Cardioprotective potential of annexin-A1 mimetics in myocardial infarction.

JPT-06737; No of Pages 19 Pharmacology & Therapeutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

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Chengxue Qin a,d, Yuan H. Yang b,1, Lauren May c,1, Xiaoming Gao a, Alastair Stewart d, Yan Tu d, Owen L. Woodman e, Rebecca H. Ritchie a,d,f,⁎

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Keywords: Ac2-26 Cardioprotection Cardiomyopathy Formyl peptide receptors Inflammation Ischemia–reperfusion

Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia Centre for Inflammatory Diseases Monash University and Monash Medical Centre, Clayton, Victoria, Australia Department of Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, and Department of Pharmacology, Monash University, Parkville, Victoria, Australia d Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, Victoria, Australia e School of Medical Sciences, RMIT University, Bundoora 3083, Victoria, Australia f Department of Medicine, Monash University, Clayton, Victoria, Australia

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Myocardial infarction (MI) and its resultant heart failure remains a major cause of death in the world. The current treatments for patients with MI are revascularization with thrombolytic agents or interventional procedures. These treatments have focused on restoring blood flow to the ischemic tissue to prevent tissue necrosis and preserve organ function. The restoration of blood flow after a period of ischemia, however, may elicit further myocardial damage, called reperfusion injury. Pharmacological interventions, such as antioxidant and Ca2+ channel blockers, have shown premises in experimental settings; however, clinical studies have shown limited success. Thus, there is a need for the development of novel therapies to treat reperfusion injury. The therapeutic potential of glucocorticoid-regulated anti-inflammatory mediator annexin-A1 (ANX-A1) has recently been recognized in a range of systemic inflammatory disorders. ANX-A1 binds to and activates the family of formyl peptide receptors (G protein-coupled receptor family) to inhibit neutrophil activation, migration and infiltration. Until recently, studies on the cardioprotective actions of ANX-A1 and its peptide mimetics (Ac2-26, CGEN-855A) have largely focused on its anti-inflammatory effects as a mechanism of preserving myocardial viability following I–R injury. Our laboratory provided the first evidence of the direct protective action of ANX-A1 on myocardium, independent of inflammatory cells in vitro. We now review the potential for ANX-A1 based therapeutics to be seen as a “triple shield” therapy against myocardial I–R injury, limiting neutrophil infiltration and preserving both cardiomyocyte viability and contractile function. This novel therapy may thus represent a valuable clinical approach to improve outcome after MI. © 2014 Published by Elsevier Inc.

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Cardioprotective potential of annexin-A1 mimetics in myocardial infarction

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Associate editor: C.G. Sobey

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Abbreviations:Aβ42, 42-amino acid beta-amyloidpeptide;ABC,ATP-bindingcassette;Ac2-26,N-terminal-derived annexin-A1peptide (acetyl-AMVSEFLKQAWIENEEQEYVVQTVK);Ac212, N-terminal-derived annexin-A1 peptide (acetyl-AMVSEFLKQAW); Ac2-6, N-terminal-derived annexin-A1 peptide (acetyl-AMVSE); ACE, angiotensin converting-enzyme; ADP, adenosine diphosphate; ALXR, aspirin-triggered lipoxin receptor; ANX-A1, annexin-A1; ARBs, angiotensin receptor blockers; Boc2, non-selective FPR antagonist; CABG, coronary artery bypass graft; cAMP, cyclic adenosine monophosphate; CD, cluster of differentiation; ChIPS, chemotaxis inhibitory protein of S. aureus; CsH, cyclosporin H; CVD, cardiovascular disease; DAMPs, Danger-Associated Molecular Patterns; ECM, extracellular matrix; ERK, extracellular signal-regulated kinases; fMLF, N-formyl-Met-Leu-Phe; FPR, formyl peptide receptor; FPRL-1, formyl peptide receptor-like receptor 1; GC, glucocorticoid; GILZ, GC-induced leucine zipper; GPCR, G-protein-coupled receptor; GRE, GC-response element; GRK, GPCR Kinase; GTPγ, guanosine triphosphate-gamma; HDAC, histone deacetylase; HMGB1, high mobility group B protein 1; ICAM-1, intercellular adhesion molecule-1; IFNγ, interferon-γ; IL, interleukin; IPC, ischemic preconditioning; I–R,ischemia–reperfusion; JAK, Janus kinase; KATP, ATP-dependent potassium channels; LAD, left anterior descending; LV, left ventricular;LXA4, lipoxin A4; Mac-1, macrophage1 antigen;MAPK, mitogen-activatedprotein kinase; MCP-1,monocytechemoattractant protein-1;MKP1,MAPKphosphatase-1; MI,myocardial infarction;MMPs,matrixmetalloproteinases; NSAIDs, nonsteroidal anti-inflammatory drugs; PCI, percutaneous coronary intervention; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PLA2, phospholipase A2; PostCon, ischemic postconditioning; PS, phosphatidylserine; PSGL-1, P-selectin glycoprotein ligand-1; ROS, reactive oxygen species; SAA, serum amyloid A; SAFE, survival activating factor enhancement; SOCS3, suppressor of cytokine signaling-3; SR, sarcoplasmic reticulum; STAT, signal transducers and activators of transcription; TLR, toll-like receptor; TNFα, tumor necrosis factor-α; uPA, urokinase plasminogen activator; VCAM-1, vascular cell adhesion molecule-1; WHO, World Health Organization; WRW4, Typ-Arg-Trp-Trp-Trp-Trp-CONH2. ⁎ Corresponding author at: Head, Heart Failure Pharmacology, Baker IDI Heart & Diabetes Institute, P.O. Box 6492, Melbourne, VIC 3004, Australia. Tel.: +61 3 8532 1392; fax: +61 3 8532 1100. E-mail address: [email protected] (R.H. Ritchie). 1 Both authors contributed equally to this work.

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http://dx.doi.org/10.1016/j.pharmthera.2014.11.012 0163-7258/© 2014 Published by Elsevier Inc.

Please cite this article as: Qin, C., et al., Cardioprotective potential of annexin-A1 mimetics in myocardial infarction, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.11.012

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Contents 1. Overview of myocardial ischemia–reperfusion injury . 2. Annexin-A1 . . . . . . . . . . . . . . . . . . . . 3. Formyl peptide receptors (FPR): the Anx-A1 receptors 4. Role of Anx-A1 and FPRs in myocardial I–R injury . . . 5. Concluding remarks . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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1.1. Contribution of inflammation to myocardial ischemia–reperfusion injury

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In the 21st century, we are fortunate to now understand a great deal about the pathophysiological mechanisms of myocardial ischemia reperfusion (I–R) injury. In particular, overproduction of reactive oxygen species (ROS), cardiomyocyte redistribution or overload of Ca2+ and infiltration of inflammatory cells into the site of injury have received considerable attention in their role as important mediators of the direct myocardial I–R injury. The contribution of ROS, and overload of Ca2+ have been extensively reviewed (Qin et al., 2009; Ibanez et al., 2011; Ertracht et al., 2014). Thus, we will focus this review on the contribution of inflammation to I–R injury, with an emphasis on the potential modulatory actions of Annexin-A1 (ANX-A1) and related formyl peptide receptor (FPR) ligands (Fig. 1).

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Cardiovascular disease (CVD), particularly myocardial infarction (MI) and stroke, is the leading cause of death and disability worldwide. The World Health Organization (WHO) has estimated that 17.3 million people died from CVD in 2008, which represented 30% of all global deaths (World Health Organization; Le Good et al., 1998). The major underlying cause of CVD is atherosclerosis, which together with thromboembolism, can result in the blockage of blood vessels, leading to ischemia. MI can be triggered by permanent or temporary blockages of the coronary blood vessels. Clinical diagnosis of MI has now been universally defined, on the basis of clinical symptoms at presentation, changes in cardiac biomarkers such as circulating troponin levels, ECG changes, regional wall motion abnormalities, and/or intracoronary thrombus detection. This has been comprehensively reviewed elsewhere (see Thygesen et al., 2012). The current treatments for patients with acute MI are revascularization with clot-busting (thrombolytic) drugs or interventional procedures (i.e. balloon angioplasty or coronary artery bypass grafting, CABG). In order to prevent tissue necrosis and retain organ function, these treatments have focused on restoring blood flow to the ischemic tissue. Restoration of myocardial reperfusion is a critical objective, as prognosis over both the immediate and longerterm is significantly worse in patients who do not receive any attempts at revascularization (as reviewed by Faxon (2007)). The re-introduction of O2 and other nutrients into the previously ischemic area on reperfusion however, results in a unique form of myocardial damage, called reperfusion injury. The manifestations of reperfusion injury include reperfusion arrhythmias, myocardial stunning, myocardial death, contractile, endothelial and microvascular dysfunction (including the no-reflow phenomenon), as well as cardiac remodeling over the longterm, as discussed previously (Jennings et al., 1995; Maxwell & Lip, 1997; Park & Lucchesi, 1999; Carden & Granger, 2000; Verma et al., 2002; Moens et al., 2005).

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(Moens et al., 2005), highlighting that myocardial I–R injury is considered to be an inflammatory condition characterized by upregulation of innate immune responses. While reperfusion is a clear prerequisite for myocardial salvage, reperfusion triggers an intense inflammatory reaction which itself promotes cardiomyocyte death, leading to left ventricular (LV) dysfunction and adverse LV remodeling (Frangogiannis et al., 1998). Following myocardial ischemia, release of various proinflammatory factors such as complement fragments, ROS and cytokines from cardiomyocytes, the endothelium and circulating mast cells activate circulating leukocytes such as neutrophils and monocytes. Clinical studies have demonstrated that higher numbers of white blood cells, and specifically of monocytes, at admission are associated with high mortality in patients with acute MI (Maekawa et al., 2002; Grzybowski et al., 2004), indicating a close association between systemic inflammation and poor prognosis post MI. We discuss below the initiation of the inflammatory response, the activation and infiltration of inflammatory cells during I–R, and the injury components mediated by neutrophil and monocyte infiltration into the myocardium.

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1.1.1. Inflammatory cell-mediated myocardial I–R injury Platelet- and neutrophil-mediated injury, as well as complement activation, are involved in the pathogenesis of myocardial I–R injury

(i) Initiation of the inflammatory response: Early studies reported that myocardial cell necrosis results in the release of subcellular mitochondrial membrane constituents capable of activating components of the complement system, including C1, C4, C2 and C3 (Pinckard et al., 1975). Upregulation of mRNA and protein levels of all components of the classical complement pathway has been observed in MI (Yasojima et al., 1998), and these are able to activate neutrophils and facilitate their adherence to endothelium. Pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα), as well as the interleukins IL1, IL-6 and IL-8, are released from ischemic myocardium; I–Rtriggered complement activation and ROS generation appear to be important factors in triggering this cytokine cascade (Ren et al., 2003). Elevated circulating levels of these proinflammatory factors after MI have been observed in numerous experimental and clinical studies (Ikeda, 2003; Tousoulis et al., 2006). In particular, activation of the inflammatory transcriptional regulator NF-κB (or nuclear factor kappa-light-chainenhancer of activated B cells), is a critical mechanism of the inflammatory cascade responsible for upregulation of cytokine and adhesion molecules in ischemic myocardium (Lenardo & Baltimore, 1989). Toll-like receptors (TLR), highly conserved transmembrane receptors with leucine-rich extracellular motif repeats, are expressed on both antigen-presenting cells and parenchymal cells, including cardiomyocytes. TLRs expressed in host cells such as cardiomyocytes bind endogenous ligands released from I–R-injured tissues to then induce the inflammatory cascade via translocation of NF-κB and subsequent proinflammatory gene transcription (Arslan et al., 2011). (ii) Activation and infiltration of inflammatory cells: Upon activation, neutrophils and monocytes/macrophages are recruited to the site of injury to initiate the healing process. Accumulation of neutrophils is observed in the myocardium in the first minutes-to-hours after MI, followed by infiltration of


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induce ICAM-1 in cultured cardiomyocytes (Frangogiannis et al., 2002). We have recently demonstrated that ICAM-1 gene expression is significantly increased as early as 6 h after permanent coronary artery occlusion in mice in vivo, which persists for up to 7 days, with marked increases in vascular cell adhesion molecule1 (VCAM-1) evident until 3 days post-MI (White et al., 2014). However, increased gene expression of ICAM-1 in the ischemic zone is evident as early as 1 h after post-ischemic–reperfusion, with enhanced global expression across the myocardium evident 24 h after reperfusion (Kukielka et al., 1993). Administration of neutralizing antibodies to ICAM-1 or CD18 after the onset of reperfusion ameliorates endothelial dysfunction, with an associated reduction in both coronary perfusion defects and in the extent of cardiac necrosis (Ma et al., 1991; Zhao et al., 1997). The inflammatory cell-derived CXC chemokine IL-8 also plays an important role in the recruitment and localization of neutrophils to ischemic tissues (Ivey et al., 1995). Importantly, although IL-8 gene expression is relatively low in the first few hours of ischemia, reperfusion induces a rapid and marked increase in its expression by 1 h after reperfusion, which remains sustained at high levels beyond 24 h post reperfusion (Kukielka et al., 1995). (iii) Neutrophil-mediated injury: Neutrophils are a major source of myocardial I–R injury, with significant contributions to microvascular injury, cardiomyocyte death and extracellular matrix (ECM) degradation and remodeling. Considerable evidence indicates that cell–cell interactions via specific adhesion molecules are critical for neutrophil-mediated pathophysiological changes. The considerable leukocyte accumulation within the microvasculature in the early stages of reperfusion, part of the “no-reflow” phenomenon described above, precedes their role in triggering inflammatory responses (Engler et al., 1986). Adhesion of neutrophils can directly damage the coronary vascular endothelium, as a result of neutrophil-derived ROS generation (Duilio et al., 2001; Seddon et al., 2007) and its subsequent impairment of endothelium-derived NO• release and bioavailability (Tsao & Lefer, 1990; Ma et al., 1993). Damage to the endothelial barrier, with increased endothelial permeability, albumin leakage and

