Cardiovascular disease resulting from atherosclerosis is one of the most common causes of death worldwide, and additional therapies for this disease are greatly needed because not all patients can be effectively treated with existing approaches. Cyclodextrin is a common FDA-approved substance that is already used as a solubilizing agent to improve delivery of various drugs. Now, Zimmer et al. have discovered that cyclodextrin can also solubilize cholesterol, removing it from plaques, dissolving cholesterol crystals, and successfully treating atherosclerosis in a mouse model. Because cyclodextrin is already known to be safe in humans, this drug is now a potential candidate for testing in human patients for the treatment of atherosclerosis.

Atherosclerosis is an inflammatory disease linked to elevated blood cholesterol concentrations. Despite ongoing advances in the prevention and treatment of atherosclerosis, cardiovascular disease remains the leading cause of death worldwide. Continuous retention of apolipoprotein B–containing lipoproteins in the subendothelial space causes a local overabundance of free cholesterol. Because cholesterol accumulation and deposition of cholesterol crystals (CCs) trigger a complex inflammatory response, we tested the efficacy of the cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (CD), a compound that increases cholesterol solubility in preventing and reversing atherosclerosis. We showed that CD treatment of murine atherosclerosis reduced atherosclerotic plaque size and CC load and promoted plaque regression even with a continued cholesterol-rich diet. Mechanistically, CD increased oxysterol production in both macrophages and human atherosclerotic plaques and promoted liver X receptor (LXR)–mediated transcriptional reprogramming to improve cholesterol efflux and exert anti-inflammatory effects. In vivo, this CD-mediated LXR agonism was required for the antiatherosclerotic and anti-inflammatory effects of CD as well as for augmented reverse cholesterol transport. Because CD treatment in humans is safe and CD beneficially affects key mechanisms of atherogenesis, it may therefore be used clinically to prevent or treat human atherosclerosis.

Here, we found that subcutaneous administration of CD profoundly reduced atherogenesis and induced regression of established atherosclerosis in mouse models. CD augmented dissolution of CCs, reducing their appearance in lesions. Furthermore, CD increased cholesterol metabolism and liver X receptor (LXR)–dependent cellular reprogramming, which resulted in more efficient reverse cholesterol transport (RCT) as well as reduced proinflammatory gene expression. The atheroprotective effect of CD was dependent on LXR expression in myeloid cells transplanted into LDL receptor (LDLR)–deficient mice. These studies suggest that CD mediates atheroprotection by increasing production of oxysterols and LXR-dependent cellular reprogramming and provide preclinical evidence that CD could be developed into an effective therapy for atherosclerosis in humans.

Genetic approaches to increase the capacity of macrophages to remove free cholesterol from atherosclerotic lesions have proven to be highly successful in preclinical trials ( 11 ). This prompted us to test whether pharmacologically increasing cholesterol solubility, clearance, and catabolism can be exploited for the prevention or treatment of atherosclerosis. 2-Hydroxypropyl-β-cyclodextrin (CD) is a U.S. Food and Drug Administration (FDA)–approved substance used to solubilize and entrap numerous lipophilic pharmaceutical agents for therapeutic delivery in humans ( 12 , 13 ). Although it has previously been shown that CD increases cholesterol solubility, promotes the removal of cholesterol from foam cells in vitro, and initiates anti-inflammatory mechanisms ( 14 – 16 ), it remains unknown whether CD can exert antiatherogenic effects in vivo.

CCs, which can result from excessive cholesterol deposition in atherosclerotic lesions, are among the proinflammatory triggers that contribute to the inflammatory response during atherogenesis ( 7 ). CCs can trigger complement activation and neutrophil extracellular trap (NET) formation, as well as induction of innate immune pathways ( 4 , 5 , 8 – 10 ). Hence, therapeutic strategies aimed at the prevention of cholesterol phase transition or the removal of CCs could reduce tissue inflammation and disease progression.

