Pharmacology & Therapeutics 186 (2018) 73–87

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

journal homepage: www.elsevier.com/locate/pharmthera

Inflammation following acute myocardial infarction: Multiple players,

dynamic roles, and novel therapeutic opportunities
Sang-Bing Ong a,b, Sauri Hernández-Reséndiz a,b, Gustavo E. Crespo-Avilan a,b, Regina T. Mukhametshina g,h,
Xiu-Yi Kwek a,b, Hector A. Cabrera-Fuentes a,b,e,f,g,1, Derek J. Hausenloy a,b,c,d,i,j,⁎,1
a
Cardiovascular and Metabolic Disorders Program, Duke-National University of Singapore, Singapore
b
National Heart Research Institute Singapore, National Heart Centre Singapore, Singapore
c
The Hatter Cardiovascular Institute, Institute of Cardiovascular Science, University College London, UK
d
The National Institute of Health Research University College London Hospitals Biomedical Research Centre, UK
e
Institute of Biochemistry, Medical School, Justus-Liebig University, Giessen, Germany
f
Escuela de Ingenieria y Ciencias, Centro de Biotecnologia-FEMSA, Tecnologico de Monterrey, Monterrey, NL, Mexico
g
Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russian Federation
h
German Center for Lung Research, Excellence Cluster Cardio-Pulmonary System, Universities of Giessen and Marburg Lung Center, Giessen, Hessen, Germany
i
Yong Loo Lin School of Medicine, National University Singapore, Singapore
j
Barts Heart Centre, St Bartholomew's Hospital, London, UK

a r t i c l e i n f o a b s t r a c t

Available online 9 January 2018 Acute myocardial infarction (AMI) and the heart failure that often follows, are major causes of death and
disability worldwide. As such, new therapies are required to limit myocardial infarct (MI) size, prevent adverse
Keywords: left ventricular (LV) remodeling, and reduce the onset of heart failure following AMI. The inflammatory response
Inflammation to AMI, plays a critical role in determining MI size, and a persistent pro-inflammatory reaction can contribute to
Acute myocardial ischemia and reperfusion
adverse post-MI LV remodeling, making inflammation an important therapeutic target for improving outcomes
injury
following AMI. In this article, we provide an overview of the multiple players (and their dynamic roles) involved
Acute myocardial infarction
Monocytes
in the complex inflammatory response to AMI and subsequent LV remodeling, and highlight future opportunities
Macrophages for targeting inflammation as a therapeutic strategy for limiting MI size, preventing adverse LV remodeling, and
Lymphocytes reducing heart failure in AMI patients.
Dendritic cells © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
Cytokines (http://creativecommons.org/licenses/by/4.0/).
Chemokines
Innate immunity

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2. The inflammatory response to acute myocardial IRI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3. Therapeutic targeting of inflammation following MI . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Abbreviations: ACS, Acute coronary syndrome; AMI, Acute myocardial infarction; AGEs, Advanced glycation end-products; BAFF, B-cell activating factor; C1-INH, C1-inhibitor; CCL2,
Chemokine ligand 2; CCL5, Chemokine ligand 5; CCR2, Chemokine receptor 2; CCR5, Chemokine receptor 5; CCR9, Chemokine receptor 5; CR1, Complement receptor 1; CINC-1, CXCL1,
GRO α, KC, Cytokine-induced neutrophil chemoattractant 1; DAMPs, Damage-associated molecular patterns; ECM, Extracellular matrix; EDA, Extra domain A; eRNA, Extracellular ribonu-
cleic acids; FN-EDA, Fibronectin-end domain A; HSPs, Heat shock proteins; hs-CRP, High-sensitivity C-reactive protein; HMGB1, High mobility group box 1; ICAM-1/ICAM-2, Intercellular
adhesion molecule; IFN-γ, Interferon-γ; IRF5, Interferon regulatory factor 5; IHD, Ischemic heart disease; IL-1, Interleukin-1; IL-8, CXCL8, Interleukin-8; LV, Left ventricular; LTB4,
Leukotriene B4; MIP-2α, CXCL2, GRO β, Macrophage inflammatory protein-2α; MMPs, Matrix metalloproteinases; MCP-1, Monocyte chemoattractant protein-1; MyD, Myeloid differen-
tiation primary response gene; MI, Myocardial infarction; NO, Nitric oxide; NLRs, NOD-like receptors; NF-κB, Nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3,
Nucleotide-binding oligomerization domain-like receptor family of cytosolic proteins; PRRs, Pattern recognition receptors; PS, Phosphatidylserine; PMN, Polymorphonuclear leukocytes;
PPCI, Primary percutaneous coronary intervention; ROS, Reactive oxygen species; RAGE, Receptor for advanced glycation end-products; Tregs, Regulatory T cells; STEMI, ST segment el-
evation myocardial infarction; TLRs, Toll-like receptors; TGF-β, Transforming growth factor-β; TNFα, Tumor necrosis factor-alpha.
⁎ Corresponding author at: Cardiovascular & Metabolic Diseases Program, Duke-National University of Singapore, 8 College Road, 169857, Singapore.
E-mail address: derek.hausenloy@duke-nus.edu.sg (D.J. Hausenloy).
1
These two authors are joint senior authors.

https://doi.org/10.1016/j.pharmthera.2018.01.001
0163-7258/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
74 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87

