Cardiovascular diseases (CVD) are the main cause of morbidity and mortality worldwide and were solely responsible for the death of about 17.7 million people in 2015, with the vast majority being due to either ischemic heart disease (IHD) or cerebrovascular disease. This has led to the exponential increase in healthcare costs with estimated increase in total medical care costs in United States (US) alone rising from $396 billion to $918 billion between the years 2012 and 2030. Additionally, the burden of CVDs has increased tremendously in developing countries and are predicted to overtake infectious disease as the leading cause of mortality by the year 2020.
Many acute cardiovascular events, such as acute ischemic stroke (AIS) and myocardial infarction (MI), are due to, e.g., unhealthy vascular states, resulting from prolonged atherosclerosis, coronary artery disease, diabetes, uncontrolled hypertension, and aging. Additional studies have suggested behavioral, psychosocial including bereavement and environmental factors can also trigger and contribute to acute cardiovascular events.
Both AIS and MI are life-threatening conditions which require prompt treatment consisting of rapid restoration of blood flow followed by subsequent management consisting of anti-inflammatory strategies and neuro- and cardio-protection for secondary prevention. However, restoration of blood supply and re-oxygenation paradoxically lead to further exaggeration of tissue injuries and tissue destruction (termed ischemia–reperfusion injury (I/RI)). To restore homeostasis, it is essential that the ensuing inflammatory and thrombotic environment is resolved. Resolution involves a tightly orchestrated series of active events and is now accepted as one of the four major outcomes for acute inflammation, the others being progression to chronic inflammation, scarring, and fibrosis. Several anti-inflammatory mediators which have pro-resolving properties have been proposed over the years, such as resolvin D1 (RvD1), RvD2, RvE1, maresins, protectin D1 and lipoxin A4 (LXA4). Another known anti-inflammatory and pro-resolving mediator, which is the focus of this review, is the glucocorticoid-regulated protein Annexin A1 (AnxA1). AnxA1 has a particular appeal for drug discovery programs as it is founded on endogenous anti-inflammatory molecular pathways and as such is attractive because it can mimic the body’s own pro-resolution effects while potentially having fewer side effects than existing therapeutic agents. This review aims to highlight the issues associated with I/RI. We also focus on AnxA1, its history, mechanism of action and its role in I/RI in different organs including brain, heart, kidney and gastrointestinal tract. Finally, we discuss the potential of AnxA1 in drug discovery programs for the treatment of I/RI.
2. Ischemia Reperfusion Injury (I/RI)
I/RI is a fundamental vascular pathobiological paradigm that contributes to the pathophysiology of various acute CVDs such as IHD and AIS, along with other pathophysiological responses such as transplantation and acute kidney injury (AKI). I/RI occurs in two distinct phases: the initial ischemic insult caused by vascular occlusion, and the subsequent reperfusion phase manifested by a profound local and systemic inflammatory response (called “reperfusion injury”). The term “ischemia” refers to deficient blood supply to tissues due to obstruction of the arterial inflow. The severity of cell dysfunction is influenced by the extent and duration of obstruction. Treatment for ischemic events is always restoration of blood flow to the affected organ at the earliest time possible (with some organs such as the brain being far more susceptible to damage: “time is brain”). However, reperfusion itself can induce and exaggerate tissue injury because it results in a gamut of inflammatory mediators including reactive oxygen species (ROS), increased cell adhesion molecules (e.g., intracellular adhesion molecule-1 (ICAM-1)), heightened production of lipid mediators including leukotriene B4 (LTB4) and platelet activating factor (PAF).
A wide range of pathological processes contribute to I/R-associated tissue damage. Prolonged occlusion triggers depletion of adenosine triphosphate (ATP) and decreases the intracellular pH as a result of anaerobic metabolism and lactate accumulation. ATP depletion inactivates ATPases (e.g., Na+/K+ ATPase), reduces active calcium ion (Ca2+) efflux and limits the reuptake of Ca2+ by the endoplasmic reticulum (ER), thereby producing calcium overload in the cell. These changes are accompanied by opening of the mitochondrial permeability transition (MPT) pore, which dissipates the mitochondrial membrane potential and further impedes ATP production. Depletion of ATP also leads to accumulation of xanthine oxidase (XO) which is formed from xanthine dehydrogenase (XD) under hypoxic conditions, allowing for a burst of superoxide and hydrogen peroxide production and initiating the overall I/RI. The re-oxygenation in reperfusion phase enhance the production ROS. During ischemic phase, electron-transport enzymes, including mitochondrial respiratory chain, are damaged. Therefore, incomplete oxidation of molecular oxygen occurs leading to generation of more ROS and exaggerating tissue injury.
