Cardiac resident macrophages in cardiovascular disease: from physiology to pathology

Origin and classification of cardiac resident macrophages

Tissue-resident macrophages (RTMs) and hematopoietic stem cells or macrophages derived from circulating monocytes have completely different genetic background, development and physiological function. RTMs are present in almost all tissues, have some tissue-specific physiological functions, and multiple phenotypes of macrophages are present in the same tissue due to their highly heterogeneity and plasticity. Resident macrophages perceive changes in environmental signals and are responsible for maintaining tissue homeostasis, immune defence, regulation of neural environment and removal of tissue waste.5 Resident macrophages ensure the proper functioning of individual tissues by exerting protective and regulatory effects. C-C chemokine receptor 2⁻ (CCR2⁻) cardiac resident cells are the dominant population of macrophages in the heart, which can be further divided into major histocompatibility complex II (MHCII)^lo and MHCII^hi subgroups. MHCII^lo CCR2⁻ cardiac resident macrophages (including the three markers TimD4⁺, Lyve1⁺ and FolR2⁺) are present in the perivascular location, and MHCII^hi cardiac resident cells are enriched around the nerve tract (figure 1).6

Figure 1

Classification of cardiac resident macrophages. CCR2⁻cardiac resident cells are the dominant population of macrophages in the heart, which can be further divided into MHCII^lo and MHCII^hi subgroups. MHCII^lo CCR2⁻ cardiac resident macrophages (including the three markers TimD4⁺, Lyve1⁺ and FolR2⁺) are present in the perivascular location, and MHCII^hi cardiac resident cells are enriched around the nerve tract. CCR2⁻, C-C chemokine receptor 2⁻; MHC, major histocompatibility complex II.

The prevailing theory is that macrophages originate during embryogenesis, starting from day 8.5 (E8.5) of the embryonic development in mice, in which erythro-myeloid progenitors (EMPs) of yolk cysts produce macrophage precursors. EMPs are programmed as implanted embryonic tissue starting from E9.0 and differentiate into tissue-specific macrophages during organogenesis. Furthermore, EMPs produce monocytes, thus contributing to replenish the tissue-specific macrophage pool.7 Most macrophages in adult tissue are long-lived and proliferate locally.

Self-renewal and proliferation of cardiac resident macrophages

Understanding the proliferation of resident macrophages is critical for determining the regulatory mechanisms of CVD. Cardiac resident macrophages undergo dynamic changes with ageing, being thus the cause of the significant decrease in their proliferation rate and self-renewal capacity. The number of heart macrophages in mice increases from approximately 18 months of age, and the number is positively correlated with age. Molawi et al found that the self-renewal capacity of mouse cardiac macrophages decreases with age and the contribution of blood monocytes to the cardiac macrophage population increases, as revealed by genetic fate mapping methods.8 Macrophages produce the proinflammatory molecules matrix metalloproteinase-9 and C-C motif chemokine ligand 2, both positively associated with the increase in the left ventricular diameter, suggesting the involvement of macrophages in cardiac ageing.9 Macrophage phagocytosis is also impaired in aged mice; macrophage produce high levels of immunosuppressive prostaglandin E2, ultimately leading to dysfunctional immune function, increased susceptibility to CVD and adverse prognosis.10

The addition of cardiac resident macrophages depends on proliferation and differentiation of peripheral blood monocytes in situ, and the proliferation of different subpopulations is different and related to cardiac status and age. CCR2-negative resident macrophage subsets with low expression of MHC-II are completely dependent on self-renewal proliferation. CCR2-negative resident macrophage subsets with high MHC-II expression depend on in situ proliferation and monocyte supplementation. CCR2-positive resident macrophage subsets with high MHC-II expression were completely supplemented by blood monocytes. Resident macrophages in tissues self-divide, renew and persist, independently of bone marrow-derived macrophages, as revealed by lineage tracing experiments.

Cardiac resident macrophage model transformation

Macrophages are immune cells that can be transformed from the inflammatory state to the repair state through a phenotypic transformation, thus participating in the repair process in the body. The regulation and formation of transcriptional profiles of resident macrophages are promoted by some signals in the tissue niche. The transformation of resident macrophages is promoted by both growth factors and cytokines in the tissue niche. However, the specialised transformation of resident macrophages does not depend on one or several specific signalling factors, but on a specific combination of growth factors or cytokines that causes the transformation.

