Search

Abstract Background The analysis of high-throughput gene expression data sets derived from microarray experiments still is a field of extensive investigation. Although new approaches and algorithms are published continuously, mostly conventional methods like hierarchical clustering algorithms or variance analysis tools are used. Here we take a closer look at independent component analysis (ICA) which is already discussed widely as a new analysis approach. However, deep exploration of its applicability and relevance to concrete biological problems is still missing. In this study, we investigate the relevance of ICA in gaining new insights into well characterized regulatory mechanisms of M-CSF dependent macrophage differentiation. Results Statistically independent gene expression modes (GEM) were extracted from observed gene expression signatures (GES) through ICA of different microarray experiments. From each GEM we deduced a group of genes, henceforth called sub-mode. These sub-modes were further analyzed with different database query and literature mining tools and then combined to form so called meta-modes. With them we performed a knowledge-based pathway analysis and reconstructed a well known signal cascade. Conclusion We show that ICA is an appropriate tool to uncover underlying biological mechanisms from microarray data. Most of the well known pathways of M-CSF dependent monocyte to macrophage differentiation can be identified by this unsupervised microarray data analysis. Moreover, recent research results like the involvement of proliferation associated cellular mechanisms during macrophage differentiation can be corroborated.

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase that has been described to be frequently mutated in gastrointestinal cancers. RNF43 downregulation was associated with distant metastasis, TNM stage and poorer survival in patients with gastric and colorectal cancers. Functional analysis has shown that overexpressed RNF43 negatively regulates Wnt signalling by ubiquitinating Frizzled receptors and targeting them for degradation and by sequestering T-cell factor 4 (TCF4) to the nuclear membrane, thereby inhibiting Wnt-mediated transcription. In the stomach, RNF43 overexpression was shown to impair stem-like properties and to be negatively correlated with expression of Wnt-target genes. In this study, we show that RNF43 knockdown enhances the tumourigenic potential of gastric and colorectal cancer cell lines in vitro and in vivo. Thus, loss of RNF43 leads to increased proliferation and anchorage-independent growth as well as increased invasive capacity. In a xenograft model, RNF43 depletion enhanced tumour growth. Furthermore, we established two mouse models in which mutations in the RING domain of RNF43 were introduced. In the intestine and colon, loss of Rnf43 did not induce changes in epithelial architecture or proliferation. In contrast, in the stomach, thickening of the mucosa, hyperplasia and cellular atypia were observed in these mice. Notably, this was independent of elevated Wnt signalling. Together, our results show that RNF43 plays a tumour suppressive role in gastric and colorectal cancer cells and that the loss of its function alters gastric tissue homeostasis in vivo., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Plasmalogens are either phosphatidylcholine (PC P) or phosphatidylethanolamine (PE P) glycerophospholipids containing a vinyl ether moiety in sn-1-position and an esterified fatty acid in sn-2 position. Multiple functions have been proposed, including reservoir of precursors for inflammatory mediators, modulation of membrane fluidity, and anti-oxidative properties. They could therefore play a role under conditions of metabolic stress. Especially enzymatically modified LDL (eLDL) and oxidatively modified LDL (oxLDL) represent modifications of LDL that are taken up by macrophages in atherosclerotic plaques. The aim of this study was to analyze plasmalogen related effects of eLDL and oxLDL in human monocyte derived macrophages, as well as the effects of HDL3 mediated deloading., Methods: Elutriated monocytes from nine healthy donors were differentiated in vitro for four days. Macrophages were then loaded with native LDL, eLDL and oxLDL for 24h and subsequently deloaded with HDL3 for another 24h. Lipidomic and transcriptomic profiles were obtained., Results: Loading of macrophages with eLDL and oxLDL led to a transient but strong elevation of lysophosphatidylcholine (LPC) most likely through direct uptake. Only eLDL induced increased levels of total PC, presumably through an induction of PC synthesis. On the other hand treatment with oxLDL led to a significant increase in PC P. Analysis of individual lipid species showed lipoprotein and saturation specific effects for LPC, PC P and PE P species. Membrane fluidity was decreased by the large amount of FC contained in the lipoproteins, as indicated by a lower PC to FC ratio after lipoprotein loading. In contrast the observed changes in the saturated to mono-unsaturated fatty acid (SFA to MUFA) and saturated to poly-unsaturated fatty acid (SFA to PUFA) ratios in PE P could represent a cellular reaction to counteract this effect by producing more fluid membranes. Transcriptomic analysis showed considerable differences between eLDL and oxLDL treated macrophages. As a common feature of both lipoproteins we detected a strong downregulation of pathways for endogenous lipid synthesis as well as for exogenous lipid uptake. Deloading with HDL3 had only minor effects on total lipid class as well as on individual lipid species levels, most of the time not reaching significance. Interestingly treatment with HDL3 had no effect on membrane fluidity under these conditions, although incubation with HDL3 was partially able to counteract the oxLDL induced transcriptomic effects. To investigate the functional effect of lipoprotein treatment on macrophage polarization we performed surface marker flow cytometry. Under our experimental conditions oxLDL was able to partially shift the surface marker pattern towards a pro-inflammatory M1-like phenotype. This is consistent with the consumption of arachidonic acid containing PE P species in oxLDL treated cells, presumably for the synthesis of inflammatory mediators., Summary: Our findings provide novel data on the lipoprotein induced, lipidomic and transcriptomic changes in macrophages. This can help us better understand the development of metabolic, inflammatory diseases as well as improve our background knowledge on lipid biomarkers in serum., Competing Interests: The authors have declared that no competing interests exist.