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monocytes/macrophages at later time points (Jung et al., 2013) (Fig. 1). Reperfusion accelerates and enhances neutrophil infiltration into the previously ischemic myocardium. Neutrophil recruitment is mediated via adhesion molecules, including Pselectin, L-selectin and intercellular adhesion molecule-1 (ICAM1). Both platelets and endothelial cells express P-selectin, and Lselectin is constitutively expressed on leukocytes (Ebnet & Vestweber, 1999). P-selectin expression is increased in the first few minutes of reperfusion (Weyrich et al., 1995), whereas ICAM-1 expression peaks later (after approximately 4–6 h from the onset of reperfusion) (Albelda et al., 1994). Recruited neutrophils produce further ROS, proteases, cytokines, and lipids, which cause direct injury to the endothelial cells (Vinten-Johansen, 2004). In turn, activated endothelial cells exhibit inflammatory phenotypes, characterized by oxidative bursts, and enhanced production of inflammatory cytokines and adhesion molecules, facilitating binding of leukocytes and platelets to the endothelial surface. Concomitantly, intercellular tight junctions are compromised, leading to endothelial barrier dysfunction and increased microvessel permeability. This promotes an influx of neutrophils and other inflammatory cells into the injured myocardial tissue (Timmers et al., 2012). Interestingly, inflammatory mediators can increase microvessel permeability even in the absence of leukocyte adhesion (Michel & Curry, 1999; Zhu & He, 2005); thus subsequent infiltration of adhesive leukocytes induces a further degree of damage to the endothelial barrier. Vascular cells are not alone in this upregulation of cell surface adhesion molecule expression; cardiomyocytes also express ICAM-1 on their cell surface in response to stimulation by proinflammatory cytokines (e.g. TNFα, IL-1, IL-6) (Frangogiannis et al., 1998; Gwechenberger et al., 1999). As a result, this upregulated cardiomyocyte ICAM-1 expression attracts the infiltration of many inflammatory cells (e.g. leukocyte). Adherence of neutrophils to cardiomyocytes is dependent on both the surface integrin macrophage-1 antigen (Mac-1, or cluster of differentiation CD11b/CD18) and ICAM-1 (Entman et al., 1992). Moreover, proinflammatory cytokines TNFα, IL-1 and IL-6 can directly

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Fig. 1. Contributing mechanism to I–R injury (see text for references) and proposed mechanism of ANX-A1-induced cardioprotection. Dash red lines indicate inhibition, and black solid lines indicate enhancement. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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Myocardial I–R injury, MI and resultant heart failure remains a major cause of death and disability in Western societies, despite clinical advances such as thrombolysis and percutaneous revascularization interventions to facilitate reperfusion. In the next decade, this morbidity will expand to all corners of the globe (Moens et al., 2005; Murphy & Steenbergen, 2008; Peart & Headrick, 2009; Thygesen et al., 2012). The most recent recommendations from the American Heart Association for patients presenting with MI include fibrinolysis within the subsequent 30 min and primary percutaneous coronary intervention (PCI) by 90 min after patient presentation (Masoudi et al., 2008; Anderson et al., 2011). The introduction of therapies including aspirin, antiplatelet drugs, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, β-adrenoceptors antagonists, Ca2+-channel antagonists, anti-thrombolytics, and glycoprotein IIb/IIIa inhibitors has improved outcomes post MI (albeit now some time ago for many of these approaches, refer to published comprehensive guidelines (Faxon, 2007; Kushner et al., 2009)). Although the majority of these pharmacotherapies remain in clinical use, MI and resultant heart failure remains a major cause of death and disability in 2014. Hence MI continues to represent an unmet clinical need and development of additional therapeutic strategies is thus essential. One of the major targets of the current pipeline for the MI therapeutics market is inflammation (Global-Data, 2011); but whether direct targeting of systemic inflammation is sufficient to manage cardiac risk post MI remains subject to debate (Nicholls & Kataoka, 2014). Many physiological and pharmacological approaches employed in a range of experimental models against myocardial I–R injury have been reviewed extensively (Qin et al., 2009; Ibanez et al., 2011; Ertracht et al., 2014), this review will focus on the pros and cons of novel anti-inflammatory strategies to limit myocardial I–R injury.

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In summary, I–R elicits release of complement, ROS, cytokines and other pro-inflammatory mediators from a range of cardiac cells that subsequently activate circulating neutrophils, monocytes and the vascular endothelium, further exacerbating I–R injury. Activation of these pro-inflammatory cell types releases additional pro-inflammatory

1.2.1. Therapy directly targeting leukocytes A range of anti-inflammatory interventions targeting on leukocytes have been examined in both experimental and clinical settings. Firstly, leukocyte depletion using filters or anti-serum containing antibodies against neutrophils have been shown to be effective for reducing I–R injury. Reperfusion with neutrophil-filtered blood reduced the size of the no-reflow region, post-ischemic arrhythmias, myocardial edema and infarct size in a dog I–R model (Engler et al., 1986; Litt et al., 1989). Neutrophil anti-serum is also effective in infarct size reduction in several species including dogs (Romson et al., 1983; Jolly et al., 1986), pigs (Hatori et al., 1991) and rats (Kin et al., 2006). Encouragingly, similar benefits have also been observed in patients undergoing CABG or open heart surgery. Reperfusion with neutrophil-depleted blood supplemented with a cardioplegia solution significantly reduces clinical and biochemical indices of myocardial reperfusion injury, improving each of hemodynamic recovery, myocardial edema, cardiac

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signals, promoting the expression of adhesion molecules on neutrophils, endothelium and cardiomyocytes, inducing neutrophil rolling and adhesion to the endothelium. Bursts of ROS released from neutrophils further damage the endothelial barrier, facilitating neutrophil and monocyte migration into the injured tissue. Direct cell–cell interactions results in regional inflammation and additional tissue damage. Neutrophils thus appear to feature prominently in this inflammatory component of post-ischemic injury, whereas monocyte/ macrophages (particularly M2 macrophages) play a reparative role, predominantly later in the healing process. Compared with permanent coronary artery occlusion without reperfusion, I–R is associated with higher neutrophil influx, but infarct-induced wall thinning, chamber dilatation and cardiac rupture are ameliorated (Chatelain et al., 1987; Vandervelde et al., 2006; Gao et al., 2012), indicating reperfusion following prolonged ischemia is necessary to rescue jeopardized myocardium and prevent additional adverse cardiac remodeling.

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interstitial edema can also occur secondary to neutrophil adhesion (Kurose et al., 1994; Reffelmann & Kloner, 2002). Moreover, the increased endothelial permeability facilitates inflammatory cell infiltration into the injured tissue. The release of cytosolic components subsequent to cardiomyocyte necrosis also triggers an inflammatory response, including considerable infiltration of neutrophils (a key source of ROS), which in turn triggers release of other pro-inflammatory mediators (Horwitz et al., 1996; Duilio et al., 2001; Seddon et al., 2007). Neutrophils are also implicated in I–R-induced myocardial apoptosis in vivo, as shown by Zhao et al. who reported a significant linear correlation between polymorphonuclear neutrophils and apoptotic cardiomyocytes (Zhao et al., 2000). Further, many studies have documented that neutrophils and macrophages are a rich source of MMP-9 and serine proteases, critical for ECM degradation and net cardiac remodeling post MI (Lindsey et al., 2001; Kolaczkowska et al., 2006; Lindsey & Zamilpa, 2012). Experimental and clinical studies have shown that high levels of MMP-9, either in the circulation or in the myocardium, are associated with severe LV dilation and higher risk of post-MI cardiac rupture (see for comprehensive review Gao et al., 2012). (iv) Monocyte infiltration-mediated injury: Circulating mononuclear cells infiltrate infarcted myocardium in the first few hours of ischemia or reperfusion, later than neutrophil infiltration. At the injury site, monocytes differentiate into macrophages, becoming the predominant phagocytic cells that remove dead cells and debris are involved in the healing process (Fig. 1). Classicallyactivated M1-phenotype macrophages are rapidly recruited by the damaged myocardium, to mediate further production of an array of pro-inflammatory mediators and cellular infiltrates (Gordon, 2003; Ma et al., 2003). After differentiation, monocytes/macrophages also promote ECM degradation, and phagocytose apoptotic neutrophils, i.e. efferocytosis (van der Laan et al., 2012). In contrast, alternatively-activated M2-phenotype macrophages recruited then differentiated at the injured site later than M1 macrophages, act to terminate inflammation and promote synthesis of growth factors for angiogenesis and fibrotic healing (Benoit et al., 2008; Odegaard & Chawla, 2011). An elegantly-designed study revealed a distinct time course of monocyte/macrophage infiltration after MI in mice. M1/Ly6Chigh monocytes predominated from day 1 to 4, whereas M2/ Ly-6Clow reparative monocytes prevailed from day 5 onward, following MI (Nahrendorf et al., 2007). Interestingly, the same biphasic monocyte response is also observed in patients with acute MI (Tsujioka et al., 2009), and increased circulating numbers of classical monocytes are associated with impaired functional outcome in MI patients (van der Laan et al., 2012). Monocyte chemoattractant protein-1 (MCP-1, also known as CC chemokine ligand-2) is a potent attractant for monocytes, especially for the M1 phenotype, as these cells highly express the MCP-1 receptor, CC chemokine receptor type 2 (van der Laan et al., 2012). Upregulated MCP-1 expression has been reported in several species following MI (Kumar et al., 1997; Ono et al., 1999; Dewald et al., 2005), induced within the first 1 h and up to 2 days after reperfusion, where it is co-localized with infiltrated inflammatory cells and small veins (Kumar et al., 1997). In mice, MCP-1 deficiency results in delayed and decreased monocyte/macrophage infiltration and impaired healing; MCP-1 neutralization elicits a similar effect on healing, without influencing macrophage infiltration following MI (Dewald et al., 2005).

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1.2.2. Inhibition of adhesion molecules P-selectin and ICAM-1 are key adhesion molecules involved in the interactions between endothelial cells, platelets, and leukocytes. Elevated circulating soluble P-selectin levels have been observed in patients in the early hours after the onset of MI, peaking 4 h after reperfusion (Shimomura et al., 1998). We have previously demonstrated that myocardial ischemia evokes early increases in circulating platelet–leukocyte adhesion in mice, mediated by P-selectin and its endogenous ligand Pselectin glycoprotein ligand-1 (PSGL-1) (Liu et al., 2011). Further, soluble PSGL-1 itself reduces myocardial infarct size (Hayward et al., 1999). Together, these findings highlight the significance of leukocyte–platelet interactions in myocardial I–R injury. Immunoneutralization of Pselectin also reduces infarct size, coronary perfusion defects, endothelial dysfunction, ROS generation and neutrophil accumulation, as observed in several pre-clinical models (Vinten-Johansen, 2004). Similarly, the anti-ICAM-1 monoclonal antibody CL18/6 preserves myocardial blood flow and significantly attenuates myeloperoxidase activity in a canine model of I–R (Fukushima et al., 2006). Following the success of these experimental studies, a very recent clinical study has shown that the recombinant monoclonal P-selectin antibody inclaclumab reduces myocardial damage, as evidenced by attenuated peak troponin I and myocardial creatine kinase levels after PCI in patients with non-STsegment elevation MI (Tardif et al., 2013). 1.2.3. Anti-platelet therapy The role of platelets in atherothrombotic disease is well established. Aggressive antiplatelet treatment is currently standard practice in patients with coronary artery disease with or without PCI. Clopidogrel and prasugrel, members of thienopyridine class of adenosine diphosphate receptor inhibitors; are able to irreversibly bind to platelet P2Y12 receptors to inhibit subsequent platelet activation and aggregation (Reinhart et al., 2009). Numerous clinical studies have demonstrated protective effects of anti-platelet interventions during ischemia or I–R, thereby ameliorating the no-reflow phenomenon and reducing adverse cardiac events (Wiviott et al., 2007; Spinler & Rees, 2009). The current rationale for routine use of the platelet P2Y12 receptor inhibitors is to prevent arterial thrombosis following coronary intervention (Raju et al., 2008). With many anti-platelet approaches however, increased risk of bleeding remains a concern (White, 2011; Roberts, 2013). We recently compared anti-platelet treatment with either clopidogrel or platelet depletion in mice subjected to MI. Both interventions inhibited platelet–leukocyte conjugation in peripheral blood, attenuated inflammatory infiltration, decreased content of matrix metalloproteinases and plasminogen activation, suppressed expression of

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inflammatory mediators in the infarcted myocardium and reduced the incidence of cardiac rupture. Moreover, clopidogrel also alleviated the extent of chronic LV dilation (Liu et al., 2011). Our findings demonstrate a previously unrecognized protective action of antiplatelet therapy in attenuation of serial inflammatory events and cardiac remodeling. GPIIb/IIIa is another type of platelet membrane receptor, clinical application of three IIb/IIIa antagonists including abciximab, tirofiban and eptifibatide effectively inhibits platelet aggregation following PCI (Hobbach & Schuster, 2003). Early use of tirofiban or abciximab significantly increases the rate of complete ST-segment resolution preand post-PCI, and improves clinical outcomes at 30 days (van't Hof & Valgimigli, 2009). Similar efficacy of eptifibatide 60 min post PCI has also been reported (Zeymer et al., 2010). As the degree of inflammatory response following MI is an important determinant of clinical outcomes (Ren et al., 2003), inhibiting inflammation remains a therapeutic target to reduce myocardial I–R injury. The mixture of favorable and unfavorable outcomes of current anti-inflammatory interventions that target different components of inflammatory processes and different timepoints within the disease process, highlight the complexity of post-ischemic inflammation. Development of novel anti-inflammatory strategies that attenuate the early inflammatory response, without impairing the reparative healing process, represents a future challenge.

