Original Article
Divergence of acetate uptake in proinflammatory and inflammation-resolving macrophages: implications for imaging atherosclerosis

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Abstract

Background

Metabolic divergence of macrophages polarized into different phenotypes represents a mechanistically relevant target for non-invasive characterization of atherosclerotic plaques using positron emission tomography (PET). Carbon-11 (11C)-labeled acetate is a clinically available tracer which accumulates in atherosclerotic plaques, but its biological and clinical correlates in atherosclerosis are undefined.

Methods and results

Histological correlates of 14C-acetate uptake were determined in brachiocephalic arteries of western diet-fed apoE−/− mice. The effect of polarizing stimuli on 14C-acetate uptake was determined by proinflammatory (interferon-γ + lipopolysaccharide) vs inflammation-resolving (interleukin-4) stimulation of murine macrophages and human carotid endarterectomy specimens over 2 days. 14C-acetate accumulated in atherosclerotic regions of arteries. CD68-positive monocytes/macrophages vs smooth muscle actin-positive smooth muscle cells were the dominant cells in regions with high vs low 14C-acetate uptake. 14C-acetate uptake progressively decreased in proinflammatory macrophages to 25.9 ± 4.5% of baseline (P < .001). A delayed increase in 14C-acetate uptake was induced in inflammation-resolving macrophages, reaching to 164.1 ± 21.4% (P < .01) of baseline. Consistently, stimulation of endarterectomy specimens with interferon-γ + lipopolysaccharide decreased 14C-acetate uptake to 66.5 ± 14.5%, while interleukin-4 increased 14C-acetate uptake to 151.5 ± 25.8% compared to non-stimulated plaques (P < .05).

Conclusions

Acetate uptake by macrophages diverges upon proinflammatory and inflammation-resolving stimulation, which may be exploited for immunometabolic characterization of atherosclerosis.

Introduction

Immunometabolism, i.e., the cross-talk of intra-cellular metabolic pathways and immune cell function, plays a pivotal role in the pathogenesis of inflammatory diseases, including atherosclerosis.1, 2, 3 For example, hypoxia, a major trigger of enhanced glycolysis in atherosclerotic plaques, promotes the production of proinflammatory cytokines, e.g., interleukin-1β, by macrophages,4, 5, 6 and expansion of the necrotic core.7 Moreover, proinflammatory stimulation of macrophages by lipopolysaccharide (LPS) enhances glucose uptake and glycolysis,8, 9, 10 which in turn accentuates the production of interleukin-1β through succinate-mediated stabilization of hypoxia-inducible factor-1α and development of a pseudo-hypoxic state.8 The unprecedented recognition of the vessel wall immunometabolic heterogeneity and its mechanistic contribution to atherogenesis provides the opportunity to explore the potential applications of metabolic imaging in cardiovascular diseases, analogous to similar efforts in oncology.11,12

Imaging of glucose utilization by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) has been instrumental in elucidating the in vivo metabolic heterogeneity of the vessel wall, supplementing the information obtained by ex vivo metabolomics techniques.1 However, the non-specificity of 18F-FDG uptake, which targets a nearly ubiquitous metabolic process upregulated in both proinflammatory and inflammation-resolving (reparative) states, makes it suboptimal for characterization of the inflammatory response as a stand-alone imaging marker.9,10,13, 14, 15 Additionally, myocardial uptake of 18F-FDG has been a major challenge for its utilization in coronary artery disease.15,16 These limitations have prompted investigators to exploit the utility of metabolic substrates other than glucose, e.g., glutamine9 and acetate,17,18 in non-invasive characterization of plaque immunometabolism. Notably, the safety of several metabolic substrates has already been established in oncological or cardiac imaging studies,17,19 which facilitates their applications in imaging atherosclerosis.

Carbon-11 (11C)-labeled acetate (11C-acetate) has been extensively studied to detect cancer-associated lipogenesis and myocardial blood flow and oxidative metabolism.20,21 More recently, pre-clinical18 and pilot clinical17 studies have demonstrated the feasibility of imaging 11C-acetate uptake in atherosclerosis. However, the biological correlates of acetate uptake in plaques and its clinical implications have not been addressed. The purpose of this study was to determine the histological correlates of acetate uptake in atherosclerotic plaques and its link to inflammatory states of macrophages, using 14C-acetate where the long half-lived β-emitting C-14 allows for ex vivo imaging via autoradiography. We hypothesized that acetate uptake in macrophage-rich plaques differentiates their proinflammatory vs inflammation-resolving polarization states. Histological correlates of acetate uptake were determined by combined immuno-histology and high-resolution 14C-acetate autoradiography in murine atherosclerotic brachiocephalic arteries. The effect of proinflammatory vs inflammation-resolving stimuli on acetate uptake was assessed in murine macrophage and human carotid endarterectomy specimens.

Section snippets

Animals

Wild-type (N = 12) and apoE−/− (N = 3) mice on a C57BL/6J background were purchased from Jackson Laboratories. Atherosclerosis was induced by feeding with a western diet (21% anhydrous fat, TD88137, Envigo, 33 weeks) in apoE−/− mice. Experiments were conducted in accordance with a protocol approved by Institutional Animal Care and Use Committee.

Cell Culture

Commercial experimental reagents are listed in Supplemental Table 1. Elicited peritoneal cells were harvested from wild-type mice through lavage, three

Uptake of 14C-Acetate by Macrophage-Rich Murine Brachiocephalic Artery Plaques

Micro-autoradiography of murine brachiocephalic arteries from western diet-fed apoE−/− mice demonstrated focal areas of increased 14C-acetate uptake in the vessel wall, corresponding to atherosclerotic plaques, as confirmed by overlaid histological (Movat’s pentachrome staining) and autoradiography images (Figure 1A). Consistent with the visual assessment, quantitative assessment of the brachiocephalic arteries trended towards higher 14C-acetate uptake in plaques compared to plaque-free regions

Discussion

As summarized in schematic Figure 6, our study demonstrated that uptake of acetate is primarily localized to macrophage-rich regions of atherosclerotic plaques. Additionally, we showed a divergence in acetate uptake by macrophages and endarterectomy specimens stimulated with proinflammatory (IFN-γ + LPS) vs inflammation-resolving (IL-4) stimuli, suggesting the potential of 11C-acetate PET in characterizing the inflammatory state of the vessel wall.

The growing appreciation of the contribution of

New Knowledge Gained

Our results demonstrate that arterial uptake of acetate is mostly localized to macrophage-rich regions of atherosclerotic plaques compared to regions enriched in smooth muscle cells. Additionally, we showed that a profound divergence in acetate uptake distinguishes the classical (proinflammatory) and alternative (inflammation-resolving) polarization states of macrophages, induced by interferon-γ + lipopolysaccharide vs interleukin-4, respectively, in two different ex vivo models, i.e., cultured

Disclosures

The authors have no conflicts of interest to disclose.

Funding

This study was supported by grants from National Institutes of Health (NHLBI, K08 HL144911) and Radiological Society of North America (RSD-1820), and a Seed Fund from University of Pittsburgh/UPMC Departments of Radiology and Medicine to S.T; as well as a grant from National Institutes of Health (NHLBI R01 HL146465) to D.G.

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