Background: 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)., Methods: 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., Results: 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., Conclusion: 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., Competing Interests: The authors have declared that no competing interests exist.

Ladder-type organoboranes featuring intramolecular N→B coordination have been prepared through hydroboration of a 2-(ortho-styryl)pyridine (PhPy) with a series of hydroboranes, including 9H-9-borabicyclo[3.3.1]nonyl (9H-BBN), BH3 ⋅THF, HBCl2 ⋅SMe2 , HB(C6 F5 )2 , and a 9H-9-borafluorene derivative. The hydroboration reaction results in highly regioselective borylation under mild conditions and gives the products in good to excellent yields. The molecular structure and electronic properties of the obtained boranes have been experimentally investigated in detail, and complemented with DFT calculations to further elucidate the origin of differences in optical and electronic properties. The electron affinity of the conjugated system can be controlled through variation of the borane, while the optical properties are likewise directly linked to the type and molecular structure of the substituents on boron. The broad substrate range shows that this preparative approach is widely applicable to introduce chemically diverse boryl groups into conjugated systems., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An unusual reactivity of 2-(1-alkenyl)-pyridines towards hydroboration with 9H-borabicyclo[3.3.1]nonane (9H-BBN) has been employed to selectively introduce two borane groups into a conjugated quaterpyridine. Quantitative conversion of the substrate was observed with exclusive regioselectivity. A molecular structure that allows intramolecular N→B coordination was generated. The effect of the ladder formation on the molecular structure and the electronic properties of the conjugated system have been investigated. The synthetic strategy demonstrated herein offers a facile access to N→B ladder-type structures from readily available substrates, and allows to simultaneously introduce several boron centers under mild conditions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The CRISPR/Cas9 system can be used to mutate target sequences by introduction of double-strand breaks followed by imprecise repair. To test its use for conditional gene editing we generated mice transgenic for CD4 promoter-driven Cas9 combined with guide RNA targeting CD2. We found that within CD4(+) and CD8(+) lymphocytes from lymph nodes and spleen 1% and 0.6% were not expressing CD2, respectively. T cells lacking CD2 carryied mutations, which confirmed that Cas9 driven by cell-type specific promoters can edit genes in the mouse and may thus allow targeted studies of gene function in vivo.

Given its fundamental role in development and cancer, the Wnt-β-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of β-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathway., (Copyright © 2015, American Association for the Advancement of Science.)

