Mildly oxidized HDL decrease agonist-induced platelet aggregation and release of pro-coagulant platelet extracellular vesicles.

Stored platelet concentrates (PLCs) for therapeutic purpose, develop a platelet storage lesion (PSL), characterized by impaired platelet (PLT) viability and function, platelet extracellular vesicle (PL-EV) release and profound lipidomic changes. Whereas oxidized low-density lipoprotein (oxLDL) activates PLTs and promotes atherosclerosis, effects linked to oxidized high-density lipoprotein (oxHDL) are poorly characterized. PLCs from blood donors were treated with native (nHDL) or mildly oxidized HDL (moxHDL) for 5days under blood banking conditions. Flow cytometry, nanoparticle tracking analysis (NTA), aggregometry, immunoblot analysis and mass spectrometry were carried out to analyze PL-EV and platelet exosomes (PL-EX) release, PLT aggregation, protein expression, and PLT and plasma lipid composition. In comparison to total nHDL, moxHDL significantly decreased PL-EV release by -36% after 5days of PLT storage and partially reversed agonist-induced PLT aggregation. PL-EV release positively correlated with PLT aggregation. MoxHDL improved PLT membrane lipid homeostasis through enhanced uptake of lysophospholipids and their remodeling to corresponding phospholipid species. This also appeared for sphingomyelin (SM) and d18:0/d18:1 sphingosine-1-phosphate (S1P) at the expense of ceramide (Cer) and hexosylceramide (HexCer) leading to reduced Cer/S1P ratio as PLT-viability indicator. This membrane remodeling was associated with increased content of CD36 and maturation of scavenger receptor-B1 (SR-B1) protein in secreted PL-EVs. MoxHDL, more potently than nHDL, improves PLT-membrane lipid homeostasis, partially antagonizes PL-EV release and agonist-induced PLT aggregation. Altogether, this may be the result of more efficient phospho- and sphingolipid remodeling mediated by CD36 and SR-B1 in the absence of ABCA1 on PLTs. As in vitro supplement in PLCs, moxHDL has the potential to improve PLC quality and to prolong storage.

Ultrastructure of skin from Refsum disease with emphasis on epidermal lamellar bodies and stratum corneum barrier lipid organization.

Classic Refsum disease (RD) is a rare, autosomal recessively-inherited disorder of peroxisome metabolism due to a defect in the initial step in the alpha oxidation of phytanic acid (PA), a C16 saturated fatty acid with four methyl side groups, which accumulates in plasma and lipid enriched tissues (please see van den Brink and Wanders, Cell Mol Life Sci 63:1752-1765, 2006). It has been proposed that the disease complex in RD is in part due to the high affinity of phytanic acid for retinoid X receptors and peroxisome proliferator-activated receptors. Structurally, epidermal hyperplasia, increased numbers of cornified cell layers, presence of cells with lipid droplets in stratum basale and reduction of granular layer to a single layer have been reported by Blanchet-Bardon et al. (The ichthyoses, SP Medical & Scientific Books, New York, pp 65-69, 1978). However, lamellar body (LB) density and secretion were reportedly normal. We recently examined biopsies from four unrelated patients, using both OsO4 and RuO4 post-fixation to evaluate the barrier lipid structural organization. Although lamellar body density appeared normal, individual organelles often had distorted shape, or had non-lamellar domains interspersed with lamellar structures. Some of the organelles seemed to lack lamellar contents altogether, showing instead uniformly electron-dense contents. In addition, we also observed mitochondrial abnormalities in the nucleated epidermis. Stratum granulosum-stratum corneum junctions also showed co-existence of non-lamellar and lamellar domains, indicative of lipid phase separation. Also, partial detachment or complete absence of corneocyte lipid envelopes (CLE) was seen in the stratum corneum of all RD patients. In conclusion, abnormal LB contents, resulting in defective lamellar bilayers, as well as reduced CLEs, likely lead to impaired barrier function in RD.

Different proliferative and survival capacity of CLL-cells in a newly established in vitro model for pseudofollicles.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B-lymphocytes that manifests in a variety of clinical courses. The accumulation of CLL-cells is primarily caused by defective apoptosis; however, a higher proliferative capacity has also been found to correlate with poorer prognostic factors. Proliferating CLL-cells are confined to specialized structures called pseudofollicles, which contain CLL-cells, T-lymphocytes, and stromal cells. We established an in vitro model for pseudofollicles to characterize the behavior of CLL-cells in relation to clinical courses with different outcomes. Only CLL-cells from progressive clinical cases were inducible to proliferate by a combination of soluble CD40L/IL-2/IL-10 in co-culture with stromal cells. Proliferating CLL-cells showed a higher and more extensive expression of antigens, which are important in T-B-cell interactions such as CD40, MHC II, and adhesion molecules. IL-4 increased interferon regulatory factor-4 expression and induced a specific immunophenotype, which may imply plasmacytic differentiation. Furthermore, it was shown that co-cultured stromal cells protected CLL-cells from apoptosis. CLL-cells from clinically indolent cases had a far worse survival rate in medium than the cells from poor prognostic cases. Thus, we can assume that not only a different resistance to apoptosis, but also proliferation contributes to the progression of CLL resulting in bone marrow failure with thrombocytopenia and anemia.

