New Insights and Novel Therapeutic Potentials for Macrophages in Myocardial Infarction

Despite advances in prevention, diagnosis, and treatment, cardiovascular disease (CVD) remains the primary cause of death worldwide, and myocardial infarction (MI) makes the greatest contribution to CVD [1]. Furthermore, the World Health Organization has predicted that annual deaths from CVD will increase from 18.1 million in 2010 to 24.2 million in 2030 globally [2]. CVDs, including hypertension, atherosclerosis, ischemic heart disease like MI, and ischemic stroke [3], result in significant death and disability [4]. Considering the poor prognosis associated with CVD, new therapeutic strategies are needed to facilitate cardiac repair following MI.

Previous studies have demonstrated that vertebrate zebrafish is capable of complete cardiac regeneration [5‐7], while some regenerative capacity of mammals can be maintained for just a few days [8, 9]. Macrophages are basic cell components of the innate immune system that infiltrate into injured myocardium during neonatal heart regeneration [8]. Accumulating evidence has revealed that inflammation plays a major role in the pathogenesis of coronary artery disease and atherosclerosis [10‐12] and is necessary for correct and timely repair [13, 14]. After MI, circulating blood monocytes rapidly infiltrate into the infarcted area and differentiate into the appropriate macrophages [15]. Based on surface markers and functions, macrophages are divided into two major subtypes: classically activated macrophages (M1), which are related to inflammatory response, and alternatively activated macrophages (M2), which are associated with regeneration and injury repair. In the inflammatory phase of MI, M1 macrophages activated by tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and lipopolysaccharide (LPS) are the leading subtypes that initially respond to the removal of dead cells and cellular and extracellular matrix (ECM) debris [16, 17]. In the proliferative phase of MI, M2 macrophages gradually predominate to facilitate the repair and regeneration of damaged cardiac tissues [18, 19]. Therefore, the correct and timely regulation of macrophage polarization is a promising therapeutic target for the treatment of MI. In addition, stem cell transplantation and nanoparticle-mediated drug delivery systems have made extensive breakthroughs. Together, macrophages modulated by all kinds of therapeutic strategies, particularly the nanoparticle-mediated drug delivery system, have become the promising therapeutic target in the field of cardiac repair.

In the present review, we discuss recent findings on the association of macrophages with the development of post-MI. More specifically, we focus on the phenotypes and functions of macrophages in a steady state and during MI, as well as possible mechanisms underlying macrophage polarization in the heart. Importantly, we discuss potential therapeutic strategies to improve injury control and functional recovery by modulating macrophage polarization, which involves self-assembly/engineered extracellular vesicles (EVs), nanomedicine, and stem cells (Fig. 1).

Macrophages are the first immune cells that develop during the development of an organism. They play a crucial role in immunity (homeostasis and inflammation) and also regulate organ development and function [20]. Macrophages primarily originate from circulating blood monocytes [21]. Monocytes pertain to the population of mononuclear leukocytes derived from hematopoietic stem cells in fetal liver, adult bone marrow, and splenic reservoir under the stimulation of some cytokines, such as M-CSF, GM-CSF, interleukin-1β (IL-1β), and IL-3 [22, 23], and are released in the bloodstream. Monocytes can be divided into two classifications after being mobilized into the peripheral circulation [24]: (1) Ly6C^l° CCR2⁻ CX3CR1^hi patrolling monocytes (CD14^l°CD16⁺ in humans), which are responsible for surveying the vascular lumen and for clearing dead cells and cellular and ECM debris, and (2) Ly6C^hi CCR2⁺ CX3CR1^l° inflammatory monocytes (CD14^hiCD16⁻ in humans), which produce proinflammatory cytokines, such as IL-1, IL-10, and TNF-α. Over the past 40 years, it has been accepted that all forms of macrophages, including resident macrophages, originate from monocytes; however, this has been challenged in the past few years [25]. Recent research has shown that tissue-resident macrophages represent a different population of cells that are derived from diverse lineages [21, 26, 27]. Studies using mouse models have revealed that many tissue-resident macrophages in the kidney, lung, skin, brain, liver, and heart originate from an embryonic lineage and are maintained throughout life free of monocyte recruitment [28‐32]. In recent years, advances in gene fate-mapping techniques have revealed the two distinct populations of tissue-resident macrophages originating from the prenatal yolk sac and fetal liver [28, 29]. It is now clear that the majority of cardiac resident macrophages originate from the yolk sac [30, 31, 33], and these cardiac resident macrophages are divided into two populations that coexist at homeostasis: MHC-II^l°CCR2⁻ and MHC-II^hiCCR2⁻ cells. The others are known as circulating monocyte-derived MHC-II^hiCCR2⁺ cardiac resident macrophages that are responsible for inflammation initiation (Table 1).