1. Introduction 2.1. The initial pro-inflammatory response to AMI

Acute myocardial infarction (AMI) and the heart failure that often Following AMI, the onset of acute myocardial ischemia induces
follows, are among the leading causes of death and disability world- cellular injury and death to different constituents of the myocardium
wide. Following an AMI, the most effective treatment for minimizing (cardiomyocytes, endothelial cells, fibroblasts and interstitium). This
acute myocardial ischemia/reperfusion injury (IRI), salvaging viable in turn, initiates an acute pro-inflammatory response through the
myocardium, and limiting myocardial infarct (MI) size, is timely myo- concerted action of several processes including complement cascade
cardial reperfusion using primary percutaneous coronary intervention activation, reactive oxygen species (ROS) production, and damage-
(PPCI). However, the process of myocardial reperfusion, can paradoxi- associated molecular patterns (DAMPs) which serve as ligands for
cally, in itself, induce cardiomyocyte death and myocardial injury, a pattern recognition receptors (PRRs), such as Toll-like receptors
phenomenon which has been termed ‘myocardial reperfusion injury’, (TLRs) and nucleotide-binding oligomerization domain-like receptor
and which can contribute up to 50% of the final MI size (Yellon & family of cytosolic proteins (NLRP3, also known as Nod-like receptors)
Hausenloy, 2007). As such, the mortality and morbidity following AMI inflammasomes. These result in the release of a variety of pro-
remain significant with 7% mortality and 22% death at one year, respec- inflammatory mediators (such as cytokines and chemokines), which
tively (Cung et al., 2015). Novel therapies are therefore required to induce the recruitment of inflammatory cells into the MI zone, and aug-
reduce MI size, and prevent adverse LV remodeling in order to reduce ment the pro-inflammatory response following AMI. By targeting the
the onset of heart failure and improve clinical outcomes following AMI viable border zone of the infarction, infiltrating leukocytes may induce
(Cabrera-Fuentes, Alba-Alba, et al., 2016; Cabrera-Fuentes, Aragones, the death of cardiomyocytes, thereby extending ischemic injury beyond
et al., 2016; Hausenloy, Botker, et al., 2017; Hausenloy, Garcia-Dorado, the original MI zone.
et al., 2017; Preissner, Boisvert, & Hausenloy, 2015).
The inflammatory response to acute IRI plays a critical role in 2.1.1. Complement cascade
determining acute MI size and subsequent post-MI adverse LV remodel- Activation of the complement cascade contributes to the acute pro-
ing, making it a potential therapeutic target for improving clinical inflammatory response following AMI (reviewed in Timmers et al.,
outcomes in AMI patients. However, the precise role inflammation 2012). It comprises 30 proteins and protein fragments, many of which
plays in the setting of AMI has been debated since the 1980s with the are circulating as pro-enzymes and are activated by proteases, through
infiltration of leukocytes or granulocytes being recognized as inflamma- 3 different pathways — classical, lectin and alternate, in response to
tory triggers, as opposed to being ‘innocent’ bystanders of the inflam- DAMPs and the release of cardiomyocyte contents during AMI
matory reaction (Engler & Covell, 1987; Engler, Dahlgren, Morris, (Timmers et al., 2012). These 3 pathways converge on the common
Peterson, & Schmid-Schonbein, 1986; Engler, Dahlgren, Peterson, (terminal) complement pathway and result in: (1) opsonization (the
Dobbs, & Schmid-Schonbein, 1986). In the 1990s, experimental studies process by which a pathogen is marked for ingestion and eliminated
questioned the role of leucocytes as mediators of cardiomyocyte death, by a phagocyte) and phagocytosis to clear foreign and damaged
suggesting they may play only a minor role in reperfusion injury material (complement C3b); (2) inflammation to attract additional
(Mullane & Engler, 1991; Ritter, Wilson, Williams, Copeland, & phagocytes (complement C3a, C4a, C5a), (3) activation of the cell-
McDonagh, 1995; Suematsu et al., 1994). However, there is an increas- killing membrane attack complex (complement C5b-9). The activation
ing amount of experimental evidence, that a number of different players of the complement cascade is regulated by various proteins, including
are involved in the inflammatory response, and they have been shown C1-inhibitor (C1-INH) and complement receptor 1 (CR1).
to contribute to the detrimental effects of acute myocardial IRI, making A number of experimental small and large animal AMI studies have
them important therapeutic targets for cardioprotection. In this article shown that MI size can be reduced by genetic or pharmacological
we provide an overview of the multiple players (and their dynamic inhibition of various components of the complement cascade including:
roles involved) in the complex inflammatory response to AMI and (1) Complement receptor 1 (Weisman et al., 1990); (2) C1 inhibitor
subsequent LV remodeling, and highlight future opportunities for (Buerke et al., 1998); (3) mannose-binding lectin (Busche, Pavlov,
targeting inflammation as a therapeutic strategy for limiting MI size Takahashi, & Stahl, 2009; Jordan, Montalto, & Stahl, 2001; Walsh et al.,
and preventing heart failure following AMI. 2005); (4) C5 and C5a (Amsterdam et al., 1995; Vakeva et al., 1998;
van der Pals et al., 2010; Zhang et al., 2007); and (5) C3b by cobra
venom factor (Gorsuch, Guikema, Fritzinger, Vogel, & Stahl, 2009). Of
2. The inflammatory response to acute myocardial IRI these therapeutic approaches for targeting the complement cascade,
C1 inhibitor therapy and antibodies directed to C5a, have both been
The onset of acute myocardial ischemia in the setting of an AMI, tested in clinical studies of AMI, although the results have been mixed
induces an initial pro-inflammatory response, the purpose of which is (see Section 3).
to remove necrotic cell debris from the MI zone. The onset of myocardial
reperfusion following PPCI, then exacerbates this pro-inflammatory 2.1.2. Damage-associated molecular patterns
response and contributes to the cardiomyocyte death and myocardial A variety of DAMPs are released from necrotic cardiac resident cells
injury characteristic of ‘myocardial reperfusion injury’ which manifests following AMI (such as ATP, mtDNA, RNA and HMBGB1) (Fig. 2). These
between 6 and 24 h post-reperfusion (Zhao et al., 2000, 2001). The ini- are known to activate cells of the innate immune system (reviewed in
tial pro-inflammatory response is then followed by an anti- van Hout, Arslan, Pasterkamp, & Hoefer, 2016). The release of ATP can
inflammatory reparative phase which allows wound healing and scar activate the P2X7 membrane receptor thereby inducing efflux of
formation to occur thereby preventing cardiac rupture. The transition intracellular potassium, the release of mitochondrial cardiolipin and
between these two phases is orchestrated by a finely regulated, but subsequent NLRP3-inflammasome activation (Iyer et al., 2013;
complex interaction, between multiple players within the heart itself Mariathasan et al., 2006). Extracellular DNA and mtDNA have been re-
(including cardiomyocytes, endothelial cells, fibroblasts, and the inter- ported to induce inflammation by binding to TLR9 (Zhang et al.,
stitium), and components of the immune response (including neutro- 2010), and are elevated in patients following AMI (Bliksoen et al.,
phils, monocytes, macrophages, dendritic cells and lymphocytes) (Fig. 2012), highlighting a potential anti-inflammatory role for DNAase fol-
1). Perturbations in both the balance and transition between the pro- lowing AMI. Similarly, the release of extracellular RNA from damaged
inflammatory and the anti-inflammatory reparative phases can exacer- cardiac resident cells has also been demonstrated to induce a pro-
bate acute myocardial IRI and contribute to post-MI adverse LV inflammatory response following AMI, through the release of TNF-α
remodeling. and subsequent NF-kB activation (Cabrera-Fuentes et al., 2014;
 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87 75

Fig. 1. Overview of the inflammatory response to acute myocardial infarction. This schema depicts the initial pro-inflammatory and the subsequent anti-inflammatory reparative phase
following AMI. Dying cardiomyocytes during acute myocardial ischemia induce the pro-inflammatory response through the production of DAMPS, ROS, and complement, which through
the release of cytokines (such as IL-1β, IL-18, IL-1α, IL-6, CCL2, CCL5), mediate the accumulation of a variety of cells including neutrophils, monocytes, macrophages, B lymphocytes and
CD8+ T cells into the infarct zone. The subsequent anti-inflammatory reparative phase, mediates the resolution of the inflammatory response through the production of anti-inflammatory
factors (such as IL-10, IL6, TGF-β), and changes in monocytes and macrophages, and the recruitment of Tregs, CD4+ T cells and dendritic cells.

Zernecke & Preissner, 2016) — whether this is mediated by binding to a Genetic or pharmacological inhibition of either TLR2 or TLR4 have
TLRs is not clear. Again, attenuating circulating levels of extracellular been shown to reduce MI size and prevent adverse LV remodeling
RNA by administering RNAse1 has been shown in experimental small following AMI (Arslan et al., 2010; Oyama et al., 2004; Shimamoto
animal studies to reduce MI size (Cabrera-Fuentes et al., 2014; Stieger et al., 2006; Shishido et al., 2003; Timmers et al., 2008), making these
et al., 2017), and provides a novel therapeutic strategy for reducing MI particular TLRs potential therapeutic targets for cardioprotection (see
size. Section 3). Interestingly, a recent experimental study has shown that
Following AMI, the nuclear factor, high mobility group box 1 the environment of the failing and infarcted myocardium was able to
(HMGB1) is released from necrotic cardiac cells, where it has been polarize resident and transplanted MSCs toward a pro-inflammatory
shown to act on several PRRs including TLR2, TLR4, TLR9 and RAGE to phenotype, and restrict their survival and reparative effects via a
activate NF-kB following AMI (Ding et al., 2013; Park et al., 2004). In TLR4-mediated mechanism (Naftali-Shani et al., 2017).
support of a pathogenic role for HMGB1, its inhibition following AMI
has been reported to be cardioprotective (Andrassy et al., 2008; Hu, 2.1.4. Inflammasomes
Zhang, Jiang, & Hu, 2013). On the other hand, in the post-AMI convales- Inflammasomes are large multiple cytoplasmic protein complexes,
cent phase, HMGB1, at low concentrations, may actually be beneficial which form in response to DAMPs released during AMI. They mediate
and contribute to recovery of LV function, tissue repair, and angiogene- the activation of pro-inflammatory cytokines such as IL-1β and IL-18
sis and regeneration (Kitahara et al., 2008; Takahashi et al., 2008), from cardiac fibroblasts, and caspase-1 dependent death of
implicating HMGB1 inhibition at the time of AMI, and HMGB1 activation cardiomyocytes (termed pyroptosis — a highly inflammatory form of
in the post-MI remodeling phase as therapeutic strategies for reducing cell death, characterized by both apoptosis and necrosis, reviewed in
MI size and preventing adverse LV remodeling. van Hout et al., 2016). Most inflammasomes typically contain 3 compo-
nents: (1) a member of the NLRP family proteins (most commonly
2.1.3. Toll-like receptors described in acute myocardial IRI is NLRP3); (2) apoptosis-associated
In response to DAMPs, TLRs, activate myeloid differentiation primary speck-like protein containing a caspase recruitment domain (ASC);
response gene (MyD)88 and NF-kB nuclear factor-kB (NF-kB), which and (3) pro-caspase-1 (van Hout et al., 2016). Common upstream
induce the release of a number of inflammatory mediators including mechanisms implicated in NLRP3 inflammasome activation during
pro-IL-1β and pro-IL-18 (Timmers et al., 2008; van Hout et al., 2016). AMI include extracellular ATP released from injured cells, potassium
The TLRs, which are present in circulating and cardiac resident cells, efflux, lysosomal destabilization, and mitochondrial ROS generation
can be divided into those that are mainly present on the cell surface (van Hout et al., 2016).
and include TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11, and act as sensors The release of IL-1β from cardiac fibroblasts, in response to AMI
to extracellular DAMPs (such as heat shock proteins [HSPs], HMGB1, requires two signals: (1) the transcription of pro-IL-1β by the TLR-NF-
fibronectin-end domain A [FN-EDA]); and those which are intracellular κB pathway, and (2) the activation of pro-IL-1β to its mature form by
such as TLR3, TLR7, TLR8 and TLR9 (which can recognize danger signals the NLRP3 inflammasome. The IL-1β then induces the initial pro-
such as microbial nucleic acids or self-DNA) (van Hout et al., 2016). inflammatory response and the release of cytokines/chemokines,
76 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87

