Macrophages in myocardial infarction

Abstract

The heart contains a population of resident macrophages that markedly expands following injury through recruitment of monocytes and through proliferation of macrophages. In myocardial infarction, macrophages have been implicated in both injurious and reparative responses. In coronary atherosclerotic lesions, macrophages have been implicated in disease progression and in the pathogenesis of plaque rupture. Following myocardial infarction, resident macrophages contribute to initiation and regulation of the inflammatory response. Phagocytosis and efferocytosis are major functions of macrophages during the inflammatory phase of infarct healing, and mediate phenotypic changes, leading to acquisition of an anti-inflammatory macrophage phenotype. Infarct macrophages respond to changes in the cytokine content and extracellular matrix composition of their environment and secrete fibrogenic and angiogenic mediators, playing a central role in repair of the infarcted heart. Macrophages may also play a role in scar maturation and may contribute to chronic adverse remodeling of noninfarcted segments. Single cell studies have revealed a remarkable heterogeneity of macrophage populations in infarcted hearts; however, the relations between transcriptomic profiles and functional properties remain poorly defined. This review manuscript discusses the fate, mechanisms of expansion and activation, and role of macrophages in the infarcted heart. Considering their critical role in injury, repair, and remodeling, macrophages are important, but challenging, targets for therapeutic interventions in myocardial infarction.

INTRODUCTION

The term macrophage was introduced in the 19th century by the Russian scientist Ilya (Elie) Metchnikoff, one of the pioneers of modern immunology, to describe a specialized cell type involved in detection and phagocytic ingestion of microorganisms that invade the body (1). Over a century later, our understanding of the role of macrophages in homeostasis and disease has expanded. Macrophages have been identified in most tissues and were found to exhibit remarkable heterogeneity. In addition to their central involvement in phagocytosis of microbes and dead cells, macrophages have also been suggested to play a broad range of roles in development, homeostasis, and disease (2). Following injury, macrophage subsets have multiple functions, not only acting as professional phagocytes, but also serving as regulators of inflammation, fibroblast activation, angiogenesis, and extracellular matrix remodeling.

In addition to cardiomyocytes, the contractile cells of the heart, the myocardium also contains large populations of vascular cells, fibroblasts and immune cells. Early descriptive studies have identified macrophages in normal human and rodent hearts (3, 4), and have documented their expansion following cardiac injury (5). Over the last twenty years, there has been an explosion in our understanding of the role of cardiac macrophages in homeostasis and disease. Important functions of normal cardiac resident macrophages have been revealed. Our knowledge on the origin and fate of macrophages in injured, healing, and remodeling hearts has been enriched. Key macrophage-activating molecular signals have been identified, and relations between the phenotypic transitions and functional roles of cardiac macrophages in cardiac repair and remodeling have been studied. This review manuscript deals with the role of macrophages in the most common form of myocardial injury, myocardial infarction. We discuss the mechanisms of expansion and activation of macrophages in the infarcted heart, and we identify macrophage-derived molecular signals involved in myocardial injury, repair, and remodeling. Finally, we discuss the challenges and opportunities of macrophage targeting in patients with myocardial infarction.

MACROPHAGES IN HEALTHY HEARTS

There are conflicting data on the abundance of myocardial macrophages in healthy mammalian hearts: some studies show a low density of cardiac macrophages (6, 7), whereas other investigations (8) suggest that large numbers of macrophages populate the myocardium. The conflicting findings likely reflect differences in the species, age, and genetic background of the subjects studied, but also different criteria and methodological approaches used for macrophage identification. Quantitative analyses in mouse and human hearts using flow cytometric and single cell transcriptomic approaches have produced more consistent results. In young adult mouse hearts, flow cytometry showed that leukocytes comprise ∼7%–10% of noncardiomyocytes; the majority of these cells were identified as macrophages (9, 10). On the other hand, single cell RNA-sequencing studies in human hearts obtained from 14 deceased organ donors (40–75 yr of age) suggested that macrophages represent <5% of ventricular myocardial cells (11).

Macrophages residing in normal hearts exhibit remarkable phenotypic and functional heterogeneity. In mice, distinct C-C chemokine receptor (CCR)2⁺ and CCR2⁻ resident macrophage populations have been identified. CCR2⁻ cells are derived from the primitive yolk sac and fetal monocyte progenitors, enter the heart during embryonic development, and their maintenance during adult life is not dependent on monocyte recruitment (12, 13). In contrast, CCR2⁺ resident macrophages are derived from hematopoietic progenitors, are recruited in the heart during the first weeks of life, and are replenished through recruitment of circulating monocytes. Studies in human hearts demonstrated similar CCR2⁺ and CCR2⁻ macrophage subpopulations (14).

Several experimental studies have suggested important roles of myocardial macrophages in cardiac development and homeostasis (Table 1). In the developing heart, CCR2⁻ macrophages have been implicated in remodeling of the primitive coronary plexus, a process required for coronary artery formation (15). On the other hand, macrophage-derived hyaluronan has been suggested to play an important role in formation of cardiac lymphatics (18). In addition to their involvement in cardiac development, macrophages have been suggested to play an important role in maintaining normal cardiac homeostasis and function. An elegant and intriguing study in mice suggested that resident macrophages serve to clear ejected mitochondria and other vesicular material secreted from cardiomyocytes, thus preventing extracellular accumulation of waste (16). The authors interpreted the findings as suggestive of a crucial role for macrophages in protecting the myocardium from systolic and diastolic dysfunction under homeostatic conditions. However, the observed effect of macrophage depletion on cardiac output was driven by changes in ventricular volumes, and not by any effects on ejection fraction, raising the possibility that the findings may be due to extracardiac effects of macrophage depletion. Unfortunately, the limited analysis of cardiac and vascular pathology performed in that study precludes definitive conclusions on the direct impact of macrophages in preserving cardiac structure and function. In another seminal study, macrophages were implicated in normal electrical conduction. Macrophage depletion experiments in mice suggested that myocardial macrophages may participate in conduction of the electrical impulse through the atrioventricular node (17). The significance of this mechanism in human homeostasis and disease is unclear.

Table 1.

Functions of cardiac macrophages in development and homeostasis of the mammalian heart

Function/Process	Findings	Method of Documentation	Mechanism of Action	Refs.
Coronary arteriogenesis	CCR2− macrophages are involved in coronary arteriogenesis	Studies in macrophage-deficient Csf1op/op mice and in CCR2−/− mice	The angiogenic effects of the macrophages were attributed to IGF secretion	(15)
Cardiomyocyte function	Cardiac macrophages are involved in preservation of metabolic stability and homeostatic function of cardiomyocytes	Macrophage depletion using the CD169DTR model	Homeostatic effects of macrophages were attributed to phagocytosis and removal of dysfunctional mitochondria ejected by cardiomyocytes	(16)
Electrical conduction	Resident cardiac macrophages facilitate conduction of the electrical impulse	Macrophage depletion using CD11bDTR and Csf1op/op mice, Macrophage-specific Cx43-deficient miceUsing the CX3CR1 Cre driver	Macrophages facilitate atrioventricular condition through coupling to conducting cardiomyocytes via connexin-43-containing gap junctions	(17)
Lymphangiogenesis	Cardiac macrophages interact with lymphatic capillaries and play a role in lymphatic vessel growth and patterning during development	Myeloid cell-deficient PU.1−/− mice , Macrophage depletion using a genetic diphtheria toxin-based model (Cx3cr1CreER/+;R26R-DTA mice)	Effects of macrophages were attributed to hyaluronan-dependent interactions with lymphatic endothelial cells	(18)

CCR, C-C chemokine receptor; CSF1, colony stimulating factor 1; CX3CL, C-X3-C chemokine ligand.

MACROPHAGES IN MYOCARDIAL INFARCTION

Myocardial infarction is caused by sudden occlusion of a coronary artery, due to rupture or erosion of a vulnerable atherosclerotic plaque. Abundant macrophages accumulate in plaques and play a central role in all stages of atherosclerosis, from initiation and expansion of the lesions to formation of the necrotic core that leads to rupture (19, 20). Moreover, macrophages have also been implicated in regression of atherosclerotic plaques. The role of macrophages in atherosclerotic plaques has been systematically discussed in several excellent reviews (21–25). Although design of therapies targeting macrophages in patients with acute myocardial infarction requires consideration of effects on plaque stability, our discussion will only focus on the functions and roles of macrophages in the infarcted myocardium.