An immune response elicits upon reperfusion and consists of both innate and adaptive immune systems. The innate immune response involves signaling events through pattern recognition molecules (especially toll like receptors (TLRs)). Ligands such as components of damaged tissue binding to TLRs lead to the activation of downstream signaling pathways, including NF-kappa-B (NF-κB), mitogen activated protein kinase (MAPK) and type I interferon pathways, resulting in the induction of pro-inflammatory cytokines and chemokines. Upon reperfusion, the ischemic area acquires multiple activated leukocyte types (e.g., neutrophils, monocytes, and lymphocytes), dominated initially by neutrophil infiltration. I/RI also activates antigen-specific T cells by mechanisms that are not yet well characterized. Recent evidence suggests both antigen-specific and independent mechanisms of activation where T cells accumulate at sites of ischemia and reperfusion. Multiple studies have shown that enhanced generation of oxidants results in the activation and deposition of complement and phospholipase A2 (PLA2)-mediated production of LTB4 and platelet activating factor (PAF). Furthermore, there is abundant evidence suggesting the role of platelets in I/RI by mediating microvascular thrombosis as well as ensuing inflammation through platelet–platelet as well as platelet–leukocyte interactions. These communications lead to the release of a broad range of pro-inflammatory molecules including high-mobility group box 1 protein (HMGB1), cell differentiation 40 ligand (CD40L), PolyP, and interleukin-1 alpha/beta (IL-1α/β) furthering the thrombo-inflammatory environment and contributing in both innate and adaptive immune responses.
AnxA1 and the Formyl Peptide Receptors (FPRs)
AnxA1 and its mimetic peptides, such as the N-terminal derived Ac2-26, bind to the formyl peptide receptor (FPR) family of seven transmembrane G-protein-coupled receptors (GPCRs). Various cell types express FPRs, especially myeloid cells, e.g., neutrophils and monocytes. Three FPR members exist in humans and they are termed: FPR1, FPR2/ALXR (also known as the LXA4 receptor), and FPR3. FPR2/ALXR shares 69% amino acid sequence homology with FPR1, and FPR3 shares 56% amino acid homology to FPR1 and 72% to FPR2/ALX. In mice, the FPR family is more complex, consisting of at least eight members. Mouse Fpr1 shares 77% sequence homology with human FPR1, and Fpr2 has 76% homology to FPR2/ALXR.
The FPRs are primarily coupled to G proteins (GIα2, GIα3). Upon the binding of a ligand, such as formyl-Met-Leu-Phe (fMLP), G proteins are activated and trigger the release of several second messengers such as Ca2+ from intracellular stores, through activation of phospholipase C (PLC), PLD and PLA2. Neutrophils predominantly sense inflammatory stimuli via FPRs to perform both pro- as well as anti-inflammatory functions, depending upon the pathophysiological status and ligand binding. In inflammatory states, neutrophil FPRs participate in various biological functions including chemotaxis, degranulation, ROS production, promoting neutrophil–platelet interactions, and enabling apoptosis and phagocytosis. The ability of FPRs to perform such wide range of biological functions is due to their ability to interact with multitude of agonists and antagonists, ranging from formylated and non-formylated proteins/peptides to small molecular weight compounds, e.g., fMLP, His-Phe-Tyr-Leu-Pro-Met (HFYLPM) (chemoattractants for monocytes and neutrophils), AnxA1, and HIV envelope proteins gp41 and gp120. A detailed description of the FPRs, their ligands and biological functions is given in Table 1 and Table 2.
Among the FPR members, FPR2/ALX is the most versatile and can interact with a multitude of ligands resulting in both anti-inflammatory and pro-resolving (AnxA1 and LXA4) as well as pro-inflammatory (serum amyloid A (SAA) and cathelicidin) functions (Table 2). These diverse effects also seem to be partly due to the ability of FPR2/ALX to facilitate biased agonism and enable different dimerization states after ligand binding. Binding of SAA and/or the cathelicidin-associated antimicrobial peptide leucine-37 (LL37) to FPR2/ALX results in neutrophil NF-κB activation, cytokine release, increased neutrophil infiltration and lifespan. However, binding of anti-neuronal nuclear antibody (AnnA1) inhibits neutrophil infiltration, promotes neutrophil apoptosis, and pushes macrophages towards a less pro-inflammatory phenotype, increasing the rate of phagocytosis by macrophages. Ongoing studies are showing that FPR1 signaling is associated with neutrophil oxidative burst by inducing a rapid and reversible increase in the mitochondrial membrane potential within neutrophils. In addition, mitochondrial-derived FPR1 ligands also function as chemotactic damage-associated molecular pattern molecules (DAMPs also known as alarmins or danger signals), activating the innate immune system.