Human aldehyde oxidase 1 interacts with ATP-binding cassette transporter-1 and modulates its activity in hepatocytes.

AOX1, a member of the cytosolic molybdenum hydroxylase family, has been identified by us earlier as an ABCA1-interacting protein. AOX1 is well-described as xenobiotic metabolizing enzyme, which upon oxidation of acetaldehyde and retinaldehyde to acetic acid and retinoic acid generates reactive oxygen species. Here we show that knock-down of AOX1 in HepG2 by small interfering RNA significantly reduced ABCA1-dependent lipid efflux and enhanced phagocytic uptake of microspheres similar to ABCA1 deficiency, without affecting ABCA1 mRNA and protein levels. ABCA1 and AOX1 are coexpressed in human hepatocytes, kidney proximal tubular epithelial cells, Leydig, and adrenocortical cells. Expression of ABCA1 and AOX1 was investigated by immunohistochemistry in liver tissue arrays. A strong AOX1 expression was found in normal liver, and in cirrhosis. In contrast, hepatocellular carcinomas showed either a complete loss or reduced expression of AOX1. Significant correlations were found between reduced AOX1 expression and tumor stage, or metastatic or regional lymph node states. Deregulation was also observed for ABCA1 expression but to a lesser extent. Our findings show that the interaction of ABCA1 with AOX1 modulates ABCA1-linked cellular functions such as lipid efflux and phagocytosis in hepatocytes, and the reduced expression of AOX1 in malignant transformed hepatocytes supports the differentiation dependent upregulation of AOX1.

The satiety factor apolipoprotein A-IV modulates intestinal epithelial permeability through its interaction with alpha-catenin: implications for inflammatory bowel diseases.

INTRODUCTION: Apolipoprotein A-IV (apoA-IV), an intestinally and cerebrally synthesized satiety factor and anti-atherogenic plasma apolipoprotein, was recently identified as an anti-inflammatory protein. In order to elucidate whether intestinal apoA-IV exerts similar repair function as its hepatic homologue apolipoprotein A-V (apoA-V), apoA-IV-interactive proteins were searched and in vitro functional studies were performed with apoA-IV overexpressing cells. ApoA-IV was also analyzed in the intestinal mucosa of patients with inflammatory bowel diseases (IBD), together with other genes involved in epithelial junctional integrity. METHODS: A yeast-two-hybrid screening was used to identify apoA-IV-interactors. ApoA-IV was overexpressed in Caco-2 and HT-29 mucosal cells for colocalization and in vitro epithelial permeability studies. Mucosal biopsies from quiescent regions of colon transversum and terminal ileum were subjected to DNA-microarray analysis and pathway-related data mining. RESULTS: Four proteins interacting with apoA-IV were identified, including apolipoprotein B-100, alpha1-antichymotrypsin, cyclin C, and the cytosolic adaptor alpha-catenin, thus linking apoA-IV to adherens junctions. Overexpression of apoA-IV was paralleled with a differentiated phenotype of intestinal epithelial cells, upregulation of junctional proteins, and decreased paracellular permeability. Colocalization between alpha-catenin and apoA-IV occurred exclusively in junctional complexes. ApoA-IV was downregulated in quiescent mucosal tissues from patients suffering from IBD. In parallel, only a distinct set of junctional genes was dysregulated in non-inflamed regions of IBD gut. CONCLUSIONS: ApoA-IV may act as a stabilizer of adherens junctions interacting with alpha-catenin, and is likely involved in the maintenance of junctional integrity. ApoA-IV expression is significantly impaired in IBD mucosa, even in non-inflamed regions.

ApoA-I-binding protein (AI-BP) and its homologues hYjeF_N2 and hYjeF_N3 comprise the YjeF_N domain protein family in humans with a role in spermiogenesis and oogenesis.