The emergence of new technologies for genetic fate mapping and lineage tracing has made possible to label and track resident cardiac macrophage origin and monitor their phenotypic transformation during tissue development. The developing ventricular myocardium contains cardiac resident macrophages of different lineages that are involved in cardiac development. The CCR2⁻ cells and CCR2⁺ cells are also restricted to different regions of the heart; the former is mainly distributed in the myocardium wall and near the coronary vessels, the latter is distributed in the endocardial trabecula. A relationship exists among CCR2⁺ macrophage abundance, left ventricular remodelling and cardiac function in patients with heart failure (HF) .11

Under the action of cytokines, macrophages change their phenotype to enhance their ability to respond to changes in the microenvironment, a process known as macrophage polarisation. According to phenotype and function, the polarisation of macrophages can be divided into classically activated M1 macrophages and alternative activated M2 macrophages. M2 macrophages were further classified into M2a, M2b, M2c and M2d subsets. On the surface of M1 macrophages, Toll-like receptor 2/4, CD80, CD86, inducible nitric oxide synthase and MHC II are expressed, which are markers of M1 macrophages. M2 macrophages will express the mannitol receptor, CD206, CD163, CD209, found in inflammatory zone protein1 and Ym 1/2 proteins. However, it is worth noting that macrophage polarisation is a dynamic process, and there is no absolute opposite distinction between M1 and M2, which are not mutually exclusive, but often coexist. Under specific conditions, M1 and M2 can also be transformed into each other, which enables macrophages to maintain tissue homeostasis and body balance when the microenvironment changes.

Effects of NLRP3 inflammasome and other mediators on macrophage

The NLRP3 inflammasome is a supramolecular protein complex composed of NLRP3, an apoptosis-related speck-like protein containing a C-terminal caspase recruitment domain (ASC) and the precursor of caspase-1. The NLRP3 inflammasome can activate caspase-1 and regulate the maturation of interleukin-1b, thereby triggering an inflammatory response. The myocardial injury at MI is accompanied by a massive immune cell infiltration, and the stimulation of macrophages can trigger the NLRP3 inflammatory body in the heart. The NLRP3 inflammasome expressed in macrophages is responsible for the detection and elimination of pathogens or pathogen-associated molecules. Yang et al found that L5-low-density lipoprotein-induced interleukin-1β production through activation of NLRP3 inflammasome in macrophages in acute ST-segment elevation myocardial infarction (MI).12 The study by Kawaguchi et al found that ASC was highly expressed in infiltrating macrophages and neutrophils in human MI samples.13 ASC expression was also detected in infiltrating macrophages and neutrophils in the mouse heart after myocardial ischemia reperfusion injury.14 In particular, ASC is expressed in almost all macrophages.

Gasotransmitters (such as nitric oxide, hydrogen sulphide and carbon monoxide) are a group of gas molecules with a variety of biological functions. There has been a remarkable increase in the understanding of the role of exogenous and endogenous gasotransmitters in immune cell types and CVD. Multiple cell populations, such as immune cells, pericytes and macrophages appear to contribute to the cardioprotective potential of gasotransmitters.15 16 Carbon monoxide, a gaseous molecule produced by cells and tissues during the catabolism of heme, can act as an anti-inflammatory molecule and a potent negative regulator of the toll-like receptor signalling pathway. Carbon monoxide inhibited the activation of caspase-1 and the secretion of interleukin-1β in bone marrow-derived macrophages under lipopolysaccharide and ATP treatment. Carbon monoxide also inhibited the generation of mitochondrial reactive oxygen species and the reduction of mitochondrial membrane potential induced.17 The exact role, function and interaction of gasotransmitters in the CVD are still being intensively studied. Among them, gas signalling molecules (gasotransmitters) produced by mammalian cells have been shown to have potential implications for the clinical treatment of ischaemia/reperfusion injury.

