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LIPID Metabolites And Pathways Strategy |
Core Laboratories and Bridges
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Administrative Core A University of California San Diego, California The Administrative Core will be responsible for the overall scientific, fiscal, and administrative leadership of the LIPID MAPS Consortium including cores, bridges and Participating Investigators. It will also be responsible for arranging and leading the meetings of the Advisory Committee, Steering Committee, and Operating Committee as well as meetings of Participating Investigators and other scientists working in this field. |
 Edward A. Dennis University of California, San Diego Department of Chemistry and Biochemistry Basic Science Building 9500 Gilman Drive m/c 0601 La Jolla, CA 92093-0601 (For deliveries, please include delivery code -- e.g. "BSB 4080")
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Bioinformatics and Data Coordination Core B/C University of California San Diego, California The LIPID MAPS project is expected to generate large volumes of data pertaining to lipids and lipid-guided interactions and networks. The management, dissemination of the data and results is an important component of the project. The Informatics Core will be responsible for a) data coordination and dissemination and b) bioinformatics analysis of the data. More specifically, the Informatics Core, will design and implement a laboratory information management system, create a pipeline for data communication and infrastructure for data dissemination and implement a web-based bulletin board for exchange of data and knowledge between different laboratories participating in the LIPID MAPS project and the community. In addition, the Bioinformatics component of the Informatics Core will be responsible for analysis of the lipidomics (mass spectrometric and other) data, creation of lipid databases and the reconstruction of lipid networks. The Bridging Project in Bioinformatics will carry out the more research-oriented aspects of LIPID MAPS project. |
 Shankar Subramaniam University of California, San Diego San Diego Supercomputing Center 9500 Gilman Drive, m/c 0505 La Jolla, CA 92037
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Macrophage Biology and Functional Genomics Core D University of California San Diego, California A Macrophage Biology and Functional Genomics Core is proposed as a component of the LIPID MAPS consortium to accomplish the following specific aims: 1. Develop standardized protocols for isolation, expansion and analysis of primary macrophages and macrophage cell lines. Standardized protocols will ensure that all LIPID MAPS cores and Participating Investigators carry out experiments under the most uniform conditions possible, allowing results in one core unit to be directly compared to results in other core units. 2. Characterize expression patterns of genes involved in lipid sensing and metabolism and responses of macrophages to bioactive lipids. The Macrophage Biology and Functional Genomics Core Facility will profile the expression patterns of genes that encode proteins involved in the production, sensing, uptake, catabolism and cellular efflux of specific lipid metabolites in macrophages under basal and stimulated conditions. The results of these studies will be correlated with studies carried out in lipid analysis core facilities, allowing regulatory, biosynthetic and degradation/export pathways to be defined. Gene expression data generated by these studies will be made available to the general scientific community through the Bioinformatics Core Facility. 3. Develop RNA interference reagents for inhibition of the expression of genes involved in lipid sensing and metabolism. The Macrophage Biology and Functional Genomics Core will develop and validate siRNA tools that will be distributed to Lipidomics cores and bridges. These reagents and/or their design will also be made available to the general scientific community. The Macrophage Biology and Functional Genomics Core will also provide facilities for initial evaluation of macrophages derived from knockout mice. |
 Chris Glass University of California, San Diego George Palade Laboratories for Cellular and Molecular Medicine Room 217 9500 Gilman Drive La Jolla, CA 92093-0651