Repair of the Infarcted Heart

The adult mammalian heart has negligible regenerative capacity and heals through formation of a collagen-based scar. Although the scar does not contribute to ventricular contraction, its formation plays a critical role in protecting the heart from catastrophic rupture. Moreover, a well-orchestrated reparative response prevents infarct expansion and attenuates adverse ventricular remodeling, a process associated with chamber dilation, progressive dysfunction and a high incidence of heart failure and arrhythmias. Thus, perturbations in the reparative response can lead to formation of a scar with low tensile strength, contributing to ventricular dilation and to the development of heart failure.

Repair of the infarcted heart can be divided into three distinct, but overlapping phases: the inflammatory phase, the proliferative phase and the maturation phase (26). Macrophages are central cellular effectors of the injurious and reparative cellular responses in the infarcted heart. During the early stages of injury, initiation of the postinfarction inflammatory response is dependent on macrophage activation. Induction of proinflammatory cytokines and chemokines triggers recruitment of abundant monocyte-derived macrophages in the infarcted heart. Infiltrating macrophages serve as professional phagocytes, clearing the infarct from dead cells and matrix debris. Ingestion of apoptotic cells by macrophages stimulates anti-inflammatory cascades, leading to suppression of inflammation and transition to the proliferative phase, which is characterized by infiltration of the infarct with activated matrix-synthetic myofibroblasts. Infarct macrophages play an important role in fibroblast and myofibroblast activation and also promote angiogenesis, which is necessary for supply of oxygen and nutrients to the metabolically active granulation tissue. Deposition of an organized network of collagenous matrix in the infarct leads to the maturation phase, associated with matrix cross-linking and with reduction of the infarct macrophage population. As the infarct heals, the noninfarcted segments exhibit hypertrophy and are subjected to pressure and volume loads that may trigger macrophage activation and subsequent stimulation of a fibrogenic program. Macrophages undergo dramatic phenotypic transitions during the three phases of cardiac repair and critically regulate inflammation, repair, matrix remodeling, angiogenesis and fibrosis.

Macrophages during the Inflammatory Phase of Cardiac Repair

The role of macrophages in initiation of inflammation following myocardial infarction.

Prolonged ischemia induces irreversible injury in cardiomyocytes, leading to their death. In mammals, a 15–20 min interval of coronary occlusion is sufficient to cause death of the most vulnerable of cardiomyocytes in the subendocardium (27, 28). Prolonged ischemia stimulates a wavefront of necrosis that extends towards the subepicardium, ultimately leading to a transmural infarct after 4–5 h of coronary occlusion (29). Necrotic cells release a wide range of damage-associated molecular patterns (DAMP) in the extracellular environment (such as HMGB1, HSPs and S100 proteins) (30–32); these mediators inform the surrounding cells of the injury, and activate downstream inflammatory and reparative cascades (33). All myocardial cells respond to these danger signals through activation of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) (34, 35), and activate downstream proinflammatory cascades. Although resident cardiac mast cells (36), cardiomyocytes (37), and vascular endothelial cells (38) have also been implicated in activation of postinfarction inflammation, several lines of evidence support a critical role for resident macrophages. First, the CCR2+ subpopulation of cardiac macrophages was found to stimulate leukocyte influx in the infarcted heart through pathways involving the cytosolic adaptor protein myeloid differentiation primary response 88 (MYD88) (39), a critical component of TLR- and IL-1-mediated responses. In contrast, CCR2⁻ resident macrophages restrained inflammatory cell recruitment (39). Second, activation of interferon regulatory factor (IRF)3 in macrophages has been implicated in induction of inflammatory cytokines and downstream recruitment of leukocytes (40). Third, activation of the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) in cardiac macrophages by phagocytosed cellular debris was found to activate IRF3, inducing inflammatory cytokine synthesis (41).

What is the fate of resident macrophages in the infarcted heart?

Studies investigating death of myocardial cells in response to an ischemic insult have traditionally focused exclusively on cardiomyocytes (42), ignoring the effects of ischemia on cell survival pathways in other cell types. Thus, our knowledge on the fate of resident cardiac macrophages following infarction is limited and is based predominantly on speculation. Parabiosis experiments suggested that resident cardiac macrophages disappear within 24 h after coronary occlusion; some descriptive data suggested that their depletion may be attributed, at least in part, to death (9). However, robust evidence supporting this notion is lacking. It is plausible to hypothesize that the fate of resident cardiac macrophages may be also dependent on the timing of reperfusion. Macrophages may be more resilient than cardiomyocytes to ischemic insults; thus, with early reperfusion a larger fraction of resident cardiac macrophages may survive. To what extent survival of resident reparative macrophages in reperfused infarcts may contribute to the beneficial actions of early reperfusion is unknown.

Dynamics of macrophage infiltration in the infarcted heart.

Depletion of resident macrophages during the inflammatory phase of infarct healing is accompanied by recruitment of an abundant population of circulating monocytes (9, 12, 43). In the cytokine-rich environment of the infarct, which contains large amounts of colony stimulating factor (CSF)-1 (43), a growth factor with a central role in macrophage expansion and differentiation (44), infiltrating monocytes differentiate into macrophages, thus replacing the resident macrophage population. Monocytes comprise 10% and 4% of human and mouse blood leukocytes respectively in the steady state (45, 46). Moreover, the spleen serves as a reservoir of monocytes that can be mobilized following injury to populate the damaged tissue (47). Following infarction, alarmins released from dead cells stimulate bone marrow-derived monocyte release and mobilize cells from the splenic reservoir, generating a large pool of activated cells available for recruitment to the site of injury. Two major monocyte types have been characterized in mice and in humans. Classical Ly6C^high mouse monocytes correspond to the CD14⁺ CD16⁻ human subpopulation and are recruited following infection or injury promoting inflammation. On the other hand, nonclassical Ly6C^lo monocytes correspond to the CD14^loCD16⁺ human cells and are also called patrolling monocytes, to reflect their homeostatic role in the normal vasculature (48, 49). The infarcted heart sequentially recruits both Ly6C^hi and Ly6C^lo monocytes (50). Ly6C^hi monocytes infiltrate the infarct during the early inflammatory phase of cardiac repair and have been suggested to differentiate into Ly6C^hi and Ly6C^lo macrophages (51). Infiltration of Ly6C^lo monocytes in the infarcted heart may play a reparative role, activating fibrogenic and angiogenic pathways (50).

It has been suggested that in addition to resident cardiac macrophages and recruited circulating monocyte-derived cells, GATA6⁺ pericardial macrophages may also infiltrate the infarcted heart, and may exert protective actions, preventing excessive fibrosis (52). This conclusion was based on in vivo experiments using cell transplantation approaches to generate bone marrow chimeras and delivery of fluorescent beads into the pericardial space to specifically label pericardial macrophages (52). However, the critical role of pericardial macrophages in cardiac repair has been challenged by recently published specific and rigorous genetic lineage tracing studies that demonstrated negligible infiltration of the infarcted heart with GATA6⁺ pericardial macrophages, and did not detect any effects of these cells in fibrotic remodeling of the heart (53).

Mediators involved in recruitment of monocytes in the infarcted myocardium.

Recruitment of monocytes in the infarct is driven by induction of adhesion molecules in the infarct microvasculature and by generation of chemotactic gradients that stimulate migration of the monocytes in the infarcted area (Table 2). Several members of the CC chemokine family are markedly upregulated in the infarcted myocardium and provide key directional signals for recruitment of monocytes into the infarct (88). The chemotactic actions of the chemokines are enhanced through binding to glycosaminoglycans expressed on the endothelial surface, or on the extracellular matrix (89). As monocytes are captured by activated endothelial cells, they roll on the endothelial surface through interactions involving the selectins, and “sense” the glycosaminoglycan-bound chemokines. This interaction stimulates monocyte integrin activation, triggering firm adhesion between the monocyte and the endothelial cell. These molecular steps are critical for migration of monocytes across the endothelial layer (90).