Atherosclerosis is characterized by arterial wall remodeling, which is initiated by the retention and accumulation of different classes of lipids in the subendothelial layer. Lipid deposition and the appearance of cholesterol crystals (CCs) have been associated with the induction of an inflammatory reaction in the vessel wall, which contributes to the pathogenesis ( 4 , 5 ). Patients with increased systemic inflammation have increased risk of cardiovascular death, and studies are under way to test whether anti-inflammatory treatment can reduce cardiovascular event rates ( 6 ).

Atherosclerosis is the underlying pathology that causes heart attacks, stroke, and peripheral vascular disease. Collectively, these conditions represent a common health problem, and current treatments are insufficient to adequately reduce the risk of disease development. Pharmacologic reduction ( 1 – 3 ) of high-cholesterol concentrations is among the most successful therapeutic approaches to reduce the risk of developing cardiovascular disease and stroke, but adequate reduction of low-density lipoprotein (LDL) cholesterol is not possible in all patients.

RESULTS

CD treatment impairs atherogenesis To investigate the efficacy of CD treatment in murine atherosclerosis, apolipoprotein E (ApoE−/−) deficient mice were fed a cholesterol-rich diet and concomitantly treated subcutaneously with CD or vehicle control for 8 weeks. Although plasma cholesterol, the main driver of atherosclerosis, remained unaffected (Fig. 1A), CD treatment profoundly reduced atherosclerotic lesions within the aortic root (Fig. 1B). Furthermore, we found reduced amounts of CCs in atherosclerotic plaques of CD-treated mice as assessed by laser reflection microscopy (Fig. 1, C and D). CD did not influence weight gain, blood pressure, heart rate, or the number of bone marrow–derived or circulating sca1/flk1-positive cells (fig. S1, A to E). Moreover, plasma concentrations of phytosterols, cholestanol, and cholesterol precursors were not influenced by CD treatment, indirectly showing that CD did not alter enteric cholesterol uptake or overall endogenous biosynthesis (fig. S1F) (17). CD also did not change the relative plaque composition, including cellularity and macrophage content (Fig. 1, E and F). However, the production of aortic reactive oxygen species (Fig. 1G) and plasma concentrations of proinflammatory cytokines were reduced by CD treatment (Fig. 1, H to J), suggesting that CD may reduce the inflammatory response during atherogenesis. Fig. 1. CD treatment impairs murine atherogenesis. ApoE−/− mice were fed a cholesterol-rich diet for 8 weeks and concomitantly treated with CD (2 g/kg) or vehicle control by subcutaneous injection twice a week (n = 7 to 8 per group). (A) Plasma cholesterol concentrations. (B) Atherosclerotic plaque area relative to total arterial wall area. (C) Plaque CC load shown as the ratio of crystal reflection area to plaque area. (D) Representative images of the aortic plaques obtained by confocal laser reflection microscopy. Red, macrophages stained with anti-CD68 antibodies; white, reflection signal of CCs; blue, nuclei stained with Hoechst. Enlarged images are the boxed areas in the left images. Scale bars, 500 μm. (E) Plaque cellularity shown as the ratio of nuclei to plaque area. (F) Plaque macrophage load shown as the ratio of CD68 fluorescence area to total plaque area. (G) Aortic superoxide production determined by L-012 chemiluminescence. ROS, reactive oxygen species; RLU, relative light units. (H to J) Plasma IL-1β, TNF-α, and IL-6 concentrations. Data are shown as means + SEM. ***P < 0.001, **P < 0.01, and *P < 0.05, control versus CD (unpaired two-tailed Student’s t test); n.s., not significant.