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ANX-A1, previously known as lipocortin-1, is a 37 kDa protein comprising 348 amino acids. First discovered as a second messenger of GC actions, ANX-A1 was initially shown to mediate the inhibitory effect of GCs on the activity of phospholipase A2 (PLA2) (Flower & Blackwell, 1979; Blackwell et al., 1980). ANX-A1 is a member of the annexin protein superfamily, which is comprised of at least 12 distinct Ca2+ and phospholipid-binding proteins. Their structure consists of a core region of 4 (as is the case for ANX-A1) or 8 homologous amino acid repeats (comprising ≥80% of the protein) attached to an N-terminal domain (Gerke & Moss, 2002). On binding of this core with Ca2+, annexins undergo a conformational change to enable their phospholipid-binding properties; this change renders exposure of their N-terminal region to the extracellular environment (Rosengarth et al., 2001; Gerke & Moss, 2002; Perretti & Dalli, 2009). These structural changes are likely to impact on the biology of annexin proteins and, in particular, on their ability to interact with potential receptors. The unique biological activities of each annexin protein are attributed to this specific N-terminal region (Flower & Rothwell, 1994; Gerke & Moss, 2002; Gerke et al., 2005). For example, the well known annexin-A5, often used as a marker for apoptosis, has protective anticoagulant and antithrombotic actions, not shared by other annexin proteins (Krikun et al., 1994).

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ANX-A1 is widely distributed in the body of adult mammals, detected in organs including lung, kidney, bone marrow, intestine, spleen, thymus and brain (Fava et al., 1989). The protein is particularly abundant in myeloid cells, and also detectable in biological fluids or an inflamed locus (Rescher & Gerke, 2004). Under conditions of inflammation such as MI or colitis, ANX-A1 is readily detectable in human serum or at the site of inflammation (Romisch et al., 1992; Vergnolle et al., 2004); the protein can also be extracellularly secreted from the prostate gland (Haigler & Christmas, 1990) and is detected in alveolar lavage fluid supernatants from both humans and animal models of inflammation (Ambrose & Hunninghake, 1990; Smith et al., 1990; Tsao et al., 1998). Under resting conditions, ANX-A1 protein is constitutively expressed at high levels in sub-cellular granules of human and mouse neutrophils, eosinophils, monocytes and macrophages, as well as in plasma, but is less abundant in mast cells (Goulding et al., 1990; Morand et al., 1995;

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enzyme release and prevalence of ventricular fibrillation on reperfusion (Hayashi et al., 2000; Civelek et al., 2003; Palatianos et al., 2004). Contrasting results have however been reported, perhaps due to differences in patient selection, leukocyte-depletion strategies and timing of intervention (as reviewed comprehensively by Warren et al. (2007) and Loberg et al. (2011)). Secondly, many experimental studies using steroidal and non-steroidal anti-inflammatory agents significantly reduce infarct size (Vinten-Johansen, 2004), via mechanisms dependent on inhibition of neutrophil activation and neutrophil-mediated postischemic injury. Other systemic anti-inflammatory strategies can however lead to worsened rather than improved outcomes in MI patients, as shown several decades ago for the broad-spectrum antiinflammatory glucocorticoid (GC) methylprednisolone, in which catastrophic impairments in cardiac wound healing and increased risk of cardiac rupture were observed (Roberts et al., 1976). More recently, a multicenter clinical trial demonstrated that an antibody to the CD11/ CD18 leukocyte integrin receptor failed to reduce infarct size in patients who underwent primary angioplasty (Faxon et al., 2002). These negative outcomes highlight the complexity of the acute inflammatory response following myocardial ischemic injury, and suggest more selective approaches are required.

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2.4.1. Effects of ANX-A1 and N-terminal peptide Ac2-26 in models of inflammation As alluded to above, ANX-A1 has been identified as an endogenous anti-inflammatory protein (Flower & Rothwell, 1994). Since ANX-A1

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2.4.2. Deficiency of ANX-A1 exaggerates inflammatory responses Much of the information regarding the anti-inflammatory actions of endogenous ANX-A1 has been derived from the consequences of ANXA1 deficiency (Yang et al., 2013a). Use of neutralizing antibodies to endogenous ANX-A1 or knockout approaches to elicit depletion of endogenous ANX-A1 exacerbate inflammatory responses both in vivo and in vitro, further supporting the anti-inflammatory role of endogenous ANX-A1. In one of the earliest such studies, we demonstrated that depletion of endogenous ANX-A1 in mice using a specific anti-ANX-A1 monoclonal antibody exacerbated antigen-induced arthritis, accompanied by increased pro-inflammatory cytokine release from inflamed synovial tissues ex vivo (Yang et al., 1999). Hannon et al. subsequently disrupted the murine ANX-A1 gene to generate the global ANX-A1−/− mouse (Hannon et al., 2003), which has enabled greater understanding of the actions of endogenous ANX-A1 protein in inflammatory and immune responses. It is now the consensus view that endogenous ANXA1 exerts a potent anti-inflammatory role in experimental models of acute and chronic inflammation. This includes models of human disease, such as endotoxemia, rheumatoid arthritis, colitis, stroke, multiple sclerosis, fibrosis, paw edema, ocular uveitis, asthma and skin contact hypersensitivity, in all of which endogenous ANX-A1 serves protective inhibitory or anti-inflammatory roles (Damazo et al., 2005; Gavins et al., 2007; Babbin et al., 2008; Yang et al., 2009; Damazo et al., 2011;

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ANX-A1 constitutes up to 2–4% of the total cellular protein content in human neutrophils (Ernst et al., 1990). Externalization of ANX-A1 to the cell surface plays an important role in its anti-inflammatory actions. This enables the protein to act as a paracrine or autocrine mediator via cell surface receptors, as a means to blunt inflammation. The precise mechanism by which ANX-A1 is secreted from cells however, remains to be fully elucidated. The protein lacks a signal sequence, and thus does not pass through the classical vesicles of the Golgi system for its secretion (Muesch et al., 1990; Christmas et al., 1991). The majority of ANX-A1 is present in granules and vesicles of neutrophils, permitting it to be rapidly externalized to the cell surface following neutrophil adhesion to endothelial monolayers (Perretti et al., 1996; Sousa et al., 2013). It can also be exported to the cell surface when neutrophils are activated under settings of inflammation (Vong et al., 2007). Cell-derived microparticle release from activated cells may also be linked to ANX-A1 secretion from neutrophils and endothelial cells (Dalli et al., 2008; Jansen et al., 2012). In non-granular cell-types, the ATP-binding cassette (ABC) and ABC-A1 transporter systems have been identified as a major non-classical pathway of ANX-A1 secretion to the cell surface in macrophages (Wein et al., 2004) and pituitary folliculostellate cells (Morris et al., 2002; Chapman et al., 2003; Omer et al., 2006). Moreover, externalization of ANX-A1 during cell death can also be detected on phosphatidylserine (PS)-rich plaques on the surface of dying cells (Arur et al., 2003; Fan et al., 2004), or during secondary necrosis (Blume et al., 2009), in which ANX-A1 cleavage to a small N-terminal peptide by “A Disintegrin And Metalloproteinase domain-containing protein”-10 induced the recruitment of monocytes to prevent inflammation (Blume et al., 2012). Factors responsible for ANX-A1 translocation are thought to include increased intracellular Ca2+, GCs, various cytokine stimuli and histone deacetylase (HDAC) inhibitors. Intracellular Ca2+-dependent translocation of ANX-A1 has been reported in activated mast cells (Kwon et al., 2012). GCs are important regulators of ANX-A1 expression, synthesis and export (Perretti & D'Acquisto, 2009). The mechanism by which GCderived ANX-A1 externalizes to the cell surface is however poorly understood. PKC-dependent serine phosphorylation on Ser27 appears to be implicated in the GC-induced transfer of ANX-A1 across the cell membrane (John et al., 2002). Although there are multiple serine phosphorylation sites present in the ANX-A1 sequence (Caron et al., 2013; D'Acunto et al., 2014), GC-induced Ser27 phosphorylation of ANX-A1 is critical for PKC-dependent ANX-A1 translocation (Yazid et al., 2009). The proinflammatory cytokine IL-6 also induces ANX-A1 secretion from human lung adenocarcinoma A549 cells, likely mediated by a CCAATenhancer-binding protein transcription factor (Solito et al., 1998). Interestingly, utilization of HDAC inhibitors, including depsipeptide (also known as FK228) and suberoylanilide hydroxamic acid, reveals that the induction of the expression and externalization of ANX-A1 at least in leukemia cells, is dependent on histone acetylation of its promoter. This ANX-A1, externalization may then in turn mediate actions of HDAC inhibitors in this context (Tabe et al., 2007).

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was first described for its inhibitory effects on PLA2 (Blackwell et al., 1980), its anti-inflammatory actions have been well-established in vivo and in vitro. Perretti and Dalli (2009) described in considerable detail the anti-inflammatory actions of the full length ANX-A1 protein as well as peptides of ANX-A1. There are a plethora of examples of these anti-inflammatory actions observed with administration of either exogenous ANX-A1 or its commonly-studied, biologically active, Nterminal peptide N-terminal-derived annexin-A1 peptide (acetylAMVSEFLKQAWIENEEQEYVVQTVK), Ac2-26, in a broad range of animal models of inflammation. These include inhibition of: (i) cytokineinduced leukocyte migration (mouse air-pouch model (Perretti et al., 1993; Perretti & Flower, 1993)); (ii) carrageenan-induced acute inflammation (rat paw edema, murine peritonitis and arthritis (Cirino et al., 1993; Perretti et al., 1993; Getting et al., 1997; Yang et al., 1997)); (iii) acute I–R injury (rat heart, kidney, murine mesenteric, brain, and intestinal microcirculation (La et al., 2001a,b; Gavins et al., 2003; Gavins et al., 2007; Souza et al., 2007; Facio et al., 2011)); and (iv) allergen (ovalbumin)-provoked inflammatory responses (rat pleurisy, including mast cell degranulation and plasma protein leakage (Bandeira-Melo et al., 2005)). Ac2-26 also enhances healing of acetic acid-induced gastric ulcers (Martin et al., 2008). Both the full-length ANX-A1 protein and f reduce endotoxin-induced inflammation, in rat ocular tissues in vivo and in cultured human ARPE-19 cells in vitro (Girol et al., 2013) as well as murine pleurisy, via induction of neutrophil apoptosis (Vago et al., 2012). Inflammation is similarly reduced in models of both bleomycin-induced pulmonary fibrosis and gut microbiotainduced colitis, the latter accompanied by NADPH oxidase subunit Nox1-dependent enhanced wound healing (Damazo et al., 2011; Leoni et al., 2013). Recent efforts have focussed on improving the potency and stability of ANX-A1 peptides. In particular, the anti-inflammatory potency of Ac2-26 is even further enhanced when delivered using collagen IV-targeted nanoparticles, as recently shown in murine models of zymosan-induced peritonitis and hind-limb I–R injury (Kamaly et al., 2013). Moreover, Perretti et al., have generated a metabolically-stable peptide, CR-AnxA12–50, which is resistant to neutrophil-mediated cleavage via human proteinase-3 (Dalli et al., 2013). This peptide exhibits enhanced potential to limit neutrophil–endothelial cell interactions, to enhance both neutrophil apoptosis and macrophage phagocytosis, and reduce myocardial infarct size after I–R injury. The ANX-A1 system thus represents an innovative therapeutic target for a range of inflammatory conditions (Alessandri et al., 2013; D'Acquisto et al., 2013; Sousa et al., 2013).

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Rescher & Gerke, 2004; Spurr et al., 2011). Although human T and Blymphocytes appear to only express low levels of ANX-A1, the natural killer cell subset of lymphocytes express high levels of ANX-A1 (Morand et al., 1995; Spurr et al., 2011). Importantly, ANX-A1 is detected in myocardium, albeit more abundantly in vascular endothelial cells than cardiomyocytes or smooth muscle cells (Hullin et al., 1989).