The screening for additional human YjeF_N domain containing proteins beside the apolipoprotein A-I interacting protein (AI-BP), identified two other genes designated hYjeF_N2-15q23 (formerly human homologue of yeast edc3) and hYjeF_N3-19p13.11 comprising the human YjeF_N family. AI-BP is ubiquitously expressed, with a predominance of these tissues where the homologues were found to be restricted including brain, mammary gland, testes and ovaries. Immunohistochemistry of human testes and ovaries showed an expression of hYjeF_N3-19p13.11 only in Leydig cells and theca cells, respectively, indicating a role in steroid hormone metabolism. Interestingly, the protein was also strongly expressed in Leydig cell tumors and in thecofibromas. The identification of hYjeF_N2-15q23 in theca cells and granulosa cells in ovaries, in human spermatids of meiotic division part II and the apical membrane of Sertoli cells in testes suggest similar functions in oogenesis and sperm maturation which is strengthened by the identification of the spermatogenesis regulator HMGA1 as a conserved transcription factor. However, in contrast to AI-BP, both homologous proteins are unable to bind apoA-I. These results relate the human YjeF_N domain containing protein family to cholesterol processing and steroid hormone metabolism in spermiogenesis and oogenesis, and AI-BP may link this function to the HDL pathway.

Lipopolysaccharide and ceramide docking to CD14 provokes ligand-specific receptor clustering in rafts.

The glycosylphosphatidylinositol-anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co-receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcgamma-receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co-clustering with Toll-like receptor 4, Fcgamma-RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand-specific cellular responses initiated by CD14.

Apolipoprotein AI and HDL(3) inhibit spreading of primary human monocytes through a mechanism that involves cholesterol depletion and regulation of CDC42.

The objective of the current study was to characterize the influence of high density lipoproteins (HDL) on processes related to the vascular recruitment of human monocytes, which may contribute to the anti-atherogenic properties of these lipoproteins. We show that HDL(3) and apo AI inhibit the following processes in primary human monocytes: (1) M-CSF induced cell spreading; (2) M-CSF stimulated expression of surface molecules involved in adhesion, migration, and scavenging; (3) fMLP induced chemotaxis. These processes are obviously modulated by the regulation of cellular cholesterol pools as indicated by the following findings. In Tangier monocytes with defective apo AI induced cholesterol efflux, apo AI had no influence on the spreading response. In control cells, stimulation of cholesterol efflux by p-cyclodextrin mimicked the effect of apo AI and HDL(3) on spreading and chemotaxis, whereas cholesterol loading with enzymatically modified LDL (E-LDL) showed the opposite effect. Finally, a similar inverse regulation by E-LDL and apo AI/HDL(3) was also observed in regard to the surface expression of beta(1)- and beta(2)-integrins as well as the hemoglobin/haptoglobin scavenger receptor CD163 and the Fcgamma-IIIaR CD16. CDC42 was identified as a potential downstream target linking changes in cellular cholesterol content to monocyte spreading and chemotaxis. Thus, CDC42 antisense markedly reduced spreading and, in parallel with their influence on monocyte spreading, HDL(3), apo AI and p-cyclodextrin down-regulated CDC42 expression while E-LDL had the inverse effect. The apo AI induced decrease of CDC42 protein expression was paralleled by the reduction of active GTP-bound CDC42. In summary, we provide evidence that HDL(3) and apo AI are able to inhibit processes in primary human monocytes, which are related to the recruitment of monocytes into the vessel wall and probably involve regulation of cellular cholesterol pools and CDC42 function.

Intracellular cholesterol and phospholipid trafficking: comparable mechanisms in macrophages and neuronal cells.

During the past ten years considerable evidences have accumulated that in addition to monocytes/macrophages, that are implicated in innate immunity and atherogenesis, neuronal cells also exhibit an extensive cellular metabolism. The present study focuses on the major protein players that establish cellular distribution of cholesterol and phospholipids. Evidences are provided that neuronal cells and monocytes/macrophages are equipped with comparable intracellular lipid trafficking mechanisms. Selected examples are presented that trafficking dysfunctions lead to disease development, such as Tangier disease and Niemann-Pick disease type C, or contribute to the pathogenesis of diseases such as Alzheimer disease and atherosclerosis.

Characterization of the ATPase cycle of human ABCA1: implications for its function as a regulator rather than an active transporter.

ABCA1 plays a key role in cellular cholesterol and phospholipid traffic. To explore the biochemical properties of this membrane protein we applied a Baculovirus-insect cell expression system. We found that human ABCA1 in isolated membranes showed a specific, Mg(2+)-dependent ATP binding but had no measurable ATPase activity. Nevertheless, conformational changes in ABCA1 could be demonstrated by nucleotide occlusion, even without arresting the catalytic cycle by phosphate-mimicking anions. Addition of potential lipid substrates or lipid acceptors (apolipoprotein A-I) did not modify the ATPase activity or nucleotide occlusion by ABCA1. Our data indicate that ATP hydrolysis by ABCA1 occurs at a very low rate, suggesting that ABCA1 may not function as an effective active transporter as previously assumed. In the light of the observed conformational changes we propose a regulatory function for human ABCA1.

