Herbert Stangl

Transport of lipids from high and low density lipoproteins via scavenger receptor-BI.

The scavenger receptor-BI (SR-BI) delivers sterols from circulating lipoproteins to tissues, but the relative potency of individual lipoproteins and the transported cholesterol has not been studied in detail. In this study, we used Chinese hamster ovary cells that express recombinant mouse SR-BI but have no functional low density lipoprotein (LDL) receptors (ldlA7-SRBI cells) to compare the fate of lipids transferred from high or low density lipoproteins to cells by SR-BI. HDL and LDL were equally effective in mediating the transfer of [3H]cholesterol to cells. Only 5% of the free cholesterol transferred to cells was esterified, in direct contrast to the findings in the cells that express LDL receptors in which 50% of the transported cholesterol was esterified. Almost all the free cholesterol transferred from lipoproteins to cells was rapidly excreted when the ldlA7-SRBI cells were switched to media containing unlabeled lipoproteins. SR-BI expression was associated with an increase in selective cholesteryl ester uptake from both lipoproteins, but HDL was a more effective donor. HDL and LDL were equally effective in delivering cholesterol to the intracellular regulatory pool via SR-BI. These data indicate that SR-BI is able to exchange cholesterol rapidly between lipoproteins and cell membranes and can mediate the uptake of cholesteryl esters from both classes of lipoproteins.

Interleukin‐13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype

Atherosclerotic lesions are characterized by the accumulation of oxidized LDL (OxLDL) and the infiltration of macrophages and T cells. Cytokine expression in the microenvironment of evolving lesions can profoundly contribute to plaque development. While the pro‐atherogenic effect of T helper (Th) 1 cytokines, such as IFN‐γ, is well established, the role of Th2 cytokines is less clear. Therefore, we characterized the role of the Th2 cytokine interleukin (IL)‐13 in murine atherosclerosis. Here, we report that IL‐13 administration favourably modulated the morphology of already established atherosclerotic lesions by increasing lesional collagen content and reducing vascular cell adhesion molecule‐1 (VCAM‐1)‐dependent monocyte recruitment, resulting in decreased plaque macrophage content. This was accompanied by the induction of alternatively activated (M2) macrophages, which exhibited increased clearance of OxLDL compared to IFN‐γ‐activated (M1) macrophages in vitro. Importantly, deficiency of IL‐13 results in accelerated atherosclerosis in LDLR−/− mice without affecting plasma cholesterol levels. Thus, IL‐13 protects from atherosclerosis and promotes a favourable plaque morphology, in part through the induction of alternatively activated macrophages.

Scavenger Receptor, Class B, Type I-dependent Stimulation of Cholesterol Esterification by High Density Lipoproteins, Low Density Lipoproteins, and Nonlipoprotein Cholesterol

Scavenger receptor, class B, type I (SR-BI) is a cell surface glycoprotein that mediates selective uptake and efflux of sterols from high density lipoproteins (HDL) to cells. A Chinese hamster ovary cell line that is deficient in functional LDL receptors, but has high expression levels of recombinant SR-BI (ldlA7-SR-BI), was used to examine the effect of SR-BI on the trafficking of sterols between lipoproteins and cells. To monitor the fate of sterols transported by SR-BI into cells, we measured the incorporation of [14C]oleate into cholesterol esters by acyl-CoA:cholesteryl acyltransferase in the endoplasmic reticulum. We show that incubation of ldlA7-SRBI cells with either LDL or HDL resulted in an equally dramatic increase in the formation of [14C]oleate-labeled cholesterol esters. The lipoprotein-stimulated, SR-BI-dependent increase in cholesterol esterification was inhibited by chloroquine. The uptake of sterols and their incorporation into cholesterol esters by SR-BI from LDL was largely a selective process. The addition of free cholesterol to ldlA7-SRBI cells also stimulated cholesterol ester formation in a chloroquine-sensitive fashion. We also show that SR-BI mediates the efflux of endogenously synthesized sterols from the cell membrane. From these studies we conclude that, in the absence of the LDL receptor, overexpression of SR-BI can mediate significant transport of sterols between lipoproteins and the endoplasmic reticulum of cells.

SR-BI-mediated High Density Lipoprotein (HDL) Endocytosis Leads to HDL Resecretion Facilitating Cholesterol Efflux *□

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that ∼0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.

HDL endocytosis and resecretion.

HDL removes excess cholesterol from peripheral tissues and delivers it to the liver and steroidogenic tissues via selective lipid uptake without catabolism of the HDL particle itself. In addition, endocytosis of HDL holo-particles has been debated for nearly 40 years. However, neither the connection between HDL endocytosis and selective lipid uptake, nor the physiological relevance of HDL uptake has been delineated clearly. This review will focus on HDL endocytosis and resecretion and its relation to cholesterol transfer. We will discuss the role of HDL endocytosis in maintaining cholesterol homeostasis in tissues and cell types involved in atherosclerosis, focusing on liver, macrophages and endothelium. We will critically summarize the current knowledge on the receptors mediating HDL endocytosis including SR-BI, F1-ATPase and CD36 and on intracellular HDL transport routes. Dependent on the tissue, HDL is either resecreted (retro-endocytosis) or degraded after endocytosis. Finally, findings on HDL transcytosis across the endothelial barrier will be summarized. We suggest that HDL endocytosis and resecretion is a rather redundant pathway under physiologic conditions. In case of disturbed lipid metabolism, however, HDL retro-endocytosis represents an alternative pathway that enables tissues to maintain cellular cholesterol homeostasis.

Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion

In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.

Visualization of the Uptake of Individual HDL Particles in Living Cells Via the Scavenger Receptor Class B Type I

The scavenger receptor class B type I (SR-BI) plays an important role in mediating selective uptake of high-density lipoprotein (HDL)-derived cholesterol and cholesteryl ester in liver and steroidogenic tissues. The molecular mechanism by which this receptor mediates selective cholesteryl ester uptake remains still enigmatic. We applied ultrasensitive fluorescence microscopy to visualize the intracellular transport routes of HDL particles taken up via SR-BI in a Chinese hamster ovarian cell line. Although diffusion of the receptor bound particles on the cell surface is slow, internalization is accompanied by a dramatic increase in the mobility of the particles. HDL particles are endocytosed as clusters and actively transported to the perinuclear region of the cell. Costaining with organelle markers confirmed the involvement of an acidic compartment and the Golgi apparatus in the uptake process; finally, resecretion of the HDL particles was observed.

Combined Light and Electron Microscopy Using Diaminobenzidine Photooxidation to Monitor Trafficking of Lipids Derived from Lipoprotein Particles

Diaminobenzidine (DAB) photooxidation is a method for conversion of fluorescent signals into electron-dense precipitates that are visible in the electron microscope. Recently, we have applied this method to analyze organelles involved in holo-high density lipoprotein (HDL) particle uptake at the ultrastructural level. In the present work we extended the spectrum of molecules visualized via photooxidation to monitor the uptake of HDL-derived lipids in HepG2 cells. By the combined light-electron microscopic method and with the aid of the DAB photooxidation technique, it became possible for the first time to visualize different intracellular pathways of lipoprotein particle-derived lipids and analyze the compartments involved at the ultrastructural level. HDL-Alexa 568 was used to visualize holo-HDL particle uptake. Reconstituted HDL particles containing the fluorescent cholesterol analogues Bodipy-cholesterol, Bodipy-cholesteryl oleate, or cholesteryl Bodipy-ester were used to visualize uptake of the HDL-associated sterol. In Bodipy-cholesteryl oleate and cholesteryl Bodipy-ester, the cholesterol moiety or the fatty acid moiety is fluorescently labeled, respectively; in contrast, Bodipy-cholesterol is an analogue of free cholesterol. The cellular compartments involved in their intracellular routes after uptake were analyzed in the fluorescence and electron microscope after DAB photooxidation. Bodipy-cholesterol was found to be localized in tubular endosomes and multivesicular bodies (MVBs), in the trans-Golgi network, and in stacked Golgi cisternae. In contrast, HepG2 cells incubated with HDL containing Bodipy-cholesteryl oleate or cholesteryl Bodipyester gave an uptake pattern comparable to that of holo-HDL particles, with MVBs being involved. Bodipy-cholesteryl oleate was also found in lysosomes. These results indicate that HDL-derived cholesterol and cholesteryl ester are transported by different intracellular pathways in HepG2 cells. Thus, the DAB photooxidation method enables the analysis of intracellular transport of lipoprotein particle-derived lipids at the light and at the ultrastructural level.

Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells

Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function.

A Combination of (ω–3) Polyunsaturated Fatty Acids, Polyphenols and L-Carnitine Reduces the Plasma Lipid Levels and Increases the Expression of Genes Involved in Fatty Acid Oxidation in Human Peripheral Blood Mononuclear Cells and HepG2 Cells

Background: Hyperlipidemia and obesity are associated with metabolic syndrome and increased risk in developing diabetes and cardiovascular disease. Nutritional supplements, e.g. L-carnitine and polyunsaturated fatty acids (PUFAs), exert lipid-lowering effects. Hence, the hypothesis that dietetic intervention reduces plasma lipid levels and metabolic enzymes in overweight hyperlipidemic subjects was tested. Subjects and Methods: In a prospective placebo-controlled double-blind study in 22 moderately hyperlipidemic obese humans consuming low-fat yoghurt enriched with a combination of low-dose PUFAs, polyphenols and L-carnitine (PPC) twice a day for 12 weeks were compared to 20 matching participants ingesting low-fat yoghurt. The effects on plasma lipids and expression of enzymes involved in regulation of fatty acid oxidation in peripheral blood mononuclear cells (PBMCs) and HepG2 cells were evaluated. Results: PPC consumption led to significantly reduced plasma free fatty acid (–29%) and triglyceride (–24%) concentrations (each p < 0.05). PPC application increased significantly peroxisome proliferator-activated receptor α (PPARα) mRNA abundances and those of PPARα target genes (carnitine palmitoyltransferases-1, CPT1A and CPT1B, carnitine acetyltransferase and organic cation transporter 2; each p < 0.05) in PBMCs. In controls, plasma lipid levels and PBMC gene expression did not change. These findings were substantiated by the results of cell culture experiments in HepG2 cells. Conclusion: Supplementation of PPC had marked lipid-lowering effects and PBMC gene expression profiles seemed to reflect nutrition-related metabolic changes.