Application of Lipid Class Ratios for Sample Stability Monitoring—Evaluation of Murine Tissue Homogenates and SDS as a Stabilizer

Lipids are a ubiquitous class of structurally complex molecules involved in various biological processes. In the fast-growing field of lipidomics, preanalytical issues are frequently neglected. Here, we investigated the stability of lipid profiles of murine liver, brain, lung, heart, and spleen homogenates by quantitative flow injection analysis using tandem mass spectrometry and high-resolution mass spectrometry. Storage of tissue homogenates at room temperature showed substantial alterations of the lipid profiles reflecting lipolytic action. Therefore, ratios of ceramide to sphingomyelin, lysophosphatidylethanolamine to phosphatidylethanolamine, lysophosphatidylcholine to phosphatidylcholine, and diglyceride to triglyceride were applied to monitor sample stability and the effect of sodium dodecyl sulfate (SDS) as a potential stabilizing agent. The addition of SDS led to a concentration-dependent stabilization of lipid profiles in liver, brain, and heart homogenates, while in lung and spleen homogenates, in particular, the lysophosphatidylethanolamine to phosphatidylethanolamine ratio increased upon addition of SDS. In conclusion, we demonstrated that lipid class ratios reflecting lipolytic activity could be applied to evaluate both the stability of samples and the influence of stabilizers

ABCA transporters and associated genes in lipid metabolism

The ATP-binding cassette transporters A1 (ABCA1) and A7 (ABCA7) are inversely regulated under loading and deloading conditions and we were interested in protein/protein interactions of these transporters. The main topic was to identify, verify and characterize these interactions. In case of ABCA7, the yeast two-hybrid approach led to false positive results, and therefore we focused on the expression and regulation of the two published isoforms. Albeit having similar tissue distribution, the two isoforms show different regulation by stimulation, indicating the use of alternative promoters. This caused us to analyze the promoter of the long isoform in more detail and we were able to map the core promoter region. Analyzes of the putative alternative promoter might enable us to understand the regulatory differences between both isoforms. Due to our initial experiments and data from the literature, we would suggest different functions for the two isoforms of ABCA7. Isoform I localizes to the plasma membrane and is upregulated during monocyte to macrophage differentiation, indicating involvment in phagocytic processes. In contrast, isoform II localizes to intracellular membranes and is upregulated together with phosphatase and tensin homolog in HL-60 cells and during terminal keratinocyte differentiation, pointing to a function in the autophagic pathway. Further experiments are necessary to confirm this hypothesis. ABCA1 regulates plasma high-density lipoprotein levels and its cellular function is mediated through various protein interactions. One aim was to identify and confirm new ABCA1 C-terminus interacting PDZ proteins. By different methods seven new PDZ candidates were identified, and four of them, namely GAIP C-terminus-interacting protein1 (GIPC1), Tax1 (human T-cell leukemia virus type I) binding protein 3 (TAX1BP3), Scribble, and Membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) were confirmed to bind to ABCA1 by different methods. Together with the known ABCA1 interacting PDZ proteins and their different tissue expression this may indicate that PDZ proteins are able to regulate ABCA1 trafficking and/or function in a tissue or even cell compartment specific manner. The cytosolic molybdo-flavoenzyme aldehyde oxidase 1 (AOX1), known as xenobiotic metabolizing enzyme, was previously suggested as an ABCA1 interacting protein. In this work it was shown that knock-down of AOX1 by siRNA significantly reduced ABCA1-dependent lipid-efflux and enhanced phagocytic uptake in HepG2 cells. ABCA1 and AOX1 were found coexpressed in certain human cell types, namely hepatocytes, kidney proximal tubular epithelial cells, Leydig cells and cells of the adrenal cortex. Deregulation of ABCA1 and AOX1 in hepatocellular- and renal cell-carcinomas was observed, suggesting that AOX1, perhaps in context with ABCA1, might be used as tumor marker. The involvement of AOX1 in metabolism of ethanol and xenobiotics is of particular interest, as it might link ABCA1 to detoxification, a process in which many ABC-transporters are involved. Finally, as ABCA1 through its interaction with Fas-associated via death domain (FADD) might influence apoptosis, we searched for apoptotic genes regulated by modified lipoproteins in a similar manner as ABCA1. Uptake of modified lipoproteins by macrophages causes foam cell formation and promotes the development of atherosclerotic lesions. Atherogenic lipoproteins exert cytotoxic effects and induce necrosis or apoptosis under certain conditions but may also enhance macrophage survival. GeneChip experiments were performed to identify genes that are regulated in macrophages treated with enzymatically modified low-density lipoprotein (E-LDL). Expression of TOSO, protecting cells against CD95- or tumor necrosis factor-mediated apoptosis, was found induced by E-LDL. Concomitantly, reduced apoptosis was detected in E-LDL loaded macrophages compared to oxidized LDL incubated cells or controls. Abundance of the caspase inhibitor FLICE-like inhibitory protein long form (FLIPL) was suggested to mediate the antiapoptotic properties of TOSO; however, FLIPL expression is induced neither in E-LDL laden macrophages nor in COS-7 cells overexpressing TOSO. E-LDL represents coreless liposome like particles and may be taken up by Fc- and complement-receptor dependent phagocytosis. Internalization of phagobeads by monocytes and macrophages upregulates TOSO but phagocytosis was not altered by TOSO in COS-7 cells. These data indicate that E-LDL-generated foam cells are protected from cell death most likely through the expression of TOSO by a mechanism independent of FLIPL. ABCA1 and ABCA7 may be related but exert distinct functions in the lipid-efflux/apoptosis/autophagy complex. While the function of ABCA1 is highly modulated by protein interactions, ABCA7 might be more affected by transcriptional regulation