CD treatment mediates regression of atherosclerotic plaques Although continuous drug administration in parallel to Western diet feeding of mice is a standard protocol to investigate potential atheroprotective substances (18), patients are generally not treated in early stages of atherogenesis. Therefore, we tested the effect of CD treatment on atherosclerosis regression. ApoE−/− mice are hypercholesterolemic even on normal or lipid-reduced chow, and thus, most murine atherosclerotic regression models rely on interventional strategies that normalize plasma lipids, such as viral gene transfer, transplantation, or infusion of high-density lipoprotein (HDL) particles (19). We adapted a less invasive regression protocol (20) in which ApoE−/− mice were first fed a cholesterol-rich diet for 8 weeks to induce advanced atherosclerotic lesions and then switched to a normal chow diet for another 4 weeks during which CD or vehicle control was administered (Fig. 2A). As expected, plasma cholesterol concentrations were decreased in both groups compared to baseline, but no difference between control and CD treatment was observed (Fig. 2B and fig. S2A). Although switching to a normal chow diet had no effect on atherosclerotic lesion size in vehicle-treated mice, CD treatment resulted in a regression of atherosclerotic plaques by about 45% (Fig. 2C). Although CC load in lesions was already decreased in vehicle-treated animals compared to the load before treatment, CC amounts were further reduced by CD treatment (Fig. 2D). Because patients with cardiovascular disease often do not adhere to the recommended lifestyle changes, which include dietary modifications, we next investigated whether CD treatment can affect atherosclerosis regression during continuous enteric cholesterol challenge. CD or vehicle treatment was started after 8 weeks of cholesterol-rich diet, which was continued for the entire 12 weeks (Fig. 2E). Although plasma cholesterol and general cholesterol metabolism were not altered (Fig. 2F and fig. S2B), atherosclerotic plaque size and CC load were decreased in CD-treated mice on continuous cholesterol-rich diet (Fig. 2, G and H). These data demonstrate that CD treatment is effective in reducing established plaques. Fig. 2. CD treatment facilitates regression of murine atherosclerosis. ApoE−/− mice were fed a cholesterol-rich diet for 8 weeks to induce advanced atherosclerotic lesions. Then, the diet was either changed to a normal chow (A to D) or the cholesterol-rich diet was continued for another 4 weeks (E to H). Mice were simultaneously treated with CD (2 g/kg) or vehicle control twice a week (n = 6 to 8 per group). (A and E) Diet and treatment schemes. (B and F) Plasma cholesterol concentrations. (C and G) Atherosclerotic plaque area relative to total arterial wall area. (D and H) Plaque CC load shown as the ratio of crystal reflection area to plaque area. Data are shown as means + SEM. ***P < 0.001, **P < 0.01, and *P < 0.05, control versus CD (unpaired two-tailed Student’s t test).

CD dissolves extra- and intracellular CCs There are several possibilities to explain the protective effects of CD treatment on both atherogenesis and established atherosclerosis. Because CD is known to form soluble inclusion complexes with cholesterol, thereby enhancing its solubility in aqueous solutions by about 150,000-fold, we tested whether CD increases the solubility of CCs. Fluorescent CD bound to the surface of CCs (Fig. 3, A and B) and CD mediated the solubilization of CCs in a dose-dependent manner (Fig. 3C). To be effective in atherosclerotic plaques, CD must also act on intracellular CCs. Macrophages rapidly internalized fluorescent CD (Fig. 3D) and concentrated it in intracellular compartments (Fig. 3E). Furthermore, incubation with 10 mM CD, a subtoxic dose (fig. S3), enhanced the dissolution of intracellular CCs over time (Fig. 3F and fig. S4). Fig. 3. CD interacts with and dissolves extra- and intracellular CCs. (A and B) CCs (1 mg) were incubated with 0.5 mM rhodamine-labeled CD or phosphate-buffered saline as control. (A) Representative images obtained by confocal laser reflection microscopy. Scale bar, 20 μm. (B) Quantification of rhodamine fluorescence on CCs by flow cytometry. (C) 3H-CCs were incubated with CD solutions of the indicated concentrations overnight with shaking at 37°C. Upon filtration through 0.22-μm filter plates, radioactivity was determined in the filtrate (filterable/solubilized) and the retentate (crystalline). (D and E) iMacs (immortalized macrophages) were loaded with 200 μg of CC per 1 × 106 cells for 3 hours before incubation with 1 mM rhodamine-labeled CD. (D) Quantification of rhodamine fluorescence by flow cytometry. (E) Representative images obtained by confocal microscopy. Red, rhodamine-labeled CD; green, laser reflection signal. Scale bars, 5 μm. (F) Intracellular CC dissolution in BMDMs treated with 10 mM CD or control for the indicated times determined by polarization microscopy. Data are shown as means ± SEM of at least three independent experiments.