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peptide has anti-inflammatory effects in the vasculature (Peshavariya, 674 et al., 2013). 675

Ng et al., 2011; Cristante et al., 2013; Girol et al., 2013; Hughes et al., 2013; Yang et al., 2013b). An interesting exception is the observation from a single study, in which a lack of ANX-A1 reduced the severity of experimental autoimmune encephalomyelitis (Paschalidis et al., 2009). This study used the MOG35–55-peptide to induce encephalomyelitis, a Th1/Th17-dependent model. It is thus possible that endogenous ANX-A1 may have pro-inflammatory role in adaptive immune models; additional studies are warranted to further investigate the impact of ANX-A1 in the context of adaptive immune responses. The possible role of endogenous ANX-A1 in the pro-inflammatory activation phase will reveal new therapeutic strategies for the development of novel anti-inflammatory agents.

2.5. Potential for anti-apoptotic actions of Anx-A1

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Whether annexin-A1 is elevated in (and/or contributes to) apoptotic processes is likely to be dependent on the cell-type and/or disease state under consideration. Another member of the annexin family, annexinA5, which has a distinct N-terminal (the region of the protein that confers unique biological actions of each family member), is often used as a marker of apoptosis (Krikun et al., 1994). Certain cellular stresses that induce intracellular Ca2+ overload lead to association of endogenous ANX-A1 with membrane ceramides (Babiychuk et al., 2008). Downstream consequences triggered by this event vary from protective anti-proliferative actions (e.g. in coronary artery disease), to pro-apoptotic actions (as observed in neutrophils and tumor cells) (Kolesnick, 2002). The latter is discussed in Section 2.6. Of relevance to the focus of this review, there is no evidence to date of proapoptotic actions of ANX-A1 in the cardiovascular system, in which pro-cell survival (albeit largely anti-necrotic) actions are observed (La et al., 2001a,b; Ritchie et al., 2005; Qin et al., 2013). Exogenous administration of Ac2-26 ameliorates spinal cord injury-induced caspase 3 activity at the site of injury, supporting a potential anti-apoptotic action of ANX-A1 mimetics in this tissue (Liu et al., 2007). Our own data reveals that Ac2-26 treatment from the start of post-ischemic reperfusion

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2.4.3. Role of ANX-A1 in the innate immune response 623 Potential anti-inflammatory mechanisms of ANX-A1 have been 624 identified at the molecular and cellular level. Neutrophils are the most 625 widely-studied cells for investigating the anti-inflammatory mecha626 nisms of ANX-A1. Administration of ANX-A1 protein or Ac2-26 induces 627 apoptosis of neutrophils (as discussed later in this review), suppresses 628 leukocyte adhesion and migration, and inhibit the activity of PLA2, pros629 Q9 taglandin E2 and myeloperoxidase (as reviewed in detail by Perretti and 630 Q10 D'Acquisto (2009), Perretti and Dalli (2009). Considerable evidence 631 suggests that ANX-A1 and Ac2-26 bind to the formyl peptide receptor 632 (FPR) family of G-protein-coupled receptor (GPCR)s to exert their bio633 logical effects, with the FPR2 subtype in neutrophils receiving the ma634 jority of research attention in this regard (Walther et al., 2000; Perretti 635 et al., 2002; Perretti, 2003; Gastardelo et al., 2009). As discussed in 636 Section 3 of this review, it is now widely accepted that formyl peptide 637 receptors (FPR) mediate the anti-inflammatory actions of ANX-A1. In 638 addition, interaction of FPR2 with the selective FPR2 ligands lipoxin A4 639 (LXA4) and antiflammin-2 elicits anti-inflammatory responses in 640 human neutrophils that are associated with subsequent ANX-A1 phos641 phorylation and externalization. Further, the anti-inflammatory proper642 ties of these FPR2-selective ligands is absent in ANX-A1−/− mice, at 643 least in this model of acute inflammation, indicating that this is an 644 ANX-A1-dependent process (Brancaleone et al., 2011). 645 There is mounting evidence for the important role of endogenous 646 ANX-A1 in the innate immune response. Firstly, it negatively regulates 647 TLR-stimulated activation of the pro-inflammatory cytokines TNFα 648 and IL-6 in macrophages, downstream of MAPK and NF-κB activation 649 (Yang et al., 2009). Moreover, endogenous ANX-A1 is released from ap650 optotic neutrophils, where it then interacts with macrophages to facili651 tate their phagocytosis of these apoptotic cells (Scannell et al., 2007); 652 this ANX-A1-mediated efferocytosis may proceed in an autocrine or 653 paracrine manner (Sousa et al., 2013). Ac2-26 also favors the resolution 654 of inflammation similar to the parent protein. Indeed, Ac2-26-induced 655 phagocytosis of apoptotic neutrophils is an FPR2-dependent process 656 (Maderna et al., 2010); in the absence of FPR2, Ac2-26 loses this 657 efferocytotic mechanism. Endogenous ANX-A1 also represents a key 658 mechanism of the anti-inflammatory actions of pharmacotherapies 659 ranging from nonsteroidal anti-inflammatory drugs (NSAIDS) to HDAC 660 inhibitors, in addition to their known second messenger role in GC ac661 tions (Flower & Blackwell, 1979; Blackwell et al., 1980; Tabe et al., 662 2007; Zhang et al., 2010b; Montero-Melendez et al., 2013). The ANX663 A1 promoter is susceptible to histone acetylation; as a consequence, 664 HDAC inhibition triggers ANX-A1 synthesis, externalization, and in 665 turn neutrophil apoptosis and macrophage phagocytosis inhibitors 666 (Tabe et al., 2007; Montero-Melendez et al., 2013). Furthermore, other 667 anti-inflammatory drugs, including NSAIDS and GCs, upregulate endog668 enous ANX-A1, which subsequently inhibits NF-κB transcriptional ac669 tivity, providing a novel molecular mechanism of drug-inducible ANX670 A1 to target inflammation (Zhang et al., 2010b). A more recent study 671 has now suggested that the Ac2-26 peptide directly inhibits TNFα672 induced NADPH oxidase derived ROS formation and attenuates the acti673 vation of the NF-κB pathway in endothelial cells, suggesting that the

2.4.4. Potential role of ANX-A1 in the adaptive immune response In contrast to the evidence implicating ANX-A1 in innate immunity, its role in the adaptive immune response is yet to be fully resolved. The anti-inflammatory properties of endogenous ANX-A1 are supported by observations in several models of autoimmune diseases, including antigen- and collagen-induced arthritis, paw edema, skin contact hypersensitivity and asthma (Yang et al., 2004; Ng et al., 2011; Yang et al., 2013b). In these settings, the mechanisms of ANX-A1-mediated negative regulation of inflammation are immune cell-type dependent. For example, the exacerbation of antigen-induced arthritis in the absence of endogenous ANX-A1 protein is associated with increased proinflammatory cytokine expression in arthritic joints. The extent of skin inflammation in ANX-A1−/− mice correlates with a switch to a Th1/ Th17 cytokine profile. In antigen-induced asthma, enhanced disease severity in the ANX-A1−/− mouse is associated with increased airway hyper-responsiveness and enhanced antibody responses. Moreover, deficiency of ANX-A1 promotes CD4+ T cell activation, as demonstrated by adoptive transfer experiments in Rag1−/− mice (which lack mature T and B cells). Further, this enhanced CD4+ T cell activation evident in the ANX-A1−/− mouse is accompanied by increases in both T cell proliferation and cytokine release [specifically interferon-γ (IFNγ), and IL-17], suggesting a potential inhibitory role of endogenous ANXA1 protein, restricting antigen-specific CD4+ T cell mechanisms in immune response (Yang et al., 2004; Ng et al., 2011; Yang et al., 2013b). However, it remains unclear whether or not the effect of endogenous ANX-A1 on CD4+ T cell is FPR-dependent. Interestingly, these findings are in direct contrast to the potential pro-inflammatory role of endogenous ANX-A1 observed in a model of autoimmune encephalomyelitis (Paschalidis et al., 2009). As discussed above, this model is associated with Th1/Th17-related T cell responses. Although it remains possible that this contrasting observation is unique to this particular model of inflammation, the findings reported are supported by in vitro evidence suggesting that impaired activation and proliferation in response to T cell receptor stimulation, and an increased Th2 phenotype, may be a contributing mechanism in ANX-A1-deficient T cells (D'Acquisto et al., 2007). A dual action of endogenous ANX-A1 may thus be present in adaptive immune responses. Further interrogation of the potential role of endogenous ANX-A1 and its exogenous mimetics in adaptive immune responses, and additional elucidation of the mechanisms involved, are clearly warranted.

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GCs have been widely and successfully used in the treatment of many inflammatory and autoimmune diseases over sixty decades (Clark & Belvisi, 2012; Vandevyver et al., 2013). They exert their actions by binding to the glucocorticoid receptor (GR), a member of the nuclear receptor family of transcription factors. GCs repress inflammation by inhibiting both the recruitment of inflammatory cells and the production of pro-inflammatory cytokines and inflammatory enzymes, as well as by the induction of anti-inflammatory mediators (Clark, 2007; Vandevyver et al., 2013). In this regard, ANX-A1 is a GC-regulated anti-inflammatory protein. As discussed above, ANX-A1 was initially identified as a second messenger of GCs (Flower & Blackwell, 1979). GCs not only augment ANXA1 synthesis, but also promote its translocation from inside the cell to the outer cellular surface, and in turn, ANX-A1 mediates the antiinflammatory actions of GCs (Perretti & D'Acquisto, 2009). Deficiency of endogenous ANX-A1 in mice results in a marked reduction in responsiveness of GCs in vivo and in vitro, further supporting the requirement of ANX-A1 for the anti-inflammatory role of GCs. A plethora of examples illustrate the critical dependence of GCs on ANX-A1 for their antiinflammatory actions. Diminished responsiveness to GCs in settings of ANX-A1 deficiency includes: (i) failure of dexamethasone to inhibit carrageenin-induced paw edema (Hannon et al., 2003); (ii) impairment of dexamethasone to blunt arthritis in two different experimental models, associated with a failure to repress synovial inflammation (Yang et al., 2004; Patel et al., 2012); (iii) failure of dexamethasone to inhibit leukocyte trafficking, cytokine synthesis, and mesenteric mast cell degranulation in acute peritonitis (Damazo et al., 2006); and (iv) impairment of dexamethasone-induced neutrophil apoptosis for resolution of LPS-induced inflammation (Vago et al., 2012). In vitro, steroid-mediated clearance of apoptotic cells by monocyte-derived macrophages requires ANX-A1 and FPR2 (Maderna et al., 2005). Deficiency of ANX-A1 in murine macrophages and fibroblasts also impairs dexamethasone resolution of inflammation, including attenuation of its ability to blunt the upregulation of pro-inflammatory cytokines (Yang et al., 2006, 2009). Similar effects are observed in human lung fibroblasts in which endogenous ANX-A1 has been silenced (Jia et al., 2013). However, the mechanisms by which GCs affect induction of ANX-A1 expression are not fully understood. Although the human ANX-A1 promoter region contains a GC-response element (GRE) (Kovacic et al., 1991; Solito et al., 1998), dexamethasone regulation of ANX-A1 expression appears independent of GRE in this region (Solito et al., 1998). Perretti and D'Acquisto (2009) have postulated that a canonical GRE does not appear to be present in the ANX-A1 promoter, although it contains a partial consensus-binding site. It is possible that the GRE in the ANX-A1 promoter region is either incomplete or is not fully functional. GCs also regulate FPR2 expression in human monocytes and neutrophils (Sawmynaden & Perretti, 2006; Hashimoto et al., 2007), indicating that GC stimulated induction of ANX-A1 may ultimately result in FPR activation, on which ANX-A1 then acts, either in an autocrine or paracrine manner. Given that GCs require both ANXA1 and FPRs for their functional activities, an important role of FPRs in GC actions will likely further emerge. This hypothesis is strongly supported by a recent study in which dexamethasone failed to repress leukocyte migration in FPR2/3 deficient mice subjected to acute inflammation (Dufton et al., 2010). Recognized contributing mechanisms to the anti-inflammatory actions of GCs include the GC-induced anti-inflammatory proteins MAPK phosphatase-1 (MKP1, or DUSP1) and GC-induced leucine zipper (GILZ) (Clark, 2007; Beaulieu & Morand, 2011; Clark & Belvisi, 2012; Newton, 2013; Vandevyver et al., 2013). Previous studies have shown