In the present paper we have performed comparative lipidomic analysis of two prototypic atherogenic LDL modifications, oxidized LDL and enzymatically modified LDL. Oxidization of LDL was carried out with different chemical modifications starting from the same native LDL preparations: (i) by copper oxidation leading to terminally oxidized LDL (oxLDL), (ii) by moderate oxidization with HOCl (HOCl LDL), (iii) by long term storage of LDL at 4°C to produce minimally modified LDL (mmLDL), or (iv) by 15-lipoxygenase, produced by a transfected fibroblast cell line (LipoxLDL). The enzymatic modification of LDL was performed by treatment of native LDL with trypsin and cholesteryl esterase (eLDL). Free cholesterol (FC) and cholesteryl esters (CE) represent the predominant lipid classes in all LDL preparations. In contrast to native LDL, which contains about two-thirds of total cholesterol as CE, enzymatic modification of LDL decreased the proportion of CE to about one-third. Free cholesterol and CE in oxLDL are reduced by their conversion to oxysterols. Oxidization of LDL preferentially influences the content of polyunsaturated phosphatidylcholine (PC) and polyunsaturated plasmalogen species, by reducing the total PC fraction in oxLDL. Concomitantly, a strong rise of the lysophosphatidylcholine (LPC) fraction can be found in oxLDL as compared to native LDL. This effect is less pronounced in eLDL. The mild oxidation of LDL with hypochlorite and/or lipoxygenase does not alter the content of the analyzed lipid classes and species in a significant manner. The lipidomic characterization of modified LDLs contributes to the better understanding their diverse cellular effects., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: 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., Methods: Elutriated monocytes from 13 healthy donors were differentiated in vitro to macrophages using rhM-CSF under serum-free conditions. Samples were taken on days 0, 1, 4 and 5 and analyzed for their lipidomic and transcriptomic profiles., Results: Gene expression analysis showed strong regulation of lipidome-related transcripts. Enzymes involved in fatty acid desaturation and elongation were increasingly expressed, peroxisomal and ER stress related genes were induced. Total plasmalogen levels remained unchanged, while the PE plasmalogen species pattern became more similar to circulating granulocytes, showing decreases in PUFA and increases in MUFA. A partial least squares discriminant analysis (PLS/DA) revealed that PE plasmalogens discriminate the stage of monocyte-derived macrophage differentiation. Partial correlation analysis could predict novel potential key nodes including DOCK1, PDK4, GNPTAB and FAM126A that might be involved in regulating lipid and especially plasmalogen homeostasis during differentiation. An in silico transcription analysis of lipid related regulation revealed known motifs such as PPAR-gamma and KLF4 as well as novel candidates such as NFY, RNF96 and Zinc-finger proteins., Conclusion: Monocyte to macrophage differentiation goes along with profound changes in the lipid-related transcriptome. This leads to an induction of fatty-acid desaturation and elongation. In their PE-plasmalogen profile macrophages become more similar to granulocytes than monocytes, indicating terminal phagocytic differentiation. Therefore PE plasmalogens may represent potential biomarkers for cell activation. For the underlying transcriptional network we were able to predict a range of novel central key nodes and underlying transcription factors using a bioinformatic approach.

Oxidized and enzymatically modified low-density lipoproteins (oxLDL and eLDL) play a key role in early stages of atherogenesis. Their uptake by recruited macrophages leads to endolysosomal phospholipidosis or foam cell formation, respectively, each of which is preceded by highly differential lipid restructuring processes. We applied (1)H-NMR spectroscopy (NMRS) to elucidate these structural rearrangements both in consequence of lipoprotein modifications and following phagocytosis. Being specifically sensitive to the mobile lipid subset, NMRS of oxLDL and eLDL revealed a partial and total immobilization of lipids, respectively. NMRS of intact macrophages showed a sixfold increase in mobile lipids in case of loading with eLDL but no significant changes for oxLDL or native LDL. This finding reflected the disparate lipid storage in lipid droplets and in multilamellar endolysosomal clusters when loaded with either eLDL or oxLDL, respectively. Moreover, a significant shift of the degree of saturation towards mainly polyunsaturated fatty acid chains was found for the mobile lipid pool in eLDL-loaded macrophages. Additional analyses of lipid extracts by NMRS and mass spectrometry (MS) reflected these changes in lipid content and in fatty acid composition only partially. In summary, in-cell NMRS represents a unique lipidomics tool to investigate structural changes within the mobile lipid pool following atherogenic triggers that can be not detected by the analysis of lipid extracts by MS or NMRS.

