0.05)

Plasma levels positively correlate with CAD complexity (p<0.0001) in UA (not in SA).MPO is an independent risk factor for complex lesions (multivariate analysis: OR 12.49; CI 3.24–48.17, p=0.0002)

Plasma levels positively correlate with IS (RR 8.188; CI 4.038–16.600; p<0.0001)

Plasma levels positively correlate with neutrophil count in UA (r=0.607, p<0.002)

Plasma levels positively correlate with CRP (r=0.685; p<0.0001) and neutrophil count (r=0.511, p<0.0001)

Plasma levels positively correlate with PMNs count (p=0–003) and CRP (p=0.02).MPO in an independent risk factor for ATS (no correlation with other CV risk factor).

Table 2: Continued




(80). Elastase is stored at high concentrations PMN azurophilic granules and is primarily detected in the shoulder region of atherosclerotic plaques (81). Elastase substrates include the major-ity of the components of the extracellular matrix. In particular, elastase-mediated digestion of elastin, collagen, fibronectin and proteoglycans has been shown to expose recognition sites that bind cellular integrin and tyrosine receptors, thus promoting leu-kocyte chemotaxis (82). More recently, neutrophil elastase has also been demonstrated to cleave the macrophage receptor for hae-moglobin-haptoglobin complexes (CD163). Considering that hae-moglobin intraplaque accumulation after intraplaque haemor-rhages promotes plaque vulnerability, neutrophil elastase may con-tribute to plaque vulnerability also through this mechanism (83). Azurophilic granules also release a human serine proteinase: pro-teinase 3 (PR-3) that may promote fibrous cap thinning by its abil-ity to degrade elastin and other macromolecules of the extracellu-lar matrix (such as fibronectin, laminin, vitronectin, and collagen types I, III, and IV) (84). Moreover, PR-3 may activate gelatinases (MMP-2 and MMP-9), thus further contributing to extracellular

matrix degradation (85) and to PMN transendothelial recruitment (86). Activated PMNs in atherosclerotic plaque exhibit an abrupt increase in oxygen consumption, known as phagocyte respiratory burst (62) that allows for reactive oxygen species (ROS) gener-ation. NADPH oxidase, the key enzyme in ROS production in PMNs, is a multicomponent complex made of proteins (gp91phox and Gp22phox) inserted in the plasma membrane and of polypep-tides located in the cytoplasm (the Gp40phox/Gp47phox/Gp67phox complex). Among the different ROS that are produced as a conse-quence of NADPH activity, hydrogen peroxide (H2O2) might also act as second messenger, which changes pH and ion flux, triggers proteolytic enzyme release and acts as a myeloperoxidase (MPO) substrate (87). MPO is the major protein in PMN primary gran-ules and catalyses the hydrogen peroxide-mediated oxidation of halide ions to hypochlorous acid (HOCl), chloramines, aldehydes, hydroxyl radicals, singlet oxygen and ozone (88). The first demon-stration of MPO within atherosclerotic plaque was in 1994 (89). More recently, Malle et al. immunohistochemically demonstrated a colocalisation of myeloperoxidase and hypochlorite-modified

Figure 1: Release of neutrophilic products in atherogenesis. IFN-γ: in-terferon-γ; TNF-α: tumour necrosis factor-α; TGF-β: transforming growth fac-tor-β; IL-: interleukin-; CXCR: CXC chemokine receptors ; IgFcR: immunoglo-bulin fragment crystallisable receptor; TLR: toll-like receptor; MPO: myeloper-

oxidase; PR-3: proteinases-3; MMP: matrix metalloproteinases; AT: angioten-sin; ET-1: endothelin-1; ROS: reactive oxygen species; VSMC: vascular smooth muscle cell; ECM: extracellular matrix.




proteins in atherosclerotic plaques (90). Subsequent studies have shown that intraplaque MPO expression is an independent predic-tor of both presence and severity of coronary artery disease (91). MPO-generated ROS might actively promote atherosclerotic plaque vulnerability through different mechanisms. In the first place, MPO-derived oxidants generate peroxided lipids (92), which are recognised by macrophage scavenger receptors (93) and promote the development of the necrotic lipid core. In addition, MPO-generated ROS contribute to extracellular matrix degrada-tion by activating MMP-7, MMP-8 and MMP-9 (94), by increasing PMN survival (95) and by promoting smooth muscle cell switch to a fibroblastic phenotype (96).