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The best established scenario demonstrating the pro-apoptotic actions of ANX-A1 and its peptide mimetics is during resolution of certain 749 types of inflammation via activation of FPR2 on neutrophils (Perretti, 750 2012). ANX-A1 has also been implicated in pro-apoptotic responses in 751 T lymphocytes (Scannell et al., 2007; Blume et al., 2009). The dual ac752 tions of ANX-A1 on promoting apoptosis and activation of inflammation 753 result in enhanced clearance of the apoptotic cells prior to their lysis 754 (Sousa et al., 2013). These products of cell lysis are now generically 755 referred to as Danger-Associated Molecular Patterns (DAMPs) or 756 alarmins, and include ATP and high mobility group B protein 1 757 (HMGB1) (Bianchi, 2007; Christia & Frangogiannis, 2013). Thus, ANX758 A1, together with lipid pro-resolving mediators such as lipoxins, 759 resolvins and protectins, are considered to lessen the burden imposed 760 by DAMPs, curtailing neutrophilic and other types of inflammation. 761 ANX-A1 has also been implicated in apoptotic processes in a range of 762 other settings including mammary gland involution following weaning 763 (McKanna, 1995) and thymocyte apoptosis (Sakamoto et al., 1996). 764 There has been great interest in the impact of ANX-A1 in tumor cell ap765 optosis, driven in part by interest in the link to FPRs role in migration 766 and by consideration of the potential impact of inflammation resolution 767 in tumor progression. For example, we have described FPR2 on human 768 breast tumor cell lines, activation of which increases cell proliferation 769 (Khau et al., 2011). Another cell type in which ANX-A1 has been impli770 cated in apoptosis is ceramide-treated bronchoalveolar cells, in which 771 caspase-3-mediated apoptosis increases in parallel with ANX-A1 ex772 pression in response to increasing ceramide concentrations (Debret 773 et al., 2003). This has implications for asthma, cystic fibrosis, and lung 774 tumors (Luthra et al., 2008). A potential role for intracellular ANX-A1 775 Q12 in TNF□-induced apoptosis has also been suggested in kidney epithelial 776 and mesangial cells. Further, the associated nuclear accumulation of 777 ANX-A1 was prevented by overexpression of the anti-apoptotic Bcl-2 778 protein (Ishido, 2005). In all of these scenarios, induction of apoptosis 779 might be considered a protective, rather than a detrimental mechanism. 780 Blockade rather than activation of FPRs may be protective in certain 781 tumor types, in contrast to the protective effect of FPR against in I–R in782 jury and rheumatoid arthritis. 783 The biochemical pathways involved in apoptosis have been explored 784 in a variety of cell types. In neutrophils, the mechanisms have not been 785 unequivocally established, but an association between FPR2 activation, 786 activation of the “executioner” pro-apoptotic Bax protein, as well as in787 hibition of pro-survival pathways Mcl-1, extracellular signal-regulated 788 Q13 kinases (ERK)1/2 and NF-κB have been established (Vago et al., 2012). 789 In circulating inflammatory cells undergoing apoptosis (e.g. neutro790 phils, lymphocytes), ANX-A1 translocates to the cell membrane, associ791 ating with discrete phosphatidylserines to facilitate their phagocytosis 792 (Scannell & Maderna, 2006). Conditioned media obtained from expo793 sure to apoptotic human neutrophils contains ANX-A1 and its peptide 794 fragments; this media alone is sufficient to promote FPR2-mediated 795 phagocytosis of apoptotic neutrophils by macrophages (Scannell et al., 796 2007). Indeed, the release of soluble ANX-A1 from apoptotic neutro797 phils has been accorded a role in maintaining an anti-inflammatory en798 vironment during resolution of inflammation (Scannell et al., 2007; 799 Pupjalis et al., 2011). Similar evidence of ANX-A1-mediated macro800 phage phagocytosis of apoptotic T lymphocytes has been reported

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(Blume et al., 2009). More recently, cell membrane-associated ANX- 801 A1 expressed on apoptotic cells has also been accorded an immunosup- 802 pressive role (Weyd et al., 2013). 803

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in isolated rat and mouse hearts in vitro increases LV Akt phosphorylation. In contrast, this is markedly suppressed in the ANX-A1−/− mouse heart subjected to I-R (Qin et al., 2013). Lastly, under some circumstances, GCs (which mediate many of their actions via ANX-A1) are thought to be anti-apoptotic (Ye et al., 2009). Together, these observations suggest that ANX-A1 has the potential to inhibit apoptosis, at least in cell types (e.g. cardiomyocytes, neurons and endothelial cells) in which this would be considered to be a protective, rather than an injurious, outcome.

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Numerous studies have established N-formyl peptides as naturallyoccurring chemoattractants produced by bacteria, including 903 L. monocytogenes and S. aureus, and the mitochondria of mammalian 904 cells (Dufton & Perretti, 2010). The tripeptide, fMLF (N-formyl-Met905 Leu-Phe, also known as fMLP), is commonly used as a chemotactic pep906 tide in studies of leukocyte migration and has approximately 1000-fold 907 Q20 greater potency at FPR1 than FPR2 (Gao & Murphy, 1993). The addition 908 of an N-formyl group increases the potency of both synthetic and en909 dogenous peptide agonists. For example when N-formylated, the syn910 thetic peptide, MMWLL (Met-Met-Trp-Leu-Leu), and the endogenous 911 neuroprotective peptide, humanin, have greater potency for each of 912 their target FPR receptors, namely FPR1 and FPR2/3 (Ye et al., 2009). 913 Formyl peptides can have a range of subtype selectivity profiles. Based 914 on the screening of peptides from various sources, Rabiet et al. (2005) 915 demonstrated that, while the bacterial protein peptide leader sequence, 916 fMLF is highly FPR1-selective, a number of mitochondria-derived mam917 malian formyl peptides, including fMYFINILTL, fMLKLIV and fMMYALF, 918 showed similar potency in elevating cytoplasmic Ca2+ levels via either 919 FPR1 or FPR2 (Rabiet et al., 2005). 920 A number of additional naturally occurring and biochemically di921 verse ligands have been suggested to bind and activate FPR2. Putative 922 naturally-occurring ligands include the GC-modulated protein, ANX923 A1, the arachidonic acid-derivative LXA4, the neuroprotective peptide 924 humanin, the 42-amino acid beta-amyloid peptide Aβ42, serum amyloid 925 A (SAA), and a peptide fragment of the aberrant human prion protein

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3.2.2. Synthetic FPR agonists The beneficial effects of FPR2 agonists in models of acute and chronic inflammation is widely recognized, generating enthusiasm for the therapeutic potential of this emerging drug class (Alessandri et al., 2013; D'Acquisto et al., 2013; Sousa et al., 2013). A high-throughput peptide library screen identified the first synthetic FPR agonists, including the hexapeptide, WKYMVm, which has greater selectivity for FPR2 than FPR1 (Baas & Berk, 1995). Additional FPR2-selective peptide agonists, including MMK-1 (LESIFRSLLFRVM), were subsequently identified using an innovative FPR2 survival-coupled approach in yeast. Yeast were transfected with a library of plasmids encoding random peptides and were also engineered to stably express FPR2 coupled to a survival pathway, allowing selection of yeast transfected with cognate peptides. Survival and proliferation of “complemented” yeast facilitated subsequent identification of the peptide sequence required to activate the receptor (Klein et al., 1998). Both MMK-1 and WKYMVm stimulate a number of established FPR2-mediated signaling pathways, including phosphoinositide hydrolysis and intracellular Ca2+ mobilization,

973 Q27

P

D

890 891

E

888 889

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886 887

C

884 885

E

883

In a striking example of convergence in function, the FPR2 receptor was discovered independently as both the receptor for the inflammation-resolving, trihydroxy-polyunsaturated fatty acid, LXA4 and named aspirin-triggered lipoxin receptor (ALXR), as well as its identification as the receptor for the GC effector, ANX-A1, initially named formyl peptide receptor-like receptor 1 (FPRL-1). For a period of time, these separate nomenclatures were maintained while the field established the single identity of the receptor for these and other diverse ligands. The FPR2 member of the human formyl peptide receptor FPR family (also comprising FPR1, and FPR3) is now identified as the receptor responsible for some of the biological activities of ANX-A1, its N-terminal peptide and LXA4 (Ye et al., 2009) and a large number of other ligands (see below). The FPR1 and FPR2 receptors are widely distributed in tissues and different cell types, being most prominently expressed on cell types involved with inflammatory processes, whereas FPR3 is thought to be highly expressed only on dendritic cells. FPR2 and FPR3 however share ≥70% level of sequence homolog (Ye et al., 2009). Reports of the expression of FPRs on different types of tumor cells are growing in number.

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926 927

R O

879 Q17 3.1. Overview of FPR receptors 880

PrP106–126 (Ye et al., 2009), and residues 88–274, the cleave product of urokinase plasminogen activator (uPA) receptor (Resnati et al., 2002). LXA4 mediates a range of cellular effects, including activation of macrophage phagocytosis and reduced neutrophil recruitment and survival, all of which contribute to the resolution of inflammation. Interestingly, the effects of LXA4 parallel those of ANX-A1. In CHO cells transfected with cDNA encoding FPR2, 3H-LXA4 has high affinity for FPR2 (LXA4 Ki of 80 nM); LXA4 increases GTPase activity and release of esterified arachidonate in this context (Fiore et al., 1994). The human FPR2 gene has a murine homolog, Fpr2. However, characterization of the pharmacological conservation between species is however currently incomplete. Nevertheless, in Fpr2−/− mice, LXA4-mediated inhibition of neutrophil migration is significantly reduced compared to wild-type littermates. However, more recently, a number of studies have questioned the ability of LXA4 to stimulate FPR2. Hanson et al. (2013) were unable to detect LXA4-mediated increases in intracellular Ca2+, ERK1/2 phosphorylation, modulation of cyclic adenosine monophosphate (cAMP) accumulation, or translocation of β-arrestin in cells heterologously expressing FPR2 (Hanson et al., 2013). Similarly, Planagumà et al. (2013) observed that15-epi-LXA4 was unable to stimulate guanosine triphosphate-gamma (GTPγ) binding or inhibit cAMP accumulation in CHO cells expressing FPR2, despite intact agonist actions of the FPR2 synthetic peptide, Trp-Lys-Tyr-Val-D-Met (WKYMVm) in this setting (Planagumà et al., 2013). An additional important consideration with respect to assessing LXA4 pharmacology within native expression systems is that LXA4 has been suggested to have affinity for both nuclear aryl hydrocarbon receptors and leukotriene B4 receptors in addition to FPR2 (Dufton & Perretti, 2010). Further confounding understanding of the mechanism of LXA4 at FPR2 is its ability to enhance FPR2 promoter activity and subsequent receptor expression in vitro (Simiele et al., 2012). Serum amyloid A (SAA) is an acute phase protein secreted predominantly by the liver following exposure to lipopolysaccharide or inflammatory cytokines such as IL-1, IL-6 and TNFα. The typical serum concentration of SAA (0.1 μM) can increase by approximately 1000fold during infection or in response to injury (Migeotte et al., 2006). Su et al. (1999) demonstrated that SAA selectively mediating an increase in Ca2+ mobilization and migration in FPR2-stably transfected HEK293 cells. In contrast, SAA was unable to stimulate intracellular Ca2+ mobilization in HEK293 cells expressing FPR1 (Su et al., 1999). SAA activation of the FPR2 receptor has been suggested to promote chemotaxis in monocytes, neutrophils, mast cells and T lymphocytes and to stimulate the production of metalloproteases, cytokines and cytokine receptors (Migeotte et al., 2006). An important consideration when interpreting results generated within endogenous FPR2 expression systems is that SAA has also been suggested to exert its action via CD36/ LIMPII analogous-1, TLR2 and TLR4 (Dufton et al., 2010).

F

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that the anti-inflammatory GC effects (including attenuation of proinflammatory cytokine interleukin (IL)-6 expression, and induction of anti-inflammatory protein MKP-1) are dependent on ANX-A1 (Yang et al., 2006). Further, the absence of endogenous ANX-A1 significantly reduces GC-induced macrophage GILZ expression (Yang et al., 2009). The inhibitory effect of ANX-A1 on pro-inflammatory cytokine expression that is absent in ANX-A1−/− cells is restored by GILZ transfection. These data suggest that deficiency of ANX-A1 enhances the inflammatory response, ANX-A1 requires GILZ for bioactivity. The reduced sensitivity to GCs in ANX-A1−/− cells can be attributed to the loss of ANX-A1driven GILZ activity. These data further suggest that MKP-1 and GILZ are key to the ability of ANX-A1 to mediate the anti-inflammatory actions of GCs.

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9


928 929 930 931 932 933 934 Q21 935 936 937 938 939 940 941 942 943 944 Q22 945 946 947 948 949 Q23 950 951 952 953 954 955 Q24 956 957 958 959 960 961 Q25 962 963 964 965 966 967 968 969 Q26 970 971 972

974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 Q28 989 990


1010 1011 1012 Q32 1013 1014 1015 1016 1017 Q33 1018 1019 1020 1021 1022 Q34 1023 1024 1025 1026 1027 1028 1029 Q35 1030 Q36 1031 1032 1033 1034 1035 Q37

1043 Q40 1044 1045 1046 1047 1048 Q41 1049 1050 Q42 1051 1052 1053 1054 1055

N

1041 1042

Synthetic peptide and small molecule antagonists have been developed for both FPR1 and FPR2. The first FPR antagonists were fMLF derivatives based on modifying the agonist using a tertiary butyloxycarbonyl group (t-BOC) in place of the formyl group (Freer et al., 1980). Two such synthetic peptides, t-Boc-Met-Leu-Phe (Boc1) and t-Boc-Phe-D-LeuPhe-D-Leu-Phe (Boc2), are now commonly used as FPR1-selective antagonists, with pIC50 values of 6.2 and 6.6, respectively (Freer et al., 1980). An FPR2-selective peptide antagonist, Typ-Arg-Trp-Trp-TrpTrp-CONH2 (WRW4), was identified through screening hexapeptide libraries using an FPR2 heterologous expression system in RBL-2H3 cell lines. At micromolar concentrations, WRW4 significantly decreases WKYMVm binding and cellular signaling at FPR2 (Bae et al., 2004). Recently, a number of synthetic tryptophan-containing dipeptide analogs were developed as novel FPR1 antagonists (Hwang et al., 2013). Lead compounds within this series inhibit fMLF-stimulated •O− 2 generation and neutrophil elastase release in human neutrophils, with pIC50 values of 6–7. Structure–activity studies identified the fragment N-benzyl-TrpPhe-OMe as a suitable core structure for the future development of antagonists that display selectivity for FPR1 over FPR2. The first small

U

1039 1040 Q39

C

1036 Q38 3.2.3. FPR antagonists 1037 1038

1096

3.3. Regulation of FPR receptors

1114 Q48

3.3.1. Potential for biased agonism at FPRs The classical notion of GPCR activation is based on the transition between two receptor states, active and inactive, with agonists promoting a functional response through stabilization of the active state (Kenakin,

1115 Q49

F

3.2.4. Annexin-A1 and its derivatives ANX-A1 was originally identified in 1979 as a protein mediator of the suppressant action of GCs on mobilization of arachidonic acid and its metabolites (Yazid et al., 2012). Although this activity on lipid mediators production has been associated with a physical interaction between ANX-A1 and the phospholipid membrane targeted by phospholipase A2, it may also involve FPR2 activation. Other actions of ANX-A1 at FPR2 include shedding of selectin from neutrophils, activation of neutrophil apoptotic pathways and promotion of macrophage phagocytosis (Dufton & Perretti, 2010). Full length ANX-A1 is proteolytically cleaved at the N-terminus, resulting in the N-terminal 26 amino acid peptide fragment Ac2-26 that is also an agonist at FPR2 in some settings (Dalli et al., 2012). As discussed above, ANX-A1 belongs to a family of ≥12 genes that are generally highly structurally homologous except at the N-terminus, leading to the suggestion that this N-terminal motif is responsible for the specific actions of each family member (Yazid et al., 2012). No other members of the annexin family aside from ANX-A1 are known to have FPR2-mediated actions.