In many different tumor entities, increased expression of tissue inhibitor of metalloproteinases-1 (Timp-1) is associated with poor prognosis. We previously reported in mouse models that elevated systemic levels of Timp-1 induce a gene expression signature in the liver microenvironment increasing the susceptibility of this organ to tumor cells. This host effect was dependent on increased activity of the hepatocyte growth factor (Hgf)/hepatocyte growth factor receptor (Met) signaling pathway. In a recent study we showed that Met signaling is regulated by Timp-1 as it inhibits the Met sheddase A disintegrin and metalloproteinase-10 (Adam-10). The aim of the present study was to elucidate whether the metastatic potential of tumor cells benefits from autocrine Timp-1 as well and involves Adam-10 and Met signaling. In a syngeneic murine model of experimental liver metastasis Timp-1 expression and Met signaling were localized within metastatic colonies and expressed by tumor cells. Knock down of tumor cell Timp-1 suppressed Met signaling in metastases and inhibited metastasis formation and tumor cell-scattering in the liver. In vitro, knock down of tumor cell Timp-1 prevented Hgf-induced Met phosphorylation. Consequently, knock down of Met sheddase Adam-10 triggered auto-phosphorylation and responsiveness to Hgf. Accordingly, Adam-10 knock down increased Met phosphorylation in metastatic foci and induced tumor cell scattering into the surrounding liver parenchyma. In conclusion, these findings show that tumor cell-derived Timp-1 acts as a positive regulator of the metastatic potential and support the concept that proteases and their natural inhibitors, as members of the protease web, are major players of signaling during normal homeostasis and disease.

Recruitment of circulating monocytes and formation of macrophage foam cells in the arterial intima are characteristic features of atherogenesis. Foam cells are formed by cellular uptake and storage of atherogenic lipoproteins, including oxidized LDL (oxLDL) and enzymatically modified LDL (eLDL). Dissection of oxLDL- and eLDL-induced cellular phenotypes indicates that these two LDL-modifications are coupled with two fundamentally different cellular responses in macrophages. Oxidized LDL preferentially up-regulates scavenger receptors required for its internalization, induces preferential lipid storage in the acidic compartment resembling drug-induced endolysosomal phospholipidosis, parallel with increased cellular content of the endolysosomal signature lipid bis(monoacylglycero)phosphate, pro-apoptotic signalling and appearance of ceramide-enriched surface membrane microdomains. By contrast, challenge of macrophages by eLDL leads to expanded cholesterol- and sphingomyelin-enriched surface membrane microdomains, up-regulation of diverse pattern recognition receptors required for phagocytosis of eLDL, parallel with extensive lipid droplet formation, increased endoplasmic reticulum (ER)-stress and membrane contact site formation for interorganelle trafficking and signalling, and enhanced cellular content of the mitochondrial lipid cardiolipin. This review focuses on biological activities of oxLDL and eLDL in human macrophages, and discusses some lipidomic considerations related to foam cell formation and phospholipidosis., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Macrophages and dendritic cells originate from a common myeloid precursor. Although several studies compared transcriptional profiles of these cells, not a single study compared their lipid profiles. Therefore, we measured and compared fatty acid (FA) and phospholipid (PL) species composition of granulocyte/macrophage colony-stimulating factor (GM-CSF) plus interleukin 4 (IL-4) and macrophage colony-stimulating factor (M-CSF) differentiated monocytes isolated from healthy volunteers. We found that these two cell types prominently differ in their FA composition. Dendritic cells (DCs) contain lower proportions of C16 and C18 mono-unsaturated FAs, but higher proportions of C20 and C22 poly-unsaturated fatty acids (PUFAs) than phagocytic macrophages. Analysis of PL species profiles revealed that GM-CSF/IL-4 differentiated cells have increased amounts of longer and more desaturated phospatidylethanolamine (PE) and phosphatidylserine (PS) species, but lower amounts of shorter and less desaturated PE and PS species than M-CSF differentiated cells. These cell type specific lipid profiles can be attributed to a differential expression and activity of stearoyl-CoA desaturase (SCD). Taken together, our results show that GM-CSF/IL-4 compared to M-CSF differentiated monocytes have less mono-unsaturated FA and PL species, which are due to lower SCD activity observed in DCs., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Monocytes are precursors of macrophages. Here we demonstrate that macrophage colony-stimulating factor (M-CSF)-dependent differentiation of primary human monocytes from healthy volunteers induces transcription of SREBP-1c target genes required for fatty acid (FA) biosynthesis and impairs transcription of SREBP-2 target genes required for cholesterol synthesis. Detailed lipid metabolic profiling showed that this transcriptional regulation leads to a dramatically increased fatty acid synthesis as driving force for enhanced phospholipid synthesis. During cell differentiation the major lipid class switches from cholesterol in monocytes to phosphatidylcholine in macrophages. Ultrastructural analysis revealed that this transcriptional and metabolic regulation is essential for development of macrophage filopodia and cellular organelles including primary lysosomes, endoplasmic reticulum, and Golgi network. Additional functional studies showed that suppression of fatty acid synthesis prevents phagocytosis representing a central macrophage function. Therefore induction of fatty acid synthesis is a key requirement for phagocyte development and function.