Table 2.

Chemotactic mediators involved in expansion/recruitment of macrophages in experimental models of myocardial infarction

Mediator	Cellular Source	Effects on Macrophages	Refs.
CC chemokines
CCL2	Endothelial cells, vascular smooth muscle cells, fibroblasts, T cells, monocyte/macrophages	1) Recruits CCR2+ monocytes 2) Stimulates expression of pro- and anti-inflammatory cytokines in the infarct 3) Mediates phagocytic removal of dead cells 4) Promotes adverse remodeling without affecting acute infarct size	(54–57, 58, 59)
CCR5 ligands (CCL3, CCL4, CCL5)	Although upregulation of CCR5 ligands has been documented in infarcts, their cellular localization has not been systematically studied	CCR5 mediates recruitment of regulatory T cells and monocytes with anti-inflammatory properties and may play a role in macrophage activation. These effects were suggested to attenuate adverse remodeling	(60, 61)
CCL5	Platelets	1) CCL5 has been implicated in recruitment of infarct macrophages 2) Platelet-derived CCL5 has been suggested to cooperate with neutrophil-derived HNP1 to attract macrophages in the infarct 3) CCL5 was reported to increase infarct size and accentuate adverse remodeling	(62, 63)
CCL7	B lymphocytes	CCL7 derived from B lymphocytes has been suggested to promote infiltration of the infarct with proinflammatory Ly6Chi monocytes. These effects accentuate adverse remodeling	(64, 65)
CCL21/CCR7	Although upregulation of CCL21 and its receptor CCR7 have been documented in infarcts, cellular localization has not been systematically studied	1) CCL21 has been implicated in recruitment of macrophages in the infarct 2) Proinflammatory effects of CCL21 were associated with worse postinfarction remodeling	(66)
CCL25/CCR9 axis	CCL25 immunoreactivity was diffusely localized in the infarct. CCR9 was localized predominantly in macrophages and T cells	1) CCR9 signaling was implicated in macrophage recruitment in the infarct 2) The proinflammatory effects of CCR9 were involved in adverse postinfarction remodeling	(67)
CX3CL1
CX3CL1/CX3CR1	Cardiomyocytes, endothelial cells	CX3CL1 was implicated in recruitment of macrophages in the infarct. CX3CL1-mediated inflammation was implicated in dysfunction. Effects on cardiac repair have not been studied	(68, 69)
Cytokines
IL-1/IL1R1 axis	Endothelial cells, monocytes/macrophages	1) IL-1α and IL-1β potently stimulate inflammatory macrophage infiltration in the infarct, either directly, or through induction of other chemoattractants. IL-1-mediated inflammation promotes adverse postinfarction remodeling 2) IL-1β was found to mobilize bone marrow leukocytes after infarction	(70, 71, 72, 73, 74)
TNF-α	In the infarct, TNF-α has been localized in macrophages, mast cells and vascular cells. Cardiomyocytes have also been suggested to be an important source of TNF-α in infarcted and remodeling hearts	TNF-α may stimulate synthesis of monocyte chemoattractants and may also induce endothelial adhesion molecule expression	(75, 76, 36)
TGF-β	Expressed in a latent form in normal hearts, and activated in infarction. De novo synthesis of all 3 TGF-β isoforms is noted in infarcts and may be localized in macrophages, vascular cells, cardiomyocytes and platelets	1) As a potent monocyte chemoattractant, TGF-β has been implicated in recruitment of monocytes in the infarct 2) The TGF-β/Smad3 cascade has been implicated in macrophage-mediated phagocytosis of dead cells, and in the transition of infarct macrophages to an anti-inflammatory phenotype. In contrast, Smad2 was not implicated in recruitment or activation of infarct macrophages	(59, 77, 78)
GM-CSF	Fibroblasts	1) GM-CSF has been implicated in recruitment of Ly6Chigh monocytes in the infarct through the induction of CCL2 production in Ly6Chigh monocytes and macrophages 2) GM-CSF stimulates the production of Ly6Chigh monocytes in the bone marrow. The proinflammatory effects of GM-CSF have been suggested to contribute to adverse postinfarction remodeling	(79)
Lipid mediators
Prostaglandin E2	Although upregulation of PGE2 has been documented in the infarct, cellular localization has not been systematically studied	1) PGE2 signaling mediated through the EP2 receptor was implicated in recruitment of macrophages in the infarct. EP2 signaling was suggested to exert reparative functions that attenuate dysfunction after infarction 2) The EP3 receptor was found to mediate reparative angiogenic functions of PGE2 in infarct macrophages	(80, 81, 82)
Leukotrienes	The cellular source of leukotrienes in the infarcted heart has not been systematically studied	5-Lipoxygenase-mediated leukotriene synthesis was found to attenuate infiltration of the infarct with proinflammatory macrophages. Whether this effect involves direct actions on macrophages is not known	(83)
Complement
C5a/C5a receptor	Although C5a receptors are upregulated in the ischemic region after reperfusion, the cellular source of C5a has not been studied	1) C5a signaling was implicated in macrophage recruitment in the infarct. The effects of C5a were suggested to increase infarct size	(84, 85)
Regenerating islet-derived proteins
Reg3b	Cardiomyocytes	1) Reg3b has been implicated in initial recruitment of monocytes in the infarct 2) Reg3b may selectively stimulate recruitment of MHC-IIhi Ly6Clo and MHC-IIlo Ly6Clo macrophages in the infarct. These effects were suggested to promote cardiac healing, improving myocardial function after infarction	(86, 87)

C5a, complement component 5a; CCL, C-C chemokine ligand; CCR, C-C chemokine receptor; CSF, colony stimulating factor; CX3CL, C-X3-C chemokine ligand; IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor.

Expression of C-C chemokine ligand (CCL) 2/Monocyte Chemoattractant Protein (MCP)-1, the best-studied CC chemokine in heart disease, is markedly upregulated in endothelial cells and leukocytes infiltrating the infarct (58, 91–94) and triggers recruitment of CCR2⁺ monocytes that serve a central role in clearance of the infarct from dead cells, but also contribute to adverse ventricular remodeling (54, 55, 95–98).

Although several other CC chemokines (including CCL3, CCL4, CCL5, CCL7, and CCL21) are also upregulated in the infarcted myocardium (88), their role in recruitment, expansion, and/or activation of macrophages is less convincingly documented. CCL5 has been suggested to contribute to the inflammatory injury in experimental models of nonreperfused infarction (62, 99). It has been suggested that formation of heteromers comprised of platelet-derived CCL5 and neutrophil-borne human neutrophil peptide 1 (HNP1) stimulate monocyte recruitment through interactions with chemokine receptors (63). Actions involving CCR1, a chemokine receptor that binds to several CC chemokines, or CCR9, the receptor for CCL25, have also been implicated in monocyte recruitment in the infarcted myocardium (67, 100).

Considering the heterogeneity of monocytes, which is determined at least in part by their chemokine receptor expression profile, some members of the CC chemokine family may exhibit selectivity by recruiting specific subsets of monocytes. For example, experiments using a genetic loss-of-function model suggested that CCR5, one of the receptors for the CC chemokines CCL3, CCL4 and CCL5, may be inhibit inflammation by recruiting anti-inflammatory monocyte subsets and regulatory T cells (60).

CXC chemokines on the other hand, predominantly act as chemoattractants for neutrophils and lymphocytes. Thus, any effects of CXC chemokines on macrophage recruitment and activation are likely to be indirect, reflecting actions on other cell types. The CX3C chemokine C-X3-C chemokine ligand (CX3CL) 1, also known as fractalkine, promotes migration of cells expressing its receptor C-X3-C chemokine receptor (CX3CR) 1. CX3CR1-expressing monocytes and macrophages are abundant in the infarcted myocardium (50, 101); their recruitment may require upregulation of CX3CL1 in the infarct zone (68, 69).