Fig. 2. The pro-inflammatory response induced by DAMPs. Following AMI, the release of damage-associated molecular patterns or DAMPs (such as ATP, mtDNA, RNA, and HMBGB1)
induce a pro-inflammatory response which mediates cardiomyocyte death through Toll-like receptors (TLRs) and the recruitment of leukocytes into the infarct zone, the release of
cytokines, mitochondrial dysfunction (calcium overload and ROS production), and NLRP3-inflammasome formation.

which recruit and activate inflammatory cells such as neutrophils and In the setting of AMI, it has been shown that the administration of
monocytes. Consistent with the role of the NLRP3 inflammasome as a recombinant HMGB1 aggravates acute myocardial IRI, suggesting the
mediator of inflammatory cell death following AMI, both genetic and involvement of RAGE in the post-MI inflammatory response (Andrassy
pharmacological inhibition of its components (caspase 1, IL-1β, ASC, et al., 2008). Mice deficient in RAGE has been shown to be protected
and NLRP3) have been demonstrated to reduce MI size (Coll et al., against AMI, an effect which was associated with less accumulation of
2015; Kawaguchi et al., 2011; Marchetti et al., 2014; Mezzaroma et al., inflammatory cells into the MI zone (Volz et al., 2012). Furthermore,
2011; Pomerantz, Reznikov, Harken, & Dinarello, 2001; Sandanger macrophages and cardiac fibroblasts subjected to simulated ischemia
et al., 2013). Interestingly, Toldo et al. (Toldo et al., 2016) found that demonstrated increased S100A8/A9-mediated RAGE activation (Volz
both MI size and myocardial NLRP3 inflammasome expression in the et al., 2012). Accordingly, combined blockade of RAGE signaling by
murine heart increased as the duration of reperfusion was extended siRNA and soluble RAGE synergistically protected from cardiac damage
over a period of 1, 3 and 24 h, suggesting that the NLRP3 inflammasome after MI (Ku et al., 2015).
may contribute to the phenomenon of ‘late reperfusion injury’, in which
reperfusion-induced cell death takes place minutes to hours following 2.1.6. Cytokines
reperfusion. Consistent with this finding, the authors found that Following AMI, a number of pro-inflammatory cytokines are
pharmacological inhibition of the NLRP3 inflammasome (using 16673- secreted by cardiac resident cells and circulating inflammatory cells.
34-0, an intermediate of glyburide substrate free of the cyclohexylurea They play a critical role in amplifying the pro-inflammatory response
moiety, involved in insulin release), reduced MI size after 24 h reperfu- to acute myocardial IRI by mediating the recruitment of inflammatory
sion, when it was administered at the onset of reperfusion or 1 h after cells into the MI zone. The major cytokine mediating the pro-
reperfusion (but not after 3 h of reperfusion) (Toldo et al., 2016), inflammatory response following AMI is interleukin-1 (IL-1). In AMI
making it possible to intervene after reperfusion has already taken experimental models, IL-1α is released by damaged cardiomyocytes,
place in AMI patients treated by PPCI. whereas IL-1β is upregulated after infarction. Both genetic and
pharmacological inhibition of IL-1 has been shown to reduce MI size
and prevent adverse LV remodeling (Abbate et al., 2008; Bujak et al.,
2.1.5. Receptor for advanced glycation end-products 2008). Small clinical trials have tested the effectiveness of IL-1 inhibition
The receptor for advanced glycation end-products (RAGE), which is in patients with AMI (see Section 3).
known to act as a receptor for advanced glycation end-products (AGEs) IL-6 has been reported to play both pro- and anti-inflammatory
in the setting of diabetes and atherosclerosis, also functions as a PRR for roles, and is released following acute myocardial IRI (Gwechenberger
a variety of ligands including HMGB1 and S100/calgranulins in the set- et al., 1999). Elevated circulating IL-6 levels have been shown to be
ting of chronic inflammation. Once it is activated, RAGE facilitates the associated with acute coronary syndromes (Empana et al., 2010). Men-
translocation of NF-kB to the nucleus for transcription of pro- delian randomization studies have demonstrated that genetic
inflammatory cytokines (Shishido et al., 2003). polymorphisms in the IL-6 receptor signaling pathway resulted in
 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87 77

