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.

Materials and Methods

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 days after intra-peritoneal injection of 1.5 mL of 3% thioglycolate.22 Adherent macrophages were cultured for 2 days in RPMI-1640 supplemented with 10% fetal bovine serum, glucose (5.5 mM), sodium pyruvate (1 mM), glutamine (2 mM), non-essential amino acids (1 ×), HEPES buffer (20 mM), penicillin (50 U·mL−1), and streptomycin (50 µg·mL−1), as described.9,10,13 Macrophages which were not activated by the addition of polarizing stimuli were considered as non-activated (Mφ0). Polarization was induced by addition of the conventional proinflammatory (M1) or inflammation-resolving (M2) stimuli: (i) MφIFNγ + LPS: recombinant murine interferon-γ (50 ng·mL−1) plus LPS (10 ng·mL−1) and (ii) MφIL4: recombinant murine IL-4 (10 ng·mL−1).

Primary aortic vascular smooth muscle cells (VSMCs) obtained from C57BL/6J mice23 were cultured for 2 days in DMEM/F12 supplemented with 10% fetal bovine serum, l-glutamine (2 mM), HEPES buffer (10 mM), penicillin (100 U·mL−1), and streptomycin (100 µg·mL−1). Polarization was induced by addition of the conventional proinflammatory or inflammation-resolving stimuli: (i) VSMCIFNγ + LPS: recombinant murine interferon-γ (50 ng·mL−1) plus LPS (10 ng·mL−1) and (ii) VSMCIL4: recombinant murine IL-4 (10 ng·mL−1).

Culture of Human Endarterectomy Specimens

Anonymized atherosclerotic specimens were collected from three patients who underwent carotid endarterectomy as the standard-of-care, under a “No Human Subject involvement” classification by Institutional Review Board. Freshly collected specimens were cut into ~ 3-mm thick slices and cultured ex vivo in complete RPMI-1640 medium.24,25 Plaques were incubated for two days in the absence (control) or presence of (i) recombinant human interferon-γ (50 ng·mL−1) plus LPS (10 ng·mL−1) vs (ii) recombinant human IL-4 (10 ng·mL−1).

Uptake of 14C-Acetate by Macrophages and VSMCs

14C-acetate uptake was measured in macrophages and VSMCs after 6, 24 or 48 hours of polarization. Immediately prior to the assay, cells were washed with phosphate-buffered saline and were incubated with an uptake buffer (NaCl 140 mM, KCl 5.4 mM, CaCl2 1.8 mM, MgSO4 0.8 mM, d-glucose 5.5 mM, HEPES 25 mM) supplemented with 2 µCi·mL−1 (74 kBq·mL−1) 14C-acetate at 37 °C for 2 hours. Subsequently, cells were washed and lysed with 0.1 mM NaOH. 14C-acetate uptake was quantified by a liquid scintillation counter (Beckman Coulter LS6500), normalized to the DNA content of the cell lysates (PicoGreen, Invitrogen), and expressed relative to the uptake values of unstimulated MФ0 or VSMCs.9,10,13

Micro-autoradiography of 14C-Acetate Uptake

Excised murine brachiocephalic arteries or human endarterectomy specimens were incubated with the uptake buffer (described above) supplemented with 2 µCi·mL−1 (74 kBq·mL−1) 14C-acetate at 37 °C for 2 hours. Specimens were then washed with a cold buffer and embedded in optimal cutting temperature (OCT) compound for cryosectioning (Leica CM1860) at 10 μm thickness. Tissues, along with known amounts of 14C-acetate blotted on papers as standards, were exposed to high-resolution phosphor screens (BAS-IP SR2025 Super Resolution, GE Healthcare). Phosphor screens were scanned by a Sapphire Biomolecular Imager (Azure Biosystems) at 10-µm resolution. Autoradiography images were adjusted using the Fire Look-Up Table and a smoothing algorithm (ImageJ). The average uptake of 14C-acetate by endarterectomy specimens stimulated with proinflammatory and inflammation-resolving stimuli was quantified (ImageJ) by drawing regions of interests around the tissues (2-3 sections per specimen at 250 µm intervals). Uptake values are expressed relative to control (non-stimulated) plaques.

