Research ArticleAtherosclerosis

Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming

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Science Translational Medicine  06 Apr 2016:
Vol. 8, Issue 333, pp. 333ra50
DOI: 10.1126/scitranslmed.aad6100
  • 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.

  • 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).

  • 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.

  • Fig. 4. CD mediates metabolism and efflux of crystal-derived cholesterol.

    (A) Macrophages loaded with CCs prepared from D6-cholesterol (D6-CC) can reduce the amount of free, crystal-derived D6-cholesterol by three main mechanisms. First, acetyl-CoA acetyltransferase (ACAT-1) can catalyze the formation of D6-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 D5-27-hydroxycholesterol, which can passively diffuse across cell membranes. Third, D5-27-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 D6-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 D6-cholesterol in cell and supernatant fractions before CD treatment (control bar) and after 48 hours of CD treatment. (C) Efflux of D6-cholesterol into supernatants of D6-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) D5-27-hydroxycholesterol in cell and supernatant fractions of iMacs loaded with 200 μg of D6-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).

  • 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).

  • 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 D6-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) D6-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 D6-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.

  • 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).

  • Fig. 8. CD impairs atherogenesis and regulates metabolic and anti-inflammatory processes in an LXR-dependent manner.

    LDLR−/− mice were transplanted with WT, LXRα−/−β−/−, or MAC-ABCDKO bone marrow. They were then fed a cholesterol-rich diet for 8 weeks and concomitantly treated with CD (2 g/kg) or vehicle control twice a week (n = 6 to 8 per group). (A to C) Plasma cholesterol concentrations of CD- and vehicle-treated animals. (D to F) Atherosclerotic plaque area relative to total arterial wall area. (G to I) Descending aortas of LDLR−/− mice transplanted with WT and LXRα−/−β−/− bone marrow were used for gene expression analysis by microarray, with subsequent filtration for the genes included in the human plaque mRNA profiling. (G) 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 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. NFκB, nuclear factor κB; GTPase, guanosine triphosphatase. (H) DE genes determined by three-way ANOVA (fold change > 1.3, P < 0.05) in aortas of LDLR−/− mice transplanted with WT bone marrow. LXR target genes are colored in red or blue. (I) The expression of genes relevant for the NLRP3 inflammasome pathway. Color bar indicates fold change. (A and F) Data are shown as means + SEM; **P < 0.01 and *P < 0.05, CD versus control (unpaired two-tailed Student’s t test).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/333/333ra50/DC1

    Materials and Methods

    Fig. S1. CD treatment does not influence general cardiovascular parameters.

    Fig. S2. CD treatment does not alter plasma sterol concentrations in atherosclerosis regression trials.

    Fig. S3. CD (10 mM) does not affect the viability of murine macrophages.

    Fig. S4. CD mediates intracellular CC dissolution.

    Fig. S5. CC loading of macrophages induces lipid droplet accumulation.

    Fig. S6. CD does not affect Cyp27a1 expression.

    Fig. S7. CD treatment induces the expression of cholesterol efflux transporters in aortic arches of atherosclerotic mice.

    Fig. S8. CD treatment does not alter murine lipoprotein profiles.

    Table S1. LXR target gene list for GSEA analysis of BMDMs from wild-type and LXRα−/−β−/− mice.

    Table S2. LXR target gene list for GSEA analysis of human atherosclerotic plaques.

    Table S3. List of additional metabolic and regulatory genes (nCounter Panel-Plus).

    Table S4. Original data for all figures (provided as an Excel file).

    References (4257)

  • Supplementary Material for:

    Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming

    Sebastian Zimmer, Alena Grebe, Siril S. Bakke, Niklas Bode, Bente Halvorsen, Thomas Ulas, Mona Skjelland, Dominic De Nardo, Larisa I. Labzin, Anja Kerksiek, Chris Hempel, Michael T. Heneka, Victoria Hawxhurst, Michael L. Fitzgerald, Jonel Trebicka, Ingemar Björkhem, Jan-Åke Gustafsson, Marit Westerterp, Alan R. Tall, Samuel D. Wright, Terje Espevik, Joachim L. Schultze, Georg Nickenig, Dieter Lütjohann, Eicke Latz*

    *Corresponding author. E-mail: eicke.latz{at}uni-bonn.de

    Published 6 April 2016, Sci. Transl. Med. 8, 333ra50 (2016)
    DOI: 10.1126/scitranslmed.aad6100

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. CD treatment does not influence general cardiovascular parameters.
    • Fig. S2. CD treatment does not alter plasma sterol concentrations in atherosclerosis regression trials.
    • Fig. S3. CD (10 mM) does not affect the viability of murine macrophages.
    • Fig. S4. CD mediates intracellular CC dissolution.
    • Fig. S5. CC loading of macrophages induces lipid droplet accumulation.
    • Fig. S6. CD does not affect Cyp27a1 expression.
    • Fig. S7. CD treatment induces the expression of cholesterol efflux transporters in aortic arches of atherosclerotic mice.
    • Fig. S8. CD treatment does not alter murine lipoprotein profiles.
    • Table S1. LXR target gene list for GSEA analysis of BMDMs from wild-type and LXRα−/−β−/− mice.
    • Table S2. LXR target gene list for GSEA analysis of human atherosclerotic plaques.
    • Table S3. List of additional metabolic and regulatory genes (nCounter Panel-Plus).
    • Legend for table S4
    • References (4257)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4. Original data for all figures (provided as an Excel file).