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Glycerolipids Core E University of Colorado Health Sciences Center Denver, Colorado The investigations proposed in the LC/Mass Spectrometric Analysis/Neutral Lipids Core involve the development of state-of-the-art approaches in mass spectrometry applied to the qualitative and quantitative analysis of lipids present in macrophage cell lines under basal and stimulated conditions. Emphasis will be placed on the development of quantitative assays of target lipids based on stable isotope dilution LC/MS/MS and homolog internal standards for neutral lipids by LC/MS. Targets of sterols and eicosanoids present in normal as well as stimulated macrophages will be isolated and structurally characterized using radiolabeled and stable isotope labeled precursors of cholesterol and eicosanoids. General methods for the isolation and class separation of major lipid substances present in the macrophage will be developed using ion exchange, normal phase, and reversed phase chromatography as critical steps in generating qualitative as well as quantitative protocols. The particular qualitative focus of this Core is the analysis of neutral lipids including molecular species of triacylglycerols, cholesterol esters, and diacylglycerols expressed in the macrophage. The molecular species of these major neutral lipids will be defined using tandem mass spectrometric techniques. Finally, quantitation of each of the individual molecular species within the major neutral lipid classes will be determined using synthetic analogs which are not naturally occurring, but for which relative response factors will be determined. These internal standards will be added to the lipid extracts prior to isolation and purification steps. The analytical figures of merit of each of the lipid assays (precision, accuracy, and sensitivity) will be determined. An assessment of the neutral lipid composition in the macrophage and alteration of neutral lipid composition with various stimuli will be the major activity of the Core after quantitative methods have been established. |
 Robert C. Murphy, Ph.D. Department of Pharmacology University of Colorado Health Sciences Center Mail Stop 8303 P.O. Box 6511 Aurora, CO 80045-0511
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Lipid Synthesis Core F(a): Lipid Standards Avanti Polar Lipids Alabaster, Alabama Core F(b): Synthesis Design Indiana University Bloomington, Indiana Core F(c): Biophysical Characterization University of California Irvine, California The Lipid Synthesis and Biophysical Characterization Core (Core F) will serve the LIPID MAPS Consortium by providing all aspects of synthetic chemistry support for each of the Lipidomics Cores as well as biophysical characterization support for all lipids of interest to the consortium. The Lipid Standards and Production Chemistry Unit, represented by Avanti Polar Lipids, Inc. will provide lipid standards to be used by investigators participating in the consortium. Avanti will synthesize a library of lipids for use as analytical standards as well as provide materials for biological assays. Novel lipids provided to the consortium by Avanti will be made available to the general scientific community for the benefit of lipidomic research. The Novel Lipid Synthetic Design Unit will provide synthetic chemistry support for all new lipids and lipid intermediates discovered by the consortium. The Unit will work close with Avanti Polar Lipids, Inc. for the transfer of technology to enable production-scale synthesis of novel lipids discovered by the consortium. This unit will also work closely with the Structural Lipidomics and Other Lipids Core (Core K) to provide chemistry support to assist structure elucidation efforts. The Biophysical Characterization Unit will use well-established methods, including calorimetry and x-ray diffraction, to study the biophysical properties of novel lipids discovered by the Lipidomics Cores. This group will also study the interaction of lipopolysaccharide (LPS) with biological membranes and model macrophage membranes in support of the LIPID MAPS Consortium. |
*Core F(a)* Walter Shaw Avanti Polar Lipids, Inc. 700 Industrial Park Drive Alabaster, AL 35007
*Core F(b)*  Mike VanNieuwenhze Indiana University Department of Chemistry 800 E. Kirkwood Ave. Bloomington, IN 47401
*Core F(c)*  Stephen H. White University of California at Irvine Dept. of Physiology and Biophysics Medical Sciences I -- D346 Irvine, CA 92697-4560
