Changes in NMR-visible lipids as consequence of loading macrophages with modified LDL.

<p>(A) Moderate mobile lipid signals were present in NMR spectra of native macrophages (control). Loading with native LDL or oxLDL did not result in any significant increase of mobile lipid intensities, whereas eLDL loading gave rise to a dominant lipid signal increase. NMR spectral parameters: 800 MHz spectrometer frequency, gradient-based water suppression pulse sequence (zgesgp) with additional water presaturation, 3.7 s repetition time, temperature: 5°C. Spectra were normalized to protein and/or glutathione (GSH) signal intensities. Spectral annotation according to dominant contributions to NMR signals. (B) The NMR-visible lipid content, i.e. the integral in arbitrary units over the deconvolved NMR signal of the terminal methyl group of fatty acid chains. (C) The average percentage of <i>bis-</i>allylic methylene per fatty acid chain in control macrophages and macrophages loaded with native LDL, oxLDL and eLDL, i.e. the ratio of NMR-visible <i>bis-</i>allylic methylene groups and methyl groups. Deconvolved peaks of (-CH = CH-CH₂-CH = CH-)- and (-CH₃)-protons were integrated. Mean and standard deviation of five samples from different donors, N = 5 except for “LDL” (N = 4). Mann-Whitney <i>U</i> test: **p<0.01.</p

ESI-MS/MS analysis of lipoprotein loaded macrophages and native LDL, oxLDL and eLDL.

<p>(A) eLDL induced significant accumulation of mono- and polyunsaturated lipid species in macrophages. oxLDL only elevated levels of monounsaturated species. (B) Lipid compositional analysis of lipoproteins. oxLDL showed substantially decreased levels of mono- and polyunsaturated lipids compared to native LDL. Saturated lipid species were decreased in eLDL, but increased in oxLDL. Mean and standard deviation of six (A) or eleven (B) replicates from different donors. Mann-Whitney <i>U</i> test: *p<0.05, **p<0.01.</p

Taqman RT-PCR analysis of enzymes mainly involved in sphingolipid biosynthesis and metabolism.

<p>Gene expression was monitored of 4 day differentiated macrophages and of 48 hours eLDL and oxLDL loaded macrophages (day 4 to day 6. RT-PCR was standardized to 18s rRNA as a reference. mean + SD. n = 3.</p

Quantitative determination of the lipid content of Lubrol rafts.

<p>Quantitative determination of the lipid content of Lubrol rafts.</p

Cell surface expression of ceramide and complex sphingolipids.

<p>Depicted is the mean fluorescence intensity as analyzed by flow cytometry using monoclonal antibodies for ceramide, lactosylceramide globotriaosylceramide and dodecasaccharideceramide, and cholera toxin subunit B for ganglioside GM1 after MCSF differentiation (d4) and lipoprotein loading (d6) with eLDL, oxLDL or control (MCSF) <b>(A)</b> ceramide, <b>(B)</b> lactosylceramide/CDw17, <b>(C)</b> globotriaosylceramide, <b>(D)</b> dodecasaccharideceramide and <b>(E)</b> GM1 ganglioside. n = 3. Data on surface ceramide and lactosylceramide have been published previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166798#pone.0166798.ref013" target="_blank">13</a>].</p

Quantitative mass spectrometric analysis of lipid classes.

<p>Lipid species composition of human blood monocytes was analyzed <b>(A)</b> on day one (d1) and four (d4) after MCSF differentiation and <b>(B)</b> following 24h enzymatically modified (eLDL) or oxidized (oxLDL) low density lipoprotein loading. Lipid classes include sphingomyelin (SM), ceramides (Cer), hexosylceramides (HexCer), lactosylceramides (LacCer), cholesteryl ester species (CE) and free cholesterol (FC). mean +/- SD. * = p< 0.05. n = 6.</p

eLDL promotes formation of cholesterol/sphingomyelin rich membrane microdomains while oxLDL induces cholesterol/ceramide microdomains.

<p><b>(A)</b> MCSF differentiation, <b>(B)</b> eLDL loading, <b>(C)</b> oxLDL loading, <b>(D)</b> eLDL loading with subsequent HDL₃-deloading and <b>(E)</b> oxLDL loading with subsequent HDL₃-deloading. Theta toxin was used for cholesterol and lysenin for sphingomyelin labeling. MID15B4 antibody was used for ceramide. Representative cells are shown.</p

oxLDL induces cell surface ceramides and SMPD1 co-localization at the membrane.

<p><b>(A)</b> Mean fluorescence intensity of MID15B4 labeled ceramide in MCSF treated, eLDL or oxLDL loaded macrophages. p < 0.05. <b>(B)</b> SMPD1 (aSM) surface expression (green) and cell surface ceramide content and distribution (red) after MCSF differentiation, eLDL or oxLDL loading. Merge: orange. Representative cells are shown.</p

Gene ontology (GO) enrichment analysis of primary monocyte-macrophage differentiation.

<p>Normalized enrichment scores (NES) indicate the distribution of Gene Ontology categories across a list of genes ranked by hypergeometrical score (HGS). Higher enrichment scores indicate a shift of genes belonging to certain GO categories towards either end of the ranked list, representing up or down regulation (positive or negative values, respectively). Lipid related catergories are generally found to be shifted upwards.</p

Results of an in silico transcription factor binding motif analysis.

<p>Motives that were enriched in the 1000 bp promoter region upstream of up-regulated lipid related genes are shown on the left, binding motives that were enriched in promoters of down regulated genes on the right and motifs that were associated with up- and down-regulated genes at the bottom. Abbreviations (as used in the Transfac database): AHRARNTHIF: aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator/hypoxia inducible factor, AP1: activator protein 1, AP2: activating protein 2, ATF CREB: activating transcription factor/cAMP response element binding, CHCH: Churchill, CNOT3: CCR4-NOT transcription complex subunit 3, E2F: E2F transcription factor, EGR: early growth response, ELF ETS: E74-like factor/E26 transformation-specific, FKLF: fetal β-like globin gene-activating Krüppel-like factor, FOX: forkhead box, GKLF: gut-enriched Krüppel-like factor, GZF1: GDNF-inducible zinc finger protein 1, HIF2A: hypoxia-inducible factor 2a, IRF: interferon regulatory factor, LXR: liver X receptor, MYCMAX: Myc/Max transcriptional complex, NFY: nuclear factor Y, p53: phosphoprotein p53, PPARG: peroxisome proliferator-activated receptor-gamma, RNF96: RING finger protein 96, SP1SP4: specificity protein 1 and 4, TATA: TATA box, TFE: Transcription Factor E (Family), USF: upstream transcription factor, ZBTB: zinc finger and BTB (broad complex, tramtrack, and bric-à-brac) domain-containing, ZFX: zinc finger X-chromosomal protein, ZNF148: zinc finger protein 148.</p