Macrophage foam cells formed during uptake of atherogenic lipoproteins are a hallmark of atherosclerotic lesion development. In this study, human macrophages were incubated with two prototypic atherogenic LDL modifications enzymatically degraded LDL (E-LDL) and oxidized LDL (Ox-LDL) prepared from the same donor LDL. To detect differences in macrophage lipid storage, fluorescent high-content imaging was used. Lipid droplets were stained using Bodipy 493/503, and the fluorescent phospholipid probe NBD-PE was used to detect endolysosomal phospholipidosis in high-content imaging assays. The phospholipidosis assay was validated using phospholipidosis-inducing cationic amphiphilic drugs. In addition, neutral lipids and phospholipidosis were determined using LipidTOX. Images of 96-well cell culture microtiter plates were captured with multichannel laser-based high-content confocal microscopy, and subsequently cell- and well-based data were analyzed. E-LDL-loaded macrophages show increased intensity of Bodipy 493/503 and LipidTOX-Green neutral lipid droplet staining and a greater mean area and number of lipid droplets per cell compared to Ox-LDL-loaded and M-CSF-differentiated control macrophages. In contrast, Ox-LDL-loaded macrophages show increased intensity of NBD-PE and LipidTOX-Red detectable phospholipidosis in the endolysosomal compartment compared to E-LDL-loaded and M-CSF-differentiated macrophages. Treatment with the peroxisome proliferator-activated receptor-gamma agonist pioglitazone leads to lipid droplet induction depending on the lipid loading state of the macrophages. These results indicate that E-LDL preferentially induces lipid droplets, while Ox-LDL provokes endolysosomal phospholipidosis in human macrophages representing two different lipid storage principles. Therefore, fluorescent high-content imaging is a useful tool to discriminate between and quantify lipid storage compartments in macrophages also in response to drugs affecting cellular lipid metabolism., ((c) 2009 International Society for Advancement of Cytometry.)

HDL functions mainly as a cholesterol scavenger, facilitating transport of cholesterol to the liver for conversion to bile acids and secretion into the bile for elimination or recycling in the enterohepatic bile acid cycle. Because of its major function in cholesterol clearance, HDL is in general considered to be atheroprotective. From cell cholesterol can be removed by efflux especially to apoA-I and HDL as extracellular acceptors which transport the cholesterol to the liver for excretion. This process is called reverse cholesterol transport. In this context the ATP binding cassette transporter protein ABCA1 facilitates cellular cholesterol and phospholipid release to apoA-I-containing HDL precursors. In addition ABCA1 plays a role in vesicular lipid transport mechanisms required for HDL particle formation. In general to maintain intracellular lipid homeostasis, sterols and associated lipids move between cellular compartments by vesicular and nonvesicular pathways. However, cholesterol sorting on vesicle formation is poorly understood. This review summarizes the current knowledge of the molecular mechanisms of HDL and associated vesicular trafficking mechanisms to mediate cellular lipid homeostasis.