O

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1006 1007

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1004 1005

1056 1057

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1002 1003 Q31

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1000 1001

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998 999

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995 996

A computational platform designed to predict novel GPCR peptide agonists cleaved from secreted proteins by convertase proteolysis later identified a novel 21-amino acid peptide agonist, CGEN-855A (TIPMFVPESTSKLQKFTSWFM-amide) that activates both FPR2 and FPR3. In murine in vivo models of myocardial I–R injury, CGEN-855A mediates protective effects via decreased polymorphonuclear leukocyte recruitment to the injured organ (Hecht et al., 2009). A recently developed peptidolipid, F2Pal16, which contains a 16-amino acid sequence corresponding to the third intracellular loop of FPR2, has been suggested to selectively activate FPR2 and subsequently mediate •O− 2 production in human neutrophils (Forsman et al., 2013). Non-peptide FPR agonists are also currently in development. A number of synthetic small molecule agonists have been identified through screening of combinatorial libraries. Some of these have pharmacological properties that make them useful as both research tools and potential FPR-targeting therapeutics. A high throughput screening approach led to the identification of a number of quinazolinone derivatives, including Quin-C1 (4-butoxy-N-[2-(4-methoxy-phenyl)-4-oxo-1,4dihydro-2H-quinazolin-3yl]-benzamide), the first non-peptide agonist targeting FPR2 (Nanamori et al., 2004). In neutrophils, Quin-C1 stimulates chemotaxis and β-glucuronidase secretion with a potency that was approximately 1000-fold lower than WKYMVm. In rat basophilic leukemia (RBL) cell lines transfected with either FPR1 or FPR2, QuinC1 selectively stimulates Ca2+ mobilization, ERK1/2 phosphorylation and β-hexosaminidase secretion in FPR2-expressing cells (Nanamori et al., 2004). Amgen have also employed high-throughput screening to identify a pyrazolone compound, Compound 43, which is orally active in a mouse model of dermal inflammation. Specifically, Compound 43 mediated a dose-dependent reduction of ear swelling is associated with inhibition of polymorphonuclear leukocyte migration (Bürli et al., 2006). Subsequent studies have demonstrated that the potent antiinflammatory properties mediated by Compound 43 in vivo are lost in mice deficient in the murine FPR2 orthologue, Fpr2. A ligand-based drug design approach, based on the structure of previously identified non-peptide FPR2 agonists, developed a series of pyridazin-3(2H)-one derivatives that were found to be either non-selective between FPR1 and FPR2 (14a), or FPR2-selective (14x) (Cilibrizzi et al., 2009; Giovannoni et al., 2013). Structure-activity studies around these compounds demonstrate that an arylacetamide moiety at N-2 of the scaffold is essential for activity. Molecular docking studies of the lead pyridazin3(2H)-one derivatives suggest these compounds recognize an overlapping binding pocket to that of fMLF and WKYMVM at FPR1 and FPR2, respectively (Giovannoni et al., 2013).

O

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molecule non-peptide FPR2 antagonist, Quin-C7, was identified in studies investigating the structure–activity relationship of the agonist, Quin-C1, at FPR2 (Zhou et al., 2007). Quin-C7 partially inhibits [125I] WKYMVm binding to FPR2-expressing RBL-2H3 cell membranes, but has no effect on [3H]fMLF binding to FPR1-expressing RBL-2H3 cells at a concentration of 10 μM. In functional assays, Quin-C7 inhibits multiple intracellular signaling events, including Ca2+ mobilization, chemotaxis, degranulation and ERK1/2 phosphorylation, stimulated by either WKYMVm and/or Quin-C1 in RBL-2H3 cells expressing FPR2 (Zhou et al., 2007). A flow cytometry-based, high-throughput screening approach has also been used to identify small molecule FPR antagonists. This approach used a WKYMVm derivative containing a fluorescein dye attached to the lysine residue, which rendered this probe non-selective between FPR1 and FPR2 (pKd of 8.9 and 8.7, respectively). Hit compounds from the screen include: a 4H-chromen-4-one derivative, 3570–0208, which has an FPR1 pKi of 7, and approximately 100-fold selectivity over FPR2; and an imidazo[1,2-a]pyrimidine derivative, BB-V115, which has an FPR2 pKi of 6.6 and approximately 20-fold selectivity over FPR1 (Pinilla et al., 2013). Interestingly, there are naturally-occurring ligands that can antagonize FPR function. Cyclosporin H (CsH) is a cyclic hydrophobic undecapeptide isolated from fungi, which acts as an inverse agonist at FPR1. Early studies demonstrated that CsH inhibits fMLF binding in Bt2cAMP-differentiated HL-60 cell membranes with a pKi of 7. Furthermore, CsH antagonizes fMLF-mediated increases in GTPase activity, intracellular Ca2+ mobilization and β-glucuronidase release (WenzelSeifert & Seifert, 1993). CsH concentration-dependently decreases [35S]GTPγS binding in Spodoptera frugiperda (Sf9) insect and HEK293 membranes expressing FPR1. These results suggest CsH can behave as an inverse agonist that preferentially stabilizes the inactive form of the receptor. A bacterial-derived 14.1 kDa protein, chemotaxis inhibitory protein of S. aureus (ChIPS), inhibits phagocyte activation and chemotaxis induced by both fMLF and the C5a complement protein fragment. The affinity (pKd) of ChIPS for FPR1 and the C5a receptor is relatively high, 7.5 and 9, respectively. Further, an N-terminal 6 amino acid ChIPS-derived peptide capable of inhibiting fMLF-induced activation of neutrophils, but without effect on the C5a receptors, has also been described (Haas et al., 2004). It has been suggested that the ability of ChIPS to inhibit C5a- and fMLF-mediated activation of phagocytes may confer virulence on S. aureus for invasion of the human host.

D

991 which in turn stimulate chemotaxis of monocytes, neutrophils and Q30992 Q29 HEK293 cells expressing human FPR2 (Hu et al., 2001; Le et al., 1999).

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1058 Q43 1059 1060 1061 1062 1063 1064 Q44 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 Q45 1075 1076 1077 1078 1079 1080 1081 Q46 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 Q47 1094 1095

1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113

1116 1117 1118 Q50


1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184

1204 Q55

3.4. Signal transduction downstream of FPRs

1226 Q56

FPR2, and to a lesser extent FPR1, have received much more attention than FPR3; we have thus restricted our focus here to the signaling cascades downstream of FPR1 and FPR2 activation only. The majority of knowledge regarding signal transduction downstream of FPRs is confirmatory of that already established for ANX-A1 and its peptide mimetics.

1227

3.4.1. FPR1 FPR1 has high affinity for its prototypic ligand fMLF; and its activation of FPR1 triggers a range of different cascades of biochemical events. FPR1 as a chemoattractant receptor on neutrophils mediates their trafficking to sites of infection for invading microorganisms as well as tissue damage. A growing body of evidence suggests that human FPR1 is important for phagocyte activation for bacterial clearance, •O− 2 production, and degranulation of neutrophils (Ye et al., 2009). Mitochondria generate fMLF during injury, triggering FPR1-dependent neutrophil migration and degranulation, in addition to enhancing neutrophil Ca2+ flux, neutrophil activation (MMP-8 and IL-8 release), and neutrophil-mediated organ injury downstream of FPR1 in vivo (Zhang et al., 2010a). Moreover, MAPK signaling pathways are required for FPR1-dependent neutrophil migration (Liu et al., 2012). For example, p38MAPK, which can

1233 Q57

O

F

3.3.3. Phosphorylation of FPRs Work to date examining FPR1 phosphorylation indicates that the intracellular C-terminus is phosphorylated on 6 sites following exposure to fMLF. Although the functional significance of this “phospho-coding” is not yet fully established (Maaty et al., 2013), separate studies manipulating β-arrestin-2 levels have suggested that receptor sequestration from the cell surface to endosomal compartments requires β-arrestins (Gripentrog & Miettinen, 2008), which are known to be recruited to GPCRs following GPCR Kinase (GRK)-dependent phosphorylation. In human neutrophils, the FPR2 C-terminus does not appear to undergo phosphorylation even with high concentrations of fMLF (Maaty et al., 2013), yet exposure of FPR2-transfected HL60 cells and an insulin secreting cell line RINm5F to the FPR2-selective agonist WKYMVm results in extensive phosphorylation (Christophe et al., 2001). The internalization of FPR2 has been shown to be dependent on the phosphorylation of three C-terminus amino acids in WKYMVm-stimulated HEK293 cells transfected with FPR2 (Rabiet et al., 2011). However, the processing of FPR2 requires further investigation in other cell types, and with native ligands, to ascertain whether receptor signaling strength is regulated by GRK-mediated FPR phosphorylation, and also whether such regulation differs among agonists that have opposing effects in some cell types, such as LXA4 and SAA.

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1134 1135

3.3.2. Desensitization of FPRs Prior to the development and characterization of antagonists, the phenomenon of receptor desensitization is often used in attempts to ascertain receptor involvement in responses to agonists that may have multiple receptor targets. Thus, the chemotactic actions of the nascent FPR2 ligand generated by uPA cleavage of its receptor, D2D388-274, are blocked by desensitization with high concentrations of fMLF in THP-1 monocytes (Resnati et al., 2002). This approach is similar to the use of fMLF to desensitize the neutrophil activating actions of SAA in both neutrophils and HEK cells expressing FPR2 (Su et al., 1999). The desensitization of GPCRs such as FPR2 is a feature that may limit the therapeutic utility of targeting by agonists, but there has been so little work on chronic FPR2 agonist treatment that this remains only a potential limitation. Receptor internalization studies in HeLa cells suggest a PKC-dependent FPR2 internalization in response to LXA4 (Maderna et al., 2010); earlier work implicates PI3K in recycling FPR2 to the membrane (Ernst et al., 2004). Until further studies have followed the impact of chronic FPR2 agonist treatment on disease states, detailed elucidation of the potential for FPRs to be desensitized is warranted.