Metabolism of crystal-derived cholesterol is increased by CD Macrophages within the arterial wall take up excessive amounts of cholesterol and transform into foam cells, a process that can impair macrophage function and promote atherogenesis (21). This can be mimicked in vitro by loading macrophages with CCs (fig. S5). After uptake of CCs into phagosomes, cholesterol is moved from the lysosome via the Niemann-Pick type C1 (NPC1) transporter to the endoplasmic reticulum, where acetyl–coenzyme A (CoA) acetyltransferase catalyzes the formation of cholesteryl esters. This mechanism turns excess free cholesterol, which forms crystals and is cytotoxic, into cholesteryl esters that can be stored in lipid droplets. A second pathway to metabolize free cholesterol is the formation of water-soluble oxysterols. Oxysterols can diffuse across cell membranes and are known to reprogram macrophages through activation of LXR, which in turn modulates the inflammatory response and supports RCT to HDL (22–24). To study how CD influences the ability of macrophages to reduce the amount of cholesterol derived from CCs, we incubated macrophages with CCs prepared from D 6 -cholesterol (D 6 -CCs) and followed D 6 -cholesterol metabolism products in cells and cellular supernatants by gas chromatography–mass spectrometry selective ion monitoring (GC-MS-SIM) (Fig. 4A). This analysis revealed that CD treatment promoted esterification of crystal-derived D 6 -cholesterol (Fig. 4B). Furthermore, CD amplified D 6 -cholesterol concentrations in supernatants while reducing the overall cellular pool of D 6 -cholesterol (Fig. 4C). Hence, CD treatment increased the cholesterol efflux capacity of macrophages, which represents an important protective factor in patients with coronary artery disease (25, 26). Active cholesterol transport is mediated primarily by the adenosine 5′-triphosphate–binding cassette transporters A1 and G1 (ABCA1 and ABCG1), which transfer free cholesterol to ApoA1 and mature HDL particles, respectively (27). In line with the observed increase in cholesterol efflux capacity, macrophages incubated with CCs had increased expression of both ABCA1 and ABCG1, which was even further enhanced by CD treatment (Fig. 4, D to F). Genes involved in driving cholesterol efflux, including Abca1 and Abcg1, are under the control of the LXR/retinoid X receptor (LXR/RXR) transcription apparatus (22, 28). Because the transcriptional activities of LXRs are positively regulated by oxysterols, we next analyzed whether CD can potentiate cholesterol oxidation. We found that CD treatment of D 6 -CC–loaded macrophages resulted in a marked 15-fold increase in D 6 -cholesterol–derived 27-hydroxycholesterol (D 5 -27-hydroxycholesterol) (Fig. 4G), although the expression of Cyp27a1 was not altered (fig. S6). Unexpectedly, CD also increased 27-hydroxycholesterol production and secretion from macrophages under normocholesterolemic conditions, meaning macrophages not treated with D 6 -CCs (Fig. 4H). Hence, CD increases the metabolism of free cholesterol and could thereby lower the potential for its phase transition into crystals. Fig. 4. CD mediates metabolism and efflux of crystal-derived cholesterol. (A) Macrophages loaded with CCs prepared from D 6 -cholesterol (D 6 -CC) can reduce the amount of free, crystal-derived D 6 -cholesterol by three main mechanisms. First, acetyl-CoA acetyltransferase (ACAT-1) can catalyze the formation of D 6 -cholesteryl esters, the storage form of cholesterol, which are deposited in lipid droplets. Second, the mitochondrial enzyme 27-hydroxylase (Cyp27A1) can catalyze the formation of D 5 -27-hydroxycholesterol, which can passively diffuse across cell membranes. Third, D 5 -27-hydroxycholesterol is a potent activator of LXR transcription factors, which in turn mediate the up-regulation of the cholesterol efflux transporters ABCA1 and ABCG1. (B and C) iMacs loaded with 200 μg of D 6 -CC per 1 × 106 cells for 3 hours were treated with 10 mM CD or vehicle control before GC-MS-SIM analysis of crystal-derived cholesterol. (B) Percentage of esterified D 6 -cholesterol in cell and supernatant fractions before CD treatment (control bar) and after 48 hours of CD treatment. (C) Efflux of D 6 -cholesterol into supernatants of D 6 -CC–loaded macrophages before CD treatment (control bar) and upon 24 hours of CD treatment. (D to F) Gene expression of Abca1 and Abcg1 and protein expression of ABCA1 in BMDMs loaded with 100 μg of CC per 1 × 106 cells for 3 hours and then incubated with 10 mM CD or medium control for (D and E) 4 or (F) 24 hours. Immunoblot in (F) is representative of three independent experiments, and densitometric analysis of all three experiments is provided for 10 mM CD and presented as ABCA1 expression relative to the loading control β-actin. Data are shown as means + SEM of at least three independent experiments. (G) D 5 -27-hydroxycholesterol in cell and supernatant fractions of iMacs loaded with 200 μg of D 6 -CC per 1 × 106 cells for 3 hours before 48 hours of treatment with 10 mM CD or medium control, determined by GC-MS-SIM. (H) 27-Hydroxycholesterol in cell and supernatant fractions of iMacs after 48 hours of treatment with 10 mM CD or medium control. ***P < 0.001 and *P < 0.05, medium versus CD (B to C); CC + control versus CC + CD (D to F); control versus CD (G and H) (unpaired two-tailed Student’s t test).