The proinflammatory cytokines Interleukin (IL)-1, Tumor Necrosis Factor (TNF)-α, and IL-6 also contribute to infiltration of the infarcted myocardium with macrophages. Genetic disruption of the type 1 IL-1 receptor (IL-1RI), the only signaling receptor for IL-1α and IL-1β, markedly attenuated macrophage recruitment in the infarcted myocardium, without affecting scar size, suggesting a central role of IL-1 signaling in mediating the macrophage-driven inflammatory response (71). Several mechanisms may mediate the effects of IL-1 on recruitment of macrophages in the infarct. First, IL-1 potently stimulates CC chemokine synthesis in several different cell types (72, 102) thus inducing expression of monocyte chemoattractants in the infarct. Second, IL-1 enhances monocyte adhesion to endothelial cells by inducing integrin expression and activation in monocytes, while upregulating synthesis of adhesion molecules in the endothelium (103, 104). Third, IL-1 may upregulate expression of proteases in macrophages, thus enhancing their migratory capacity (105). Fourth, IL-1 also acts on the bone marrow, accentuating generation and release of monocytes (73). In comparison to the very extensive evidence on the role of IL-1 signaling cascades in recruitment of infarct macrophages, the data on the role of TNF-α and IL-6 are much more limited. Studies using genetic models demonstrated contrasting effects of TNF receptor (TNFR)1 versus TNFR2 signaling on recruitment of activated macrophages in the infarct. TNFR1-mediated infiltration of the infarcted myocardium with macrophages; in contrast, TNFR2 restrained macrophage recruitment (106). Whether these effects are due to direct actions of TNF receptors on macrophages or reflect epiphenomena has not been investigated. In vitro studies suggest that, in addition to its proinflammatory effects, TNF-α is also a central regulator of macrophage survival, promoting necroptosis and pyroptosis (107). The in vivo effects of IL-6 on macrophages are also complex, and likely reflect the balance between canonical (mediated through binding of the cytokine to cell surface IL-6 receptor/IL-6R) and trans signaling (involving binding to soluble IL-6R and subsequent activation of gp130 cascades in cells lacking surface IL-6R) (108). In a model of myocardial infarction, pharmacologic inhibition experiments demonstrated that IL-6 trans signaling may mediate macrophage infiltration (109).

Activation of the complement cascade has also been implicated in recruitment of monocytes in the infarcted myocardium (84, 110). Myocardial cell necrosis results in the release of subcellular membrane constituents capable of activating the complement cascade (111, 112). mRNA and proteins for all the components of the classical complement pathway are overexpressed in myocardial infarcts (113) and trigger monocyte chemotaxis, acting predominantly during the early phase of leukocyte infiltration (59). Complement-mediated monocyte recruitment may involve, at least in part, Complement component 5a (C5a)-stimulated induction of CC chemokine synthesis in microvascular endothelial cells (114). In addition to its chemotactic effects, activation of the C5a/C5a receptor (C5aR) axis in macrophages may also contribute to proinflammatory activation, by generating reactive oxygen species and by inducing and activating proinflammatory IL-1β (115).

The functional role of infarct macrophages during the inflammatory phase: phagocytosis and efferocytosis.

The best documented functions of macrophages during the inflammatory phase of infarct healing are phagocytosis, the clearance of necrotic cells and matrix fragments, and efferocytosis, the removal of apoptotic cells. In vivo studies demonstrating the presence of phagocytosed cells within infarct macrophages (116), and evidence showing that perturbed phagocytic functions of macrophages are associated with defective clearance of necrotic or apoptotic cardiomyocytes and neutrophils (54, 77, 116, 117) support the central role of the macrophages as professional phagocytes that remove dead cells from the infarcted heart. Some experimental evidence suggested that myofibroblasts may also exhibit phagocytic capacity (118), thus contributing to removal of dead cells from the infarct; however, the significance of this mechanism is unclear, as phagocytosis of dead cells in the healing infarct typically precedes myofibroblast conversion (94).

Effective phagocytosis requires activation of pathways that mediate recognition and engulfment of dying cells by the macrophages (Fig. 1), as well as expression of “don’t eat me” signals by live cells to prevent inappropriate removal of uninjured bystanders (119). In the infarcted myocardium, death of up to a billion cells poses a major challenge for the relatively small population of phagocytic macrophages located in the normal cardiac interstitium. Recruitment of abundant monocytes in response to the release of DAMPs provides a large pool of macrophages, capable of rapidly phagocytosing the dying cells in the infarct zone. Following transmigration to the area of the infarct, macrophages sense “find-me” signals released by dying cells, which direct the phagocyte to the site of cell death. Lysophospholipids (such as lysophosphatidylcholine and sphingosine-1-phosphate) and nucleotides [such as Adenosine Tri-Phosphate (ATP), Adenosine Mono-Phosphate (AMP), and Uridine Tri-Phosphate (UTP)] are the best characterized find-me signals; however, their potential role in guiding macrophages to the dying myocardial cells has not been studied. Once the macrophages have migrated to the site of cell death, they recognize “eat-me” signals expressed on the surface of the dying cells. The best characterized and most important of the “eat-me” molecules is phosphatidylserine (PtdSer). PtdSer is ubiquitously expressed by all cells, and is normally confined to the inner surface of the cell membrane. In apoptotic cells, PtdSer is externalized on the cell surface and is detected by the transmembrane receptors of the TIM (T-cell immunoglobulin and mucin domain-containing molecule) and TAM [tyrosine-protein kinase receptor (TYRO) 3, AXL, and myeloid-epithelial-reproductive receptor tyrosine kinase (MerTK)] families, expressed on the macrophage membrane (119). Although TIM synthesis is restricted to specific macrophage subpopulations, MerTK exhibits broad expression in phagocytic macrophages in all tissues and has been implicated in efferocytosis in the infarct.

Figure 1.

Molecular signals involved in efferocytosis-mediated clearance of apoptotic cells by macrophages in the infarcted myocardium. Apoptotic cells expose “eat me” signals, such as phosphatidylserine on their surface. Macrophages detect phosphatidylserine through the TAM receptors (Tyro3, Axl, and MerTK), using activating ligands, such as GAS6/Protein S, which bind to phosphatidylserine. ADAM17 inhibits macrophage phagocytosis by cleaving the extracellular domain of MerTK. The soluble glycoprotein MFGE8 also contributes to phagocytosis, by binding to phosphatidylserine on the surface of dead cells and to macrophage αV integrin. Macrophage CD36 has been suggested to contributes to dead cell recognition through interactions with thrombospondin-1. In the infarct, CD47 expression by apoptotic cells has been suggested to interact with macrophage SIRPα, inhibiting phagocytosis. ADAM, a disintegrin and metalloprotease; MerTK, myeloid-epithelial-reproductive receptor tyrosine kinase; MFGE8, milk fat globule-EGF factor 8; TSP-1, thrombospondin 1.

Global and macrophage-specific loss-of-function studies demonstrated that MerTK mediates clearance of apoptotic cells from the infarct (116, 120). MerTK-dependent efferocytosis plays a central role in acquisition of an anti-inflammatory and reparative phenotype in infarct macrophages (121, 122). The extracellular domain of MerTK is cleaved by a disintegrin and metalloprotease (ADAM) 17 (123), a protease that is markedly induced in the infarcted myocardium (124), generating soluble Mer, and reducing the levels of functional MerTK on the macrophage surface. MerTK cleavage in the infarcted heart perturbs efferocytosis and accentuates adverse remodeling following myocardial infarction (120). On the other hand, the AXL receptor tyrosine kinase was found to exert proinflammatory actions, directing a switch to glycolytic metabolism and inducing IL-1β expression (125).

Much like the other members of the TAM family, MerTK does not bind directly to PtdSer, but interacts with activating ligands, such as GAS6 and PROS1, to recognize PtdSer and to mediate signaling (119). Through a similar bridging mechanism, the soluble glycoprotein milk fat globule-EGF factor 8 (MFGE8) can also contribute to macrophage-mediated efferocytosis in the infarcted heart (77, 121). MFGE8 promotes efferocytosis by bridging PtdSer on the apoptotic cell surface with macrophage αV integrins. The pattern recognition receptor CD36 may also be involved in recognition of apoptotic cells by macrophages through interactions with thrombospondin-1 (126). In the infarcted heart, myeloid cell expression of CD36 has been implicated in early phagocytosis of dying cardiomyocytes and in activation of a reparative program (127).