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1132 1133

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1130 1131

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1128 1129 Q53

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1126 Q52 1127

R

1125

R

1123 1124

N C O

1121 1122 Q51

2011). However, it is now known that GPCRs are highly dynamic proteins that fluctuate between many different conformations, each with the potential to mediate diverse intracellular signaling outcomes (Kenakin & Miller, 2010). As such, chemically-distinct ligands acting at the same receptor and in the same cellular background have the potential to stabilize distinct subsets of GPCR conformational states that preferentially couple to distinct functional outcomes; a phenomenon termed biased agonism (Kenakin, 2011). Such biased agonism can be advantageous, as ligands may be developed to selectively stimulate therapeutically relevant signaling pathways over those that contribute to unwanted effects (Kenakin, 2011). A prominent and noteworthy feature of FPR2, the FPR that has received the majority of attention in the drug discovery arena to date, is its interaction with multiple and structurally diverse naturally occurring agonists, including lipids, proteins and peptides. Furthermore, drug discovery programs have also identified a number of synthetic smallmolecular weight compounds that are agonists at FPR2. Structurallydistinct FPR2 agonists have been suggested to mediate biased agonism. For example, LXA4 and SAA appear to mediate anti-inflammatory and pro-inflammatory responses through differential activation of the FPR2, respectively. In human neutrophils, SAA activation of FPR2 promotes the secretion of the pro-inflammatory cytokine IL-8 and to a lesser extent, TNFα. SAA-mediated IL-8 secretion is pertussis toxinsensitive (suggesting a Gi/o mechanism of action), and dependent on intracellular Ca2+ mobilization, ERK1/2 phosphorylation and p38MAPK activation (He et al., 2003). Similar results are observed in HeLa cells heterologously expressing FPR2, supporting the suggestion that SAA can mediate pro-inflammatory effects through this receptor. Interestingly, LXA4, an anti-inflammatory mediator, reduces SAA-mediated phosphorylation of ERK1/2 and p38MAPK and IL-8 levels. It has been suggested that the lack of coupling of LXA4 to FPR2-mediated ERK1/2 phosphorylation in neutrophils may explain the inhibitory effects observed on this cell type. However, the exact mechanisms of biased agonism at the FPR2 that can give rise to the differential, ligand-dependent, intracellular signaling events are currently unknown. A further challenge (and opportunity) in exploiting biased signaling lies in the potential for different cell phenotypes to manifest contrasting bias profiles. For example, LXA4-mediated FPR2-dependent ERK1/2 phosphorylation can be observed in airway (Buchanan et al., 2013) and breast epithelia (Khau et al., 2011), human mesangial cells (McMahon et al., 2000), human lung fibroblasts (Wu et al., 2006), but not in neutrophils (Bae et al., 2003). Ligand bias at FPR2 is likely to result from the recognition of divergent binding domains by structurally distinct agonists. Bena et al. (2012) have used chimeric FPR1 and FPR2 receptors to investigate the regions involved in the binding of Anx-A1, SAA and Compound 43 (Bena et al., 2012). ANX-A1 requires interactions with the N terminus and/or extracellular loop II, SAA recognized extracellulars loop I and II, whereas Compound 43 was suggested to penetrate deeper within the transmembrane domains interacting with extracellular loop I and transmembrane domain II. An additional study has suggested LXA4 predominantly interacts with extracellular loop III and transmembrane domain VII. Combined, these studies suggest that endogenous and synthetic agonists interact with different domains within FPR2. This heterogeneity of binding may explain the ability of structurally distinct ligands to stabilize distinct subsets of FPR2 conformations, resulting in biased agonism. As there are ligands that appear to have opposing effects on cell activation acting through the same receptor to generate distinct and inhibitory signals, it will be useful to ascertain whether such interactions can occur through individual receptors simultaneously binding multiple ligands at the above-described distinct regions of the FPR2. Alternatively or additionally, different populations of receptors engaging with different ligands may interact to determine the signaling outcome. The potential for ligand bias in other members of this family has not been investigated.

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1119 1120

11


1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203

1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225

1228 1229 1230 1231 1232

1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246

1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269

1270 Q58 3.4.2. FPR2 1271 As outlined above, FPR2 (previously also known as ALXR and FPRL-

1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311

4. Role of Anx-A1 and FPRs in myocardial I–R injury

1342 Q59

4.1. ANX-A1 protection in I–R

1343 Q60

The protective actions of ANX-A1 and its peptides have been reported in I–R injury in several vascular beds, including heart, kidney, gut and brain (D'Amico et al., 2000; La et al., 2001a,b; Gavins et al., 2005, 2006, 2007; Peskar et al., 2009; Facio et al., 2011). This evidence has been obtained using both exogenous administration of ANX-A1 and its peptide mimetics, as well as mice deficient in endogenous ANX-A1. For example, after I–R ANX-A1−/− mice exhibit increased leukocyte adhesion in cerebral venules compared to their littermate controls, with larger infarcts and worse neurological scores. Importantly, the large majority of these alterations in inflammation and tissue injury were rescued by exogenous administration of human recombinant ANX-A1 or the short ANX-A1 peptide derivative Ac2-26 (Gavins et al., 2007). Ac2-26 also inhibits neutrophil extravasation and macrophage infiltration into renal epithelial cells in kidney I–R injury, without affecting lymphocyte migration (Facio et al., 2011). As a result, Ac2-26 maintained kidney function, including glomerular filtration rate and urinary osmolality, as well as preventing development of acute tubular necrosis (Facio et al., 2011). Similar protection is seen in the myocardium. as discussed in detail below. ANX-A1 thus offers broad protection against I–R injury in a range of tissues, although much of this evidence to date has focused on its ability to limit inflammatory responses in this context, rather than direct effects on the organ in question (i.e. those potential protective actions independent of circulating inflammatory cells).

1344

4.2. Anti-inflammatory protection of Anx-A1 in myocardial I–R in vivo: key role of FPR2

1367 Q62 1368

ANX-A1 is cardioprotective in both rat and mouse myocardial I–R injury models in vivo (D'Amico et al., 2000; La et al., 2001a,b; Gavins et al., 2005; Dalli et al., 2013). These in vivo studies have focused predominately on the anti-inflammatory actions of Ac2-26 in this context

1369

T

C

E

1283 1284

R

1281 1282

R

1280

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1278 1279

C

1276 1277

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1274 1275

1) has been identified as a receptor for ANX-A1 and its N-terminal peptides (Walther et al., 2000; Perretti et al., 2002). Current evidence indicates that both ANX-A1 and Ac2-26 interact with FPR2 and FPR1 for their bioactivity although perhaps with differences in selectivity (Hayhoe et al., 2006). FPR2 is regarded as somewhat of a promiscuous receptor. Among the ligands, ANX-A1, its N-terminal peptides, and LXA4 bind to FPR2 for their anti-inflammatory bioactivities, while the binding with other agonists such as SAA permits FPR2 to act as proinflammatory receptor, supporting the likelihood that ligand-bias occurs at FPR2, as outlined above (Ye et al., 2009; Li et al., 2011). As a result, ligand interactions with FPR2 are able to promote diverse signaling pathways, including Ca2+ influx as well as activation of PLA2, PI3K, and MAPKs in human neutrophils (Selvatici et al., 2006). SAA binding to FPR2 also triggers a variety of signaling pathways, including Ca 2+ mobilization, chemotaxis and production of both MMPs and pro-inflammatory cytokines (Ye et al., 2009). SAA-stimulated neutrophil recruitment is lost in FPR2-deficient mice (the murine mixed-Fpr2/3 knockout) (Dufton et al., 2010). As alluded to above, post-transcriptional modifications of ANX-A1, via phosphorylation and SUMOYlation, affect its anti-inflammatory mechanisms. Interestingly, it has been suggested that these post-transcriptional modifications may occur downstream of FPR2 and its stimulation by the ANXA1 mimetic, Ac2-26 (Caron et al., 2013; Girol et al., 2013). FPR2 activation can also trigger changes in other signaling pathways. ANXA1 release from apoptotic neutrophils is accompanied by upregulated STAT3 activation and reduced pro-inflammatory cytokine secretion in human monocytes, each of which are FPR2-dependent actions (Pupjalis et al., 2011). Exogenous Ac2-26 peptide similarly regulates STAT3 activation downstream of the FPRs and Janus kianse (JAK) activation in monocytes. This then in turn induces SOCS3 (suppressor of cytokine signaling-3), blunting pro-inflammatory IL-6/TNFα signaling (Pupjalis et al., 2011). As described above, LXA4 is a selective ligand for FPR2, and its activation mediates a range of anti-inflammatory activities including inhibition of leukocyte migration as well as promotion of macrophage phagocytosis of both infective agents and apoptotic leukocytes (Chiang et al., 2006; Ye et al., 2009). In recent years, an endogenous anti-inflammatory loop has also been proposed, in which externalization of ANX-A1 has been suggested as a major downstream effector of both LXA4, and antiflammin 2 (a nonapeptide corresponding to residues 246–254 of full-length ANX-A1). This externalization of

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1312 1313

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ANX-A1 then negatively regulates leukocyte recruitment during inflammation, likely via ANX-A1-FPR2 interaction (Brancaleone et al., 2011). Much of the information regarding the endogenous role of FPR2 has been derived from the development of an FPR2−/− mouse model (Dufton et al., 2010). Although this model is actually deficient in both murine Fpr2 and Fpr3 (i.e. may be considered as an mFpr2/3−/− mouse), mFpr2 and mFpr3 share relatively strong structural homology with human FPR2. The increased susceptibility of this FPR2−/− mouse to some settings of experimental inflammation (including arthritis and mesenteric I–R injury) highlights the anti-inflammatory actions normally mediated by FPR2 activation, at least in these disease contexts (Dufton et al., 2010; Dalli et al., 2013). Responses to both full length ANX-A1 and its peptides, across macrophage ERK1/2 phosphorylation in vitro as well as leukocyte migration in air-pouch and zymosan peritonitis models of inflammation, were also reduced (Dufton et al., 2010; Dalli et al., 2013). In contrast, a reduced severity of ovalbumininduced airway inflammation was observed (in direct contrast to the mFpr2/3−/− mouse above) (Chen et al., 2010). More recently, the same investigators observed that the inflammatory responses in the early stages of experimental colitis were both accelerated and prolonged in the mFpr2−/− mouse, with increased susceptibility to subsequent lethality compared to wild type mice (Chen et al., 2013). The inconsistency is the findings suggests that the inflammation resolving actions of ANX-A1 via FPR may be limited to inflammatory conditions, in which clearance of neutrophils by macrophage is an important aspect for limiting the inflammatory impact of the disease. Taken together, it is thus likely that mouse Fpr2/3 at least (and its human homolog FPR2) is a key receptor for ANX-A1 and its derivatives; whether the same applies to mouse Fpr2 is unknown.

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phosphorylate FPR1, inhibits GRK2-mediated FPR1 desensitization to facilitate neutrophil migration. In contrast, ERK1/2-mediated signals enhance GRK2 activity, in turn, inhibiting FPR1-dependent neutrophil migration. These differential post-receptor mechanisms may thus explain the ability of a number of FPR1 agonists (including ANX-A1and its derivatives), such as Ac2-26 and Compound 43, to act as anti-inflammatory mediators yet elicit ERK1/2 activation (Dufton et al., 2010). Interestingly, Walther et al. (2000) suggesting that the anti-inflammatory actions of ANX-A1-derived peptides observed at lower concentrations may, at much higher concentrations, switch to exhibit pro-inflammatory, neutrophil-activating properties, at least in vitro. In mice, the importance of endogenous FPR1 in host defense has now emerged. Mice deficient in Fpr1 exhibit increased susceptibility to bacterial Listeria monocytogenes infection (Gao et al., 1999) and to experimental induction of colitis (Farooq & Stadnyk, 2012), supporting the general consensus that endogenous FPR1 are indeed anti-inflammatory. Interestingly, there is now evidence in an intestinal mucosal injury model to suggest that ANX-A1-mediated wound repair uses epithelial Nox1-dependent ROS generation downstream of FPR1 (Leoni et al., 2013), although deficiency of FPR1 is protective at earlier stages of diseases in the same model. The role of FPR1 in the development and regression of inflammation at least in the intestine is thus complex and not yet fully resolved.

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Fifteen years ago, we provided the first evidence of the direct protective action of Ac2-26 on the myocardium, independent of inflammatory cells (Ritchie et al., 1999). We have now shown that Ac2-26 completely prevents the response of the myocardium to the inflammatory mediators endotoxin and IFN-γ, on both inotropic responsiveness and induction of inflammatory cyclooxygenase COX2 gene expression, in myocardial preparations in vitro (Ritchie et al., 1999, 2003). Moreover, these effects were not only comparable to cardioprotection with the GC dexamethasone, but the latter were abolished by an antibody to ANX-A1. Importantly, these powerful protective effects were exerted in the absence of neutrophils. Thus, these observations demonstrate that both endogenous ANX-A1 and exogenous Ac2-26 can directly protect the myocardium from cytokine-induced depression, and provide the first evidence that ANX-A1 can improve LV contractile function (Ritchie et al., 2003). Although ANX-A1 is detected in the heart (albeit at low levels constitutively), fibroblasts and vascular endothelium (Dreier et al., 1998), it is markedly increased after I–R and oxidative stress (La et al., 2001). In order to study the potential for neutrophil-independent, cardioprotective actions of Ac2-26, we developed a cardiomyocyte model of the metabolic component of I–R injury, in which adult rat cardiomyocytes are subjected to glucose deprivation and acidosis, prior to recovery in normal medium (Gordon et al., 2003). Ac2-26 protects against metabolic inhibition-induced cardiomyocyte injury, as assessed by cardiac enzyme activity (particularly creatine kinase and lactate dehydrogenase), whether given before, during, or after the insult (Ritchie et al., 2005). Furthermore, these cardioprotective Ac2-26 actions are comparable to the benefit induced by either adenosine, ischemic preconditioning or ischemic postconditioning (Gordon et al., 2003), and are dependent on both FPRs (as demonstrated by sensitivity to Boc2) and cardiomyocyte RISK signaling (Ritchie et al., 2005). Ac2-26 is regarded as a non-subtype selective FPR agonist as discussed earlier (Hayhoe et al., 2006; Dufton et al., 2010; Gavins, 2010). We have now confirmed that the ability of exogenous Ac2-26 to preserve the viability of cardiomyocytes from I–R injury is also evident in a second experimental model, hypoxia–reoxygenation in vitro (Qin et al., 2013). The concentrations of Ac2-26 shown to be effective in our myocardial in vitro studies to date (0.3–1 μM) (Ritchie et al., 2003, 2005; Qin et al., 2013) are in line with the published Kd's for the peptide at FPR1 and FPR2 (Hayhoe et al., 2006; Dufton et al., 2010; Gavins, 2010). There remains a paucity of information regarding the molecular mechanisms utilized by ANX-A1 and its derivatives in the heart. Inhibition of PKC, p38MAPK or ATP-dependent potassium channels (KATP) channels blunts the protective actions of Ac2-26 in cardiomyocytes in vitro (Ritchie et al., 2005), intracellular signals previously implicated in the protective actions of IPC. Thus, exogenous Ac2-26 can directly protect the myocardium from I–R injury, even in the absence of circulating inflammatory cells. Until recently, the only interrogation of the impact of ANX-A1 and its peptide mimetics had been our report in rat papillary muscles studied in vitro (Ritchie et al., 1999, 2003); no attempts at elucidating their impact on contractile function in the intact heart had been sought. Given our promising findings of Ac2-26 cardioprotection in neutrophil-free settings (Ritchie et al., 1999, 2003, 2005), our next step was to demonstrate that Ac2-26 elicited robust preservation of the recovery of LV