Macrophages, as the basic cell component of the innate immune system, are involved in all stages of atherosclerosis. Macrophages exhibit extensive functional plasticity that is dependent on activation (in vitro) or microenvironmental milieu (in vivo). Macrophages not only possess the essential functions of phagocytic killing of pathogens and antigen presentation activating an adaptive immune response but also maintain tissue homeostasis by eliminating dead cells, cellular debris, and ECM debris and by promoting adaptive remodeling of the ECM [34]. Macrophages acquire a proinflammatory or an anti-inflammatory subtype under proper environmental stimuli [34]. Therefore, macrophages have been divided into M1 and M2 [35, 36] based on the type of in vitro stimulation, surface molecule expression pattern, secretory profile, and function. Although this distinction has deficiencies in adequately including the entire macrophage biological complexity, it provides a common scheme to classify macrophage function. In addition, the switch of macrophage polarization is tightly regulated by signaling networks at the transcriptional and translational levels (Figs. 2 and 3).

M1 macrophages are characterized by increased microbicidal or tumoricidal capacity and secrete high amounts of proinflammatory cytokines and mediators. Classically activated M1 macrophages are triggered by IFN-γ and LPS and are characterized by upregulated biomarkers, such as IL-1β, IL-6, IL-12, and IL-23; inducible nitric oxide synthase (iNOS); TNF-α; chemokine (C-C motif) ligand 2 (CCL2), CCL15, and CCL20; C-X-C motif chemokine 9 (CXCL9), CXCL10, and CXCL11; and CD80 and CD86 [37‐39]. M1 macrophages detect and recognize damage-associated molecular patterns (DAMPs) present in the debris of necrotic cells and pathogen-associated molecular patterns (PAMPs) such as LPS and chitin through toll-like receptors (TLRs) located on the surface of macrophages to promote inflammation response. During an innate immune response, TLR agonists engage the MyD88-dependent pathway, including IRAK4, TRAF6, and IKKβ, ultimately leading to the activation of NF-κB to induce M1 polarization [40]. In addition, a TLR ligand can induce the transcription of TNF through an MyD88-dependent manner, further cooperating with IFN-γ in an autocrine manner to activate these macrophages [34]. IFNs mediate the activation of IRF/STAT signaling pathways via the JAK/STAT signaling pathway, favoring M1 polarization [41, 42]. IFN-γ, which is produced by natural killer (NK) cells responding to stress and infections, can promote macrophages to produce proinflammatory cytokineswhich in turn increase the killing ability of NK cells [43]. Typically, research has shown that mice and humans who lack IFN-γ expression are more susceptible to protozoal and certain kinds of bacterial or viral infections [44]. Furthermore, activated M1 macrophages by LPS via an exogenous TLR ligand can clear the parasite completely [34]. Finally, M1-secreted proinflammatory cytokines play an important role in host defense but also can cause extensive damage to the host.