Histology

Histological analysis of the brachiocephalic arteries was performed after completion of the autoradiography by Movat’s pentachrome staining per standard protocols. Smooth muscle cells and macrophages were identified by immunostaining with anti-mouse α-smooth muscle actin (αSMA) and anti-mouse CD68, respectively (Supplemental Table 1). Immunostaining for inducible nitric oxide synthase (iNOS) and CD206 was performed as conventional markers of M1 and M2 polarization, respectively. Slides were mounted using ProLong™ Gold Antifade with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear counter-staining. Tissues were photographed using an AxioVert Microscope (Zeiss) equipped with a digital camera, controlled by ZEN software (Zeiss).

High-Resolution Quantification of 14C-Acetate Uptake

AzureSpot software was used to place a 5 × 5 µm grid on top of overlayed autoradiography and immunostaining (both CD68 and αSMA) (Supplemental Figure 1). 14C-acetate intensity data were transferred to FlowJo 10.5.3 software. Each 5 × 5 µm box was ranked from the lowest to the highest based on its 14C-acetate uptake and assigned a quartile (Q1 = lowest, Q4 = highest). CD68+ and αSMA+ percent areas were determined in each quartile of 14C-acetate uptake (averaged from 2 to 3 tissues sections per mouse).

Gene Expression Analysis

Messenger RNA (mRNA) extraction and reverse transcription were performed on cultured mouse macrophages and endarterectomy specimens 2 days after stimulation using TRIzol® reagent and QuantiTect® Reverse Transcription Kit, per standard protocols.9,10,13 Quantitative PCR (qPCR) was performed using a QuantStudio-3 Real-Time PCR System (ThermoFisher) and TaqMan® gene expression assays (Supplemental Table 2). Transcript amplification data were analyzed using QuantStudio v1.4.3 software (Applied Biosystems) and were normalized to the expression level of 18S ribosomal RNA (Rn18s), as the housekeeping gene. mRNA expression levels are expressed as relative to the level of expression in Mφ0 macrophages and summarized as a heat map. Transcript levels of macrophage polarization markers in endarterectomies are expressed relative to the expression of CD68 to account for variations in the macrophage contents of different specimens.

Statistical Analysis

Data were expressed as mean ± standard error of the mean. Statistical analyses were performed with Prism-8 software (GraphPad). Student t test or analysis of variance (followed by Fisher’s exact post hoc test) was used to test the differences between two or multiple groups, respectively. P < .05 was considered statistically significant.

Results

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 (Figure 1B). To determine the cellular composition of plaque regions with focal 14C-acetate uptake, we performed immunofluorescent staining of brachiocephalic arteries using CD68 and αSMA antibodies, delineating macrophages and smooth muscle cells, as the two most abundant cells within the plaques. As demonstrated in representative images of combined autoradiography and immunostaining (Figures 2A-D), CD68+ macrophages were highly abundant in plaque regions with high 14C-acetate uptake. Quantification of CD68+ and αSMA+ areas (Figure 2E) confirmed that macrophages were the predominant cells in regions with the highest quartile (Q4) of 14C-acetate uptake (CD68+ macrophages: 41.2 ± 0.8% vs αSMA+ smooth muscle cells: 18.1 ± 3.5%, P < .01). On the other hand, vessel wall regions with the lowest quartile (Q1) of 14C-acetate uptake were predominantly comprised of αSMA+ smooth muscle cells (43.3 ± 1.3%) compared with CD68+ macrophages (30.5 ± 2.1%) (Figure 2E, P < .01). Of note, 14C-acetate uptake within the macrophage-rich regions of plaques was heterogeneous, and CD68+ regions consisted of areas with high (marked by the red oval shape in Figures 2A and D) and low (marked by the red box in Figures 2A and D) 14C-acetate uptake. On the other hand, smooth muscle cells both within the plaques (blue arrows in Figures 2B and D) and normal regions of arteries (blue arrowheads in Figures 2B and D) had relatively low 14C-acetate uptake.