Increased histidine decarboxylase expression during in vitro monocyte maturation; a possible role of endogenously synthesised histamine in monocyte/macrophage differentiation.

OBJECTIVE: In this study the expression of histidine decarboxylase (HDC), the pivotal enzyme in histamine formation and the effect of endogenously produced histamine on differentiation antigens was examined during in vitro differentiation of human monocytes. MATERIAL AND TREATMENT: Human elutriated monocytes from healthy volunteers were incubated with macrophage colony stimulating factor (M-CSF) and the expression of HDC was followed at both mRNA and protein levels. To study the possible function of histamine we followed the expression of some cell surface markers (CD14, CD16, CD91, CD49d and CD11c) relevant for phagocytic differentiation upon incubation in the presence of different histamine inhibitors, an HDC inhibitor: S(+)-alpha-fluoromethyl-histidine HCl, (alphaFMH), a compound that disturbs the interaction of histamine with intracellular cyp450 moieties: N,N-diethyl-2-[4-(phenylmethyl) phenoxy]-ethanamine HCI, (DPPE); and H1 and H2 receptor antagonists, Triprolidine and Cimetidine. RESULTS: During in vitro culture of elutriated human monocytes, in the presence of M-CSF, the gene expression and biosynthesis of HDC was considerably increased. The various antihistamine agents decreased the expression of the cell surface markers examined in this study. CONCLUSIONS: These data support the elevation of HDC expression during human monocytic differentiation and the possibility that monocyte-derived histamine is partially involved in regulation of M-CSF induced in vitro human monocyte/macrophage phagocytic differentiation.

Adipophilin is a sensitive marker for lipid loading in human blood monocytes.

Adipophilin, a marker of lipid accumulation initially described in adipocytes, was recently shown to be induced in macrophage foam cells. We found that even freshly isolated blood monocytes express adipophilin and that the amount of adipophilin protein is variable in monocytes from different healthy individuals. However, the physiological expression of adipophilin does not correlate with the levels of free fatty acids, cholesterylesters or free cholesterol. Enzymatically modified low-density lipoprotein (E-LDL) induces rapid foam cell formation in monocytes and upregulates adipophilin mRNA and protein within 2 h of incubation. This rapid induction of adipophilin is accompanied by a significant increase of free fatty acids in monocytes incubated with E-LDL. Adipophilin facilitates the uptake of free fatty acids, and here we demonstrate that free fatty acids increase is related to the early upregulation of adipophilin expression in blood monocytes. Fatty acids are ligands for peroxisome proliferator-activated receptor-gamma (PPARgamma), and the upregulation of adipophilin mRNA by PPARgamma agonists like 15d-PGJ(2) and ciglitazone indicates that PPARgamma may mediate the induction of adipophilin expression in human blood monocytes.

Inhibition of effects of endogenously synthesized histamine disturbs in vitro human dendritic cell differentiation.

Histamine, a principal mediator in various physiological and pathological cell functions is synthesized from L-histidine exclusively by histidine decarboxylase, an enzyme, which is expressed in many tissues of mammalian organism. Histamine plays a role in various cellular functions, including cell differentiation. The aim of this study was to determine the presence and to characterize the role of the endogenously produced histamine during in vitro dendritic cell (DC) differentiation induced by interleukin-4 (IL-4) and granulocyte-monocyte colony stimulating factor (GM-CSF). The changes in intracellular histamine content, biosynthesis and gene expression of histidine decarboxylase were investigated during this process. One also studied how histamine receptor antagonists and a histamine synthesis blocker influence the expression of differentiation antigens on the DC during in vitro maturation. During in vitro differentiation parallel culture incubations were performed by adding H1 receptor antagonist triprolidine, H2 receptor antagonist tiotidine, the tamoxifene derivate DPPE which blocks the intracellular binding of histamine, and an irreversible blocker of histidine decarboxylase, alpha-fluoromethyl histamine (alpha-FMH). The results show simultaneous increase in both histidine decarboxylase level and histamine content during differentiation of elutriated monocytes toward DC. Both blockade of de novo histamine production (by alpha-FMH) and inhibition of histamine binding (by H1 and H2 receptor antagonists, triprolidine and tiotidine, respectively) markedly decreased CD40 expression and that of CD45 from the 3rd day of treatment. DPPE by disturbing intracellular interaction of histamine with cytochrome P-450 moieties was able to decrease the expression of CD45, CD86, HLA-DR, CD33, CD40 and CD11c. Based on the data it is suggested that endogenous histamine is actively synthesized during cytokine-induced in vitro DC differentiation. The functional relevance and autocrine and paracrine action of endogenously produced histamine is supported by the data showing that inhibition of histamine synthesis by HDC, blocking of histamine binding by both 'extracellular' histamine receptors (by specific antagonists, triprolidine and tiotidine) and 'intracellular' antagonists (DPPE) disturb the differentiation of DC. This conclusion is supported by the fact, that by the inhibition of histamine acting in an autocrine/paracrine way, the expression pattern of differentiation markers on DC is markedly changed.