Interaction between neutrophils and other atherogenic cells

PMNs were shown to influence the atherosclerotic inflammatory response also through their interaction with both circulating im-

mune cells and vascular cells (▶ Figure 2). Such interactions are mediated through a variety of membrane-bound receptors for en-dothelial adhesion molecules, extracellular matrix proteins, cyto-kines and chemokines. Activated PMNs also synthesise and release cytokines and chemokines (such as TNF-α, CXCL-8, interferon [IFN]-γ), CCL3, CCL4, and IL-17), thus recruiting other circu-lating inflammatory cells or promoting intraplaque cell differenti-ation (97). For instance, IL-17 producing T-lymphocytes, named Th17, show a marked pro-inflammatory activity (98), which ulti-mately leads to atherosclerotic lesion progression (99, 100), and also might favor PMN recruitment and activation (101). In turn PMNs exhibit a remarkable chemotactic activity on Th17 (102). Despite a still unclear role in atherogenesis (103), IL-17 might rep-resent a soluble mediator linking T lymphocytes with neutrophils. TNF-α has been shown increase PMN survival and PMN antigen presenting cell properties within inflamed tissues (104, 105). Addi-tionally, the neutrophil product MPO is able to bind to the man-nose receptors (MMRs) on resident macrophages, leading to ROS

Figure 2: Interactions between neutrophils and immune and vascu-lar cells in atherogenesis. MPO: myeloperoxidase; ROS: reactive oxygen species; TNF-α: tumour necrosis factor-α; IL-: interleukin-; IFN-γ: interferon-γ; MIP-: macrophage inflammatory protein; GM-CSF: granulocyte-monocyte

colony-stimulatory factor; APC: antigen presenting cells; G-CSF: granulocyte colony-stimulatory factor; EC: endothelial cell; VSMC: vascular smooth muscle cell; ECM: extracellular matrix.




release (106-108). PMNs also contribute to atherothrombosis by promoting superficial erosion of the endothelial monolayer, which has been estimated to underlie about 40% of coronary athero-thrombotic events (109). In particular, endothelial dysfunction ap-pears to contribute to determine vasoconstriction and activation of the coagulation cascade, of platelets, and of anti-fibrinolysis ac-tivities. PMNs promote endothelial dysfunction by releasing ROS and proteases (110). MPO might induce endothelial dysfunction via two distinct mechanisms: i) reduction of nitric oxide (NO) lev-els (NO is used as substrate for MPO catalytic activity) (111, 112); ii) generation of NO-derived oxidants that stimulate the activation of tissue factor pathways and promote apoptosis together with MPO-generated HOCl (113). Hazell et al. indeed identified HOCl-modified proteins accumulated at ruptured or eroded sites of human coronary atheromas (114). More recently, Sugiyama et al. showed that MPO-generated HOCl may directly affect the en-dothelial cells (ECs) (115). Matrix-degrading proteases also par-ticipate in endothelial cell dysfunction by degrading the basement membrane and type IV collagen (116). Some common biomarkers (i.e. auto-antibodies or chemokines) activating both neutrophils and monocytes to increase atherosclerotic plaque vulnerability have been recently identified in both humans and mice (117). However, Soehnlein et al. showed that some neutrophil granule proteins might favour monocyte infiltration within atherosclerotic plaques in mice (118), thus potentially increasing vulnerability. Previous studies had showed that neutrophil cathepsin G and alpha-defensin were able to potently attract human and mouse monocytes both in vitro and in vivo (119, 120). As better discussed in detail in the next paragraph, neutrophils have been described to highly interact also with platelets mainly in the phase of thrombus formation. However, elevated levels of neutrophil-platelet aggre-gates have been observed in peripheral blood of patients with un-stable as compared to stable angina (121). Whether this patho-physiological event observed in a low number of subjects (n=50) might directly increase plaque vulnerability is still a matter of dis-pute.

