Macrophage Biology in Cardiovascular Diseases

Abstract

Macrophages have key functional role in the pathogenesis are various cardiovascular diseases, such as atherosclerosis and aortic aneurysm. Their accumulation within the vessel wall leads to sustained local inflammatory responses characterized by secretion of chemokines, cytokines, and matrix protein degrading enzymes. Here, we summarize recent findings on macrophage contribution to cardiovascular disease published in ATVB. In this issue, we focus on the origin, survival/death, and phenotypic switching of macrophages within vessel walls.

Introduction

Atherosclerotic cardiovascular disease and aortic aneurysm are two separate disease entities. Atherosclerosis is characterized by the presence of lipid-rich plaques in the subintimal space while aortic aneurysm involves medial destruction.^1,2 Clinically, these two major vascular disorders shared several risk factors that include familial predisposition male sex, smoking, high blood pressure, and hypercholesterolemia.^3,4 Interestingly, diabetes, a well-established comorbidity of atherosclerosis, is thought to be protective of aortic aneurysm.^5,6

Accumulation of macrophages in the vessel wall is a major pathological feature shared by atherosclerosis and aortic aneurysm.^7–9 Broadly speaking, macrophages contribute to the sustained local inflammatory response by secreting chemokines/cytokines as well as factors leading to oxidative stress.^7,10,11 addition, macrophages engaged in a dynamic cross-talk with vascular cells and are responsible for the phenotypic changes of vascular smooth muscle cells.^12,13 Macrophages are a major source of matrix protein degrading enzymes and; thus; play a critical role in both the rupture of atherosclerotic plaques and the destruction of elastin integrity in aneurysmal aorta.¹⁴

Recent publications in ATVB continued the investigation of macrophage-mediated inflammation in atherosclerosis and aortic aneurysm by exploring the molecular mechanisms ranging from macrophage recruitment, proliferation, and survival to functional phenotypes. These publications noticeably expanded our knowledge regarding macrophage biology in the context of cardiovascular disease.

Origins of Lesion Macrophages

Monocyte recruitment from the circulation to the vessel wall is a critical step in the pathogenesis of atherosclerosis and aortic aneurysm. Experimentally, strategies that target monocyte recruitment, such as deletion of the chemokine receptor CCR2¹⁵, diminishes vascular inflammation and inhibits formation of atherosclerosis and aneurysm. However, evidence provided by Robbins et al. suggests that macrophage turnover is rapid, particularly after 4 weeks of high fat diet feeding of ApoE^−/− mice.¹⁶ The authors elegantly showed that replenishment of macrophages, at least in mice, depends primarily on local macrophage proliferation rather than recruiting new monocytes. The notion of local macrophage proliferation is supported by recent analysis of human carotid plaques that were found to contain Ki-67 positive macrophages at the border between the lipid rich necrotic core and overlying fibrous cap.¹⁷ In line with this recent report, Zhang et al. demonstrated a role for the small GTPase Rheb (Ras homolog enriched in brain 1) in regards to macrophage proliferation in atherosclerotic lesions.¹⁸ By crossing Rheb-floxed mice with F4/80-Cre mice, the authors were able to establish that macrophage-specific knockout of Rheb in mice reduced diet-induced atherosclerotic lesion size by 32%. While Rheb is likely to contribute to atherogenesis through multiple mechanisms, including promotion of lipid uptake, local macrophage proliferation is found to be inhibited in the conditional Rheb knockout mice bred into the ApoE null background, likely due to suppressed mTORC1.

Traditionally, macrophages were believed to give rise to foam cells via the uptake of lipids. However, recent lineage tracing studies demonstrated that vascular smooth muscle cells (VSMCs) can undergo phenotypic changes to assume a macrophage-like phenotype in response to pro-atherosclerotic stimuli.¹⁹ In their recent ATVB publication, Wang and colleagues provided convincing evidence that 60% to 70% foam cells in mouse plaques are of SMC origin²⁰, which is consistent with their previous report evaluating human atherosclerotic plaques.²¹ However, not all experimental data support the notion of SMC-to-macrophage phenotypical change. Utilizing lineage-tracing along with single cell RNA-sequencing, Wirka et al. analyzed the fate of SMCs during atherogenesis and found them to assume mostly a “fibromyocyte” instead of macrophage phenotype.²²

The concept of phenotypic change was also explored in aortic aneurysm by Salmon and colleagues using KLF4 deficient mice, a transcription factor known to regulate SMC phenotype.²³ Lack of KLF4 in SMCs was sufficient to confer aneurysm protection in both elastase and angiotensin II models. Previously, Muratoglu et al. reported that the VSMC-specific deletion of Lrp1 (low-density lipoprotein receptor-related protein 1) led to spontaneous aortic aneurysms.²⁴ Subsequently, Au et al. discovered that LRP1 is responsible for maintaining the contractile phenotype in SMCs by regulating calcium signaling events that protect against aortic aneurysm.²⁵ In their recent ATVB publication, Clement and colleagues reported their multicolor lineage tracing analysis of SMCs during the development and progression of angiotensin II–induced aortic aneurysm.²⁶ The authors provided evidence of clonal expansion of a subset of SMCs in the media, which can outgrow into the adventitia of the aneurysmal wall.