Cardiac resident macrophages and arrhythmia

The electrical conduction system of the heart is essential for maintaining the normal heart rate and function is ensured by the electrical conduction system. The classic idea that cardiomyocytes (CMs) are the only cell unit able of transmitting electrical impulses from the heart has been revised. Indeed, it has been recently demonstrated that cardiac macrophages are involved in electrophysiology and arrhythmogenesis, having antiarrhythmic and proarrhythmic effects (figure 2), while the mechanisms they use to influence electrophysiology are inconsistent, including direct and indirect interactions with other heart cells.18

Figure 2

Regulatory mechanisms of cardiac resident macrophages in arrhythmia. Cardiac resident macrophages derived from yolk sac are involved in myocardial electrical conduction and arrhythmia through the coupling of connexin and atrioventricular node cardiomyocytes. ARGE, amphiregulin; AV, atrioventricular; Cx43, connexins 43; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; MHC-II, major histocompatibility complex II; MEK, mitogen-activated extracellular signal-regulated kinase.

Hulsmans et al found that many macrophages are also concentrated in the atrioventricular node endings and atrioventricular bundles in addition to the presence of macrophages in the normal left ventricular myocardium.19 The mean cell surface area of macrophages in the atrioventricular node is comparable to that of macrophages in the left ventricular free wall. This also demonstrates for the first time that cardiac resident macrophages can alter electrical conduction, being also important mediators in this process. To investigate macrophages’ presence and spatial distribution, De Smet et al optically cleared and imaged entire AV nodes of Cx3cr1 GFP/+ mice, an extensively validated reporter strain in which green fluorescent protein identifies cardiac macrophages. The results found an abundant amount of macrophages in the distal atrioventricular node and suggested that they may contribute to cardiac conduction.20 The resting membrane potential of macrophages is from −35 mV to −3 mV, with a membrane resistance of 2.2±0.1 G Ω and a capacitance of 18.3±0.1 pF, suggesting that macrophages are involved in CM depolarisation.21 Resident macrophages express potassium channels, including Kv1.3, Kv1.5 and Kir2.1 involved in several inward and outward rectifying currents. Individual macrophages have no spontaneous electrical depolarisation,22 but once they are cultured with neonatal mouse CMs, a synchronous action potential of their cell membrane and cardiac cells is recorded on day 3. Among the macrophages linked to CMs, 23% of the resting membrane potential is more negative than that of other macrophages and 23% have irregular depolarisation. Macrophage depolarisation is directly consistent with its coupled CMs.23

Connexins 43 (Cx43) are distributed throughout the entire heart, representing important mediators in the transmission of electrical signals among cells. The Cx43 expression region of the distal atrioventricular node in both mouse and humans contains a particularly dense population of macrophages. Nahrendorf et al found that atrioventricular nodal macrophages acted on CMs through gap junctions containing Cx43 and accelerate CM repolarisation and electrical conduction, as demonstrated by an optical clearing technology combined with a specific macrophage reporter cell line.24 The impaired atrioventricular node conduction observed in macrophage-specific Cx43 knockout mice suggests the functional role of Cx43 in the mouse atrioventricular node.25 Moreover, macrophage ablation can also seriously affect the function of the atrioventricular node and triggering arrhythmia. Hitscherich et al also found that M2 macrophages enhance the partial release of Ca²⁺ from CM using a simplified in vitro coculture model of macrophages and induced pluripotent stem cell-derived CMs.26 Monnerat et al demonstrated that cardiac resident macrophages dysregulate the electrical activity of CMs, probably due to the paracrine release of interleukin-1β, which induces oxidative stress in neighbouring cells, consequently triggering arrhythmic events. These findings highlight the impact of macrophages on CMs.

Cardiac resident macrophages and myocardial remodelling

Myocardial remodelling is an important pathophysiological process in the progression of CVD. It consists of adaptive changes in the structure, function, number and genetic phenotype of CMs and non-CMs to adapt to the increased cardiac load by activating neurohumoral mechanisms. It has been recently discovered that macrophages are also involved in myocardial remodelling. Cardiac resident macrophages ensure cardiac stability, with a strong regenerative capacity and organ specificity. Resident cardiac tissue macrophages are involved in the regulation of inflammation and the remodelling process after MI, thus redefining their function in the heart (table 1) .