lower plasma levels of high-sensitivity C-reactive protein (hs-CRP) and determinants of reperfused-induced cardiomyocyte death following
reduced cardiovascular risk (Swerdlow et al., 2012), suggesting that IL-6 AMI.
may be detrimental in patients with coronary artery disease. However,
genetic or pharmacological modulation of IL-6 in the experimental set- 2.1.9. Neutrophils
ting of AMI have produced mixed results. Mice deficient in IL-6 have Neutrophils normally provide the first line of defense against invading
been reported to sustain either similar MI size (Fuchs et al., 2003; microorganisms and tissue injury (reviewed in Prabhu & Frangogiannis,
Kaminski et al., 2009) or smaller MI size (Jong et al., 2016) when com- 2016). Following AMI, polymorphonuclear leukocytes (PMNs) in the
pared to wild-type mice. Similarly, it has been shown that administering bone marrow mobilize into the blood, and are the first inflammatory
the IL-6 receptor antibody, MR16-1, prior to reperfusion and weekly for cells to arrive at the injured myocardium, being present within hours
1 month, actually worsened adverse LV remodeling in a reperfused mu- following AMI, peaking at days 1–3, and starting to decline at day 5.
rine AMI model (Hartman et al., 2016), suggesting a beneficial role for Neutrophils tend to target the border zone of the MI and their accumula-
IL-6 in this setting. This illustrates the challenges in targeting the differ- tion is accentuated at reperfusion. The PMNs are recruited into the in-
ent components of the inflammatory response to AMI with respect to jured myocardium in response to a high concentration of chemotactic
the discordant effects which can arise depending on the timing of the in- factors, such as macrophage inflammatory protein-2α (MIP-2α, CXCL2,
tervention. Despite the mixed experimental data, clinical studies have GRO β), leukotriene B4 (LTB4), cytokine-induced neutrophil
been performed targeting IL-6 in the clinical setting (see Section 3). chemoattractant 1 (CINC-1, CXCL1, GRO α, KC), interleukin 8 (IL-8,
CXCL8), and complement 5a. Here, they leave the circulation and infil-
trate the injured myocardium across the endothelium of post-capillary
2.1.7. Chemokines
venules in 3 sequential steps (a process termed extravasation):
The chemokines are a large family of chemoattractant cytokines
which are secreted in response to pro-inflammatory cytokines — these (1) The PMNs first adhere to and roll on endothelial cells by binding
play an important role in selectively recruiting monocytes, neutrophils, to P-selectin, E-selectin, intercellular adhesion molecules
and lymphocytes. The CC chemokine, monocyte chemoattractant (ICAMs), and vascular cell adhesion molecules expressed on
protein-1 (MCP-1)/chemokine ligand 2 (CCL2), is rapidly upregulated activated endothelial cells.
in the infarcted heart, and acts as a potent chemoattractant to mononu- (2) Firm adhesion then occurs by interaction of the integrins, αLβ2
clear cells (Kumar et al., 1997). Genetic ablation of MCP-1 or its and αMβ, present on PMNs with their ligands ICAM-1 and
receptor, chemokine receptor 2 (CCR2), reduced recruitment of pro- ICAM-2 on endothelial cells.
inflammatory monocytes, and decreased cytokine expression in the (3) Finally, trans-endothelial migration of the PMNs across the
infarct zone, and prevented adverse remodeling following AMI endothelial cells is also mediated by the integrins αLβ2 and
(Dewald et al., 2005). Injection of anti-MCP-1 into the skeletal muscle αMβ2, ICAM-1, and ICAM-2.
one month following a non-reperfused MI prevented adverse LV
Once in the injured myocardium, the PMNs play multiple roles including
remodeling and reduced mortality (Hayashidani et al., 2003). The CC
phagocytosis of cellular debris, degradation of extracellular matrix
chemokine, chemokine ligand 5 (CCL5), also plays a critical role as a
through the release of granules containing matrix metalloproteinases
chemoattractant for neutrophils and macrophages following AMI. It
(MMPs), generating ROS, and secreting factors that are chemotactic to
has been shown that treatment with an anti-mouse CCL5 monoclonal
monocytes. Excessive PMN infiltration and/or their delayed removal
Ab following a non-reperfused infarct reduced MI size, decreased circu-
may exacerbate myocardial injury by prolonging the pro-inflammatory
lating levels of chemokines, attenuated reduction of neutrophil and
response.
macrophage infiltration within the infarcted myocardium, prevented
Interestingly, Ma et al. (2016) have recently suggested that infiltrat-
adverse LV remodeling and reduced mortality (Montecucco et al.,
ing neutrophils may be polarized following AMI, opening up opportuni-
2012). However, it has also been shown that mice deficient in chemo-
ties for therapeutic modulation of neutrophil polarization as a strategy
kine receptor 5 (CCR5) (Dobaczewski, Xia, Bujak, Gonzalez-Quesada, &
for preventing inflammation and cardioprotection. They found that
Frangogiannis, 2010) exhibited enhanced myocardial inflammation,
neutrophils harvested from myocardium at day 1 following AMI had
enhanced matrix metalloproteinase expression, and worsened adverse
high expression of pro-inflammatory markers and were polarized by
LV remodeling following reperfused MI, findings which were associated
TLR4 activation (termed N1 neutrophils and induced by lipopolysaccha-
with impaired recruitment of anti-inflammatory CCR5+ foxp3+ regula-
ride and interferon-γ), whereas those collected from the heart at days
tory T cells (Tregs). More recently, the role of the CC chemokine recep-
5–7 post-MI, were anti-inflammatory (termed N2 neutrophils and
tor 9 (CCR9) (mainly expressed in lymphocytes, dendritic cells and
induced by interleukin-4).
monocytes/macrophages) has been investigated in the setting of AMI
Loss of the CD11/CD18-integrin receptor (which allows neutrophils
(Huang et al., 2016). Mice deficient in CCR9 had less mortality following
to bind to and traverse the endothelium) has been demonstrated to re-
AMI, smaller MI size and less adverse LV remodeling, effects which were
duce acute MI size in small and large animal AMI models (Arai et al.,
associated with attenuated inflammation and decreased inflammatory
1996; Aversano, Zhou, Nedelman, Nakada, & Weisman, 1995; Lefer
signaling through NF-κB and MAPK pathways (Huang et al., 2016).
et al., 1993; Tanaka et al., 1993), making it a therapeutic target, a strategy
Finally, pharmacological inhibition of the neutrophil attracting CXC
which has been tested in the clinical setting of AMI (see Section 3).
chemokine during myocardial ischemia reduced MI size by preventing
CXC chemokine-induced neutrophil recruitment and reactive oxygen
2.1.10. Monocytes and macrophages
species production following AMI (Montecucco et al., 2010).
Monocytes produced in the bone marrow and spleen enter the blood
after AMI and are recruited to the injured myocardium in 2 phases. The
2.1.8. Endothelial cells first phase is dominated by inflammatory Ly-6chigh monocytes (peaking
Following AMI, DAMPs released by dying cardiomyocytes induce day 3–4 post MI), and the second phase by anti-inflammatory Ly-6clow
activation of endothelial cells within the heart — this is characterized monocytes (peaking day about day 7 post-MI). The infiltrating mono-
by increased production of ROS and pro-inflammatory cytokines, and cytes then differentiate into M1 macrophages responsible for clearing
enhanced expression of adhesion molecules which mediate the binding cell debris from the MI zone. Subsequently, cytokines, chemokines and
of Leukocytes and platelets (Prabhu & Frangogiannis, 2016). Intercellu- growth factors secreted by M1 macrophages influence the reparative
lar tight junctions between endothelial cells become compromised fol- phase co-ordinated by M2 macrophages. However, the prolonged pres-
lowing acute myocardial IRI resulting in leaky coronary endothelium ence of M1 macrophages can extend the pro-inflammatory phase and
and increased coronary microvascular permeability dysfunction, key cause expansion of the infarcted area, thereby delaying the reparative
78 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87

phase and formation of scar tissue mediated by M2 macrophages and which in turn induces mobilization from the bone marrow of pro-
exacerbating adverse LV remodeling. As such, therapeutic modulation inflammatory Ly6Chi monocytes (Zouggari et al., 2013). Genetic
of macrophage polarization may provide a novel treatment strategy depletion of B lymphocytes using specific antibodies to CD20 or against
for reducing MI size and preventing adverse LV remodeling (reviewed B-cell activating factor (BAFF) (a B lymphocyte survival factor) was
in ter Horst et al., 2015). It has been shown in experimental studies shown to attenuate systemic inflammation (measured by systemic
that targeting pro-inflammatory monocytes or M1 to suppress the IL-1β, TNF and IL-18), reduce MI size and improve LV function by
pro-inflammatory phase post-MI, is cardioprotective (Courties et al., day 14 post-AMI (Zouggari et al., 2013), implicating B cells as a new
2014; Harel-Adar et al., 2011; Leuschner et al., 2011). Conversely, pro- therapeutic target for preventing inflammation post-AMI.
moting M2 macrophage polarization has been demonstrated to
facilitate the resolution of inflammation and prevent adverse LV 2.1.12. Cardiac fibroblasts
remodeling following AMI (Weirather et al., 2014; Zhou et al., 2015). In addition to their contribution to scar formation and matrix
remodeling, cardiac fibroblasts play an important role in the inflamma-
2.1.11. T and B lymphocytes tory response to AMI and subsequent LV remodeling (reviewed in
Experimental small and large animal models have demonstrated in- Shinde & Frangogiannis, 2014). They are known to be activated by
filtration of both T and B lymphocytes into the MI zone following AMI DAMPs released by damaged cardiomyocytes. In the first 24–72 h fol-
(Frangogiannis et al., 2000; Yan et al., 2013; Zouggari et al., 2013). It lowing AMI, the cardiac fibroblasts act as pro-inflammatory mediators,
has been shown that circulating cytotoxic T (CD8) lymphocytes increase activating the inflammasome and producing cytokines, chemokines
one week following AMI, and when harvested from rats subjected to and exhibiting matrix-degrading properties, thereby helping to clear
AMI, they were found to exert a cytotoxic effect on healthy neonatal the wound of dead cells and remove matrix debris — this process is fa-
cardiomyocytes (Varda-Bloom et al., 2000). Whether infiltrating T cilitated by specific pro-inflammatory cytokines (such as interleukin-1
cells are able to exacerbate acute ischemic injury in vivo is not clear. which inhibits α-SMA expression by fibroblasts), which delay the
The role of T cells in mediating acute myocardial IRI has recently been transformation of fibroblasts into myofibroblasts.
investigated in ST-segment elevation myocardial infarction (STEMI)
patients treated by PPCI. It was demonstrated that at 90 min after reper- 2.2. The anti-inflammatory reparative phase following AMI
fusion there was a reduction in circulating T cells (predominantly
CD8+), most pronounced in patients with microvascular obstruction The anti-inflammatory reparative phase (days 4–7) following AMI is
on cardiac MRI, an effect which coincided with a peak elevation in the orchestrated by suppression, resolution and containment of the initial
expression of the fractalkine receptor CX3CR1 (Boag et al., 2015). In pro-inflammatory response (Fig. 3). This is driven by the activation of
contrast, another study has shown that circulating levels of CD8 T cells specific endogenous inhibitory pathways that suppress inflammation,
in AMI patients were associated with short-term (6 months) cardiovas- and dynamic changes in the roles of infiltrating leukocytes within the
cular mortality, suggesting that they might contribute to acute coronary MI zone.
events via their pro-inflammatory cytotoxic effects.
It has been shown that following AMI, mature B lymphocytes 2.2.1. Neutrophils
infiltrate into the MI zone (peaking at day 5 post-AMI), and augment Apoptosis of neutrophils and their subsequent clearing from the MI
the pro-inflammatory response by secreting the chemokine CCL7, zone is a hallmark of inflammation resolution and the reparative