Alterations of plasma lysophosphatidylcholine species in obesity and weight loss

Background Obesity and related diseases of the metabolic syndrome contribute to the major health problems in industrialized countries. Alterations in the metabolism of lipid classes and lipid species may significantly be involved in these metabolic overload diseases. However, little is known about specific lipid species in this syndrome and existing data are contradictive. Methods In this study, we quantified plasma lipid species by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in obese subjects before and after 3 month weight loss as well as in a control group. Results The comparison of obese subjects with control subjects before weight loss revealed significantly lower lysophosphatidylcholine (LPC) concentrations in obesity. LPC concentrations did not significantly increase during the observed period in the weight loss group. Analysis of LPC species revealed a decrease of most species in obesity and negative correlations with C-reactive protein (CRP) and body mass index (BMI). Correlating BMI ratio before and after weight loss with the ratio of total LPC and individual LPC species revealed significant negative relationships of LPC ratios with BMI ratio. Conclusions Our findings contribute to the contradictive discussion of the role of LPC in obesity and related chronic inflammation strongly supporting pre-existing data in the literature that show a decrease of LPC species in plasma of obese and a potentially anti-inflammatory role in these subjects

ATP-binding cassette transporters in immortalised human brain microvascular endothelial cells in normal and hypoxic conditions

Background Rapid reperfusion following ischemia is the most effective therapy in stroke therapy. However, the success may be compromised by ischemia & reperfusion (I/R) injury and at the human blood-brain barrier (BBB), therefore the effects on transendothelial transport are of special interest. Current studies suggest the ATP-binding cassette (ABC) transporters to be regulated upon ischemic stroke in a way that impedes the effects of drug therapy. The immortalised human brain microvascular endothelial cell line hCMEC/D3 provides most of the unique properties of the BBB with respect to transport and might be a reliable in vitro model to study transendothelial transport after I/R. Methods We exposed hCMEC/D3 cells to 24 hours of hypoxia alone and to hypoxia followed by 60min of reoxygenisation as a in vitro model for I/R. Western blot showed mild upregulation of hypoxia inducible factor (HIF-1alpha) after hypoxia alone and RNA lysates were analysed with a well-established real-time RT-PCR-based TaqMan low-density array detecting 47 of 48 known human ABC transporters. Results No significant increases of ABC mRNA expression levels were detected neither in hypoxic nor in I/R samples. However, slight decrease of ABCC1 in hypoxic and I/R samples and of ABCA10 and ABCD3 in I/R samples was observed. Conclusion Our data suggests that hCMEC/D3 cell line and - at the moment - in vitro models in general are a poor basis for stroke research but may be enhanced by co-culturing more cells of the neurovascular unit including neuronal stem cells inducing an overall ischemic response at the BBB

oxLDL and eLDL Induced Membrane Microdomains in Human Macrophages.