Cardiac resident macrophages in HF

At present, the pathogenesis and clinical treatment of HF still face many problems that need to be solved. The entire progression of HF is accompanied by an inflammatory response. Most studies on the mechanisms that cause cardiac hypertrophy and failure have focused on CM-specific signalling. Monocyte-derived macrophages and T cells are involved in the transition from compensatory hypertrophy to HF.27

Non-ischaemic cardiomyopathy caused by an increased stressful afterload is an important cause of HF (figure 3). Liao et al have demonstrated that early resident macrophages proliferate in a Kruppel-like factor 4-dependent manner and stimulate angiogenesis, which is a necessary condition for the adaptive response of the heart, as revealed by the myocardial hypertrophy model of pressure-loaded hypertensive mice.28 The number of cardiac macrophages in mouse transection aortic contraction (TAC) models increases after 3 days, peak at approximately 1 week, and return to baseline levels after 2 weeks. The number of cardiac macrophages increases after TAC due to recruitment of CCR2⁺ monocytes and the expansion of resident CCR2⁻ macrophages. Cardiac resident macrophages promote the repair after injury, while recruited monocytes and monocyte-derived macrophages induce inflammation and oxidative stress. Fujiu et al found that cardiac pressure overload in the mouse TAC model induces the production of CSF2 and amphiregulin in renal tubular epithelial cells through the sympathetic nerve, thereby promoting the proliferation of cardiac resident macrophages, limiting ventricular dilation and improving cardiac function.29

Figure 3

Regulatory mechanisms of cardiac resident macrophages in TAC. (A) Dynamic changes in the number of cardiac resident macrophages after TAC and MI; (B) the ARGE production and CCR2, CX3CR1 signalling in TAC regulate cardiac macrophage proliferation to cause cardiac hypertrophy. ARGE, amphiregulin; CCR2, C-C chemokine receptor 2; CX3CR1, chemokine C-X3-C motif receptor 1; CSF2, colony-stimulating factor 2; MI, myocardial infarction; TAC, transverse aortic constriction.

Wong et al demonstrated that CCR2⁻resident macrophages in a chronic HF model of dilated cardiomyopathy maintain cardiac output by promoting left ventricular enlargement and dilation of the coronary system.30 Moreover, cardiac resident macrophages reduce the progression of HF by regulating cardiac angiogenesis probably by interacting with CMs through the adhesion plaque complex, being activated in response to mechanical stretching through the transient receptor potential vanillin-14-dependent pathway, promoting the release of the coronary angiogenic factor insulin-like growth factor-1.31 The depletion of cardiac CCR2-resident macrophages in mice compromises the adaptive remodelling and coronary neovascularisation and accelerates death.32

Cardiac resident macrophages in MI

MI leads to myocardial remodelling and severe cardiac dysfunction, with macrophages being the core participants in the cardiac inflammatory response after MI. Resident macrophages engulf and clear the apoptotic and necrotic CMs present after MI, reduce inflammatory cell infiltration in MI area, secrete anti-inflammatory factors, promote the production of angiogenic factor, and coordinate the repair response through the interaction with other cell types.33 Macrophages are involved in cardiac remodelling and transition to HF after MI. Most cardiac macrophages recruited after MI are monocytes derived from the site of haematopoiesis. Studies on the effects of blood-derived monocytes on cardiac repair revealed that these cells are recruited to the infarct site early along with neutrophils and induce a strong inflammatory response, leading to tissue damage and the formation of fibrotic scars.34 Sager et al recently reported a 2.9-fold increase in the number of macrophages in the distal myocardium of 8 weeks after establishing a MI model in mice with permanent ligation of the proximal left anterior descending coronary artery caused by the renewal of local macrophages and the recruitment of blood monocytes.35 The silencing of five endothelial cell adhesion molecules (intercellular adhesion molecule 2, vascular cell adhesion molecule 1, E-selectin and P-selectin) inhibits monocyte extravasation into the heart, reduces macrophage number and improves cardiac physiology, suggesting the contribution of macrophages to the adverse remodelling of the distal myocardium.

The results indicate that immunotherapy performed by macrophages is of great significance in the treatment of ischaemic heart disease. Dead CMs after acute MI (AMI) release endogenous damage-associated molecular patterns, which activate inflammatory signalling pathways by the interaction of toll-like receptors, recruit and activate immune cells including mononuclear macrophages to infiltrate the infarct site, and exacerbate the inflammatory response. Macrophages undergo phenotypic changes during the advanced stages of cardiac injury, consequently mediating tissue repair responses. Notably, an imbalance between inflammatory and repair responses contributes to pathological cardiac remodelling. Recent studies showed that the expression of genes related to cardiac hypertrophy, fibrosis and inflammation inhibits the harmful transcription of harmful genes and promotes that of beneficial genes by regulating transcription, effectively improving cardiac function after heart injury.36 The number of macrophages in the infarcted site increases significantly after AMI, and their infiltration into the infarct site occurs in three consecutive phases (figure 4).