Fig. 3. The anti-inflammatory reparative phase following acute myocardial infarction. Following the pro-inflammatory response of AMI, the anti-inflammatory reparative phase allows the
resolution of inflammation. (1) Bone marrow and circulating monocytes are reported to differentiate into dendritic cells that prevent LV remodeling by the exosome activation of CD4+
leukocytes. (2) PS expression of apoptotic neutrophils induces M2 macrophage polarization and the secretion of anti-inflammatory and pro-fibrotic cytokines such as IL-10 and TGF-β that
suppress inflammation and promote tissue repair. (3) A switch from pro-inflammatory Ly6Chi monocytes and M1 macrophages localized at the MI zone in response to increased
myocardial CCL-2/MCP-1 expression during the initial pro-inflammatory phase to anti-inflammatory Ly6Clow monocytes and M2 macrophages, possibly mediated by Nr4a1 and in the
case of macrophages mediated by IRF5.
 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87 79

phase. It is an active process that requires the recruitment of a number (immunosuppressive) role in the setting of AMI through the secretion
of inhibitory pathway cascades (Mantovani, Cassatella, Costantini, & of anti-inflammatory cytokines (such as TGF-β and IL-10), and cell-
Jaillon, 2011; Serhan, Chiang, & Van Dyke, 2008). The expression of contact-dependent interaction with other cell types (reviewed in
phosphatidylserine (PS) facilitates the ingestion of apoptotic neutro- Meng et al., 2016; Wang et al., 2016). Tregs constitute a specific subset
phils by macrophages, resulting in M2 macrophage polarization and of T lymphocytes with immunosuppressive capacity, and make up
the secretion of anti-inflammatory and pro-fibrotic cytokines such as 5–10% of circulating CD4+ T lymphocytes under physiological condi-
IL-10 and TGF-β that suppress inflammation and promote tissue repair tions (Wang & Alexander, 2013).
(Ortega-Gomez, Perretti, & Soehnlein, 2013). Furthermore, the release Following AMI, the infiltration of Tregs into the MI zone has been
of anti-inflammatory mediators such as pro-resolving lipid mediators demonstrated to have several beneficial effects in terms of reducing
(e.g., lipoxins and resolvins), annexin A1, and lactoferrin, act to prevent MI size and preventing adverse LV remodeling. Experimental studies
neutrophil transmigration and entry, and promote neutrophil apoptosis have demonstrated that mice deficient in Tregs experience larger MI
and the phagocytic uptake of apoptotic neutrophils by macrophages size and more adverse LV remodeling (Hofmann et al., 2012) whereas
(Ortega-Gomez et al., 2013). the infusion of this cell population reduced MI size and preserved
myocardial function in murine AMI models (Matsumoto et al., 2011).
2.2.2. Monocytes and macrophages The protective effects of Tregs following AMI has been attributed to
It has been demonstrated that dynamic changes in the polarization of the following:
monocytes and macrophages from proteolytic and pro-inflammatory
1. Inhibiting the recruitment of inflammatory cells (neutrophils, mono-
Ly6Chi and M1 phenotypes (peak day 3–4 following AMI), respectively,
cytes, CD4+ T lymphocytes etc.) and suppress local expression of
to anti-inflammatory (e.g., IL-10, TGF-β, and vascular endothelial
pro-inflammatory cytokines such as TNF-α and IL-1β (Hofmann
growth factor) Ly6Clo and M2 phenotypes (peak day 7 following AMI)
et al., 2012; Tang et al., 2012);
are critical to the reparative phase following AMI (Nahrendorf et al.,
2. Promoting polarization of macrophages to an anti-inflammatory M2
2007; Nahrendorf, Pittet, & Swirski, 2010). The current paradigm sug-
phenotype, and inhibiting polarization to a pro-inflammatory M1
gests that pro-inflammatory Ly6Chi monocytes infiltrate the MI zone
phenotype (Weirather et al., 2014);
in response to increased myocardial CCL-2/MCP-1 expression during
3. Inhibiting the transdifferentiation of fibroblasts into myofibroblasts
the initial pro-inflammatory phase, and then the Ly6Chi monocytes
and downregulation of pro-fibrotic MMPs, thereby preventing
switch their phenotype to anti-inflammatory Ly6Clow monocytes, in
adverse LV remodeling (Saxena et al., 2014);
the reparative phase. The factors mediating this transition in monocyte
4. Preventing cardiomyocyte apoptosis (Tang et al., 2012);
phenotype remain unclear although Nr4a1 has been suggested to play a
role (Hanna et al., 2011; Hilgendorf et al., 2014). A number of factors Modulation of Tregs by pharmacological cardioprotective interventions
have been implicated as mediating the polarization changes from M1 such as rosuvastatin (Ke, Fang, Fan, Chen, & Chen, 2013) and FTY720 (an
to M2 macrophages following AMI, such as interferon regulatory factor analogue of sphingosine-1-phosphate) (Wang et al., 2014) has been
5 (IRF5) (Courties et al., 2014). Pharmacological modulation of reported in experimental AMI models. Whether Tregs mediate the
monocyte and macrophage polarization to their anti-inflammatory cardioprotection induced by endogenous strategies such as ischemic
phenotypes, may therefore provide a therapeutic strategy for augmenting preconditioning and postconditioning is not known and remains to be
the reparative phase following AMI. tested.
Interestingly, patients presenting with an AMI have been demon-
2.2.3. Dendritic cells strated to have decreased levels of circulating Tregs, compared with
Following tissue injury, bone marrow and splenic precursors and control patients (Mor, Luboshits, Planer, Keren, & George, 2006;
circulating monocytes are reported to differentiate into dendritic Sardella et al., 2007). Furthermore, it has been shown that low levels
cells — these exert various influences on the immune system at the in- of Tregs at baseline are associated with a higher risk for future AMI
flammatory site, such as priming of antigen-specific immune responses, (Wigren et al., 2012). The reduction in Tregs following AMI has been at-
induction of tolerance, and chronic inflammation. It has been shown tributed to a number of factors including: (1) accumulation of Tregs in
that following an AMI, dendritic cells migrate from the bone-marrow MI zone (Saxena et al., 2014); (2) attenuated production of Tregs by
into the ischemic zone accumulating in the MI and border zones thymus (Zhang et al., 2012); and (3) increased apoptosis of Tregs
(peaking at 7 days post-MI). Anzai et al. (2012) demonstrated that de- (Zhang et al., 2012). These clinical studies, suggest a protective role of
pletion of dendritic cells from the bone marrow in mice extended the Tregs in MI, and they may therefore, present an important therapeutic
pro-inflammatory phase following AMI to 7 days as evidenced by: target for reducing MI size and preventing adverse remodeling in AMI
(1) sustained elevation of pro-inflammatory cytokines such as IL-1β, patients. Preliminary clinical studies have reported ex vivo expanded
IL-18, TNF-α, and CCL2 and inhibition of anti-inflammatory cytokines human TREG cells were safe and effective in the prevention and
such as IL-10 and CX3CL1; (2) increased pro-inflammatory Ly6Chigh treatment of graft-versus-host disease or type 1 diabetes mellitus
monocyte but decreased anti-inflammatory Ly6Clow monocyte infiltra- (Brunstein et al., 2011; Marek-Trzonkowska et al., 2014; Trzonkowski
tion into the MI zone; (3) enhanced infiltration of pro-inflammatory et al., 2009). Whether this therapeutic approach can be applied to
M1 macrophages and attenuated recruitment of anti-inflammatory prevent adverse LV remodeling following AMI remains to be tested.
M2 macrophages into the MI zone. These pro-inflammatory effects
were associated with worse adverse LV remodeling following AMI as 2.2.5. Other T lymphocyte subpopulations
evidenced by accelerated cardiac dilatation and deterioration of LV Other subpopulations of T lymphocytes may also contribute to the
function. These results suggested a protective role of dendritic cells in reparative phase following AMI. CD4+ helper T cells have been shown
the inflammatory response to AMI and subsequent LV remodeling. to be activated following AMI (probably in response to released cardiac
Interestingly, a recently published study has found that exosomes autoantigens). They have been demonstrated to contribute to resolution
produced by dendritic cells may recruit CD4+ helper T cells into the of inflammation and wound healing with collagen matrix formation and
MI zone and help prevent adverse LV remodeling post-AMI (Liu et al., scar formation to prevent adverse LV remodeling (Hofmann et al.,
2016). 2012). Invariant natural killer lymphocytes have been reported to be
activated following AMI, and reduce leukocyte infiltration, lessen myo-
2.2.4. Regulatory T lymphocytes (Tregs) cardial injury, and prevent adverse LV remodeling, beneficial effects
Emerging evidence suggests that CD4+CD25+FOXP3+ regulatory which were shown in part, to be due expression of anti-inflammatory
T lymphocytes (Tregs) may play an anti-inflammatory cytokines such as IL-10 (Homma et al., 2013; Sobirin et al., 2012).
80 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87