Figure 1
figure 1

Focal uptake of 14C-acetate by murine atherosclerotic plaques. Micro-autoradiography (A, left), Movat’s pentachrome (A, middle), and their superimposed (A, right) images demonstrate that vessel wall 14C-acetate uptake colocalizes with brachiocephalic artery atherosclerotic plaques of apoE−/− mice fed with a western diet. Plaque uptake of 14C-acetate (B) trended higher in arterial regions with plaques rather than plaque-free regions

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Figure 2
figure 2

Colocalization of 14C-acetate uptake and macrophage-rich regions of atherosclerotic plaques. Combined micro-autoradiography and immunofluorescent staining for CD68 (macrophage marker) and αSMA (smooth muscle cell marker) (A-D) demonstrates that areas of focal uptake of 14C-acetate primarily correspond to macrophage-rich regions of murine brachiocephalic artery atherosclerotic plaques. Of note, 14C-acetate uptake within the macrophage-rich regions of plaques was heterogeneous. Some macrophage-rich regions demonstrated high 14C-acetate uptake (marked by the red oval shape in A and D), while other regions had low uptake (marked by the red box in A and D). Smooth muscle cells both within the plaque (blue arrows in B and D) and normal regions of artery (blue arrowheads in B and D) had relatively low 14C-acetate uptake. Quantification of CD68+ and αSMA+ areas in vessel wall regions with increasing quartiles of 14C-acetate uptake (E) confirms that macrophages are the most abundant cells in regions with higher 14C-acetate uptake, while smooth muscle cells comprise the most abundant cells in regions with lower 14C-acetate uptake (N = 3 mice, 2-3 tissue sections per each mouse). **indicates P < .01 for within quartile comparison of abundance of macrophages and VSMCs; P values above the brackets indicates between quartiles comparisons of the abundance of macrophages and VSMCs

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Divergence of 14C-Acetate Uptake upon M1 and M2 Polarization of Macrophages

The striking heterogeneity of 14C-acetate uptake in CD68+ regions of plaques led us to examine the association between acetate uptake and different polarization states of macrophages. We determined through immunofluorescence staining that both M1-like (CD68+/iNOS+) and M2-like (CD68+/CD206+) macrophages were present in murine brachiocephalic plaque (Supplemental Figure 2).

We utilized a conventional approach to induce proinflammatory (M1) and inflammation-resolving (M2) polarization states in murine macrophages using IFN-γ plus LPS (MφIFNγ + LPS) and IL-4 (MφIL4), respectively, over a 2-day period. Successful conversion of macrophages into different polarization states was confirmed by markedly different transcript levels of a panel of conventional M1 (Arg2, Cxcl9, Cxcl10, Il1b, Nos2, and Tnf) and M2 (Arg1, Cd36, Chil3, Mrc1, Tfrc, Tgfb) markers (Figure 3).

Figure 3
figure 3

Distinct inflammatory profiles of Mφ0, MφIFNγ + LPS and MφIL4. Heat map illustration of macrophage polarization transcript levels (relative to Mφ0) confirms the development of distinct polarization phenotypes upon 2-day culture of murine macrophages with by IFN-γ + LPS vs IL-4 (N = 5)

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The uptake of 14C-acetate by macrophages was determined at 6, 24, and 48 hours after the induction of polarization. As demonstrated in Figure 4A, there was a steady decline in 14C-acetate uptake in MφIFNγ + LPS, which was noticeable as early as 6 hours after stimulation (59.5 ± 3.7% of the baseline level, P < .001) and reached to its minimum at 48 hours (25.9 ± 4.5% of the baseline level, P < .001). On the other hand, MφIL4 demonstrated a significant, but delayed, increase in 14C-acetate uptake, reaching to 164.1 ± 21.4% of the baseline level at 48 hours after stimulation (P < .01). The 14C-acetate uptake by VSMCs, however, was essentially unchanged upon stimulation by either IFN-γ + LPS or IL-4 (Supplemental Figure 3).