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Perretti et al., 2001; Gavins et al., 2003, 2005, 2006; Dufton et al., 2010; Maderna et al., 2010; Dalli et al., 2013). The first report of ANX-A1 cardioprotection by D'Amico et al. (2000) demonstrated that intravenous administration of human recombinant ANX-A1 at the onset of reperfusion following 25 min of left anterior descending (LAD) coronary artery occlusion offered a marked reduction (~50%) in both infarct size and myeloperoxidase (MPO) activity, determined after 2 h reperfusion. ANX-A1 cardioprotection was dosedependent, was abolished by prior protein denaturation and was not mimicked by the structurally-related protein, ANX-A5 (D'Amico et al., 2000). Further, the cardioprotective effects induced by ANX-A1 in this model are associated with reduced recruitment of blood-borne neutrophils into the damaged tissue. Using a chimeric protein comprised of the ANX-A5 core (which itself was without protective effects) and the N-terminal tail of ANX-A1, these investigators demonstrated that this N-terminal region is responsible for the inhibition of inflammatory cell trafficking in this model (D'Amico et al., 2000). This was followed by the report from the same laboratory that two N-terminal-derived ANX-A1 peptides, Ac2-26 and Ac2-12, mimicked the effects of the parent protein in cardioprotection against the same conditions of I–R injury in vivo (La et al., 2001a,b). The shorter ANX-A1 peptide Ac2-6 was however without benefit, as were scrambled versions of the longer peptides. Again, cardioprotection was associated with reduced LV MPO activity and IL-1β levels. Excitingly, Ac2-26 cardioprotection remained effective when administered 60 min into the 2 h reperfusion period, highlighting the translational potential of this work. Ac2-26 was similarly cardioprotective in a mouse model of I–R (Gavins et al., 2005), as was a longer ANX-A1 derived peptide Anx-A12–50 and its cleavage-resistant form CR-Anx-A12–50 (Dalli et al., 2013). Moreover, the non-selective FPR antagonist Boc2 significantly attenuated the cardioprotective actions of Ac2-26 in rodents, implicating the FPR family in the mechanism of ANX-A1 cardioprotection (La et al., 2001a,b; Gavins et al., 2005). The influence of endogenous ANX-A1 and its N-terminal cleavage product may arise from cardiac or non-cardiac sources of ANX-A1. Even though constitutive expression of ANX-A1 in the heart and vascular endothelium is relatively low, ischemia induces ANX-A1 (Dreier et al., 1998; La et al., 2001a,b), further increasing our interest in a potential role for cardiac-derived ANX-A1 in I–R injury. Although a global ANX-A1−/− mouse has been available for a number of years, surprisingly the impact of ANX-A1−/− on cardiac I–R injury in vivo remains to be established. The generation of subtype-selective FPR knockout mice, and of subtype-selective FPR agonists and antagonists, has now enabled further investigation of the mechanisms of ANX-A1 cardioprotection. Gavins and colleagues reported that the cardioprotective actions of Ac2-26 in vivo remained intact in the FPR1−/− mice, implicating FPR2 in ANX-A1 mediated cardioprotection (Gavins et al., 2005), particularly that dependent on neutrophil-mediated injury in the first few hours of reperfusion. Similar studies are however yet to be reported in FPR2−/− mice, or after administration of FPR2-selective antagonists in vivo. Following the early studies with ANX-A1 and its peptide mimetics, Hecht and colleagues recently reported that the FPR2-selective agonist, CGEN-855A, elicts dose-dependent protection against myocardial I–R injury, reducing p-nitro-blue tetrazolium-determined infarct size in mouse (25 min LAD occlusion/2 h reperfusion) and rat heart in vivo (30 min LAD occlusion/3 h reperfusion) by roughly 30% when administered at the onset of reperfusion, with reduced migration of neutrophil to the site of insult (Hecht et al., 2009). Plasma troponin I levels after 2 h reperfusion were also significantly lower in CGEN-855A-treated mice; the cardioprotective effect of CGEN-855A on infarct size in rats was comparable to that elicited by PostCon (Hecht et al., 2009). Similar evidence favoring FPR2 cardioprotection from I–R injury in vivo has recently been described, using the FPR2-selective agonist LXA4. Administration of LXA4, prior to or following 30 min LAD occlusion in adult male

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rats elicited powerful protection from reperfusion-induced arrhythmias, as well as levels of serum cytokines and troponin I after 2 h reperfusion (Zhao et al., 2013). In summary, evidence to date implicates FPR2 in particular as the receptor responsible for the infarct-reducing actions of ANX-A1 and its mimetics in the intact heart. Whether these cardioprotective actions are associated with concomitant preservation of post-ischemic myocardial function remains unresolved.

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Other hypotheses for the mechanism of ANX-A1 actions, separate from its activation of FPRs, have been suggested. The first example relates to the potential for ANX-A1 cleavage. In hearts subjected to I–R injury, endogenous ANX-A1 appears as a characteristic doublet, with bands detected at both 34 kDa and 37 kDa. Treatment with Ac2-26 reduces this doublet such that the 37 kDA band predominates (La et al., 2001). The enzyme responsible for this putative catabolism of ANX-A1 has not been resolved, but the 34 kDa fragment appears to lack antiinflammatory activity (Smith et al., 1990). Perretti's group has reported that an ANX-A1 mimetic that is resistant to this cleavage, CR-AnxA12–50, remains cardioprotective (Dalli et al., 2013). It is possible that peptide Ac2-26 may compete with intact ANX-A1 at the enzyme level, reducing endogenous ANX-A1 catabolism, which may contribute to the net Ac2-26 cardioprotective action. It is also possible that Ac2-26, by reducing leukocyte extravasation, reduces the total amount of

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GCs have been shown to attenuate myocardial I–R injury in animal models in vivo (Enc et al., 2006). However, the dose-dependent systemic side-effects of GCs in relation to the endocrine, cardiovascular, musculoskeletal and immune system remains an ongoing concern, especially for long-term use (Beck et al., 2009). Alternative cardioprotective approaches are thus required. In addition, treatment with the GC methylprednisolone elicited catastrophic impairments in cardiac wound healing and resulted in an increased risk of cardiac rupture (Roberts et al., 1976). ANX-A1, as a downstream mediator of GC, is considered to potentially offer minimal systemic side effects (Perretti and D'Acquisto, 2009). The pro-resolving properties of ANX-A1 (Fig. 1), especially the ability to mediate neutrophil apoptosis and macrophage efferocytosis via FPRs (Chiang et al., 2006), suggests that ANX-A1 might promote the removal of infiltrating neutrophils by macrophages, thereby contributing to the resolution of inflammation (Perretti and D'Acquisto, 2009). Importantly, ANX-A1 not only improves wound healing, but also has limited systemic effects, and thus may offer cardioprotective effects superior to those of existing GC therapies.

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Following myocardial I–R injury, loss of both cardiac contractility and muscle viability are evident. Rescue of cardiac contractile function in addition to preservation of cell viability, could offer an effective therapeutic strategy for myocardial infarction. ANX-A1-based therapies might potentially be seen as a “triple shield” therapy in myocardial I– R injury, limiting neutrophil infiltration and preserving both cardiomyocyte viability and LV contractile function. We propose that ANX-A1 thus represents a novel candidate for treating myocardial inflammatory disorders. Moreover, anti-inflammatory ANX-A1 peptides represent a novel therapeutic approach for several reasons: I–R injury induces myocardial inflammation; ANX-A1 is naturally-occurring; and there is excellent potential to regulate its levels in the body.

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Conflict of interest statement

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ANX-A1 available for protein degradation in the inflamed tissues (Gavins et al., 2006). In addition, as annexins are a family of Ca2+ binding proteins, ANX-A1 may influence cardiac excitation–contractile coupling (Camors et al., 2005; Gavins et al., 2006); myocardial I–R injury impairs this coupling and recovery of LV contractile function is impaired as a result. The precise Ca2+ regulatory proteins involved in cardiac excitation–contraction coupling that are affected by I–R injury and/or ANX-A1 remains to be resolved, but the possibility that a component of the intracellular mechanism of actions of ANX-A1 may be linked to altered Ca2+ handling remains, similar to that associated with the actions of GCs such as dexamethasone (Reilly et al., 1999). Further studies investigating the impact and regulation of both potential mechanisms of ANX-A1 actions in the heart and elsewhere are thus warranted to further understand the potential for the existence of non-FPR mechanisms.

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contractile function in isolated, Langendorff-perfused adult rat and mouse hearts studied ex vivo (Qin et al., 2013). Following 30 min global ischemia with 30 min reperfusion, parameters of LV function were reduced to ~60% of their pre-ischemic values in untreated rat hearts; similar dysfunction was evident in the mouse heart following 22.5 min global ischemia with 40 min reperfusion. Administration of Ac2-26 from the onset of reperfusion (pharmacologically mimicking PostCon) significantly restored LV function comparable to pre-ischemic values within 10–20 min after the start of reperfusion in both models (Qin et al., 2013). Interestingly, Ac2-26-mediated cardioprotection was abolished by cotreatment with either the FPR-nonselective antagonist Boc-2 or the FPR1-selective antagonist CsH, but was relatively insensitive to the FPR2-selective antagonist Quin-C7, suggesting that the activation of FPRs, and perhaps FPR1 rather than FPR2, may be implicated in the contractile function preservation of Ac2-26 (Qin et al., 2013). Ac2-26 cardioprotection was also associated with preservation of Akt activity and enhanced phospholamban phosphorylation. We have detected all 3 FPR subtypes in mouse, rat and human heart: FPR1 is the major subtype present (by approximately 5-fold) in these 3 species, relative to both FPR2 and FPR3, which have a much lower level of constitutive expression (Qin and Ritchie, unpublished observations). This is in direct contrast to inflammatory cells such as neutrophils and macrophages, where FPR2 expression is higher than FPR1, and FPR3 is barely detectable. We anticipate however that in the heart subjected to myocardial I–R injury in vivo, where circulating inflammatory cells are recruited to the site of injury, that FPR2 (whether upregulated in cardiomyocytes by the insult or introduced to the heart by these infiltrating cells) would likely contribute to myocardial viability and function. Interestingly in the normoxic rat heart, Ac2-26 is not a potent inotrope: for example, LV developed pressure and LV + dP/dt (parameters of systolic function) are modestly enhanced, by approximately 20% (Qin et al., 2013). Lastly, these studies of the role of ANX-A1 mimetics have also revealed that endogenous ANX-A1 contributes significantly to the maintenance of normal cardiac physiology. ANX-A1−/− mice exhibit a marked exaggeration of the myocardial I–R injury response across a range of parameters of LV contractile function, accompanied by reduced phosphorylation of the cell survival kinase Akt and a marked downregulation of LV FPR1 (but not FPR2) expression (Qin et al., 2013). There is no impact of ANX-A1 deficiency on cardiac function at baseline per se in the absence of a stressor, nor is the response to I–R injury a sexspecific phenomenon. Thus, together with the previous reports in vivo (D'Amico et al., 2000; La et al., 2001a,b; Gavins et al., 2005; Dalli et al., 2013), these results clearly demonstrate that ANX-A1 and Ac2-26 can prevent myocardial injury even in the absence of neutrophils (Ritchie et al., 1999, 2003, 2005; Qin et al., 2013), and further support the development of ANX-A1 derivatives as potential treatments for the clinical management of ischemic cardiomyopathy.

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The authors declare that there are no conflicts of interest.

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Acknowledgments

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This work was supported in part by National Health and Medical Research Council (NHMRC) of Australia project grants, including ID1045140 (to RHR, XMG and YHY) and ID1067547 (to AGS), and supported in part by the Victorian Government's Operational Infrastructure Support Program. RHR is an NHMRC Senior Research Fellow (ID472673 and ID1059960).

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Cardioprotective Potential of Polyphenolic Rich Green Combination in Catecholamine Induced Myocardial Necrosis in Rabbits.

Cardioprotective effects of traditional Chinese medicine Guanmaitong on acute myocardial infarction.

Caspase inhibition via A3 adenosine receptors: a new cardioprotective mechanism against myocardial infarction.

Cardioprotective actions of Notch1 against myocardial infarction via LKB1-dependent AMPK signaling pathway.

Potential of lipoproteins as biomarkers in acute myocardial infarction.

beta-adrenoceptor blocker, carvedilol, in two models of myocardial infarction in the rat.

Cardioprotective effect of fimasartan, a new angiotensin receptor blocker, in a porcine model of acute myocardial infarction.

Randomised controlled trial of cardioprotective diet in patients with recent acute myocardial infarction: results of one year follow up.