The function of FPR3 is less well characterized, although it is expressed on eosinophils, monocytes, macrophages, and dendritic cells. Evidence suggests that FPR3 plays role in allergic reaction and dendritic cell maturation.
The Role of Annexin A1 in AIS
AnxA1 has been shown to act in various ways to mitigate cerebral I/RI. Our work has shown a significant effect of AnxA1 (and its peptide mimetics) on diminishing leukocyte adhesion and trafficking (mainly by acting in an in an autocrine/paracrine fashion to decrease leukocyte–endothelial interactions), inhibiting the release of pro-inflammatory and pro-thrombotic cytokines, and regulating neutrophil–platelet interactions. Human recombinant AnxA1 and its peptide mimetics have shown to markedly reduce the lesion size, clinical score and markers of leukocyte infiltration in murine middle cerebral artery occlusion (MCAo) model. Recently, we showed that exogenous administration of AnxA1 N-terminal peptide Ac2-26 attenuated neutrophil and platelet activation and neutrophil–platelet aggregation in the murine cerebral microvasculature after induction of cerebral I/RI in mice. Ac2-26 has also been shown to be protective in rats, as demonstrated by intracerebroventricular administration of the peptide reducing infarct size and cerebral edema two hours post cerebral ischemia. McArthur et al. demonstrated that animals lacking the AnxA1 receptor Fpr2/ALX (i.e., Fpr2/ALX knock-out or Fpr2/ALX−/− mice) showed markedly greater BBB leakage post-ischemia than their wild-type (WT) counterparts. Additionally, AnxA1 plays a role in mediating the permeability of the BBB. Cristante et al. showed that AnxA1 knock-out (AnxA1−/−) mice possess a disorganized actin cytoskeleton due to increase in activity of RhoA small GTPase and a disruption of occludin and VE-Cadherin. Furthermore, it is not just blood cells that express Fpr2/ALX, but also brain resident cells such as Microglia and as such are potential targets for the anti-inflammatory and pro-resolving effects of Ac2-26 and its parent compound.
The Involvement of AnxA1 in IHD
AnxA1 and its N-terminal peptides have both been shown to be cardioprotective in various I/RI models. D’Amico et al. demonstrated a decrease in infarct size (50%) upon infusion of recombinant AnxA1 and the protective effects of AnxA1 were further confirmed by La et al. as evidenced by the administration of the AnxA1 N-terminal peptide, Ac2-26, to decrease infarct size and reduce MPO and IL-1β content in infarcted hearts. Treatment with AnxA1 also attenuated loss of fiber organization, decreased MPO activity, reduced TNF-α and macrophage inflammatory protein (MIP-1α) levels and leukocyte extravagation in cardiac tissues. Peptide Ac2-26 has been shown to preserve cardiomyocyte contractile function and reduce cardiac myocyte injury, with protection, at least in part, being associated with activation of PKC, p38-MAPK and KATP channels. Additionally, Qin et al. demonstrated that exogenous administration of Ac2-26 at the onset of reperfusion increased cardiomyocyte viability and recovery of LV function, which was associated with FPR1/Akt signaling.
Hematopoietic progenitor stem cell (HPSC) mobilization and differentiation has been shown to be regulated by AnxA1, e.g., AnxA1 deficiency results in increased cardiac necrosis, inflammation, hypertrophy and fibrosis resulting in exaggerated cardiac infarct size after eight days of myocardial infarction. These effects were linked to a greater expansion and altered mobilization of HPSC, with increased circulating neutrophils and platelets and altered macrophage inflammatory phenotype and function.
More recently, there has been a large focus on the pharmacological concept of biased-agonism (multiple active conformations of a receptor exist and elicit distinct signals yielding to multiple functional outcomes). The small-molecule FPR1/FPR2/ALX agonist, Compound 17b (Cmpd17b), has been shown to have biased agonism, by enabling FPR based pro-resolving biology without changing FPR1/2-mediated calcium mobilization, hence providing superior cardio-protection. Cmpd17b, similar to AnxA1, attenuates both early and late inflammatory responses after reperfusion in MI, with ERK1/2–Akt kinases seeming to play a significant role.