2.2.6. Cardiac fibroblasts the anti-inflammatory reparative phase which follows, a potential
During the proliferative phase of MI healing, the loss of pro- therapeutic strategy for limiting MI size and preventing adverse LV
inflammatory signals such as IL-1β and Interferon-γ-inducible Protein remodeling, is to attenuate the initial pro-inflammatory response, and
(IP)-10, allows cardiac fibroblasts within the MI border zone to upregulate the subsequent anti-inflammatory reparative response.
transdifferentiate into myofibroblasts (reviewed in Shinde & Separate to the role of inflammation in AMI, it has been postulated
Frangogiannis, 2014). These express the contractile protein, α-smooth that persistent or chronic inflammation following an AMI, may result
muscle actin (α-SMA), and exhibit an extensive endoplasmic reticulum, in adverse LV remodeling, providing an additional therapeutic target
and are capable of secreting large amounts of matrix proteins. Factors for preventing post-MI heart failure (Westman et al., 2016). A number
contributing to the trans-differentiation of cardiac fibroblasts into of therapeutic approaches aimed at targeting the pro-inflammatory
α-SMA myelofibroblasts include TGF-β signaling, modulation of the response following AMI have been investigated, many of which have,
matrix environment (expression of ED-A fibronectin) and deposition unfortunately, failed to demonstrate any benefit on reducing MI size
of non-fibrillar collagens (such as collagen VI), expression of proteo- or improving clinical outcomes (see Table 1 for a summary of the
glycans by cardiac fibroblasts, and mechano-sensitive signaling. major clinical studies), further details of which will be discussed in
The formation of a mature scar signifies the end of the proliferative more detail in Section 3.6. Therefore, novel therapeutic strategies are re-
phase and is associated with matrix cross-linking and the progressive quired to target the inflammatory response to AMI, and some of these
clearance of myelofibroblasts by apoptosis. The mechanisms underlying are highlighted in this section.
this process are unclear but may involve the withdrawal of growth
factors, specific inhibition of the TGF-β-driven fibrotic response, and 3.1. Corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDS)
alterations in the composition of the matrix such as the small leucine-
rich proteoglycan biglycan. Early experimental studies using non-specific anti-inflammatory
In the non-infarcted remote myocardium, cardiac fibroblasts may agents such as corticosteroids and NSAIDs showed a reduction in MI
remain chronically activated in response to pressure overload inducing size (Kirlin et al., 1982; Libby, Maroko, Bloor, Sobel, & Braunwald,
early activation of matrix synthetic pathways, associated with cardiac fi- 1973). Although this therapeutic strategy was initially associated with
brosis and diastolic dysfunction, a process which is followed by activa- adverse effects such as impaired myocardial repair, myocardial thinning
tion of matrix-degrading signals, chamber dilatation and systolic and cardiac rupture in the clinical setting (Hammerman, Kloner, Hale,
dysfunction. In contrast, volume overload is primarily associated with Schoen, & Braunwald, 1983), later studies showed that prolonged
matrix loss and cardiac dilation. The mechanisms which regulate these corticosteroid therapy-induced aneurysm formation was related to
differential matrix response to volume and pressure overload remain inhibition of the reparative phase, as well as the intended acute pro-
unclear, although stretch and humoral triggers of remodeling concur- inflammatory phase (Garcia, Go, & Villarreal, 2007; Jugdutt, Khan,
rent with loss of contractile tissue and heart failure may be involved Jugdutt, & Blinston, 1995).
(Burchfield, Xie, & Hill, 2013; Dorn, 2009). Elucidation of these regulato-
ry pathways may identify novel therapeutic targets for modulating the 3.2. Complement cascade inhibition
fibrotic response to AMI and help to prevent post-MI adverse
remodeling. A number of experimental studies have reported that C1 and C5/C5a
inhibition of the complement cascade following AMI can reduce MI size.
2.3. The contribution of persistent or chronic inflammation to post-MI However, several small and large clinical trials have investigated the ef-
adverse LV remodeling fect of Pexelizumab, an Anti-C5 Complement Antibody, but have failed
to find any beneficial effects on clinical outcomes in either reperfused
Following AMI, the LV undergoes geometric and functional changes, STEMI (Mahaffey et al., 2003) or CABG patients (see Table 1 for details).
with hypertrophy of the non-infarcted segments and dilatation/ Preliminary experience with intravenous C1 inhibition infusion therapy
thinning of the infarcted segments resulting in reduced LV ejection has reported reduced myocardial injury in STEMI patients treated by
fraction — a process termed adverse LV remodeling, and the occurrence thrombolysis or emergency CABG surgery (see Table 1 for details).
of which is associated with worse clinical outcomes. There is preliminary Whether C1 inhibition therapy would be effective in STEMI patients
evidence that an excessive, persistent and expanded pro-inflammatory treated by PPCI is not known.
response following AMI may worsen post-MI adverse LV remodeling
through the following processes: activating proteases (Dobaczewski 3.3. Neutrophils
et al., 2010); increasing cytokine expression which may induce
cardiomyocyte apoptosis and suppress contractility; increasing matrix Loss of the CD11/CD18-integrin receptor (which allows neutrophils
deposition which may result in a stiffer ventricle and causes diastolic to bind to and traverse the endothelium) has been demonstrated to re-
dysfunction (Bujak et al., 2008); and the activation of cardiac fibroblasts duce acute MI size in small and large animal AMI models (Arai et al.,
in the infarct border zone which may expand fibrosis into viable tissue. 1996; Aversano et al., 1995; Lefer et al., 1993; Tanaka et al., 1993). How-
Experimental studies have demonstrated that myocardial expression ever, several clinical trials have demonstrated that the administration of
levels of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) at 8 and monoclonal antibodies directed to the CD11/CD18-integrin receptor
20 weeks after AMI in a rat model, were significantly associated with failed to reduce acute MI size and improve short-term clinical outcomes
left ventricular end-diastolic volume (LVEDV) (Ono, Matsumori, Shioi, at 30 days in STEMI patients treated by reperfusion therapy (see
Furukawa, & Sasayama, 1998). Similarly in STEMI patients, IL-1β levels Table 1) (Baran et al., 2001; Faxon et al., 2002).
measured pre-, 2, and 7 days post-PPCI were able to predict indexed
left ventricular end-systolic volume (LVESV) and LVEDV values 3.4. P-selectin Ab
measured by CMR at 1 year (Orn et al., 2012). Therefore, modulating
the persistent or chronic inflammatory response to AMI, may provide In the Select-ACS trial, Inclacumab, a recombinant monoclonal anti-
therapeutic targets for preventing adverse LV remodeling following AMI. body against P-selectin, administered prior to PCI in NSTEMI patients,
has been found to reduce peri-procedural myocardial injury, as evi-
3. Therapeutic targeting of inflammation following MI denced by less Troponin I and CK-MB (Tardif et al., 2013). Whether
this therapeutic approach would be effect in reducing MI size and pre-
Given the detrimental effects of an excessive and persistent pro- vent adverse LV remodeling in STEMI patients undergoing PPCI is not
inflammatory response to AMI, and the beneficial healing effects of known and remains to be tested.
 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87 81

Table 1
Major clinical studies investigating an anti-inflammatory therapeutic strategy to protect the myocardium against acute ischemia/reperfusion injury.