CD induces LXR target gene expression in macrophages The drastic CD-mediated increase in oxysterol production upon D 6 -CC loading and the unanticipated finding that CD can increase oxysterols in normocholesterolemic macrophages prompted us to comprehensively investigate whether CD influences the expression profiles of LXR-regulated genes. Wild-type or LXRα−/−β−/− macrophages were exposed to CD, CC, or CC and CD, and gene expression was assessed by genome-wide mRNA profiling. To investigate whether CD changes LXR target gene expression in macrophages, we performed gene set enrichment analysis (GSEA) (29) with a set of 533 of previously identified LXR target genes (30) (Fig. 5A and table S1). Enrichment of LXR target gene sets was identified when wild-type macrophages were incubated with CCs (Fig. 5B), presumably because of cholesterol overloading of macrophages. Consistent with the strong induction of CC-derived 27-hydroxycholesterol and the observed increase in cholesterol efflux by CD, LXR target gene sets were enriched when CD was added together with CCs (Fig. 5B). CD treatment alone also resulted in LXR gene set enrichment under normocholesterolemic conditions, which correlates with the observed induction of cellular 27-hydroxycholesterol (Fig. 4H). In LXRα−/−β−/− macrophages, none of the conditions resulted in significant enrichments of LXR target gene sets (Fig. 5C). Furthermore, these findings could be confirmed for the key LXR target genes ABCA1 and ABCG1 in wild-type and LXRα−/−β−/− macrophages on the mRNA and protein levels (Fig. 5, D to F) (31). Fig. 5. CD induces LXR target gene expression in wild-type macrophages. (A) BMDMs from wild-type (WT) and LXRα−/−β−/− mice were loaded with 100 μg of CC per 1 × 106 cells for 3 hours and incubated with 10 mM CD for 4 hours for microarray analysis. GSEA for the LXR target gene sets described by Heinz et al. (30) (table S1) was performed on gene expression data. DB, database. (B and C) GSEA results for (B) WT and (C) LXRα−/−β−/− BMDMs presented as volcano plots of normalized enrichment score (NES) and enrichment P values. Red circles show positively and significantly enriched gene sets (NES > 1, P < 0.05). (D to F) Gene expression of (D) Abca1 and (E) Abcg1, and (F) protein expression of ABCA1 in BMDMs from WT and LXRα−/−β−/− mice loaded with 100 μg of CC per 1 × 106 cells for 3 hours and then incubated with 10 mM CD for (D and E) 4 or (F) 24 hours. The synthetic LXR agonist T0901317 (10 μM) was used as a positive control for ABCA1 protein induction. Immunoblot in (F) is representative of two independent experiments. Data are shown as means + SEM of two independent experiments. *P < 0.05, CC + control versus CC + CD (unpaired two-tailed Student’s t test).

