Macrophage heterogeneity complicates reversal of calcification in cardiovascular tissues

Macrophages are highly heterogeneous cells exhibiting a wide range of protein markers, tissue locations, and functions. Often thought of as host defense cells with phagocytic, innate/adaptive immune system and pro-inflammatory roles, macrophages contribute to a variety of tissue functions including: adipose thermogenesis, tissue repair, splenic and hepatic iron recycling, neural synaptic pruning, and cardiac electrical conduction. In the current issue of Circulation Research, Chinetti-Gbaguidi et al.¹ enhance our understanding of the diverse roles of macrophages in vascular disease by reporting that macrophages near calcified regions of human atherosclerotic lesions display low mineral resorption potential. This study ties macrophage osteoclastogenesis to vascular calcification and identifies disrupted signaling pathways that may help to develop anti-calcification therapies.

A traditional view proposed mechanisms for macrophage polarization induced by pro-inflammatory stimuli such as lipopolysaccharide (LPS) and interferon-γ (IFN-γ) (so-called classically activated or M1 macrophages) or anti-inflammatory factors, e.g., interleukin 4 (IL-4) (alternatively activated or M2 macrophages). Recent evidence has suggested that macrophage heterogeneity is more multidimensional². We recently demonstrated that M1-like human primary macrophages elicited by IFN-γ contain subpopulations, suggesting a more complex pattern of heterogeneity than a traditional theory of macrophage dichotomy³. This diversity brings complexity to the pathogenesis of vascular disease. In atherosclerotic plaques, macrophages contribute to disease progression by secreting pro-inflammatory and cytotoxic factors, and releasing matrix metalloproteinases resulting in extracellular matrix breakdown and plaque instability⁴. These phagocytes also have beneficial roles such as removal of lipoproteins and apoptotic cells, and resolution of inflammation. Similarly, the balance of various macrophage functions may promote or inhibit cardiovascular calcification. Cardiovascular calcification is one of the strongest risk factors associated with negative clinical outcomes in cardiovascular disease⁵, with vascular calcification causing arterial stiffness, microcalcification contributing to plaque rupture, and mineralization of aortic valve leaflets leading to heart failure. Mineralization of cardiovascular tissues is an active process resulting from calcium deposits, largely in the form of hydroxyapatite in the vascular wall and valve. The mechanisms behind this process are still being resolved. Smooth muscle and valve interstitial cells play major roles in driving the formation of extracellular calcium deposits, but several macrophage related factors may contribute to the progression of the calcification in cardiovascular tissue⁶. Our laboratory has demonstrated a role of pro-inflammatory, classically activated macrophages in this process through the secretion of calcifying extracellular vesicles⁷, as well as through poor mineral resorption potential of macrophages in calcified valve tissue⁸. Blocking osteogenic differentiation of cardiovascular cells provides opportunities for prevention and inhibition of calcific disease progression, while enhancing cardiovascular mineral resorption through macrophage differentiation to osteoclast-like cells may induce calcific disease regression. As macrophages can contribute both to the production of cardiovascular mineral deposition and its clearance, they are a uniquely attractive therapeutic target for cardiovascular calcification.

In human atherosclerotic plaques, Chinetti-Gbaguidi et al. observed macrophages surrounding calcium deposits that expressed carbonic anhydrase type II, a molecule associated with osteoclast differentiation and bone resorption⁹, and relatively low cathepsin K (CTSK), a major enzyme involved in osteoclast bone resorption activity¹⁰. These macrophages expressed mannose receptor (MR), a marker typically associated with alternatively activated or anti-inflammatory macrophages. As mentioned, macrophage heterogeneity seems more multidimensional than the traditional M1 vs. M2 paradigm². Nevertheless, this theory of macrophage dichotomy involves clear relationships between stimuli and downstream responses and thus may remain useful to identify specific mechanisms as therapeutic targets. Monocytes/macrophages can undergo osteoclastogenesis in vitro by treatment with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In the present study, when Chinetti-Gbaguidi et al. treated monocytes in vitro with the traditional anti-inflammatory cytokine IL-4 in addition to RANKL and M-CSF, differentiated cells showed low CTSK expression, similar to that observed in macrophages near calcified human atherosclerotic tissue. Additionally, when compared to M-CSF and RANKL treatment alone, IL-4 treated cells had low tartrate-resistant acid phosphatase (TRAP) expression, a gene associated with osteoclastogenesis and resorption function¹¹. The authors identified an ERK-c-fos-NFATc-1 signaling pathway defect as a mechanistic explanation for how IL-4 treatment blocked mineral resorption when monocytes were differentiated to osteoclast-like cells. Therefore, in addition to classically activated macrophages, alternatively activated macrophages may also contribute to cardiovascular calcification via incomplete differentiation to osteoclast-like cells that instead maintain low calcium resorption potential. Based on previous reports^7,8 and the present study by Chinetti-Gbaguidi et al., we propose a working model for the role of macrophages in the development of cardiovascular calcification (Figure 1).