Macrophage Survival and Death

A large necrotic core is a major pathological feature of advanced atherosclerotic plaques. Secondary necrosis, consequential to failed clearance of apoptotic macrophages, has been an established contributing process to the expansion of necrotic core.²⁷ The importance of macrophage cell death is further demonstrated by the recent ATVB brief report in which Chipont et al. showed that reducing survival of Ly-6C^lo monocytes by knocking out microRNA-21 increases necrotic core of advanced plaques in ApoE^−/− mice.²⁸ However, evidence provided in another ATVB publications highlights the beneficial effects of diminishing macrophage survival through selective deletion of Akt isoforms in diet induced atherosclerosis.²⁹ This may be explained by the fact that Akt2 deletion leads to a substantial decrease in circulating monocytes and thus recruitment into the vascular tissue was impaired.

Necroptosis, a form of programmed cell-death that resembles necrosis, depends on a unique molecular pathway mediated by receptor interacting protein kinase 1 (RIPK1) and RIPK3.³⁰ The involvement of necroptosis in atherosclerosis was first demonstrated by Lin et al. who showed that RIPK3-dependent necrosis is not a post-apoptotic event. Knocking out RIPK3 in bone marrow of hypercholesterolemia mice reduced the necrotic core size of advanced lesions.³¹ Necroptotic cell death of macrophages was further demonstrated in human advanced atherosclerotic plaques by Karunakaran and colleagues.³² Using the RIPK1 inhibitor necrostatin-1, these authors illustrated that necroptosis could be targeted in experimental atherosclerosis for both therapeutic and diagnostic interventions. In a recent ATVB publication, the same investigative group showed that knockdown of the RIPK3 substrate MLKL decreased both programmed cell death and the necrotic core in the atherosclerotic plaques of Apoe null mice.³³

Karshovska et al. investigated whether mitochondrial function of macrophages such as oxidative phosphorylation and reactive oxygen species production may cause necroptosis and subsequently promote necrotic core formation.³⁴ The authors showed that in inflammatory bone marrow-derived macrophages, deletion of Hif1a, the gene encodes for hypoxia-inducible factor-1a (HIF-1a), inhibited necroptosis likely secondary to reduction in reactive oxygen species. In vivo, myeloid-specific Hif1a knockout reduced macrophage necroptosis and necrotic core size in the ApoE atherosclerosis model. Mechanistically, the group demonstrated that in inflammatory macrophages HIF1a promotes necroptosis via regulating microRNA-mediated ATP deletion.

Markers of necroptosis are also found to be elevated in human and experimental aortic aneurysmal tissues.^35,36 Inhibition of necroptosis in mice with existing aortic aneurysm with inhibitors to RIPK1 and RIPK3 attenuates aneurysm growth and promotes inflammatory resolution.^36,37 However, SMCs appear to be the major cell type affected by necroptosis during aneurysm progression.³⁵ Consistently, SMCs in human as well as mouse aneurysmal tissues express elevated levels of the key don’t eat me molecule CD47 compared to normal aortic tissues.³⁸ Mice treated with proefferocytic anti-CD47 antibodies developed smaller aneurysmal dilations in two experimental models of aortic aneurysm.

Macrophage Phenotypes

In response to different stimuli, macrophages can differentiate in a spectrum of polarization with different functions.³⁹ The two polarizing ends are the M1 (classically activated) and M2 (alternatively activated macrophages). In the context of cardiovascular disease, the general view is that M1 macrophages are pro-inflammatory and M2 macrophages are anti-inflammatory. While this view is somewhat supported by experimental evidence, the regulatory mechanisms underlying inflammation initiation/maintenance and resolution in cardiovascular disease are likely to be complex.

Since the chemokine-like receptor ChemR23 is critical in chronic inflammation, van der Vorst and colleagues examined the effect of ChemR23-deficiency on macrophages as well as dendritic cells in ApoE deficient mouse model of atherosclerosis.⁴⁰ ChemR23^−/− mice showed diminished plaque formation in response to western diet despite increased M2 accumulation within the lesion while reducing the total number of macrophages. This work suggests that the overall function of ChemR23-mediated response favors M1 polarization.

In their recent studies, Hung and colleagues showed the long noncoding RNA PELATOM is highly expressed in macrophages around the shoulders and necrotic core of human plaque section.⁴¹ Knocking down PELATOM significantly reduced macrophage phagocytosis, lipid uptake, and reactive oxygen species production. The reduction is phagocytic oxLDL uptake is thought to be mediated by diminished CD36 expression.