Role of neutrophils in atherothrombosis and coronary neo-intima formation after percut-aneous transluminal coronary angioplasty (PTCA)Recently, atherothrombosis emerged as a highly complex event, in which not only activated platelets (PLTs), but also PMNs have emerged as critical players within the coronary thrombus (122). PLTs were shown to promote PMN adhesion and activation en-hancing chemokine release (CXCL4, CXCL7 and CCL5) and ex-pression of inflammatory receptors, co-stimulatory molecules and scavenger receptors in vitro and in a mouse model of athero-sclerosis (123, 124). The CD40-CD40L co-stimulatory pathway has also been suggested to be involved in the interaction between PLTs and PMNs. Through soluble CD40L release, PLTs stimulate PMNs to release ROS, which, in turn, activate PLTs (125). Specifi-cally, this mechanism was proposed to contribute to neointima

formation after arterial injury (126). PMNs might also activate PLTs by a novel mechanism only recently described as a part of the innate immune response: the formation of neutrophil extracellular traps (NETs), a meshwork of extracellular DNA fibers comprising histones and antimicrobial proteins. Fuchs et al. have demon-strated the role of NETs in PLT activation, adhesion and aggre-gation in vitro (127), further supporting a direct role for PMNs in atherothrombosis.

A potential protective role for PMNs in neointima formation after both balloon angioplasty and stenting has recently been pro-posed. PTCA is a clinically effective method for improving blood flow through stenosed or occluded arteries (128). However, reste-nosis, resulting from neointimal hyperplasia, negative remodell-ing, and elastic recoil limit the long-term success of this procedure (129). PTCA frequently induces a severe injury that would expose to the blood flow the neointima composed of a mixture of smooth muscle cells and inflammatory cells (130). Although their role is still under debate, an early inflammatory response to arterial in-jury is characterised by PMN influx. Similarly to early atherogen-esis, the pathophysiological recruitment of PMNs within neointi-ma might be related to the dysregulation of the CXCL12/CXCR4 axis that might influence neutrophil-mediated pro-atherosclerotic activities (59). Studies on β2 integrins (131, 132) and P-selectin (133) defective mice, showing a reduction in neointima thickness, suggested that PMN adhesion promotes restenosis. However, sub-sequent studies demonstrated that both β2-integrins and P-selec-tin are poorly specific for PMNs (134). Likewise, the blockade of the chemokine receptor CXCR2 (critical for neutrophil chemo-taxis towards CXCL1) was found to delay, rather than promoting, endothelial recovery after arterial injury (135, 136). Recently, Xing et al. assessed the role of PMNs in neointima formation by admin-istering IL8RA- and/or IL8RB-transduced endothelial cells (ECs) to a rat of vascular injury. An accelerated re-endothelialisation of the injured area in association with downregulated expression of inflammatory mediators, PMN infiltration, and neointima formation were observed (137). Potential direct interactions be-tween PMNs, ECs and EPCs have been recently demonstrated by Soehnlein et al. PMN-derived cathelicidin (LL-37) promotes re-endothelisation and thereby limited neointima formation after stent implantation. Drawing from these findings, the authors gen-erated a LL-37-coated stent. LL-37-trigger EPC-mediated function resulting in pro-angiogenic factors release (VEGF and EGF) and enhancing EC migration and proliferation and reducing their apoptosis (138). Overall, a potential protective role for PMNs in PTCA and stenting appears highly likely, with these cells possibly acting as potential promoters of re-endothelisation. However, these conclusions still await further confirmation.

Post-infarction cardiac remodelling

Following infarction, the myocardium undergoes major changes both in its function and structure (139). Dying cardiomyocytes to-gether with damaged extracellular matrix (ECM), activate a repar-ative response and an immune reaction, the latter being initially