This review summarizes the current knowledge of endolysosomal and cytoplasmic lipid storage in macrophages induced by oxidized LDL (Ox-LDL), enzymatically degraded LDL (E-LDL) and other atherogenic lipoprotein modifications, and their relation to the adapter protein 3 (AP-3) dependent ABCA1 and ABCG1 cellular lipid efflux pathways. We compare endolysosomal lipid storage caused either through drug induced phospholipidosis, inheritable endolysosomal and cytosolic lipid storage disorders and Ox-LDL or E-LDL induced phagosomal uptake and cytosolic lipid droplet storage in macrophages. Ox-LDL is resistant to rapid endolysosomal hydrolysis and is trapped within the endolysosomal compartment generating lamellar bodies which resemble the characteristics of phospholipidosis. Various inherited lysosomal storage diseases including sphingolipidosis, glycosphingolipidosis and cholesterylester storage diseases also present a phospholipidosis phenotype. In contrast E-LDL resembling coreless unesterified cholesterol enriched LDL-particles, with a multilamellar, liposome-like structure, lead to rapid phagosomal degradation and cytosolic lipid droplet accumulation. As a consequence the uptake of E-LDL through type I and type II phagocytosis leads to increased lipid droplet formation and moderate upregulation of ABCA1 and ABCG1 while uptake of Ox-LDL leads to a rapid expansion of the lysosomal compartment and a pronounced upregulation of the ABCA1/ABCG1/AP-3 lipid efflux pathway.

Several techniques are available to purify circulating blood monocytes for research. CD14-containing MicroBeads are suitable and reliable tools to reproducibly isolate human monocytes with a high purity but are quite expensive. This report describes that a comparable number of highly pure monocytes can be isolated from samples using up to tenfold lower amounts of CD14-MicroBeads. MicroBeads are widely used to isolate different cell populations and with this report more researchers may be encouraged to use this highly efficient, low-cost and thus affordable method to pursue their scientific goals.

Purpose of Review: Lipid membrane microdomains are involved in major types of disease, ranging from vascular and metabolic diseases to neurodegeneration, autoimmunity, infectious and inflammatory diseases, and cancer. This review provides an update of membrane microdomain abnormalities., Recent Findings: Lipid membrane microdomains are dynamic assemblies of sphingolipids, cholesterol and proteins that dissociate and associate rapidly and form functional clusters. Membrane microdomain clustering is the key to how membrane microdomains can form lipid-protein platforms in cell membranes, functioning in membrane trafficking, cell polarization and signalling. Clustering of membrane microdomains can be modified, for example by dietary lipids and pharmacological agents., Summary: Metabolic overload through a cholesterol-rich and fat-rich diet can trigger metabolic learning, which is associated with membrane microdomain persistence, persistent signalling and disturbed vesicular traffic. Detailed characterization of lipid membrane microdomains and dynamics at the molecular level is necessary and will help to identify new dietary and pharmacological therapeutic targets for the treatment and prevention of lipid membrane microdomain related diseases.

Background: The analysis of high-throughput gene expression data sets derived from microarray experiments still is a field of extensive investigation. Although new approaches and algorithms are published continuously, mostly conventional methods like hierarchical clustering algorithms or variance analysis tools are used. Here we take a closer look at independent component analysis (ICA) which is already discussed widely as a new analysis approach. However, deep exploration of its applicability and relevance to concrete biological problems is still missing. In this study, we investigate the relevance of ICA in gaining new insights into well characterized regulatory mechanisms of M-CSF dependent macrophage differentiation., Results: Statistically independent gene expression modes (GEM) were extracted from observed gene expression signatures (GES) through ICA of different microarray experiments. From each GEM we deduced a group of genes, henceforth called sub-mode. These sub-modes were further analyzed with different database query and literature mining tools and then combined to form so called meta-modes. With them we performed a knowledge-based pathway analysis and reconstructed a well known signal cascade., Conclusion: We show that ICA is an appropriate tool to uncover underlying biological mechanisms from microarray data. Most of the well known pathways of M-CSF dependent monocyte to macrophage differentiation can be identified by this unsupervised microarray data analysis. Moreover, recent research results like the involvement of proliferation associated cellular mechanisms during macrophage differentiation can be corroborated.