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Fatty Acyls Core G University of California San Diego, California The Eicosanoid/Fatty Acid Core will determine the levels of free fatty acids and fatty acid metabolites (FAM) in the macrophage cytosol and secreted by these cells. FAMs include, but are not restricted to, the prostanoids, hydroxyl- and hydroperoxy- eicosaenoic acids, leukotrienes, epoxyeicosatrienoic acids, free fatty acids, and fatty acid amides. Resting cells contain and secrete very low levels of FAMs. These compounds are usually generated in response to a signal, are produced for a specific function, and are often potent second messengers. As part of the Consortium's "discovery approach" to lipidomics, this Core will identify all FAMs present in and secreted by macrophages and will quantitate the levels of all FAMs in these cells in the resting state and after various stimulations. The first step in the pathways leading to FAM generation is the liberation of the fatty acid from lipids. One of the primary enzymes that carries out this reaction is phospholipase A2 (PLA2), a superfamily of fourteen different enzymes. The fatty acids thus released can enter myriad metabolic pathways that, in the end, produce hundreds of FAMs, many of which are highly bioactive. The number of pathways involved, coupled with the fact that FAMs have such similar chemical structures and reactivities, suggests that there is a complex flow of FAMs through these metabolic networks. Because of the high probability that "Lipid Networks" are operative in this class of lipid, the Eicosanoid Core will collaborate closely with the LIPID MAPS Networks Bridge to develop and test techniques for detecting and identifying such networks. The subcellular location of both the enzymes and the substrates involved in these pathways appear to play an important role in how FAM biosynthesis is controlled in vivo. This Core will explore, in conjunction with the Lipid Subcellular Localization Bridge and the Glycerophospholipid Core, the relationship between FAM generation and the subcellular location of the substrates for these enzymes. The Dennis laboratory has found evidence that, in P388D1 cells, sphingolipids participate in regulating eicosanoid production. It also appears that rafts and caveolae may play a role in this process also. Thus, the Eicosanoid Core will develop projects in conjunction with the Sphingolipid and Sterol Cores to explore these interactions further. This core will also collaborate with the Sterol Core to explore the role that P450 enzymes play in eicosanoid production. |
Edward A. Dennis University of California, San Diego Department of Chemistry and Biochemistry Basic Science Building 9500 Gilman Drive m/c 0601 La Jolla, CA 92093-0601 (For deliveries, please include delivery code -- e.g. "BSB 4080")
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Glycerophospholipids Core H Vanderbilt University Nashville, Tennessee The primary goal of the Glycerophospholipid Core of the LIPID MAPS Project is to develop a state of the art approach to qualitative and quantitative analysis of phospholipids. We will also be part of a larger consortium of investigators using identical instrumentation (QqTOF) with the goal of developing a highly integrated methodology that will achieve comprehensive analysis of cellular lipid species. The goal of resolving, identifying, quantitating and analyzing what is likely to exceed 1000 distinct species of lipids in the macrophage is formidable. Yet recent advances in mass spectrometry provide an opportunity for such a technical achievement. This type of advance will also require the focused efforts of several accomplished groups working in a coordinated manner. In essence, Lipidomics is a subdivision of Metabolomics and as such there are opportunities to learn much about cellular processes and the course of pathological states from monitoring lipid profiles. Significant progress has already been made by several groups in qualitative analysis of phospholipids by mass spectrometry, including investigators in this consortium. However, to successfully achieve quantitative analysis of a broad spectrum of cellular phospholipids there needs to be new methods developed and synthesis of an array of new stable isotopic internal standards. Chromatographic separation prior to mass spectrometry allows measurement of low abundant species that have not previously been accurately measured for changes in absolute mass amounts (e.g. PI4,5P2 and PI3,4,5P3). Measurement of these phospholipids are critical to another major goal of the Glycerophospholipid Core and the overall project, that is, to determine the cascade of membrane signaling events that occurs in response to lipopolysaccaride (LPS) stimulation of the macrophage. This includes a coordinated analysis using lipid metabolism oriented gene arrays by the Cell Biology Focus Area and contextual proteomic studies of the LPS signaling pathway. These studies will likely focus on a number of phospholipases, lipid kinases, and lipid phosphatase enzymes, as well as other targets. We anticipate that two areas of central importance will include the poly-phosphate