CD increases in vivo RCT To test whether CD-induced LXR reprogramming of macrophages improves macrophage cholesterol efflux in vivo, bone marrow–derived macrophages (BMDMs) from wild-type or LXRα−/−β−/− mice were loaded with D 6 -CCs ex vivo and injected into the peritoneum of wild-type mice. The mice carrying crystal-loaded macrophages were then treated with CD or vehicle control, and D 6 -cholesterol excretion into the feces and urine was monitored by GC-MS-SIM (Fig. 6A). CD increased RCT of crystal-derived D 6 -cholesterol from wild-type and, to a lower extent, LXRα−/−β−/− macrophages (Fig. 6B). Of note, CD treatment not only induced D 6 -cholesterol excretion into the feces but also promoted urinary D 6 -cholesterol elimination (Fig. 6C), a process that is normally not observed during RCT. Prior work on NPC disease, a rare genetic disorder in which cholesterol cannot escape the lysosome, has shown that CD can mobilize lysosomal cholesterol and activate LXR-dependent gene expression (32, 33). NPC1-deficient patients receive weekly injections of CD with the aim of overcoming this cholesterol transport defect. To investigate whether CD can also stimulate urinary cholesterol excretion in humans, we monitored urinary cholesterol excretion of patients with NPC1 mutations after CD infusion over time. CD, which is primarily excreted through the urinary tract, resulted in a time-dependent cholesterol excretion into the urine (Fig. 6D). These data suggest that CD enhances in vivo RCT from macrophages, partially in an LXR-dependent manner, but can also directly extract and transport cholesterol for excretion. Fig. 6. CD facilitates RCT in vivo and promotes urinary cholesterol excretion. (A) BMDMs from WT or LXRα−/−β−/− mice were loaded with 100 μg of D 6 -CC per 1 × 106 cells and injected into the peritoneum of WT mice. Subsequently, mice were treated subcutaneously with CD (2 g kg) or vehicle control (n = 4 per group). (B and C) D 6 -cholesterol content in feces and urine collected every 3 hours over 30 hours after CD injection. Data are shown as total area under the curve (AUC) of excreted D 6 -cholesterol pooled from the mice within a group per time point. (D) Urine samples collected from three individual NPC1 patients upon intravenous application of CD for specific treatment of NPC. Urine cholesterol concentration was determined by GC-MS-SIM and normalized to urine creatinine excretion.