Figure 4
figure 4

Divergence of 14C-acetate uptake in MφIFNγ + LPS and MφIL4. Stimulation of murine macrophages with IFN-γ + LPS vs IL-4 resulted in the divergence of 14C-acetate uptake (A), characterized by a progressive decline by ~ 4-folds in MφIFNγ + LPS and a delayed ~ 64% increase in MφIL4. mRNA level of Slc16a1 (B) is increased in both MφIFNγ + LPS (3.9-fold) and MφIL4 (6.3-fold). Expression of Slc16a3 transcript (C) was also increased in MφIFNγ + LPS (6.1-fold); but the apparent overexpression of Slc16a3 in MφIL4 (3.3-fold) did not reach statistical significance (P = .14). The expression of Slc16a7 (D) was not significantly changed by the polarization state of macrophages (N = 7 for uptake assays and = 5 for gene expression assays)

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The transport of acetate through cell membrane occurs through members of the monocarboxylate transporter family.26 Therefore, we determined the mRNA expression of Slc16a1, Slc6a3, and Slc16a7, encoding monocarboxylic acid transporter-1, -3, and -2, respectively (Figures 4B-D). The expression of Slc16a1, representing the most abundant of the three transporters,26,27 was 3.9 ± 0.3 (P = .05) and 6.3 ± 1.6 (P < .01)-fold higher in MφIFNγ + LPS and MφIL4 compared to Mφ0, respectively (P < .001). The expression of Slc16a3 was also 6.1 ± 1.3 (P < .01) and 3.3 ± 1.2 (P = .14)-fold higher in MφIFNγ + LPS and MφIL4 compared to Mφ0, respectively. The expression of Slc16a7, the least abundant transporter, was comparable among the different polarization states of macrophages.

14C-Acetate Uptake by Human Carotid Plaques Stimulated by IFN-γ + LPS vs IL-4 Mirrors the Changes in Polarized Macrophages

In order to determine the effect of different polarizing agents on acetate uptake in the vessel wall micro-environment, we utilized a previously validated technique of ex vivo culture of human carotid endarterectomy specimens.24,25 Induction of different polarization states in endarterectomy specimens was confirmed by the increased expression of M1 polarization markers, interleukin-1β (IL-1β) and nitric oxide synthase-2 (NOS2), by IFN-γ + LPS (Figure 5A) vs M2 polarization markers, CD163 and mannose receptor C-type 1 (MRC1), by IL-4 (Figure 5B). Consistent with our observation in polarized murine macrophages, stimulation of human endarterectomy specimens with IFN-γ + LPS led to reduction of 14C-acetate uptake to 66.5 ± 14.5% of non-stimulated plaques over a 2-day period (Figures 5C and D). On the other hand, stimulation by IL-4 increased 14C-acetate uptake to 151.5 ± 25.8% (Figures 5C and D) (P < .05).

Figure 5
figure 5

Divergence of 14C-acetate uptake by human atherosclerotic plaques upon proinflammatory vs inflammation-resolving stimulation. Induction of distinct polarization states in human carotid endarterectomy specimens is confirmed by quantification of M1 polarization markers [interleukin-1β (IL-1β) and nitric oxide synthase-2 (NOS2), panel A] vs M2 polarization markers [CD163 and mannose receptor C-type 1 (MRC1), panel B]. Quantitative autoradiography demonstrates that stimulation of plaques with IFN-γ + LPS leads to a significant decline in 14C-acetate uptake, while IL-4 stimulation increases 14C-acetate uptake (C-D). N = 3 endarterectomy specimens (2-3 tissue sections per each specimen quantified)

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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.