phosphatidylinositol containing lipids and production of oxidative species of phospholipids. As such, we are fortunate to have two distinguished colleagues, Dr. Lew Cantley at Harvard Medical School and Dr. Jack Roberts at Vanderbilt Medical Center, as Participating Investigators in this Core. As for other classes of lipids a central goal is to identify those lipid species involved in LPS signaling as well as other cell surface receptor pathways to be identified as the studies progress. We anticipate that these efforts will lead to identification of novel lipid species and illuminate new functions for known phospholipids species. Specifically, bioinformatics analysis will cluster changes in individual phospholipid species against defined macrophage functions (i.e. respiratory bursts and bacterial phagocytosis). This will establish metabolic interconnections between classes of lipids (e.g. precursor-product relationships between phospholipids and glycolipids) and generate Lipid Arrays that will both identify species and direct future studies of molecular processes in the macrophage. |
 H. Alex Brown, Ph.D. Vanderbilt Univ School of Medicine Department of Pharmacology 442 Preston Research Building 23rd Ave South & Pierce Nashville, TN 37232-6600 Delivery Address for samples: 406 Preston Research Building
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Sphingolipids Core I Georgia Tech Atlanta, Georgia This LIPID MAPS Core will develop methods to analyze sphingolipids (sphingomyelins, ceramides, sphingoid bases, sphingoid base 1-phosphates and related lyso-species and metabolites, such as the N-methyl-sphingoid bases) and glycosphingolipids (glucosylceramide, lactosylceramide and other neutral glycosphingolipids, and gangliosides) by mass spectrometry, and evaluate their occurrence, metabolism, and interactions with other lipid signaling pathways in macrophages.. The new methods will allow quantitative analysis of the sphingolipid/glycosphingolipid class by both head group and the nature of the ceramide" backbone (i.e., the sphingoid base and amide linked fatty acid); in addition, the methods will allow analysis of sphingolipids/ glycosphingolipids made from stable isotope precursors such as 13C-palmitic acid. Development of the methods will utilize LC ESI- and MALDI-MS/MS and two state-of-the-art instruments (the ABI 3000 triple quadrupole MS/MS and the QSTAR-XL, a quadrupole time-of-flight instrument) to analyze the major (glyco)sphingolipids of macrophages (sphingomyelins, glucosylceramides, lactosylceramides, ganglioside GM3's and the lipid backbones). An additional mass spectrometric approach (evaluation of connectivity by MS3 using an ion trapping instrument, the Q-TRAP), will facilitate analyses of ganglioside GM3 and more complex gangliosides and neutral glycosphingolipids. Because these instruments generate a large amount of data (> 103 data points per experiment) the data output will be organized by collaboration with the Bioinformatics Core. These MS/MS methods are highly sensitive (typically requiring only 106 cells) and will be used to determine how agonists (such as LPS, oxidized LDL and others) affect sphingolipid metabolism in intact cells. Using complementary biochemical and genetic tools, it will become possible to clarify how sphingolipid metabolism is regulated, to elucidate the role(s) of sphingolipids as modulators and mediators of cellular responses to these agents, and to understand how sphingolipid metabolism is connected to other lipid metabolic (and signaling) pathways. |
 Alfred Merrill, Jr. School of Biology Georgia Institute of Technology Atlanta, GA 30332-0230
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Sterol Lipids Core J UT Southwestern Medical Center Dallas, Texas Sterols play important structural, signaling, and regulatory roles in macrophage biology. Cholesterol, together with saturated and unsaturated fatty acids, is required to maintain the integrity of the plasma membrane and to modulate fluidity and phase transitions in this essential barrier. Cholesterol and a second class of lipids, the sphingomyelins, also form specialized plasma membrane rafts or caveolae, which concentrate tyrosine kinase receptors and other molecules that transmit signals into the cell. The regulatory roles of sterols in macrophages are illustrated by their abilities to serve as agonists for nuclear hormone receptors, as suppressors of cholesterol biosynthesis, and as products of cholesterol excretion and turnover. The diverse functions of sterols, together with their structural complexity, suggest that novel sterols and biological roles remain to be discovered. The initial goal of the Sterol Core