It has been suggested that clearance of apoptotic cells is regulated not only by molecules that promote efferocytosis, but also by protective “don’t eat me” signals, which mark healthy cells, and protect them from engulfment by the activated macrophages. Overexpression of the transmembrane protein CD47 has been proposed as a mechanism that may explain the resistance of leukemic progenitor cells to macrophage-mediated death (128). Whether induction of “don’t eat me” signals plays a role in protecting border zone cardiomyocytes from death has not been investigated. CD47 inhibition with neutralizing antibodies has been reported to exert beneficial actions in an infarction model by facilitating clearance of dead cells (129), suggesting a maladaptive role of this molecule in cardiac repair. However, the broad range of effects of CD47 on many different cell types (130) and the lack of data using cell-specific approaches preclude definitive conclusions.

Macrophages during the Proliferative Phase of Cardiac Repair

Proliferation contributes to macrophage expansion.

Phagocytosis of dying cells triggers acquisition of an anti-inflammatory phenotype by infarct macrophages, marking the end of the inflammatory phase of cardiac repair, and leading to the transition to the proliferative phase. Despite the reduced recruitment of monocytes, due to the downregulation of chemokines, the infarct macrophage population continues to expand through proliferation (43, 51, 131). Proliferation of infarct macrophages may involve myocardial induction of CSF-1 (43). Moreover, during the proliferative phase, infarct macrophages exhibit dramatic phenotypic changes and release a broad range of mediators that orchestrate repair, but may also be involved in the pathogenesis of maladaptive fibrosis.

During the proliferative phase, infarct macrophages acquire an anti-inflammatory, reparative and fibrogenic M2-like phenotype.

The M1/M2 macrophage polarization paradigm suggests that macrophages can acquire two polarized phenotypes: proinflammatory M1 cells or anti-inflammatory M2 cells with reparative and fibrogenic properties (132). Although infarct macrophages exhibit a range of nuanced phenotypes between these two extremes, identification of macrophages with M1-like or M2-like cytokine expression profiles has been very useful in understanding the temporal dynamics of the cellular response in myocardial inflammation and repair. Studies combining flow cytometry and gene expression analysis suggest that M1-like cells are prominent during the inflammatory phase of infarct repair, whereas M2-like macrophages dominate the proliferative phase (131, 133). This temporal sequence of M1 versusM2 cell expansion in the infarcted heart is also supported by single cell RNA-sequencing studies (134). During the peak of the proliferative phase of cardiac repair, the most prominent population of macrophages in the infarcted myocardium are M2-like cells that exhibit expression of genes encoding reparative proteins, such as Pdgfb and Igf1 (134). In contrast, the anti-inflammatory cytokine Il10 was only expressed in a small number of infarct macrophages. This finding is consistent with observations in a canine model of reperfused infarction, showing that IL-10 protein is predominantly expressed in T cells infiltrating the infarct, and only in a small subset of macrophages (135).

The mechanisms and signals involved in anti-inflammatory transition of infarct macrophages.

Several distinct cellular mechanisms have been implicated in anti-inflammatory transition and in acquisition of an M2-like reparative phenotype in infarct macrophages. First, as discussed in the previous section, efferocytosis potently stimulates expression of anti-inflammatory mediators by macrophages. Second, other immune cell types, including neutrophils and lymphocytes, have been implicated in regulation of the inflammatory profile of infarct macrophages; their effects may involve secreted anti-inflammatory mediators, or contact-dependent mechanisms. Neutrophils have been suggested to stimulate a reparative macrophage phenotype in healing infarcts through secretion of neutrophil gelatinase-associated lipocalin (NGAL), and subsequent accentuation of efferocytosis (136). Recruitment of anti-inflammatory T cells (such as regulatory T cells/Tregs) (137) may also play a role in stimulating an anti-inflammatory and reparative macrophage phenotype (138) by secreting IL-10 (60, 139), or by exerting contact-mediated effects (139). Third, cytokine activation in the infarct microenvironment induces endogenous anti-inflammatory signals in infarct macrophages that restrain proinflammatory cascades. Upregulation of IL-1 receptor-associated kinase (IRAK)-M in infarct macrophages has been demonstrated to inhibit TLR- and IL-1-driven inflammatory activity, protecting the infarcted heart from adverse remodeling (140). Fourth, several different anti-inflammatory cytokines, such as transforming growth factor (TGF) -βs, IL-4, IL-10, and IL-13, are induced in the infarct and cooperate to suppress proinflammatory macrophage activation (141). TGF-β signaling cascades are activated in the infarcted heart, both through increased de novo synthesis (94) and activation of latent stores (142), and may play an important role in mediating the anti-inflammatory actions of macrophages (143–145). The anti-inflammatory effects of TGF-β on infarct macrophages were found to involve activation of Smad3, but not Smad2 signaling (77, 78). Fifth, the effects of secreted mediators on the phenotypic and functional profile of infarct macrophages may involve modulation of transcription factors of the IRF family (146). For example, IRF5 expression was found to be downregulated in infarct macrophages during the proliferative phase of cardiac repair and may play a central role in suppression of their proinflammatory activity (147). The endogenous signals responsible for de-activation of IRF-mediated pathways in the healing infarct have not been identified. Sixth, macrophage phenotype may be also regulated through release of extracellular vesicles (exosomes) containing miRNAs with pro- or anti-inflammatory properties (148). Most of the evidence implicating exosomes in regulation of infarct macrophage phenotype is derived from cell therapy studies. In these studies, infusion of a broad range of reparative cell types promotes cardioprotective, reparative, or anti-inflammatory macrophage phenotypes through exosome release (149, 150). Ischemic cardiomyocytes, fibroblasts, and T cells are also capable of secreting miRNA-containing exosomes that may modulate macrophage phenotype; however, the relative significance of this endogenous mechanism has not been convincingly demonstrated.

Macrophages critically regulate cardiac repair.

Considering their central role in phagocytosis of dead cells and their involvement in activation and recruitment of reparative cells, such as fibroblasts and vascular cells, it is not surprising that macrophages are key regulators of postinfarction repair. Several experimental studies using liposomal clodronate administration to deplete monocytes and macrophages demonstrated a central protective role of macrophages in the healing infarct (Table 3). Macrophages are required for replacement of dead cardiomyocytes with granulation tissue and protect the infarcted heart from adverse remodeling (151, 153, 154). Whether the reparative functions of macrophages involve specific subsets is less convincingly demonstrated, due to the challenges in implementing strategies with subset-specific effects. Two different depletion strategies suggested an important role for M2-like macrophages in repair of the infarcted heart. Deletion of the adaptor protein tribbles homolog 1 (TRIB1), which is critically implicated in differentiation of M2-like macrophages (159), resulted in depletion of M2-like infarct macrophages, leading to defective repair, and a high incidence of cardiac rupture after myocardial infarction (160). Similar perturbations were noted in infarcted mice that received treatment with a CSF-1 receptor kinase inhibitor (161). Blockade of the effects of CSF-1 depleted M2 macrophages, disrupting repair and worsening adverse remodeling following infarction (161).

Table 3.