Figure 6
figure 6

Schematic summary of the study. 14C-acetate uptake is primarily localized within the macrophage-rich regions of atherosclerotic plaques in brachiocephalic arteries of apoE−/− mice. Ex vivo polarization of murine peritoneal macrophages and human carotid endarterectomy specimens into proinflammatory and inflammation-resolving states, by IFN-γ + LPS vs IL-4, respectively, leads to a marked divergence in their uptake of 14C-acetate, suggesting a potential avenue for immunometabolic characterization of atherosclerotic plaques by PET

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The growing appreciation of the contribution of plaques’ immunometabolic heterogeneity to the vessel wall biology1 underscores the potential role of metabolic imaging in non-invasive characterization of atherosclerosis, which may improve the risk stratification of patients and monitoring the response to preventive or therapeutic interventions.15,16 To further enhance the translational potential of this study, we used murine brachiocephalic plaques, rather than aortic plaques, as the former more accurately models the complexities of human plaque pathophysiology.28,29,–30

Despite initial promising results indicating an association between 18F-FDG uptake and the proinflammatory state of vessel wall macrophages,15,16,31 emerging evidence supports that glucose uptake, as a stand-alone marker, does not reveal sufficient immunometabolic information to allow discrimination of plaques macrophages from smooth muscle cells14,32 or proinflammatory from inflammation-resolving macrophages.5,9,10,13

A promising approach to overcome the non-specificity of 18F-FDG in elucidating vessel wall immunometabolism is a combined imaging approach to target other key metabolic pathways, which may aid in differentiation of different immune cell phenotypes. For example, combined imaging of glutamine and glucose uptake improves the identification of different polarization states of macrophages.9 Considering the central roles of acetate in various immunometabolic pathways, including tricarboxylic acid cycle, lipogenesis, and protein acetylation,3311C-acetate PET represents a promising, but largely unexplored, approach in inflammatory diseases. Notably, 11C-acetate uptake has been reported in ~ 30% of atherosclerotic plaques.17 However, biological/immunometabolic correlates and clinical implications of acetate uptake in atherosclerosis remained to be determined. In this study, we showed that acetate uptake is primarily localized to macrophage-rich regions of atherosclerosis.

In order to identify the immunometabolic implications of acetate uptake, we determined the time-course of changes in cultured macrophages over a 2-day period of ex vivo polarization. We identified a progressive decline in acetate uptake by proinflammatory macrophages, but a delayed increase in acetate uptake in inflammation-resolving macrophages. The distinct temporal pattern of acetate uptake in polarized macrophages may reflect differences in IFN-γ + LPS vs IL-4-induced cellular metabolic reprogramming, particularly in M2-like macrophages which rely on mitochondrial biogenesis for activation and subsequent upregulation of specific metabolic pathways (e.g., tricarboxylic acid cycle, β-oxidation, oxidative phosphorylation).10,34,35 By contrast, IFN-γ + LPS stimulation affects macrophage metabolism by more rapid regulation of flux through certain pathways, including enhanced glycolysis and suppression of the tricarboxylic acid cycle and oxidative phosphorylation cycle.8 This divergence of acetate uptake in differentially polarized macrophages highlights the potential role of 11C-acetate PET in non-invasive discrimination of inflammatory states of atherosclerotic plaques. As a proof-of-principle ex vivo experiment, we confirmed that IFN-γ + LPS reduces, while IL-4 increases, the acetate uptake in human endarterectomy specimens.