Figure 4

Regulatory mechanisms of cardiac resident macrophages in MI. The number of macrophages in the infarcted site increases significantly after AMI, and their infiltration into the infarct site occurs in three consecutive phases. Cardiac resident macrophages efficiently remove and degrade apoptotic cardiomyocytes through various pathways to ensure inflammation clearance and tissue repair. AMI, acute myocardial infarction; CCR2, C-C chemokine receptor 2; CCL2, C-C motif chemokine ligand 2; CX3CR1, chemokine C-X3-C motif receptor 1; FIZZ1, found in inflammatory zone 1; IL, interleukin; NF-κB, nuclear factor kappa-B; TNF, tumour necrosis factor; TRAF6, tumour necrosis factor receptor-associated factor 6; SIRT, sirtuins; TLR, Toll-like receptor.

The pericardial cavity is equally populated by macrophages. Cardiac and pericardial macrophage subsets have been employed to contribute to the homeostasis and remodelling environment of the heart (figure 5). The Gata 6⁺ pericardial macrophages (GPCMs) in the mouse pericardial fluid are involved in the reparative immune response. In mice with permanent ligation of the left anterior descending coronary artery, Deniset et al revealed that the pericardial cavity is an important source of macrophages that migrate to the heart after ischaemic injury to prevent the detrimental repair from excessive fibrosis.37 GPCMs in the intact pericardial cavity directly regulate the adverse remodelling of the distal region of the heart, leading to cardiac stiffness and diastolic dysfunction, while resident cardiac macrophages regulate cardiac fibrosis in a local environment. It is probable that GPCMs mediate the changes in cardiac function through a similar paracrine effect according to their location near the epicardial surface of the infarct, but those remaining in the pericardial gap may also contribute to this effect. It is important to note that this study lacks a specific approach to fate mapping, limiting the significance of the work. The development of this tool for macrophages expressing Gata 6 is imperative to evaluate the localisation and effector functions of these cells in the pericardial lumen and cardiac tissue after cardiac injury.

Figure 5

Pericardial macrophage populations in mice and humans. The pericardial cavity of mice consists of two main populations of macrophages: Gata6⁺ pericardial macrophages and MHCII⁺ pericardial macrophages. Samples taken from the pericardial cavity of patients undergoing cardiac surgery revealed the presence of GATA6⁺expressing macrophages, including populations expressing CD163^hi and CD163^lo. MHCII, major histocompatibility complex II.

Cardiac resident macrophages in myocardial damage induced by antitumour drug

Doxorubicin induces cardiomyopathy, which is a common clinical manifestation after doxorubicin chemotherapy, causing left ventricular expansion and poor function in the absence of abnormal load conditions, leading to cardiac systolic dysfunction. Cardiomyopathy is associated to cardiac inflammation, which in turn causes severe pathological changes, including myocardial apoptosis, myocardial wall thickening and myocardial dysfunction. Heart function is improved by the suppression of heart inflammation, although the underlying mechanism is unclear. Macrophages are the most numerous and diverse leucocyte type in the body, highly heterogeneous and involved in the pathogenesis of heart disease by coordinating the systemic inflammatory response. There are different functional phenotypes involved in the cardiovascular inflammatory response. Class A1 scavenger receptor (SR-A1, also known as macrophage scavenger receptor 1 (MSR 1) and CD204) is an innate immune recognition receptor mainly expressed in macrophages and involved in several diseases by regulating the macrophage activity. SR-A1 activates macrophage proliferation in atherosclerotic lesions, while its deficiency inhibits the proliferation of the intrinsic reparative macrophages and exacerbates cardiac injury in a mouse cardiomyopathy model induced by doxorubicin.38