M2 macrophages are characterized as having wound healing and proliferative properties. M2 macrophages, which are activated by the stimulation of IL-4, IL-13, or IL-21, are involved in wound repair, homeostasis, and tumor metastasis and tumor promotion. They also secrete anti-inflammatory cytokines [45‐47]. M2 macrophages are characterized by decreased expression levels of biomarkers, such as TGF-β, CD163, CD206, chil3 chitinase-like 3 (known as Ym-1), resistin-like-α (known as Fizz1), arginase 1 (Arg-1), and IL-10 to promote cell proliferation, collagen formation, and tissue repair [47]. Aberrant activation of M2 macrophages is associated with tissue fibrosis. Accumulating evidence has indicated that macrophages lacking expression of IL-4 receptor (IL-4R) are incapable of promoting wound healing. In terms of mechanism, M2 macrophages can be triggered by IL-4/L-13 and IL-10/IL-21, depending on the activation of the IRF/STAT signaling pathway via STAT6 and STAT3 [48‐50]. IRF4, PPARγ, Krüppel-like factor 4 (KLF-4), and HIF-2α also mediate the induction of the M2 phenotype [40]. M2 macrophages are further divided into different phenotypes denoted by the stimulus and effector function. IL-4 and IL-13 can induce M2a polarization, whereas Fc-γ receptors and TLR stimulation can trigger M2b macrophages, and GC, IL-10, or TGF-β ligands are responsible for M2c activation [37]. In function, both M2a and M2c can enhance the adaptive immune response, whereas M2b plays a key role in suppressing and regulating inflammation and immunity [51].

Cardiac resident macrophages, which account for 6% to 8% of the noncardiomyocyte population according to the data from healthy adult mouse heart, are indispensable for maintaining the cardiac homeostasis and neonatal heart regeneration [52]. The long-held perspective is that the majority of cardiac resident macrophages originate from peripheral blood monocytes and present an M2 polarization profile [53]. Recently, numerous researchers have demonstrated that cardiac resident macrophages consist of a heterogeneous population that includes resident macrophages derived from the yolk sac: MHC-II^l°CCR2⁻ and MHC-II^hiCCR2⁻ cells, macrophages derived from fetal monocytes and macrophages derived from adult monocytes [15, 33, 54, 55]. The populations seed the heart at specific developmental stages so that they can maintain cardiac homeostasis.

As noted, yolk sac–derived CCR2⁻ macrophages play a major role in the coronary development and cardiac repair. Deletion of CCR2⁻ macrophages in the embryonic period could cause abnormal remodeling, diminished LV systolic function, larger LV chamber dimensions, and increased akinetic myocardium [56]. Circulating monocyte-derived CCR2⁺ cardiac resident macrophages are abundant in proinflammatory genes involved in inflammation initiation and exhibit an M1 polarization profile to maintain cardiac homeostasis. Interestingly, more substantial contributions of circulating monocytes have been observed in the aging heart, indicating that circulating blood monocytes may differentiate into CCR2⁻ macrophages [57, 58], which seemingly coincide with the decreased self-renewal ability of yolk sac–derived resident CCR2⁻ macrophages with age, as demonstrated by Molawi et al. [58].