characterised by the involvement of innate immunity cells. Multiple innate immune pathways (such as toll-like receptors [140, 141]), receptors for Advanced Glycation End-product (AGEs) (142), complement system (143) and ROS (144, 145) were shown to be active in this phase. Through intracellular signalling cas-cades, these signals converge to activate nuclear factor (NF)-κB (146), which, in turn, induces the synthesis of inflammatory cyto-kines, such as TNF-α (147), IL-1β (148), IL-6, and chemokines, such as CCL2, CCL4, CXCL1, CXCL8 (149). Although strongly re-cruited at the infarction site, PMNs are unlikely to have an impor-tant role in late remodelling processes: from 3 to 7 days after the acute event, PMN infiltration resolves in both mouse and canine models (150). In addition, no significant long-term effects of neut-rophil depletion in animal models of acute MI could be detected (151, 152). Indeed, PMNs may contribute to the healing response: the release of apoptotic mediators (such as annexin A1 and lacto-ferrin) was shown to inhibit further PMN recruitment (153) and to promote the release of anti-inflammatory molecules, such as IL-10 and TGF-β (57). On the other hand, neutrophils have also been shown as pivotal players in post-infarction healing by poten-tially favouring the recruitment of inflammatory monocytes (118), instead of the reparatory monocytes (154). In fact, Soehnlein et al. showed that PMN components (i.e. PMN-derived LL-37 and he-parin-binding protein [HBP/CAP37/azurocidin]) might directly activate inflammatory monocyte extravasation in mice (118). Al-though highly speculative, the early release of these neutrophilic mediators in mice might affect the cardiac recruitment of different monocytes subsets (Ly-6C[high] and Ly-6C[low] mononuclear cells, respectively) in the first week post-infarction (154). The rel-evant activation of monocytes in the first phases after an acute is-chaemic event (such as stroke of acute MI) was recently confirmed by Dutta et al., showing that a marked increased monocyte intra-plaque recruitment might accelerate atherogenesis in Apoe-/- mice submitted to an acute MI (155). Vice versa, the detrimental role of PMN infiltration in the first hours after cardiac ischaemia is well known. The reperfusion of the ischaemic myocardium is necessary to rescue tissue from death (156). However, at the same time, the restored blood flow halts the ischaemic process by supplying oxygen and nutrients, but triggers a cascade of inflammatory and toxic mediators (157). Frame et al. (158) and Farb et al. (159) found more important signs of myocardial injury after reperfusion than after prolonged ischaemia, while Rochitte et al. (160) docu-mented a progressive increase in tissue damage and microvascular obstruction during the hours after reperfusion. Over the last two decades, experimental evidence suggested that PMNs may also di-rectly damage cardiomyocytes through the release of toxic prod-ucts, such as reactive oxygen species and proteolytic enzymes (161). In particular, compelling evidence for a key role of PMNs in ischaemia-reperfusion-mediated myocardial damage stems from studies of pharmacologic and genetic interventions aimed to pre-vent PMN recruitment or function. For instance, Liehn et al. in-vestigated the role of CXCL12/CXCR4 axis in an experimental model of acute MI in mice (162). Using heterozygous (+/-) CXCR4 animals (characterised by a lower expression of CXCR4 on bone marrow–derived mononuclear cells) (163), the authors showed

that these mice developed a reduced scar area at four weeks post-MI when compared to wild type littermates (162). Importantly, this beneficial remodelling was associated with reduced infarct size and leukocyte infiltration (i.e. neutrophils and monocytes) in the early hours after the acute MI (162). This article confirmed that early interference with PMN recruitment after an acute MI might be a promising therapeutic approach to reduce infarct size and adverse cardiac remodelling after an acute MI (162).

Treatments targeting PMN activities in coronary atherogenesis and post-infarction cardiac remodellingStatins