Studies investigating the effects of macrophages in myocardial infarction using depletion strategies

Species/Strategy/Model of MI	Effects on Macrophage Population	Role of Macrophages	Mechanism	Ref.
Mouse/liposomal clodronate depletion/cryoinjury	Infiltration of the injured heart with F4/80+ macrophages was reduced by ∼85%	Macrophages reduce mortality and protect from dilative remodeling following infarction	Protective effects of macrophages were attributed to clearance of cell debris and to activation of a reparative program, leading to myofibroblast infiltration and increased angiogenesis	(151)
Mouse/liposomal clodronate depletion/nonreperfused MI	CD11b+Ly6G− monocytes were reduced by 69% in the infarcted heart	Macrophages enhance postinfarct sympathetic nerve sprouting. Effects on myofibroblast infiltration are also suggested	The effects of macrophages on nerve sprouting are attributed to secretion of NGF	(152)
Mouse/liposomal clodronate depletion/ nonreperfused MI	Efficient macrophage depletion was achieved; however, quantitative data are not provided	Macrophages reduce mortality after infarction and protect from dilative remodeling	Not studied	(153)
Mouse/liposomal clodronate depletion/nonreperfused MI Mouse/genetic macrophage depletion using LysMCreDTR mice/nonreperfused MI	In clodronate-treated mice, CD11b+Ly6G− monocytes were reduced by 81% No data are reported on the efficiency of depletion using the diphtheria toxin model	Clodronate depletion experiments suggested that macrophages reduce mortality after infarction and protect from formation of LV thrombus. Genetic depletion only suggested effects on thrombus formation without effects on mortality	Protective effects of macrophages were attributed to clearance of cell debris and to activation of a reparative program, leading to replacement with granulation tissue	(154)
Mouse/liposomal clodronate depletion/nonreperfused MI	CD11b+Ly6G− monocytes were reduced by ∼50% in the infarcted heart	Macrophages in neonatal mouse hearts enhance cardiac regeneration	The regenerative effects of neonatal macrophages are mediated through stimulation of angiogenesis	(155)
Mouse/liposomal clodronate depletion/genetic model of cardiomyocyte death	CCR2− MHC-IIhigh embryonic-derived macrophages were reduced by ∼80% in the injured heart	Macrophages in neonatal mouse hearts enhance cardiac repair	CCR2− MHC-IIhigh embryonic-derived macrophages promote cardiac repair through cardiomyocyte proliferation and angiogenesis. stimulation of angiogenesis	(156)
Mouse/liposomal clodronate depletion/ reperfused MI	CD45+CD68+ macrophages were reduced by ∼77% in blood and by ∼71% in spleen	Macrophage depletion does not affect the infarct size and cardiac function 48 h after ischemia-reperfusion	Not studied	(149)
Mouse/liposomal clodronate depletion/nonreperfused MI	CD11b+F4/80+ macrophages were reduced by 44% in spleen and by 15.3% in the infarcted heart	Macrophages produce bioactive resolving mediators, such as lipoxygenase, and suppress inflammation	Not studied	(157)
Mouse /genetic depletion using CCR2DTR mice to deplete CCR2+ cells and CD169DTR mice to eliminate CCR2− resident macrophages/nonreperfused MI, reperfused MI, genetic model of cardiomyocyte death and heart transplantation that mimics ischemic injury	In CCR2DTR mice, 4 days after diphtheria toxin treatment, circulating CCR2+ cells and cardiac dendritic cells were repopulated, whereas CCR2+ cardiac resident macrophages remained depleted In CD169DTR mice, CCR2− cardiac resident macrophages were depleted	CCR2+ cardiac resident macrophages increase proinflammatory leukocyte infiltration in the infarct and promote adverse remodeling. In contrast, CCR2− resident macrophages exert protective actions, attenuating adverse remodeling	CCR2+ cardiac resident macrophages increase monocyte recruitment through an MYD88-dependent pathway	(39)
Mouse/ Genetic depletion of resident macrophages using Cx3cr1CreER–YFP:R26 Td / DTR mice. Tamoxifen was discontinued for 6 wk to achieve selective depletion of resident cardiac macrophages /nonreperfused MI	Cardiac resident macrophages were selectively depleted	Resident cardiac macrophages protect the infarcted heart from adverse postinfarction remodeling	Not studied	(158)

CCR, C-C chemokine receptor; CX3CR, C-X3-C chemokine receptor; MI, myocardial infraction; NGF, nerve growth factor.

Macrophage-derived mediators that negatively regulate inflammation.

A broad range of macrophage-derived secreted mediators have been implicated in the anti-inflammatory effects of M2-like macrophages in the infarcted heart. Induction of TGF-βs in infarcted hearts is dependent, at least in part, on macrophage infiltration; however, the relative significance of macrophage-derived TGF-βs (151) versus other cellular sources and preformed stores is unclear. IL-10, IL-4, and IL-13 are also upregulated in subpopulations of infarct macrophages (135, 162); however, considering the high levels of synthesis of these cytokines in T cells, the relative significance of macrophage-specific expression is unknown. Vascular endothelial growth factor (VEGF)-C has been recently identified as a macrophage-derived mediator with anti-inflammatory properties that may be induced on efferocytosis, suppressing the postinfarction inflammatory response (163). Insulin-like Growth Factor 1 (IGF1) is also highly expressed by M2-like infarct macrophages (134) and may act, at least in part, through anti-inflammatory actions (164). It should be emphasized that macrophage-derived IGF1 may exert a broad range of actions on myocardial cells that may include activation of a hypertrophic program in cardiomyocytes (165). Infarct macrophages can also suppress and resolve inflammation through secretion of specialized proresolving mediators (SPM), such as lipoxins, resolvins, protectins, and maresins (166, 167). However, the role of macrophage-derived endogenous SPMs in repair and remodeling of the infarcted heart has not been investigated.

Macrophages in regulation of fibroblast function.

Cardiac repair is dependent on timely fibroblast activation and conversion to myofibroblasts, the main matrix-producing cells in the infarcted heart (168, 169). Although the central role of macrophages in stimulating myofibroblast activation is strongly supported by macrophage depletion experiments (Table 3), the specific macrophage-derived mediators involved in the reparative fibrogenic actions are poorly understood. TGF-βs are highly expressed in infarct macrophages (54, 77) during the proliferative phase of cardiac repair and are potent activators of myofibroblast conversion and extracellular matrix protein synthesis (170). Other fibrogenic cytokines and growth factors upregulated in infarct macrophages, such as IL-6 (36), IL-4, IL-13, and platelet-derived growth factors (PDGFs) (134, 171) may also contribute to macrophage-mediated fibroblast activation (Graphical abstract). Neuregulin 1, a member of the epidermal growth factor (EGF) family, is also upregulated in M2 macrophages infiltrating the infarct, and has been suggested to promote fibroblast expansion by attenuating their apoptotic death (172). Macrophages can also trigger fibroblast activation by producing proteins with matricellular properties, such as thrombospondin (TSP)-1 (173), secreted protein acidic and rich in cysteine/SPARC (174), and osteopontin (175, 176). These secreted glycoproteins are minimally expressed in normal hearts, but enrich the extracellular matrix during the proliferative phase of infarct healing, and play an important role in stimulation of fibroblasts and myofibroblasts, by locally releasing bioactive cytokines and growth factors, and by binding to receptors on the surface of fibroblasts, thus transducing downstream activating cascades (177, 178). In addition to cytokine and growth factor-mediated effects, it has been suggested that infarct macrophages may also regulate fibroblast phenotype by producing exosomes enriched in miRNAs (179, 180). However, the profile and properties of the extracellular vesicles secreted by infarct macrophages has not been systematically studied.

Some studies have suggested that in addition to their paracrine fibrogenic actions, macrophages may produce significant amounts of collagens (181) or may even directly contribute to the expansion of myofibroblasts in the infarcted heart, by converting to fibroblasts (182). However, the evidence supporting this notion is weak. Robust lineage tracing studies accompanied by rigorous methodology for fibroblast identification have demonstrated negligible conversion of myeloid cells into fibroblasts in the infarcted heart (183, 184). Moreover, single cell RNA-sequencing studies showed negligible expression of collagen genes by infarct macrophages (134).

Macrophages as a source of matrix metalloproteinases.

The role of macrophages in reparative fibrosis following infarction is not limited to the expression of fibroblast-activating mediators. Macrophages may not secrete significant amounts of collagens but have profound effects on the composition of the matrix by producing and secreting proteases and anti-proteases that regulate matrix metabolism. Combined transcriptomic and proteomic studies have demonstrated that macrophages are a major cellular source for many different members of the MMP family in the infarct, including MMP2 (185), MMP7 (186), MMP10, MMP11, MMP12, MMP14, MMP16, MMP24, MMP25, MMP27, and MMP28 (187–190). Several of these proteases may have effects beyond their role in matrix degradation, regulating the levels of bioactive cytokines and chemokines, or processing proteins involved in cardiomyocyte contraction (191) and in electrical conduction (186). For example, MMP12, predominantly localized in infarct macrophages, has been suggested to play a role in resolution of postinfarction inflammation (188), presumably through inactivation of several distinct proinflammatory substrates (192).