Functions of lesion macrophages is influenced by the environmental cues including signals directly released by endothelial cells. In their investigation of microRNA(miR)-92a, Chang and colleagues tested the EC-macrophage communication in the context of atherosclerosis. By co-culturing macrophages and ECs with miR-92a overexpression or knockdown, the investigators demonstrated that ECs can promote the atheroprone phenotypes of macrophages through miR-92a.⁴² While miR-92a is enriched in ECs as well as in circulation under atheroprone condition, it remains unclear how miR-92a synthesis and secretion is upregulated in atherogenesis.

To determine mechanisms responsible for the regulation of macrophage polarization, Zhang et al showed that mice double deficient in ApoE and IgE had reduced expression of M1 macrophage markers (CD68, MCL1, TNFa, IL-6 and iNOS) but increased expression of M2 macrophage markers (Arg-1 and IL-10).⁴³ In vitro, adding IgE to macrophages under M1 and M2-polarizing conditions significantly increased expression of M1 markers but diminished the expression of M2 markers. Finally, IgE deficiency reduced atherosclerotic lesion inflammation and protected mice from diet-induced plaque formation.

Macrophages are an attractive therapeutic target. To address whether manipulation of macrophages stabilizes established atherosclerotic plaques, Rinne and colleagues fed ApoE^−/− mice with high fat diet (HFD) for 12 weeks and treated the HFD-fed mice with palmitoylethanolamide for 4 weeks.⁴⁴ Palmitoylethanolamide is an endogenous fatty acid with anti-inflammatory properties. The authors demonstrated that plaques in palmitoylethanolamide treated mice exhibited characteristics of stable plaques: reduced necrotic core size, increased collage deposition and downregulation of M1 macrophage markers.

Similarly, both M1 and M2 macrophages are found in aneurysmal tissues and are assumed to play opposing roles in inflammation associated with aortic aneurysm.⁴⁵ M1 macrophages are thought to contribute to the pathogenesis of aortic aneurysm through section of various chemokines/cytokines and MMPs. The idea that M1 macrophages are pro-inflammatory and promote vascular destruction through secretion of their cytokines/chemokines was further supported by Batra et al.⁴⁶ They found that infusion of TNFα^−/− macrophages that were forced into M1 polarization via IFN-γ and LPS inhibited growth of aortic aneurysms. Total body knockout of TNFα in addition to utilizing its inhibitor, infliximab, has previously been shown to attenuate aneurysm progression.⁴⁷ Thus, these findings suggest that M1 polarized macrophages lead to aneurysm formation through TNFα secretion. However, a study by Sharma et al. using Il12p40^−/− mice countered the traditional concept that M2 macrophages are beneficial in aortic aneurysm formation.⁴⁸ Interleukin 12 (Il12), a heterodimeric cytokine composed of p35 and p40 subunits, has been shown to be a key regulator of macrophage polarization. Previous studies have reported that macrophages deficient in IL12p40 are biased towards an M2 profile.⁴⁹ Sharma et al. found that IL12p40 depletion promoted the development of abdominal aortic aneurysm by facilitating the recruitment of M2-like macrophages. Thus, the idea of M2 macrophages being beneficial in aneurysms may not necessarily be as clear as once thought.

In the context of cardiovascular disease, it is increasingly appreciated that macrophages exist in a spectrum of activation states rather than the simple M1 and M2 polarization states. The recent advances in single-cell RNA sequencing have improved the understanding the snapshot of macrophage heterogeneity (as well as other cell populations) in human and experimental plaques. While macrophages in normal mouse aortas appear to cluster within a single population, macrophages in aortic plaques of HFD-fed hypercholesterolemia mice display multiple distinct transcriptome profiles.^50–52 Similarly, Fernandez et al. reported that human atherosclerotic plaques contain at least 6 macrophage clusters.⁵³ The complex gene expression patterns of these macrophage clusters further highlight the multi-functionality and plasticity of macrophages. Interestingly, macrophages in the aortic tissues in aneurysm models appeared to be less diverse than macrophages found in atherosclerotic plaques. Only one to two macrophage clusters were reported in mouse aneurysmal tissues.^54,55 It is unclear whether the time of tissue sampling (4 or 5 weeks after angiotensin II treatment) obscures the true spectrum of macrophage functional phenotypes in aneurysm.

Conclusion

Macrophages are critical players in the development of atherosclerosis and aortic aneurysm. Recent publications in ATVB highlight the complexity of the origins of lesion macrophages as well as the plasticity of these cells. Being a central contributor to both atherosclerosis and aortic aneurysm, great potential exists for a pharmaceutical agent that diminishes macrophage- This study was supported by the National Institute of Health R01HL088447 (BL), R01HL122562 (BL), and T32HL110853 (MK). challenging, increasing understanding of macrophages in human disease facilitated by recent technical advances provide optimism.