Atherosclerosis is considered to be a chronic inflammatory disease. Perturbation of lipid metabolism and local inflammation are the two major causes in the pathogenesis of atherosclerosis, including cells such as platelets, which play an initiating role in the development of atherosclerosis and the atherosclerotic plaque [59], monocytes and endothelial cells, connective-tissue elements, lipids, and debris. First, low-density lipoproteins (LDLs) accumulate in the intima, activating endothelial cells to initiate atherosclerosis formation [60]. This accumulation of lipoproteins is located in the subendothelial space and is modified by reactive oxygen species and enzymatic cleavage to be involved in the inflammatory process [61]. LDL, especially oxidized LDL (ox-LDL), and activated platelets are responsible for recruiting circulating monocytes into the endothelial space where they are further differentiated into macrophages that engulf ox-LDL and LDL to form foam cells, which finally produces proinflammatory cytokines (e.g., TNF-α, IL-1, and IL-6) and exacerbate local inflammation [62]. In addition, Lindemann et al. reported that progenitor cells adhere to lipid-laden platelets and turn into macrophages that internalize the lipid-rich platelets and develop into foam cells [63]. Next, macrophages, mast cells, and T cells infiltrate the atherosclerotic plaques, exhibit signs of activation, and secrete inflammatory cytokines responding to proinflammatory cytokines [10]. The apoptosis of foam cells in the lipid core is a key reason for atherosclerosis progression. Next, these apoptotic foam cells are removed primarily by macrophages, a process known as efferocytosis. Further inflammation, necrosis, and thrombosis originating from the gradual accumulation of apoptotic debris in the lipid core have occurred when the balance in which efferocytosis internalizes all dead cells is disturbed [64].

MI is defined as pathologically myocardial cell death resulting from prolonged myocardial ischemia. MI is the leading cause of mortality and disability worldwide [1] and results in a major socioeconomic burden. Such events result from the imbalance between myocardial oxygen supply and demand. Although multiple other mechanisms have been reported to contribute to MI, coronary thrombosis promoted by rupture of the atheroma plaque accounts for most of the cases of MI [65]. Cardiomyocyte necrosis resulting from coronary artery ischemia triggers both a systemic inflammatory response and a local reaction to recruit circulating monocytes into the infarcted site. The post-MI repair response includes three sequential stages: inflammation, tissue replacement, and healing or maturation. In the early inflammatory stage, infiltrated neutrophils and recruited circulating monocytes and cardiac resident macrophages contribute to clear the dead cells and matrix debris. Next, these inflammatory responses are gradually replaced by proliferative monocytes and macrophages, which results in angiogenesis and myofibroblast differentiation. Finally, in the healing phase, fibroblasts, immune cells, and microvasculature form a mature scar [66]. In the following phases, we analyze the role of macrophages in the inflammatory and healing stages after MI.

As noted, the most timely and effective treatment involves amelioration of myocardial ischemia and restriction of the size of MI. Ischemia/reperfusion (I/R) such as percutaneous coronary intervention and intravenous thrombolysis has so far been the principal or only strategy for MI treatment, thereby promptly restoring blood supply [76]. However, deteriorated ischemic damages and further swelling of the infarct size will be accompanied with sudden reperfusion, which results in secondary cascade damages, known as myocardial I/R injury [77]. I/R injury may trigger all kinds of pathological changes, including local acute inflammatory reactions, metabolic disorders, and cell apoptosis or necrosis, even resulting in cardiac dysfunction. Macrophages, a major type of inflammatory cells, have a crucial effect on myocardial ischemic injury with reperfusion [78] and have multiple roles because of their specific phenotypes and the stage of disease.

Macrophages play an indispensable role in the mammalian heart and respond to both the post-MI regeneration and repair by mediating inflammation and immunity. M1 and M2 macrophages are indispensable in repairing the myocardium and in retaining functional architecture. Different populations of macrophages respond to a special stage after MI. Several studies have focused on the mechanism of macrophage polarization that is related to the cardioprotective potential. With advancement in therapeutic approaches, especially the burgeoning NP-mediated drug delivery system, modulating macrophage activation to guide cardiac repair and regeneration is now a promising therapeutic strategy. These strategies are also available to other CVDs associated with macrophages, such as atherosclerosis and myocarditis.

As this review highlights, the roles of macrophages in terms of cardiac repair and regeneration are complex. To better understand the mechanisms of macrophages during the various stages of acute and chronic myocardial disease, further research is warranted. Moreover, although specially designed nanocarriers have been applied to improve the efficiency of therapeutic agents and a major breakthrough has been reached, many challenges remain, including the off-target delivery and security issues, which require additional investigation.