Cardiac resident macrophages in septic cardiomyopathy

Cardiac recovery in sepsis is performed by cardiac-resident macrophage subsets. The Mac1 subgroup has distinct endocytotically enriched transcriptome profile, and Mac1 cells with a high expression of TREM 2 (TREM2^hi) actively remove dysfunctional mitochondria expelled by CMs. Loss of TREM2 impairs the ability of the Mac1 subgroup to self-renew and affects the removal of damaged mitochondria, resulting in excessive inflammatory responses in the heart, exacerbating cardiac dysfunction and reducing survival. Cardiac function is improved by an intraperitoneal injection of TREM2^hi Mac1 cells, preventing septic cardiomyopathy. Thus, regulatory TREM2^hi Mac1 cells can be used as a therapeutic strategy to cure septic cardiomyopathy, providing a new direction in the development of septic cardiomyopathy immunotherapies targeting TREM2.39

Immunomodulatory approaches targeting specific cell types may help to timely resolve and prevent CVD. A matrix metallopeptidase 2/9 responsive-based intelligent hydrogel system consisting of PEG and CTL4 gene nanocarrier, which responds to highly expressed matrix metallopeptidase 2/9 after MI, enables targeted and controlled CTL4 release. CTL4 was taken up by macrophages and became more polarised towards M2, and the released transforming growth factor-β, interleukin-10 increased cardiomyocyte function.40 Flores et al developed a macrophage-specific nanotherapy based on single-wall carbon nanotubes loaded with chemical inhibitors of anti-phagocytic CD47-SIRP α signalling axis. The study found that single-walled carbon nanotubes accumulated within atherosclerotic plaques, reactivated pathological phagocytosis and reduced plaque burden in atherosclerotic apoE^−/− mice without compromising safety.41 Strategies for targeting specific cardiac macrophages remain challenging despite the interest and investigation of the role of cardiac macrophages in cardiac development and disease. Indeed, most clinical attempts did not meet the expectations from preclinical data despite the highly promising results from the experimental model.

Traditional Chinese medicine has obvious efficacy in regulating inflammatory response, maintaining body homeostasis and preventing and treating CVD. Lu et al found that Qishen granules could recruit macrophages from the spleen to the heart in the MI model and play a role in improving cardiac function.42 In a mouse model of ischaemia-reperfusion injury, salvianolic acid B treatment reduced the proportion of M1/M2 macrophages after 3 days of reperfusion, alleviated cardiac collagen deposition after 7 days of reperfusion, and improved cardiac function.43 Zhang et al used the posterior limb ischemia model constructed by femoral artery ligation in mice to explore the role and mechanism of Shexiang Baoxin pill in promoting angiogenesis under inflammatory or ischaemic pathological conditions. The results showed that Shexiang Baoxin pill could promote endothelial cell proliferation, migration and tubule formation by activating macrophages to release vascular endothelial growth factor A (VEGF-A) proangiogenic factor.44 At present, the effect of traditional Chinese medicine on regulating the function of macrophages to improve CVD is still focused on the inflammatory response, and it tends to study the M1 and M2 polarisation of macrophages. Traditional Chinese medicine has the characteristics of multicomponent, multitarget and multilink effect and plays different roles in different stages of disease development through compatibility. Targeting the functional diversity of cardiac macrophages at different stages of the disease, its therapeutic potential remains to be further exploited. This review summarises the recent studies on macrophages as targets of CVD (table 2), to provide a reference for a subsequent research on the treatment of CVD (figure 6) .

Figure 6

Modulating effect of macrophages in cardiovascular with active ingredients. Aiming at the diversity of cardiac macrophages in different cardiovascular diseases, targeted regulation of macrophages can improve myocardial damage in cardiovascular diseases. CCR2, C-C chemokine receptor 2; CDC, cardiosphere-derived cells; CX3CR1, chemokine C-X3-C motif receptor 1; Erk1, extracellular signal-regulated kinase1; EV-YF1, Y RNA fragment; IRF5, interferon regulatory factor 5; IMPs, immune-modifying microparticles; LAMP1, lysosomal-associated membrane protein 1; lncRNAs, long noncoding RNAs; MEK, mitogen-activated extracellular signal-regulated kinase; NF-κB, nuclear factor kappa-B; Nrf2, nuclear factor erythroid 2-related factor 2; NPs, nanoparticles; SIRP, signal regulatory protein-α.