Target Clinical study Patient Treatment Outcome Mechanism

population

Neutrophils Baran et al. 394 STEMI IV rhuMAb CD18 0.5 mg/kg No effects on coronary blood flow, rhuMAb CD18 is a monoclonal antibody to
(2001) LIMIT thrombolysed or 2.0 mg/kg prior to MI size (SPECT), or ST-segment the CD18 subunit of the β2 integrin adhesion
AMI b12 h thrombolysis resolution receptors to prevent neutrophil adhesion
Neutrophils Faxon, Gibbons, 420 STEMI PPCI IV Hu23F2G 0.3 mg/kg or No effects on MI size (SPECT), Hu23F2G (LeukArrest), a humanized MAb to
Chronos, Gurbel, b6 h Per-PPCI 1.0 mg/kg prior to PPCI corrected TIMI frame count or the neutrophils integrin receptor CD11/CD18
and Sheehan TIMI ≤1 clinical events at 30 days
(2002) HALT MI
Complement de Zwaan et al. 22 STEMI IV C1-inhibitor 48 h Reduction in MI size (CK-MB or Cetor is a monoclonal antibody to human
cascade C1 (2002) thrombolysed infusion initiated 6 h after Trop T) but small study C1-inhibitor which inhibits activation of the
b12 h reperfusion complement cascade.
Complement Thielmann et al. 57 STEMI IV C1-inhibitor 6 h infusion Reduction in peri-operative Berinert is a monoclonal antibody to human
cascade C1 (2006) emergency initiated 10 min prior to myocardial injury size (Trop I) C1-inhibitor which inhibits activation of the
CAG b12 h reperfusion (unclamping of complement cascade.
aorta)
Complement Fattouch et al. 80 STEMI IV C1-inhibitor 3 h infusion Reduction in peri-operative A monoclonal antibody to human C1-inhibitor
cascade C1 (2007) emergency initiated 10 min prior to myocardial injury size (Trop I) which inhibits activation of the complement
CAG b12 h reperfusion (unclamping of cascade.
aorta)
Complement Mahaffey et al. 943 STEMI IV Pexelizumab 2.0-mg/kg No effects on MI size (CK-MB or Pexelizumab, is an Anti-C5 Complement
cascade C5 (2003) COMPLY thrombolysed bolus, or 2.0-mg/kg bolus Trop I) or clinical events Antibody which inhibits activation of the
b6 h plus 0.05 mg/kg/h for 20 h complement cascade.
prior to or soon after start
of thrombolysis
Complement Granger et al. 960 STEMI IV Pexelizumab 2.0-mg/kg No effects on MI size (CK-MB). Pexelizumab, is an Anti-C5 Complement
cascade C5 (2003) COMMA PPCI b6 h bolus plus 0.05 mg/kg/h for However, reduction in mortality Antibody which inhibits activation of the
20 h prior to PPCI at 90 days. complement cascade.
Complement Verrier et al. 3099 IV Pexelizumab 2.0-mg/kg No effects on peri-operative MI Pexelizumab, is an Anti-C5 Complement
cascade C5 (2004) CABG ±valve bolus plus 0.05 mg/kg/h for size (CK-MB or Trop I) or clinical Antibody which inhibits activation of the
PRIMO-CABG 20 h prior to or soon after events complement cascade.
start of thrombolysis
Complement Armstrong et al. 5745 STEMI IV Pexelizumab 2.0-mg/kg No effects on mortality at 30 days. Pexelizumab, is an Anti-C5 Complement
cascade C5 (2007) APEX MI PPCI Ant/Inferolat bolus plus 0.05 mg/kg/h for Antibody which inhibits activation of the
b6 h 24 h prior to PPCI complement cascade.
Fibrin Atar et al. 234 STEMI IV FX-06400 mg in 2 49% reduction in 7 day AUC hsCRP FX06 is a naturally occurring peptide
(2009) FIRE PPCI divided doses at time of non-significant 21% (P = .21) fragment of fibrin which prevents binding
PPCI reduction in MI size (LGE MRI on to an endothelial specific molecule,
day 5). No difference in 48 h VE-cadherin, thereby reducing plasma
troponin. leakage into tissues and acting as an
anti-inflammatory agent.
IL-1 Abbate et al. 10 STEMI PPCI Subcutaneous IL-1receptor Smaller increase in index LVESV at Anakinra (Kineret™ from Amgen) is a
(2010) VCU-ART antagonist (IL-1ra, 100 mg) 10–14 weeks assessed by MRI. humanized anti-IL-1R antibody.
or placebo daily for
14 days.
IL-1 Abbate et al. 25 STEMI PPCI Subcutaneous IL-1receptor Failed to show a statistically Anakinra (Kineret™ from Amgen) is a
(2013) pooled analysis antagonist (IL-1ra) or significant effect on indexed humanized anti-IL-1R antibody.
VCU-ART2 placebo for 14 days. LVESV, LVEDV or LVEF.
IL-1 Morton et al. 182 NSTEMI Subcutaneous IL-1receptor 49% reduction in 7 day AUC Anakinra (Kineret™ from Amgen) is a
(2015) MRC-ILA undergoing PCI antagonist (IL-1ra) or hsCRP. However, there was an humanized anti-IL-1R antibody.
Heart Study placebo for 14 days. increase in MACE (death, stroke,
and new MI).
P-selectin Tardif et al. 322 NSTEMI IV Inclacumab (20 mg/kg) 24% and 34% reduction in peak Inclacumab is a humanized antibody that
(2013) undergoing PCI initiated prior to Trop I at 16 and 24 h post-PCI inhibits P-selectin, an adhesion molecule
SELECT-ACS angiography for 1 h (borderline significant) involved in interactions between endothelial
cells, platelets, and leukocytes.
IL-6 Kleveland et al. 117 NSTEMI IV Tocilizumab (20 mg/ml) 52% reduction in median AUC Tocilizumab is a humanized anti-IL-6R antibody
(2016) undergoing PCI initiated prior to hsCRP and 22% reduction in that binds to both membrane-bound and
angiography for 1 h median AUC hsTropT soluble (s) IL-6R

3.5. Therapeutic targeting of inflammatory cytokines Table 1) (Abbate et al., 2010, 2013). The ongoing VCU-ART3 study is cur-
rently investigating the effect of Anakinra on serum CRP levels and post-
Inflammatory cytokines constitute intriguing therapeutic targets be- MI LV remodeling in a larger population of 99 STEMI patients
cause of their pleiotropic effects on immune responses. Despite the (ClinicalTrials.gov Identifier NCT01950299). In NSTEMI patients undergo-
promising results shown in animal models, however, clinical studies ing PCI, serum hs-CRP levels were again suppressed, although there was
of therapies targeting inflammatory cytokines and chemokines have no effect on PCI-related myocardial injury. There was however, a signifi-
produced mixed results. cant increase in death, stroke and non-fatal MI at one year — 18.9% with
IL-1 inhibition versus 5.4% in control (Morton et al., 2015), questioning
3.5.1. IL-1β this therapeutic approach following AMI.
Two small clinical trials (VCU-ART 1 and 2) have shown that 2 weeks Interestingly, it has been recently shown in a large phase III
daily subcutaneous administration of the IL-1 inhibitor, Anakinra, was Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS)
safe and suppressed serum CRP levels in the post-MI period, although clinical trial (NCT01327846), that quarterly subcutaneous injections of
the effects on preventing adverse LV remodeling were mixed (see ACZ885 (also known as canakinumab, a monoclonal antibody against
82 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87