Cellular transport of acetate is primarily through monocarboxylate transporters.26 Consistent with prior reports, Slc16a1 and Slc16a3 were the most abundant transporters at the transcriptional level.26,27 Interestingly, Slc16a1 was ~ 6-fold over-expressed in MφIL4, consistent with their higher acetate uptake, compared to Mφ0. There was also ~ 4- and ~ 6-fold increase in the expression of Slc16a1 and Slc16a3 in MφIFNγ + LPS, despite their markedly reduced acetate uptake, which likely reflects their role in transport of other substrates, particularly lactate, which is required for glycolytic reprogramming of macrophages in response to LPS.36

Intra-cellular metabolic fate of various substrates, e.g., glucose and glutamine, plays crucial role in phenotypic alterations of macrophages in response to different stimuli.8,37,38,–39 Through acetyl-CoA synthetase-mediated conversion into acetyl-CoA and subsequent condensation with oxaloacetate to generate citrate, intra-cellular acetate is at the cross-road of metabolic pathways and may contribute to lipogenesis and tricarboxylic acid (TCA) cycle.18 Analysis of rabbit atherosclerotic arteries administered with 14C-acetate has revealed that acetate contributes to both aqueous (e.g., glutamate) and fat-soluble (e.g., triglycerides and cholesterol esters) metabolites.18,40,41 Our study demonstrated profound divergence of overall uptake of acetate by polarized murine macrophages and human atherosclerotic plaques. However, potential alterations in intra-cellular utilization of acetate by its differential fluxes through different pathways and the potential impact of these metabolic changes on macrophage biology and atherogenesis remained to be determined. Importantly, the dichotomy of IFN-γ + LPS (i.e., M1-like) and IL-4 (i.e. M2-like) stimulated macrophages fails to capture the profound and nuanced complexity of macrophage phenotypes and their unique metabolic and functional profiles.42

In this study, we took advantage of the higher spatial resolution of lower-energy β− decay through 14C-acetate autoradiography, in contrast to high-energy low-resolution β+ by 11C-acetate, to determine the histological distribution of acetate uptake within the vessel wall. The long half-life of 14C-acetate was also compatible with the lengthy tissue processing steps required for micro-autoradiography, which would have been challenging for the short half-life of 11C (~ 20 minutes). Although the use of 14C-acetate eliminated the possibility of PET, the feasibility of in vivo detection of 11C-acetate uptake in atherosclerosis has been previously demonstrated17; hence, our primary purpose was to determine the histological correlates of acetate uptake to unravel its association with plaque biology and eventually its clinical implications. Future clinical studies are needed to correlate in vivo 11C-acetate uptake with molecular and histological markers of plaque vulnerability, like the macrophage burden, the abundance of different polarization states of macrophages, and pro/anti-inflammatory cytokine production, and disease outcome.

While acetate is a promising molecular imaging probe for atherosclerotic plaques,17 certain limitations common to other immune-metabolic tracers, including low spatial resolution with respect to plaque size and low signal-to-background, remain roadblocks for translational application. Additionally, high 11C-acetate uptake by metabolically active organs, particularly heart and liver, may limit its usefulness in imaging coronary artery disease.17,43,44,–4511C-acetate PET may therefore be more suited for assessment of atherosclerosis in larger arteries, particularly carotid arteries, to predict and monitor response to conventional, e.g., statin, or emerging immunomodulatory therapies.

In summary, our data demonstrate that acetate uptake within the vessel wall primarily correlates with macrophage-rich atherosclerotic plaques. Moreover, classical proinflammatory polarization of macrophages and atherosclerotic plaques significantly reduces their uptake of acetate, while alternative activation upregulates the uptake. Together, these data suggest a potential link between the enhanced uptake of acetate and the induction of an inflammation-resolving state in atherosclerotic plaques. Further in vivo imaging studies are required to determine if 11C-acetate PET, alone or in combination with other metabolic substrates, e.g., 18F-FDG and 18F-fluoroglutamine, may play a role in identifying vulnerable plaques and in monitoring the response to anti-inflammatory interventions, such as interleukin-1β blocking antibody, canakinumab.46

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 murine macrophages and human endarterectomy specimens. These data provide a new potential venue for in vivo immunometabolic characterization of plaques by non-invasive imaging.