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

Maximum-likelihood Pearson fit of the EM-densities, for EM number 3 in (a) and number 12 in (b)

The corresponding four moments are () = 1.4, () = 1.0, skewness() = -0.95 and kurtosis() = 4.0 for (a) and () = -0.84, () = 1.0, skewness() = 0.49 and kurtosis() = 4.4 for (b).<p><b>Copyright information:</b></p><p>Taken from "Analyzing M-CSF dependent monocyte/macrophage differentiation: Expression modes and meta-modes derived from an independent component analysis"</p><p>http://www.biomedcentral.com/1471-2105/9/100</p><p>BMC Bioinformatics 2008;9():100-100.</p><p>Published online 17 Feb 2008</p><p>PMCID:PMC2277398.</p><p></p

Histograms and expression profiles of an untransformed (A) and logarithmic corrected (B) microarray expression data set

<p><b>Copyright information:</b></p><p>Taken from "Analyzing M-CSF dependent monocyte/macrophage differentiation: Expression modes and meta-modes derived from an independent component analysis"</p><p>http://www.biomedcentral.com/1471-2105/9/100</p><p>BMC Bioinformatics 2008;9():100-100.</p><p>Published online 17 Feb 2008</p><p>PMCID:PMC2277398.</p><p></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

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

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

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

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