Statins are a class of lipid-lowering drugs more effectively through their action on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), the rate-limiting enzyme of the mevalonate pathway for cholesterol synthesis (164). The beneficial effects of statins are clearly related to the reduction in cholesterol levels they induce, but also to their complex “pleiotropic” anti-inflammatory activity. Dunzendorfer (165) and Kaneider (166) were the first to docu-ment the ability of statins to induce leukocyte apoptosis and in-hibit leukocyte transendothelial migration (167-171). Statins were shown to inhibit PMN migration and activation in vitro. Cerivas-tatin inhibited adhesion molecule expression (ICAM-1, P-selectin, and E-selectin) on endothelial cells, thus preventing neutrophil ad-hesion to endothelial cells (172, 173). Bandoh et al. (17)] and Kow-alski et al. (175) reported the anti-oxidant effect of fluvastatin and atorvastatine, which reflects the ability of these drugs to scavenge ROS and to inhibit NADPH-dependent ROS generation. The metabolic effects of statins are not limited to cholesterol, but also affect cholesterol intermediate products that are involved in sev-eral cell-signalling pathways. Isoprenoids, such as geranylgeranyl pyrophosphate (GGP) and farnesyl pyrophos-phate (FPP), pro-mote the activation of GTPases, including RhoA (176). This pro-tein, together with its effector Rho kinase, is thought to control cy-toskeleton rearrangement, cell migration and ROS generation (177) in inflammatory cells. Statins were shown to modulate the levels of these metabolites and, as a result, to dampen PMN mi-gration (178) and platelet-PMN interaction (179). Finally, Naka-mura et al. showed that fluvastatin treatment in atherosclerotic mice suppressed atherosclerotic plaque rupture by reducing MMP-9 expression, endothelial adhesion molecule expression and PMN infiltration thus confirming pleiotropic anti-inflammatory properties for statins. Such effects may reflect, at least in part, a di-rect anti-inflammatory activity of fluvastatin on PMNs (180).

HDL and HDL apolipoproteins

High-density lipoprotein (HDL)’s beneficial role is complex and likely involves several mechanisms, including anti-inflammatory effects (181). HDL and apolipoprotein(apo)A-I regulate PMNs ac-tivation, as detected by CD11b expression, by decreasing lipid rafts abundance (182), specialised plasma membrane areas with a role




in oxidative burst, calcium channel trafficking (183) and NADPH oxidase activity (184). In addition, apo lipoproteins inhibit PMN-mediated release of TNF-α, IL-1, IL-8, IL-1 receptor antagonist (IL-1Ra) and degranulation (185, 186). In vivo, infusion of HDL or apoA-I significantly attenuates PMN intima infiltration in rabbits (187, 188), by acting both on endothelial cell activation (188, 189) and circulating PMNs (190-192). This potential protective role of HDL on leukocyte-mediated pro-atherosclerotic activities was partially confirmed by Yvan-Charvet et al., who recently showed that transplantation of bone marrow knockout for adenosine trip-hosphate-binding cassette (ABC) transporters (Abca1 and Abcg1) into apolipoprotein A-1 transgenic mice (animal with elevated HDL) was associated with a reduced leukocytosis, and accelerated atherogenesis (193). This study indicated that ABCA1, ABCG1 in presence of elevated HDL levels might abrogate both conditions. More recently, Murphy et al. using the same knockout mice (char-acterised by leukocytosis due to the high proliferation of hemato-poietic progenitor cells in the bone marrow) identified a specific role for the proteoglycan-bound ApoE on these leukocyte progeni-tors to promote cholesterol efflux via ABCA1/ABCG1, resulting in a final abrogation of leukocytosis and atherosclerosis in mice (194). Finally, Westerterp et al. showed that the increase in HDL levels was inversely associated with haematopoietic progenitor cell mobilisation in the same mouse model (knockout for ABCA1, ABCG1 and ApoE) characterised by atherosclerosis and concomi-tant leukocytosis (195). Taken together, these interesting studies from animal models might support cholesterol efflux pathways as potential therapeutic targets to reduce neutrophil-mediated activ-ities in atherosclerosis.

Treatments that selectively target PMN chemokines

Tissue reoxygenation during reperfusion triggers an inflammatory reaction that is characterised by high levels of cyto-chemokines and consequent leukocyte recruitment, potentially causing greater damage to affected organs than ischaemia itself. Chemokine-in-duced PMN recruitment and activation within ischaemic organs is believed to be a pivotal mechanism of reperfusion injury. CXC chemokines, including CXCL1, 2, 3, 5 (ligands of CXCR2) and CXCL6–8 (ligands of both CXCR1 and CXCR2) were shown to at-tract neutrophils in both human and mouse atherosclerotic plaques and infarcted tissues (196). On the other hand, also CC chemokines have been considered as critical neutrophil chemoat-tractants in certain inflammatory micronevironments mimicking the atherosclerotic plaque and the ischaemic myocardium (197-199). Thus, selective blockade of CC and CXC chemokines has been evaluated as a therapeutic strategy to improve the out-comes of MI in several animal models. Monoclonal antibody treat-ments against CXCL8 (200) or CCL5 (201), chemokine-binding proteins (202), as well as CXCR1/2 competitive antagonists (203) were recently investigated both in vivo and in vitro with promising preliminary results. Widdowson et al. firstly illustrated the thera-peutic potential of CXCR1/2 blockade using IL-8 inhibiting mol-ecules that were identified from phenolic urea series (203). Mahler et al. documented the efficacy of monoclonal antibodies against