Macrophages and infarct angiogenesis.

Formation of neovessels in the healing infarct plays an important role in repair of the infarcted myocardium by supplying oxygen and nutrients to the metabolically active wound (193). Moreover, angiogenesis in the border zone of the infarct may be required to meet the increased metabolic needs of viable cardiomyocytes that need to compensate for the lost contractile capacity of dead myocardium. Macrophages play a central regulatory role in the angiogenic response following myocardial infarction (Fig. 2). Several studies have demonstrated that macrophages are a major source of the potent angiogenic factor VEGF-A in the infarcted myocardium (77, 81, 194). Experiments using myeloid cell-specific knockout mice demonstrated the critical role of macrophage-derived VEGF-A in stimulating infarct angiogenesis, and in mediating repair of the infarcted heart (121). Moreover, macrophage-derived VEGF-C is involved in stimulation of lymphangiogenesis after myocardial infarction (163). Several pathways including efferocytosis (121), Smad-3 signaling (77), and activation of the Prostaglandin E2 (PGE2)/EP3 axis (81) have been implicated in stimulation of an angiogenic phenotype in infarct macrophages. In addition to secretion of angiogenic growth factors, macrophages can also stimulate angiogenesis by producing matrix metalloproteinases (MMPs), necessary to cleave and remodel the extracellular matrix, providing conduits for endothelial cells.

Figure 2.

Angiogenic effects of macrophages in the infarcted myocardium. Macrophages are crucial cellular effectors in infarct angiogenesis. Efferocytosis stimulates macrophage-derived release of growth factors, such as VEGF-A and VEGF-C, contributing to angiogenesis and lymphangiogenesis. Annexin a1, TGF-β, and Prostaglandin E2 have also been suggested to promote VEGF-A release by macrophages. Macrophage-derived MMPs may also play an important role in neovessel formation. Moreover, macrophage subsets can serve as a major source of PDGF-BB, a growth factor involved in mural cell recruitment and vascular maturation. MMP, matrix metalloproteinases; PDGF, platelet-derived growth factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.

Macrophages in the Maturation Phase of Infarct Healing

Infarct maturation, the most enigmatic phase of cardiac repair, ultimately leads to formation of a mature scar, containing cross-linked collagen fibers. As the infarct matures, myofibroblasts convert to matrifibrocytes, a population of fibroblast like-cells that do not express α-SMA, but synthesize cartilage and tendon genes, and likely serve in maintaining the structural integrity of the mature scar (195). During the maturation phase of cardiac repair, the infarct microvasculature also undergoes dramatic changes, as neovessels acquire a mural cell coat (196, 197). Recruitment of mural cells by infarct microvessels involves activation of a platelet-derived growth factor receptor (PDGFR) β-mediated pathway and contributes to scar stabilization, restraining inflammatory activity and hemorrhagic infiltration of the infarct (198).

The potential contribution of infarct macrophages to the cellular events leading to scar maturation has not been investigated. Experiments in a mouse model of infarction, using macrophage-specific antibodies and reporter mice showed that despite a reduction in macrophage density during the transition from the proliferative to the maturation phase, a significant population of macrophages remains in mature scars (101). The fate and functional role of these cells is unknown. Macrophages may be an important source of matrix-crosslinking enzymes, such as lysyl-oxidase (199) and transglutaminase-2 (200), and may also contribute to vascular maturation by synthesizing and secreting PDGF-BB (134).

MACROPHAGES IN THE REMODELING NONINFARCTED HEART

As the infarct heals, the surviving remote myocardium remodels, exhibiting compensatory hypertrophic changes, accompanied by interstitial fibrosis. Moreover, the infarct border zone exhibits changes in the biochemical characteristics of the matrix, associated with denaturation of the collagenous network, which may reflect increased mechanical stress in the interface between viable myocardium and noncontractile scar (189). Thus, macrophages in the remodeling noninfarcted myocardium may exhibit phenotypic alterations similar to the changes noted in pressure or volume overload models of noninfarctive heart failure. Unfortunately, the information on the fate and functional role of macrophages in viable noninfarcted segments is very limited. Experiments in a mouse model demonstrated that 8 wk after myocardial infarction, the macrophage population in remote remodeling myocardial segments is increased (201); the local expansion of macrophages was attributed to both increased proliferation and monocyte recruitment (201). It is plausible to hypothesize that activation of macrophages in the remote remodeling myocardium may reflect the increased wall stress in these segments. Experiments in a model of nonischemic heart failure suggested that macrophages sense mechanical stress through mechanosensitive ion channels, such as transient receptor potential vanilloid (TRPV) 4, and become activated, expressing and secreting growth factors (202). However, whether similar mechanosensitive pathways activate macrophages populating the viable remodeling myocardial segments of the infarcted ventricle has not been investigated. Another mechanosensitive ion channel, TRPV2 was found to be overexpressed in macrophages infiltrating the peri-infarct zone (203). Although studies in a model of global germline loss suggested that TRPV2 may promote inflammatory injury after myocardial infarction (204), cell-specific studies examining the role of TRPV2 in regulation of infarct macrophage phenotype are lacking.

MACROPHAGES AND POSTINFARCTION ARRHYTHMOGENESIS

Experimental evidence has implicated macrophages in the pathogenesis of infarct-related arrhythmias. Descriptive and associative studies have suggested that macrophages form gap junctions with cardiomyocytes in the infarct border zone and that M1 macrophages may activate KCa3.1 in cardiomyocytes, thus facilitating arrhythmia generation (205). A recent study in a mouse model of myocardial infarction combined with hypokalemia (to stimulate arrhythmogenesis) showed that depletion of monocyte-derived macrophages in CCR2 KO mice, or of all macrophages (in mice receiving a CSF1R inhibitor) promote electrical storm, suggesting a protective role for macrophages in postinfarction arrhythmogenesis (206). Whether these protective effects involve primary actions on cardiomyocyte electrophysiologic properties, or simply reflect the consequences of perturbed clearance of dead cardiomyocytes and impaired repair (54, 151) in macrophage-deficient infarcts is unclear.

SEX-SPECIFIC DIFFERENCES IN MACROPHAGE PHENOTYPE AND THEIR ROLE IN POSTINFARCTION REMODELING

Sex is a significant modulator of the immune response to myocardial infarction and may affect prognosis and the development of chronic heart failure. Although the reduced incidence of myocardial infarction in women is well documented, convincing clinical evidence on sex-specific patterns of postinfarction remodeling is lacking (207). In a small clinical study male sex was associated with worse early and late remodeling after ST elevation myocardial infarction (STEMI) (208). In contrast, in a larger study of STEMI patients treated with primary percutaneous intervention (PCI) and optimal pharmacotherapy, men and women had comparable remodeling patterns (209). Because sex also affects the prevalence of several other comorbid conditions that affect the remodeling response (such as diabetes, hypertension, smoking, etc.), identification of sex-specific patterns of adverse remodeling in clinical studies is challenging.

In animal models, most experimental studies have demonstrated that males develop worse postinfarction remodeling than females, in response to comparable initial ischemic insults (210, 211). Moreover, in mice male, sex is associated with a higher incidence of left ventricular free wall rupture after nonreperfused infarction (212). Increased rupture and accentuated remodeling in male animals may be due to sex-related differences in macrophage phenotype (213). However, the evidence supporting this notion is associative and has generated conflicting results. Some studies have suggested that female mice have more infiltrating macrophages in the infarct, with a higher abundance of reparative monocytes (210, 211). In contrast, other investigations failed to detect significant differences in the inflammatory gene expression profile of myeloid cells harvested from male versusfemale mouse infarcts (212).