IL-1β) in combination with standard of care prevented recurrent studies, the therapeutic agent was given at a different time-
cardiovascular events (CV death, non-fatal MI, and non-fatal stroke) point compared to the experimental studies (in relation to the
over a median follow-up of 3.8 year among 10,061 people with prior myo- phases of acute ischemia and reperfusion). The timing of the
cardial infarction and with a high-sensitivity C-reactive protein (hsCRP) anti-inflammatory agent is crucially important, depending on
level of ≥ 2 mg/L. Whether the administration of canakinumab to AMI the intended target (i.e. neutrophils or cytokines and so on),
patients to target IL-1β to dampen the pro-inflammatory response to and in this regard, further work is required to determine the op-
AMI can reduce MI size and prevent adverse LV remodeling is not known. timum timing of the therapeutic agent. Another issue is the het-
erogeneity of patients in clinical trials, which of course is not
3.5.2. IL-6 reproduced in the experimental animal studies, and this may im-
A recently published clinical study has demonstrated that an intra- pact on sample size for showing an effect with a therapeutic
venous infusion of Tocilizumab (humanized anti-IL-6R antibody that agent in the clinical setting. Finally, patient selection is critical
binds to both membrane-bound and soluble IL-6R), reduced peri- with patients presenting with shorter ischemic times (b3–4 h)
procedural myocardial injury in NSTEMI patients undergoing PCI in most likely to benefit from a cardioprotective therapy adminis-
terms of less serum high-sensitive Troponin-T and CRP (Kleveland tered at the time of reperfusion (Hausenloy, Botker, et al., 2017;
et al., 2016) (Table 1). The ongoing ASSAIL-MI clinical study is cur- Kloner et al., 2006).
rently investigating the effects of this therapeutic approach on MI
size in STEMI patients treated by PPCI (ClinicalTrials.gov Identifier: As such, in order to improve the translation of future therapeutic strat-
NCT03004703). egies for preventing inflammation in AMI into the clinical setting more
clinically relevant animal AMI models should be used and one should
3.6. Why have so many anti-inflammatory therapies failed to protect in ensure that the therapy can adequately suppress the immune response
AMI? following AMI. In addition, a multicenter blinded randomized placebo-
controlled approach should be adopted in the pre-clinical assessment
The reason why so many anti-inflammatory therapeutic strategies of potential cardioprotective strategies, in order to improve the transla-
have failed to reduce MI size or improve clinical outcomes in AMI pa- tion of cardioprotective for patient benefit (Hausenloy, Garcia-Dorado,
tients, despite showing beneficial effects in small and large animal et al., 2017). One such initiative, the Consortium for preclinicAl assESs-
models of AMI, is not clear. A number of potential factors have been ment of cARdioprotective therapies (CAESAR) was established to im-
suggested: prove the pre-clinical evaluation of cardioprotective therapies using a
multi-center (3 sites with small and large animal models) blinded ran-
(1) Animal models: The animal AMI models used to test potential domized controlled studies with standardized acute IRI protocols and
cardioprotective therapies in the experimental setting are far re- centralized analysis of data (Jones et al., 2015; Lefer & Bolli, 2011). How-
moved from the typical AMI patient (reviewed in Ferdinandy, ever, with the exception of ischemic preconditioning, the CAESAR net-
Hausenloy, Heusch, Baxter, & Schulz, 2014; Lecour et al., 2014). work failed to demonstrate multi-center cardioprotection with either
For example, the use of a rodent model in the study of inflamma- sodium nitrite (Lefer et al., 2014) or sildenafil citrate (Kukreja et al.,
tion is cautioned as the rodent has a much greater and more re- 2014), despite these agents showing cardioprotection in single-center
silient inflammatory response than other species (Seok et al., studies. The reasons for the discordancy between the single and multi-
2013; Warren et al., 2010). Furthermore, the experimental center studies, with respect to these 2 interventions, are not clear, but
models tend to use young healthy animals of same gender and may either be due to a true lack of cardioprotection in the multi-
similar genetic background, and induce acute coronary artery oc- center setting with these 2 agents, or it may reflect the difficulties and
clusion in a health coronary artery. In contrast, the typical AMI challenges of standardizing animal husbandry and acute IRI protocols
patient is middle aged, has one or more co-morbidities (such as across the 3 different sites. As such, for any such multi-center
diabetes, hypertension, and obesity which may interfere with cardioprotection research network to work in the future, will require
cardioprotective therapies), is on co-medications (such as careful co-ordination, rigorous selection of the cardioprotective agent
statins, beta-blockers, anti-platelet P2Y12 inhibitors which may to be tested, meticulous standardization of animal selection and care,
interfere with cardioprotective therapies), and acute coronary and the acute IRI protocols.
artery occlusion occurs due to plaque rupture in an inflamed
and diseased coronary artery; 3.7. Future therapeutic approaches to target inflammation
(2) Anti-inflammatory efficacy: The therapy may have failed to ade-
quately suppress the pro-inflammatory response in AMI patients, 3.7.1. Multi-targeted therapies
despite showing beneficial effects in animal AMI models. For ex- The reasons for the many failures to target inflammation as
ample, the C5B inhibitor, Pexelizumab, failed to inhibit assembly a strategy for cardioprotection are unclear but may relate to the
of the terminal complement complex in STEMI patients undergo- limitations of using a single-targeted approach directed to only the
ing PPCI, which may in part explain the lack of effect on clinical pro-inflammatory proponent of acute myocardial IRI. This approach
outcomes in the APEX-AMI trial (Martel et al., 2012). does not address the other components of acute myocardial IRI (for
(3) Inflammation as a therapeutic target: It has been suggested that example mitochondrial dysfunction, calcium overload, oxidative stress,
the pro-inflammatory response to AMI may not contribute to is- microvascular obstruction, other components of the inflammatory
chemic cardiomyocyte death. Experimental studies in mice defi- response and so on). As such, a multi-targeted approach and combining
cient in P-selectin and intercellular adhesion molecule-1 (Briaud anti-inflammatory agents with mitochondria and endothelial protective
et al., 2001), MCP-1 (Dewald et al., 2005), and animals with de- therapies may be a better approach to reducing MI size in reperfused
fective IL-1 signaling (Bujak et al., 2008), did not sustain a reduc- STEMI patients (Hausenloy, Garcia-Dorado, et al., 2017).
tion in MI size, when compared to wild-type, despite having a
suppressed inflammatory response following AMI. 3.7.2. TLR2
(4) Study design and protocol: Selecting the right dose for testing in Experimental studies have demonstrated that administration of
clinical studies is challenging, especially, when in many cases, anti-TLR2 antibodies (OPN-301 or OPN-305) reduced MI size when ad-
phase 2 clinical studies were not undertaken. It is difficult to ex- ministered at the time of reperfusion, in both small and large animal
trapolate doses shown to be effective in small and large animal AMI models (Arslan et al., 2010, 2012). A recent phase I study, has
studies, into clinical studies. Furthermore, in some clinical demonstrated safety with OPN-305 administered to healthy volunteers
 S.-B. Ong et al. / Pharmacology & Therapeutics 186 (2018) 73–87 83

(Reilly et al., 2013), and phase II trials are currently underway to test the Acknowledgments
efficacy of OPN-305 in patients with myelodysplastic syndrome
(ClinicalTrials.gov Identifier: NCT02363491) and patients undergoing Sang-Bing Ong is supported by the Ministry of Health — Singapore
renal transplantation (ClinicalTrials.gov Identifier:NCT01794663). National Medical Research Council under its Open Fund —Young
It would be interesting to investigate whether targeting TLR2 with Individual Research Grant (OF-YIRG) — NMRC/OFYIRG/0021/2016
OPN-305, administered at the time of reperfusion can reduce myocardi- as well as a Khoo Postdoctoral Fellowship Award (KPFA) — Duke-
al inflammation and limit MI size in patients presenting with AMI. NUS-KPFA/2016/0010 from the Estate of Tan Sri Khoo Teck Puat,
Singapore. Derek Hausenloy is supported by the Ministry of Health —
Singapore National Medical Research Council (NMRC CSASI16may002,
3.7.3. NLRP3 inflammasomes CGAug16C006), Ministry of Education — Singapore Tier 2 grant
Experimental rodent studies have shown that genetic and pharma- (MOE2016-T2-2-021), British Heart Foundation (FS/10/039/28270),
cological inhibition of the NLRP3 inflammasome may reduce MI size Duke-NUS Medical School, and the National Institute for Health Re-
and prevent adverse LV remodeling (Coll et al., 2015; Kawaguchi et al., search University College London Hospitals Biomedical Research Centre.
2011; Marchetti et al., 2014; Mezzaroma et al., 2011; Pomerantz et al., Part of the work presented by Hector Cabrera-Fuentes was supported by
2001; Sandanger et al., 2013). Crucially, pharmacological inhibition of the Russian Government Program for competitive growth of Kazan
the NLRP3 inflammasome (using 16673-34-0) has been shown to re- Federal University, Kazan (Russian Federation) and by the 2016 ISHR-
duce MI size in the murine heart, when administered at time of reperfu- ES/Servier Research Fellowship. This work was supported by the
sion, and even up to 60 min after the onset of reperfusion (Toldo et al., European Cooperation in Science and Technology (COST Action
2016), making it an attractive therapeutic target in STEMI patients treat- CA16225/EU-CARDIOPROTECTION).
ed by PPCI. A more specific small molecule inhibitor of the NLRP3
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Conflict of interest
recombinant tissue plasminogen activator for acute myocardial infarction: Limitation
of myocardial infarction following thrombolysis in acute myocardial infarction (LIMIT
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