CXCL8 in reducing inflammation, and therefore the symptoms, in patients with chronic obstructive pulmonary disease (COPD) (200). However, the authors did not focus on cardiovasuclar out-ocomens in this study. In 2010, in a mouse model of acute MI/rep-erfusion injury, we demonstrated that treatment with the CXC chemokine-binding protein Evasin-3 was associated with a reduc-tion in myocardial infarct size and post-ischaemic inflammation. These beneficial effects were closely related with the inhibition of cardiac neutrophil recruitment and ROS production. On the other hand, in contrast with previously reported data (204), Evasin-3 was ineffective in a Langendorff perfused heart model, an ex vivo experimental system characterised by absence of circulating leuko-cytes (202).

In 2012, evidence from our laboratory suggested a pivotal role for another neutrophilic chemokine (i.e. CCL5) in two mouse models of chronic cardiac ischaemia and transient ischaemia fol-lowed by reperfusion. This CC chemokine has been shown as a crucial chemoattractant for macrophages both in mouse athero-genesis and MI (198, 199) via the binding to two main transmem-brane receptors (such as CCR1 and CCR5). Since several intra-plaque and circulating cell subsets (including platelets, macro-phages) are might release CCL5, multiple sources of this chemo-kine might be considered in atherogenesis. For instance, Drechsler et al. showed that platelet-derived CCL5 was deposited on en-dothelial cells of the in arteries but not in veins in hypercholestero-laemic mice (60). In addition, the selective activation of receptors has been shown to play different activities in cardiovascular dis-eases. For instance, Liehn et al. showed that the main receptor for CCL5-mediated neutrophil recruitment after MI was CCR1 and not CCR5 (205). Considering these premises, we firstly confirmed that CCL5 was upregulated both locally (within the infarcted myocardium) and systemically early after the onset of myocardial chronic ischaemia onset (201) and during reperfusion (198). Then, in the first 24 hours, we focused on the pharmacological inhibition of CCL5. This approach potently reduced neutrophil recruitment and inflammation within infarcted hearts, improving infarct size when compared to control vehicle. At 21 days of chronic ischae-mia, the pharmacologic inhibition of CCL5 (in the first three days after ischaemia onset) was associated with improved mouse sur-vival, cardiac myocyte mass and function (201).

More recently, evidence from our research group also showed that treatment with FK866 (a nicotinamide phosphoribosyltrans-ferase [Nampt] inhibitor that lowers NAD+ levels) potently re-duces CXC chemokine serum levels, neutrophil infiltration in the infarcted heart and myocardial injury following ischaemia-reper-fusion in mice. This treatment approach also ROS formation in the heart (likely as a consequence of the reduced PMN infiltration of the ischaemic tissue), thus suggesting reducing the levels of CXC chemokines may induce benefits that are similar to those achieved with strategies that inhibit chemokine bioactivity instead (206). The mechanisms underlying FK866 ability to prevent CXC chemokine production appears to entail a reduced activity of SIRT6, an NAD+-dependent deacetylase which we have recently shown to promote inflammatory cytokine expression by boosting



511

Ca2+ responses and Nuclear Factor of Activated T-cells (NFAT) ac-tivity (207).

Conclusions and perspectives

Amongst the different inflammatory cell types, PMNs are now in-creasingly appreciated as pivotal players in coronary plaque vul-nerability, neointima formation and the first phases of myocardial reperfusion injury and adverse remodelling. Compelling in vivo studies have demonstrated a strong therapeutic potential for PMN-targeting treatments, including those aimed to dampen che-mokine levels or bioactivity. Although human clinical studies are still lacking, selective treatments targeting chemokine-mediated PMN recruitment and activation within atherosclerotic and in-farcted myocardium are predicted to have a strong impact on car-diovascular disorders and to help improve clinical outcomes.

Conflicts of interestNone declared.

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