ABC transporters in cellular lipid trafficking.

ATP-binding cassette (ABC) transporters constitute a group of evolutionary highly conserved cellular transmembrane transport proteins. Recent work has implicated ABC transporters in cellular transmembrane lipid transport and hereditary diseases have been causatively linked to defective ABC transporters translocating lipid compounds. The emerging concept that a defined subset of ABC transporters is intimately involved in cellular lipid trafficking has recently been substantiated convincingly by the finding that ABCA1 plays a central role in the regulation of HDL metabolism and macrophage targeting to the RES or the vascular wall. Differentiation dependent expression of a large number of ABC transporters in monocytes/macrophages and their regulation by sterol flux render these transporter molecules potentially critical players in atherogenesis and other chronic inflammatory diseases.

Multi-color analysis of monocyte and dendritic cell precursor heterogeneity in whole blood.

The coexpression analysis of the 55-kDa lipopolysaccharide receptor (CD14) and the Fc gamma-receptor III (CD16) reveals a broad heterogeneity of blood monocytes which in our previous work could be divided into five subpopulations based on correlated differences in expression of the pan-myeloid antigen CD33 and the adhesion antigens CD11a, CD11b and CD56. An even larger complexity of myeloid cells with antigen presenting capacity in peripheral blood is suggested by the description of small populations of immature and mature precursors of dendritic cells which rapidly develop potent costimulatory activity and a dendritic morphology in in vitro culture. The identity of the subsets of cells which have been described based on heterogeneous analytical approaches, however, remains unclear. The goal of this study, therefore, was the correlated analysis of monocyte subpopulations and dendritic cell precursors in a quantitative whole blood assay. This was achieved based on simultaneous expression analysis of the monocyte markers CD14 and CD16 with antigens such as CD33, HLA-DR, the integrin CD11c, and the interleukin-3 receptor alpha-chain (CD123) which in absence of lineage-related antigens have been used for description of dendritic cell precursors. The selected marker panel revealed identity of cells previously described as CD33bright CD14dim dendritic cell precursors with CD11c+lin-HLA-DR+ cells. Dendritic cell precursors considered to be less mature which have been alternatively described as CD33+ CD14dim CD16- cells or CD123hi dendritic cell precursors, however, were shown to differ in phenotype from each other with regard to expression density of CD33 and expression of CD14. In summary, our study revealed a complex heterogeneity of monocytes and dendritic cell precursors in peripheral blood and indicates that a direct comparison of the analytical approaches of different authors is needed to further clarify the ontogeny of human monocytes and dendritic cells.

Identification of a novel human sterol-sensitive ATP-binding cassette transporter (ABCA7).

We report the identification of the full-length cDNA for a novel ATP-binding cassette (ABC) transporter from human macrophages. The mRNA is of 6.8 kb size and contains an open reading frame encoding a polypeptide of 2146 amino acids with a calculated molecular weight of 220 kDa. The predicted protein product is composed of two transmembrane domains and two nucleotide binding folds indicating that it pertains to the group of full-size ABC transporters. The novel transporter shows highest protein sequence homology with the recently cloned human cholesterol and phospholipid exporter ABCA1 (54%) and the human retinal transporter ABCR (49%), both members of the ABC transporter subfamily A. In accordance with the currently proposed classification, the novel transporter was designated ABCA7. ABCA7 mRNA was detected predominantly in myelo-lymphatic tissues with highest expression in peripheral leukocytes, thymus, spleen, and bone marrow. Expression of ABCA7 is induced during in vitro differentiation of human monocytes into macrophages. In macrophages, both the ABCA7 mRNA and protein expression are upregulated in the presence of modified low density lipoprotein and downregulated by HDL(3). Our results suggest a role for ABCA7 in macrophage transmembrane lipid transport.