Apart from its role as a risk factor in arteriosclerosis, plasma cholesterol is increasingly recognized to play a major role in the pathogenesis of Alzheimer's disease (AD). Moreover, alterations of intracellular cholesterol metabolism in neuronal and vascular cells are of considerable importance for the understanding of AD. Cellular cholesterol accumulation enhances the deposition of insoluble beta-amyloid peptides, which is considered a hallmark in the pathogenesis of AD. In order to test the hypothesis, whether exogenous beta-amyloid peptides (Abeta42, Abeta40) might contribute to cellular cholesterol accumulation by opsonization of lipoproteins, we compared the binding and uptake of native LDL, enzymatically modified LDL (E-LDL), copper oxidized LDL (Ox-LDL) and HDL as control, preincubated either in the absence or presence of Abeta42 or Abeta40, by human monocytes or monocyte-derived macrophages. Incubation of monocytes and macrophages with Abeta-lipoprotein-complexes lead to increased cellular free and esterified cholesterol when compared to non-opsonized lipoproteins, except for HDL. Furthermore, the cellular uptake of these complexes regulated Abeta-receptors such as FPRL-1 or LRP/CD91. In summary, our results suggest that Abeta42 and Abeta40 act as potent opsonins for LDL, E-LDL and Ox-LDL and enhance cellular cholesterol accumulation as well as Abeta-deposition in vessel wall macrophages.

Hyperlipidemias and small dense LDLs in patients with high-triglyceride low-HDL syndromes lead to a prolonged half life of apoB-containing particles. This is associated with reactive oxygen species (ROS) activation and leads to formation of oxidized LDL (Ox-LDL). Generators of ROS in macrophages (MACs) include myeloperoxidase (MPO)-mediated respiratory burst and raft-associated NADPH-oxidase. The intracellular oxidant milieu is involved in cellular signaling pathways, like ion-transport systems, protein phosphorylation, and gene expression. Lipid oxidation through ROS can amplify foam cell formation through Ox-LDL uptake, leading to formation of ceramide (Cer)-rich lipid membrane microdomains, and is associated with expansion of the lysosomal compartment and an upregulation of ABCA1 and other genes of the AP3 secretory pathway. Ox-LDL may also affect cell-surface turnover of Cer-backbone sphingolipids and apoE-mediated uptake by LRP-family members. In contrast, HDL-mediated lipid efflux causes disruption of lipid membrane microdomains and prevents foam cell formation. Oxidation of HDL through MPO leads to a failure of lipid efflux and enhancement of MAC loading. Therefore, lipid rafts and oxidation processes are important in regulation of MAC foam cell formation and atherosclerosis, and the balance between oxidant and antioxidant intracellular systems is critically important for efficient MAC function.

Uptake of modified lipoproteins by macrophages causes foam cell formation and promotes atherosclerosis. Atherogenic lipoproteins are cytotoxic and induce cell death under certain conditions but may also enhance macrophage survival. Macrophages treated with enzymatically modified LDL (E-LDL) were subjected to GeneChip analysis and the antiapoptotic gene TOSO was found induced. TOSO mRNA is upregulated and apoptosis is reduced in E-LDL but not in oxidized LDL (Ox-LDL) loaded macrophages. FLIP(L) abundance was suggested to mediate the antiapoptotic properties of TOSO; however, FLIP(L) was not changed. Ox-LDL is internalized predominantly by scavenger receptors such as CD36 while E-LDL particles are preferentially internalized by Fc- and complement-receptor dependent phagocytosis and internalization of phagobeads by macrophages upregulates TOSO. In COS-7 cells however, phagocytotic activity was not affected by TOSO. These data indicate that E-LDL-generated foam cells are protected from cell death most likely through the expression of TOSO by a FLIP(L) independent mechanism.