AGING-ASSOCIATED MACROPHAGE PERTURBATIONS IN THE PATHOGENESIS OF POSTINFARCTION REMODELING

Patients aged 65 yr and over account for the majority of hospital admissions and deaths from acute myocardial infarction (214) and exhibit an increased incidence of adverse remodeling and postinfarction heart failure (215). Worse prognosis in older patients cannot be attributed only to increased susceptibility of senescent cardiomyocytes to ischemia, resulting in larger infarcts (216), but may also involve dysregulation of inflammatory and reparative responses (217, 218). Senescent mice have reduced and delayed macrophage infiltration in the infarct, associated with perturbed removal of dead cardiomyocytes, impaired resolution of inflammation and attenuated activation of reparative fibroblasts (219). Several macrophage-dependent mechanisms may explain the reparative impairment and accentuated adverse remodeling in senescent subjects. First, aging is associated with perturbations in the normal resident cardiac macrophage population, leading to progressive replacement of resident embryonic origin macrophages with monocyte-derived cells (220) that may exhibit attenuated reparative capacity. Second, aging results in overactive baseline inflammatory gene expression in macrophages, accompanied by diminished oxidative responses to activating signals (221). Thus, in older subjects macrophages may exhibit decreased phagocytic activity (222) that may delay removal of dead cardiomyocytes from the healing infarct, leading to a prolonged inflammatory response. Third, in models of tissue repair, aging is associated with reduced macrophage-derived expression of reparative and angiogenic growth factors (223). Attenuated activation of reparative fibroblasts and impaired infarct angiogenesis may disrupt repair, increasing dilative remodeling of the infarcted ventricle.

TARGETING MONOCYTE-DERIVED MACROPHAGES IN MYOCARDIAL INFARCTION

Experimental evidence suggests that macrophages have both deleterious and reparative functions in the infarcted heart. Several studies in mouse models support the notion that newly recruited CCR2⁺ monocyte-derived macrophages may accentuate inflammation, extending injury following infarction, whereas resident CCR2⁻ macrophages may exert protective actions (39, 54, 158). Pharmacologic interventions targeting proinflammatory CCR2⁺ monocytes and macrophages were found to have beneficial effects in animal models of myocardial infarction. CCR2 targeting using a nanoparticle-based siRNA approach was reported to reduce recruitment of proinflammatory leukocytes in a broad range of pathologic conditions and was suggested to reduce injury in myocardial infarction (224). Treatment with a CCL2 inhibitor attenuated dysfunction in a model of reperfused myocardial infarction (95). Administration of lipid micelles loaded with a CCR2 inhibitor and surface decorated with anti-CCR2 antibody to specifically target proinflammatory CCR2⁺ macrophages, attenuated inflammation in a model of myocardial infarction; these anti-inflammatory effects were associated with a trend towards attenuated dysfunction (98). Treatment with the chemokine-binding protein evasin-4 selectively inhibited CC chemokine effects and was found to attenuate injury in a model of myocardial infarction (99). Although these findings suggest therapeutic promise of strategies inhibiting CC chemokine-driven macrophage activation in myocardial infarction, implementation in patients poses several major challenges. First, the effects of proinflammatory macrophages are not uniformly detrimental. Myocardial infarction requires induction of chemokine-mediated inflammation to clear the infarct from dead cells and to initiate the reparative response (54). Thus, overzealous inhibition of macrophage-mediated inflammation may perturb repair. Second, studies in young adult mice with robust inflammatory responses may not accurately reflect the effects of targeting proinflammatory signals in elderly subjects suffering myocardial infarction. Senescence is associated with suppressed and prolonged inflammatory responses after myocardial injury (219). Thus, in elderly subjects with myocardial infarction acute inhibition of inflammatory signaling may perturb repair; instead approaches attenuating chronic inflammation and restoring normal patterns of resolution of the inflammatory response may be more effective in protection from adverse remodeling. Third, no animal model can recapitulate the pathophysiologic heterogeneity of postinfarction remodeling in human patients. In animal models, a reductionist approach is typically used, which is optimal for dissection of mechanistic pathways (225). Healthy, sex-, and age-matched animals with identical genetic backgrounds are studied in a well-standardized model of cardiac injury, and in the absence of other therapeutic interventions. In contrast, patients suffering myocardial infarction are highly heterogeneous, exhibiting differences in remodeling responses, due to genetic variability, the presence or absence of comorbidities, or distinct atherothrombotic and myocardial disease patterns. These differences greatly affect the characteristics of the postinfarction inflammatory and reparative response. A subset of patients may exhibit perturbed suppression and resolution of the postinfarction inflammatory response, leading to excessive protease-driven matrix degradation, dilative remodeling, and systolic heart failure. Other patients may have accentuated fibrogenic pathways, resulting in excessive collagen deposition, increased myocardial stiffness and development of heart failure due to diastolic dysfunction. Biomarkers and imaging strategies are needed for pahophysiologically driven stratification of patients with myocardial infarction to identify those likely to benefit from targeted anti-inflammatory approaches (226, 227).

DO MACROPHAGES HOLD THE KEY FOR CARDIAC REGENERATION?

Adult human hearts exhibit a very limited capacity for self-renewal (228), that is overwhelmed by the massive loss of hundreds of millions of cardiomyocytes following myocardial infarction. Despite intense efforts, no therapeutic strategy has shown consistent regenerative effects in adult mammalian hearts. It has been suggested that macrophages may hold the key for cardiac regeneration; however, supporting evidence is predominantly derived from experimental studies in models of cardiac regeneration in fish, amphibians, or neonatal mice. In zebrafish, macrophage subpopulations have been suggested to play a central role in resolution of the scar, an essential step in remuscularization of the injured myocardium (229). Moreover, a subset of macrophages expressing Wilms tumor 1B (WT1b) was implicated in stimulating proliferation of zebrafish cardiomyocytes following injury (230). In the salamander, macrophages were required for myocardial regeneration following cryoinjury; however, their effects were not related to activation of a proliferative program in cardiomyocytes, but rather to effects on fibroblasts and the extracellular matrix that lead to resorption of the provisional matrix network and protect from permanent scarring (231). In neonatal mice, a unique profile of resident macrophages may explain the regenerative capacity of neonatal hearts following myocardial infarction (155, 156). Macrophage depletion experiments suggested that the proregenerative effects of neonatal macrophages may be due to their enhanced angiogenic actions and may not involve direct effects on cardiomyocyte proliferation (155). In contrast, other studies suggested that neonatal macrophages may activate a proliferative program in cardiomyocytes by secreting oncostatin M, thus activating gp130 pathways (232). Considering that oncostatin M (233) and other gp130 cytokines (234) are markedly upregulated in adult infarcted hearts in the absence of regeneration, macrophage-derived cytokines are unlikely to be sufficient to activate a regenerative program in the injured adult myocardium. Myocardial regeneration in neonatal mammals likely requires plasticity and unique phenotypes of several different myocardial cell types.

Conclusions

Macrophages are critically implicated in injury, repair, and remodeling of the infarcted heart. Over the last 20 yr, emerging concepts in fundamental immunology and the availability of genetic models to study macrophage phenotype and function in myocardial disease have greatly advanced our understanding on the fate, mechanisms of expansion and role of macrophages in the infarcted and remodeling heart. Single cell transcriptomic studies have revealed a remarkable heterogeneity of macrophages in the healing infarct (134, 235). Development of new therapies to improve repair and attenuate adverse remodeling in patients with myocardial infarction is dependent on a deeper understanding of the role of macrophage subpopulations in the reparative response and in heart failure progression. The role of specific macrophage subpopulations needs to be dissected by studying the functional role of clusters with distinct transcriptomic signatures. The identity and role of macrophage-derived molecular signals needs to be studied. Moreover, translation into therapy will also require characterization of the changes in macrophage phenotype in human patients with myocardial infarction and pathophysiologic stratification of infarction patients into subgroups with distinct inflammatory and reparative responses.

GRANTS

Dr Frangogiannis’ laboratory is supported by National Institutes of Health grants R01 HL76246, R01 HL85440, and R01 HL149407 and by US Department of Defense grants PR181464 and PR211352. Dr Kubota is supported by the Japan Heart Foundation.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.K. prepared figures; A.K. and N.G.F. drafted manuscript; A.K. and N.G.F. edited and revised manuscript; A.K. and N.G.F. approved final version of manuscript.