CD modifies human plaque cholesterol metabolism and gene expression To test whether the protective functions of CD on murine macrophages are also exerted in human atherosclerotic plaques, we next performed lipid and genomic analyses on biopsy specimens obtained from carotid endarterectomies (Fig. 7A). Comparable to our findings in murine macrophages, incubation of human atherosclerotic plaques with CD resulted in a transfer of cholesterol from plaques to supernatants (Fig. 7B). Moreover, we observed an increase in the production of 27-hydroxycholesterol, which was mainly released into the supernatants of the CD-treated plaques (Fig. 7C). Gene expression profiling of a large panel of human immunology–related genes and selected LXR target genes (table S3) was performed in resting or treated plaque tissue. These gene expression data were analyzed by several bioinformatics approaches. First, we performed gene ontology enrichment analysis (GOEA) using the genes differentially expressed (DE) after treatment with CD or vehicle control. Consistent with our lipid results, we found that genes involved in lipid transport, storage, metabolism, and efflux were up-regulated upon CD exposure. Conversely, genes known to regulate immune responses, represented by terms such as “regulation of immune responses in lymphocytes,” “regulation of leukocyte-mediated immunity,” or “interleukin response, T cell, and natural killer cell regulation,” were down-regulated after CD treatment (Fig. 7D). Further interrogation of the GOEA revealed that CD treatment of human plaques affected many key genes in the GO term “regulation of inflammatory response” (GO:0050727). These included innate immune receptors, such as Toll-like receptors (TLRs) 2, 3, 4, 7, and 9; the TLR adapter MyD88; the inflammasome sensor NLRP3; and the inflammasome-dependent proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 (Fig. 7E). Because we observed that CD increased the endogenous LXR agonist 27-hydroxycholesterol, we next analyzed whether CD regulates the expression of LXR target genes in human atherosclerotic plaques. GSEA revealed an enrichment of LXR target genes after CD treatment when compared to control-treated plaques (Fig. 7F and table S2). Additionally, many LXR target genes were found among the most DE genes (Fig. 7G, red or blue gene labels). Of note, the inflammasome sensor NLRP3 and the inflammasome inhibitor HSP90 (34) are both LXR target genes (24) and CD treatment resulted in NLRP3 down-regulation and an up-regulation of HSP90 when compared to control (Fig. 7H). Together, these data show that CD activates LXR-dependent transcriptional programs in human plaques, influencing both cholesterol transport and several inflammatory processes, which are relevant to the pathogenesis of atherosclerosis. Fig. 7. CD induces cholesterol metabolism and an anti-inflammatory LXR profile in human atherosclerotic carotid plaques. (A) Human atherosclerotic carotid plaques obtained by carotid endarterectomy (n = 10) were split into two macroscopically equal pieces and cultured for 24 hours with 10 mM CD or control. Half of the plaque tissue was used for mRNA profiling with nCounter Analysis System (NanoString Technologies), and the other half and the culture supernatant were analyzed by GC-MS-SIM. (B) Cholesterol efflux from plaque tissue into supernatants displayed as percent of total cholesterol per sample. (C) Distribution of 27-hydroxycholesterol relative to cholesterol in plaque and supernatant. (D) GOEA of DE genes (fold change > 1.3, P < 0.05) visualized as GO network, where red nodes indicate GO term enrichment by up-regulated DE genes and blue borders indicate GO term enrichment by down-regulated DE genes. Node size and border width represent the corresponding false discovery rate (FDR)–adjusted enrichment P value (q value). Edges represent the associations between two enriched GO terms based on shared genes, and edge thickness indicates the overlap of genes between neighbor nodes. Highly connected terms were grouped together and were annotated manually by a shared general term. (E) Heat map of genes involved in the GO term “regulation of inflammatory response” (GO:0050727). Color bar indicates fold change. (F) Volcano plot of NES and enrichment P values based on GSEA for the LXR target gene set (table S2). Red circle indicates positive and significant enrichment of the LXR target gene set (NES > 1, P < 0.05). (G) Top DE genes determined by three-way analysis of variance (ANOVA) (fold change > 1.5, P < 0.05). LXR target genes are colored in red or blue. (H) The expression of genes relevant to the NLRP3 inflammasome pathway. Color bar indicates fold change. (B and C) Data are shown as means ± SEM. ***P < 0.001 and *P < 0.05, CD versus control (paired two-tailed Student’s t test).