Extravasation of macrophages and formation of lipid-laden foam cells are key events in the development and progression of atherosclerosis. The degradation of atherogenic lipoproteins subsequently leads to alterations in cellular lipid metabolism that influence inflammatory signaling. Especially sphingolipids and ceramides are known to be involved in these processes. We therefore analyzed monocyte derived macrophages during differentiation and after loading with enzymatically (eLDL) and oxidatively (oxLDL) modified low-density lipoproteins (LDL).Primary human monocytes were isolated from healthy, normolipidemic blood donors using leukapheresis and counterflow elutriation. On the fourth day of MCSF-induced differentiation eLDL (40 μg/ml) or oxLDL (80 μg/ml) were added for 48h. Lipid species were analyzed by quantitative tandem mass spectrometry. Taqman qPCR was performed to investigate transcriptional changes in enzymes involved in sphingolipid metabolism. Furthermore, membrane lipids were studied using flow cytometry and confocal microscopy.MCSF dependent phagocytic differentiation of blood monocytes had only minor effects on the sphingolipid composition. Levels of total sphingomyelin and total ceramide remained unchanged, while lactosylceramides, cholesterylesters and free cholesterol decreased. At the species level most ceramide species showed a reduction upon phagocytic differentiation. Loading with eLDL preferentially increased cellular cholesterol while loading with oxLDL increased cellular ceramide content. Activation of the salvage pathway with a higher mRNA expression of acid and neutral sphingomyelinase, neutral sphingomyelinase activation associated factor and glucosylceramidase as well as increased surface expression of SMPD1 were identified as potentially underlying mechanisms. Moreover, flow-cytometric analysis revealed a higher cell-surface-expression of ceramide, lactosylceramide (CDw17), globotriaosylceramide (CD77), dodecasaccharide-ceramide (CD65s) and GM1 ganglioside upon oxLDL loading. ApoE in contrast to apoA-I preferentially bound to the ceramide enriched surfaces of oxLDL loaded cells. Confocal microscopy showed a co-localization of acid sphingomyelinase with ceramide rich membrane microdomains.eLDL leads to the formation of lipid droplets and preferentially induces cholesterol/sphingomyelin rich membrane microdomains while oxLDL promotes the development of cholesterol/ceramide rich microdomains via activation of the salvage pathway

Monocyte to Macrophage Differentiation Goes along with Modulation of the Plasmalogen Pattern through Transcriptional Regulation

Dysregulation of monocyte-macrophage differentiation is a hallmark of vascular and metabolic diseases and associated with persistent low grade inflammation. Plasmalogens represent ether lipids that play a role in diabesity and previous data show diminished plasmalogen levels in obese subjects. We therefore analyzed transcriptomic and lipidomic changes during monocyte-macrophage differentiation in vitro using a bioinformatic approach

Quantitative determination of the lipid content of Lubrol rafts.

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

Combined Expression of HGFR with Her2/neu, EGFR, IGF1R, Mucin-1 and Integrin α2β1 Is Associated with Aggressive Epithelial Ovarian Cancer

Hepatocyte growth factor receptor (HGFR), also known as c-mesenchymal–epithelial transition factor (c-MET), plays a crucial role in the carcinogenesis of epithelial ovarian cancer (EOC). In contrast, the mechanisms contributing to aberrant expression of HGFR in EOC are not fully understood. In the present study, the expression of HGFR with its prognostic and predictive role was evaluated immunohistochemically in a cohort of 42 primary ovarian cancer patients. Furthermore, we analyzed the dual expression of HGFR and other druggable biomarkers. In the multivariate Cox regression analysis, high HGFR expression was identified as an independent prognostic factor for a shorter progression-free survival (PFS) (hazard ratio (HR) 2.99, 95% confidence interval (CI95%) 1.01–8.91, p = 0.049) and overall survival (OS) (HR 5.77, CI95% 1.56–21.34, p = 0.009). In addition, the combined expression of HGFR, human epidermal growth factor receptor 2 (Her2/neu), epithelial growth factor receptor (EGFR), insulin-like growth factor 1 (IGF1R), Mucin-1 and Integrin α2β1 further significantly impaired PFS, platinum-free interval (PFI) and OS. Protein co-expression analyses were confirmed by transcriptomic data in a large, independent cohort of patients. In conclusion, new biomarker-directed treatment targets were identified to fight poor prognosis of primary EOC

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