Figure 1. The role of macrophages in cardiovascular calcification.

In addition to established roles of macrophages in atherosclerotic lesion progression and instability, macrophages promote calcification deposition vs. resorption and may contribute to poor clinical outcomes. Dyslipidemia, diabetes, chronic kidney disease, genetic factors, and age may cause vascular and aortic valve calcification. Macrophages can be driven to a classically activated phenotype by stimuli such as IFN-γ or an alternatively activated phenotype through factors including IL-4. Monocytes/macrophages can become osteoclast-like cells by the addition of RANKL and M-CSF in vitro; however, they may not differentiate into fully functional osteoclasts in vivo. This incomplete differentiation may result from the presence of either a pro-inflammatory (Nagy et al.⁸) or anti-inflammatory environment (Chinetti-Gbaguidi et al.¹). A heterogeneous macrophage population may drive cardiovascular calcification though the coexistence of a low potential for mineral resorption and enhanced release of calcific extracellular vesicles (New et al.⁷) contributing to mineral deposition.

The next major step is to ascertain how and if macrophages can be targeted to safely improve clinical outcomes for patients with cardiovascular calcification. This goal presents multiple questions and complex challenges. 1) Can it be accomplished without simultaneously stimulating, or better yet while inhibiting pathways in macrophages, smooth muscle and valve interstitial cells that contribute to cardiovascular calcification through cell osteogenic differentiation? 2) Can macrophage osteoclast-like activity be induced without losing the beneficial roles of macrophages such as in inflammation resolution, while limiting undesired macrophage functions in atherosclerotic lesions in order to reduce lesion progression and promote plaque stability? 3) Can osteoclastogenesis stimulation be done in a way that avoids long-term safety concerns involving bone, as any target elevating cardiovascular osteoclast-like activity should avoid consequential induction of osteoclast activity in the skeletal system and bone resorption? 4) Can targeted pathways be examined in vivo noninvasively to demonstrate regression of the cardiovascular tissue mineral deposits that present the greatest risk for negative clinical outcomes? Cardiovascular calcification can be easily observed in excised tissue through multiple histological methods including Von Kossa and Alizarin Red staining as well as scanning electron microscopy and OsteoSense near-infrared fluorescence microscopy, but the field is currently limited in the availability of high-resolution noninvasive in vivo calcification imaging tools¹². X-ray, ultrasound, fluoroscopy, and CT scanning identify advanced vascular calcification; however, evidence suggests that early microcalcifications may be responsible for atherosclerotic plaque rupture¹³, and while near-infrared fluorescence molecular imaging is able to visualize microcalcifications, it has a limited tissue penetration. It is thus important to identify the effects of macrophages on signs of plaque rupture inducing early microcalcifications, in addition to advanced calcification that could have both tissue stiffness inducing and plaque stabilization roles. A promising way to visualize microcalcifications in addition to advanced calcification is positron-emitting isotope imaging and CT scanning in combination with¹⁸F-fluoride¹⁴, which binds to growing hydroxyapatite crystals. Further work, however, is needed to validate the specificity of the microcalcification readout obtained by this imaging method.

In summary, emerging evidence suggests that macrophage heterogeneity contributes to low osteoclastic activity in human calcified atherosclerotic plaques that may lead to increased mineral deposition, thus supporting further investigation into this area of high unmet medical need.