Background: For understanding cholesterol and phospholipid efflux pathways there is a need for cellular fluorescence-based high-content screens (HCS) to investigated the cholesterol and phospholipid content in human macrophages., Methods: Making use of fluorescence imaging based on HCS we have developed a tool to evaluate new agents that can act as inducers of cholesterol efflux. The fluorescence assay is based on the different staining patterns of cholesterol-loaded (E-LDL) and deloaded (HDL3) differentiated monocytes by the saturated, fluorescent lipid probe (1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine)-tetramethyl-rhodamin., Results: Morphologic examination and statistical evaluation of the staining pattern such as gray value, threshold area, shape factor and the spot size distribution provides evidence for a significant pattern change when cholesterol enriched and cholesterol depleted differentiated monocytes were imaged., ((c) 2006 International Society for Analytical Cytology.)

Atherosclerosis is characterized by the generation of lipid-loaded macrophage-derived foam cells. To study the effect of different types of atherogenic lipoproteins, human macrophages were loaded with enzymatically degraded low density lipoprotein (E-LDL) or oxidized LDL (Ox-LDL). Cellular cholesterol content was increased by E-LDL, whereas Ox-LDL increased the ceramide content. Cell surface expression analysis by flow cytometry and confocal microscopy revealed that Ox-LDL increased ceramide and lactosylceramide expression compared to E-LDL loading and induced ceramide rafts, whereas loading with E-LDL induced cholesterol-rich microdomains. Formation of different rafts may have consequences for raft associated signaling in cholesterol homeostasis and apoptosis in human macrophages., ((c) 2006 International Society for Analytical Cytology.)

Vitamin A and its naturally occurring derivatives 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA) exert a variety of biological effects including immunomodulation, growth, differentiation, and apoptosis of normal and neoblastic cells. In order to directly study the effects of these retinoids on macrophage gene expression and lipid metabolism, primary human monocytes and in vitro differentiated macrophages were stimulated with beta-carotene, 9-cis RA, and ATRA and global gene expression profiles were analyzed by Affymetrix DNA-microarrays and differentially regulated genes were verified by quantitative TaqMan RT-PCR. Among others, we have identified a strong up-regulation of a cluster of genes involved in cholesterol metabolism including apolipoproteins (apoC-I, apoC-II, apoC-IV, apoE), the scavenger receptor CD36, steroid-27-hydroxylase (CYP27A1), liver X receptor alpha (LXRalpha), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Since the CYP27A1 gene displayed the strongest up-regulation on the mRNA level, we cloned various deletion constructs of the promoter region and analyzed the response to retinoids in macrophages. Thereby, a novel retinoic acid-responsive element could be located within 191 bp of the proximal CYP27A1 promoter. To further assess the functional consequences of retinoid receptor action, we carried out phospholipid and cholesterol efflux assays. We observed a strong induction of apoA-I-dependent lipid efflux in stimulated macrophages, implicating an important role for retinoids in cellular functions of macrophages.

The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I-dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX-insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains.

The highly homologous genes NIPSNAP3 and NIPSNAP4, with 87% amino acid identity, are members of the NIPSNAP family with putative roles in vesicular trafficking. NIPSNAP3 mRNA and NIPSNAP4 mRNA and protein were detected in multiple tissues and cells at varying degrees. Interestingly, NIPSNAP3 is most highly expressed in skeletal muscle, where NIPSNAP4 has a low mRNA abundance. NIPSNAP4 was found associated with membranes and partly localized in rafts. The ubiquitous expression of the highly conserved NIPSNAPs and their association with membranes further support an important cellular function of these proteins probably linked to vesicular trafficking. The NIPSNAP3 and NIPSNAP4 genes are located in close proximity to the 3' end of the ATP-binding cassette transporter A1 (ABCA1), whose mutations cause familial high-density lipoprotein deficiency syndromes. The adjacent genomic location and the finding that ABCA1 is a regulator of vesicular trafficking may indicate a functional relation of these proteins, even though NIPSNAP4 does not interact directly with ABCA1 nor is its expression altered in cells with mutated ABCA1.