The Association of AnxA1 in Renal I/RI
The role of AnxA1 in kidney I/RI is in its nascent stage. However, some studies have shown that AnxA1 may exert a protective role, e.g., in a rat model of renal I/RI, exogenous administration of peptide Ac2-26 reduced: (a) tubular necrosis; (b) the influx of phagocytic cells (neutrophil and monocytes); (c) sodium and potassium excretion; and (d) GFR, thereby improving functional recovery and outcome.
Cyclosporine is an immunosuppressant and is used to avoid organ transplant rejection. The major side effect of Cyclosporine treatment is nephrotoxicity, as the compound decreases renal blood flow and increases renal vascular resistance (both common conditions in renal I/RI). However, exogenous administration of Ac2-26 has been shown to reverse these side effects.
Diabetes is metabolic syndrome which affects many organs including the kidneys, with 30–40% of diabetic patients developing nephropathy. AnxA1 has been shown to afford protection in murine diabetic nephropathy by decreasing p38, ERK and JNK, and activating Akt signaling, suggesting that AnxA1 could be a potential therapeutic strategy for treating renal dysfunction caused by diseases such as diabetes.
The Protective Role of AnxA1 in GI-Associated I/RI
AnxA1 has been shown to play a protective role in gastrointestinal I/RI. We have shown that intravenous administration of Ac2-26 inhibits leukocyte adhesion and emigration in a mouse mesenteric I/RI model, as induced by clamping of the superior mesenteric artery, followed by reperfusion. The loss of epithelial cell barrier is major characteristic of intestinal I/RI. The effect of AnxA1 or its peptides on epithelial cell barrier has not been studied in intestinal I/RI models but they have afforded protection in other gastrointestinal inflammatory murine models including colitis and gastric ulcers. Leoni et al. demonstrated that Ac2-26, when delivered locally in the intestine using polymeric nanoparticles, enhanced the epithelial cell restoration in colitis. Martin et al. showed the positive effect of peptide Ac2-26 in enhancing the healing process of gastric mucosal damage and reducing the ulcer areas. Furthermore, this same group also demonstrated that hemorrhagic lesions were greater in AnxA1−/− mice upon induction of gastric ulcers. Leoni et al. suggested that AnxA1 exhibits an FPR1/NOX1-dependent positive role in intestinal wound healing: upon binding to FPR1, AnxA1 activates Src and downstream NOX-1. ROS produced by NOX-1 causes rapid and reversible oxidation and subsequent inactivation of phosphatase PTEN and PTP-PEST. This inactivation of phosphatases leads to activation of focal adhesion protein involved in cell movement, FAX and Paxillin, thereby increasing the intestinal epithelial cell movement and wound repair.
Intestinal I/RI is also a major cause of acute lung injury (ALI). Treatment with exogenous Ac2-26 has been shown to reduce pulmonary vascular leakage, decrease neutrophil infiltration and MPO content in lung tissues following intestinal I/RI, possibly by inducing release of anti-inflammatory cytokine IL-10 and decreasing the release of pro-inflammatory cytokine TNF-α. Babbin et al. also showed in a dextran-sulfate sodium (DSS)-induced colitis model that AnxA1 was able to regulate intestinal mucosal injury and inflammation via engagement with FPR2/ALX. In addition, AnxA1 deficient mice failed to regain weight and showed no improvement in disease activity index and mucosal injury upon withdrawal of disease in the DSS-induced colitis model, advocating the crucial and beneficial role of AnxA1.
8. Concluding Remarks
In summary, CVDs are the leading cause of morbidity and mortality worldwide, with MI and IS being the primary causes of death. The majority of CVD related complications are due to I/RI. Hence, it has become imperative to develop preventative as well as therapeutic strategies to mitigate these life-threatening complications. In this review, we focus on AnxA1 and its peptide mimetics as possible therapeutic strategies for the treatment of I/RI, based on their ability to alleviate thrombosis and inflammation and promote resolution (Figure 1). Indeed, drug development programs focused on endogenous anti-inflammatory and pro-resolving agents, such as AnxA1-based pharmacologic strategies, offer great therapeutic potential as they are devoid of metabolic side effects because they mimic the way inflammation naturally subsides in the body. However, the development of novel therapeutics should also be mindful of ascertaining not only if the drug is anti-inflammatory, but also if it is resolution-toxic (i.e., deranges or impairs timely and/or complete resolution), which could ultimately outweigh the overall therapeutic efficacy in treating I/RI and other inflammatory disorders.