will be to develop methods for the analysis and quantitation of sterols in macrophages. Working in collaboration with the LC/MS Analysis Core, mass spectrometry procedures will be devised to extract, identify, and measure the major and minor sterols of the resting macrophage. These methods will be applied to determine how the macrophage sterol complement changes in response to lipopolysaccharide, cytokine and phagocytic challenges. The second objective is to identify the enzymes responsible for the synthesis of novel sterols in macrophages. Candidate cDNAs will be identified based on the regulation of their encoding genes by sterol response element binding proteins (SREBPs), which are transcription factors that coordinately regulate lipid biosynthesis. Working with two participating investigators of the Sterol Core, Drs. Michael S. Brown and Joseph L. Goldstein, expression of SREBP-regulated cDNAs in cultured cells, mass spectrometry, and interference RNA will be employed to identify a candidate enzyme's sterol substrate and product. The third aim is the identification of the lipid substrates of macrophage cytochrome P450 enzymes. The combined expertise of the LIPID-MAPS consortium will be exploited together with expression of P450 cDNAs, mass spectrometry, and interference RNA to determine the substrates and products of a large class of enzymes known to be crucial for lipid synthesis and catabolism. The proposed research of the Sterol Core will provide new insight into the roles of sterols and other lipids in the macrophage, a cell type that is relevant to immunity, inflammation, and atherosclerosis. |
 David Russell UT Southwestern Medical Center 5323 Harry Hines Blvd. L5.272 Dallas, TX, 75390
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Prenols and Other Lipids Core K Duke University Durham, North Carolina The overall goal of the Structural Lipidomics/Other Lipids Core is to provide comprehensive methodological approaches to the isolation and identification of novel minor lipids present in macrophage cell lines under basal and lipopolysaccharide stimulated conditions, as well as with other agonists. The strategy will involve the purification of novel lipids by preparative column chromatography, followed by state of the art mass spectrometry and NMR spectroscopy of pure compounds. As a prototype for methods development, the Core will reinvestigate the previously described, but not fully characterized, accumulation of lyso-phosphatidylinositol in the RAW 264.7 macrophage cell line following exposure to lipopolysaccharide. The Core will collaborate at the earliest possible stage with all other lipidomics cores of the consortium as they encounter new lipid species that they cannot assign. Furthermore, the Core will work with the Lipid Synthesis and Characterization Focus Area in a timely manner to ensure that novel structural proposals are validated by synthesis. The Structural Lipidomics Core will therefore provide "glue" to the other units from the beginning of the project and throughout its entire course. In the coming grant period, the Core has the following goals: 1)The development of general large-scale procedures for fractionating macrophage lipids into their major and minor components. Purified compounds will be evaluated by a combination of mass spectrometry and NMR spectroscopy, followed by synthesis. 2) The complete structural characterization of the lyso-phosphatidylinositol that accumulates in RAW cells stimulated with lipopolysaccharide. 3) Development of stable isotopic labeling methods for determining which lipids are newly synthesized or are derived from pre-existing species following macrophage stimulation. 4)The development of techniques for extracting, identifying and quantifying known lipids not covered by the other cores. |
 Chris Raetz 243 Nanaline Duke Building Research Drive Durham, NC 27710 919-684-5178 919-668-2667
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LIPID MAPS Networks Bridge A University of California San Diego, California The scope of the LIPID MAPS project is to obtain context specific knowledge of genes, proteins and lipids involved in well-defined cellular responses which will then enable us to reconstruct lipid metabolic and signaling networks. Expression profile data accruing from specific mammalian cells in addition to providing data on the differences between normal and abnormal biological function, can provide in principle a partial list of genes expressed in that cell. For the macrophage cells that are the subject of network and modeling study in the LIPID MAPS project, gene expression data will be generated by investigators in the project. Proteomics, i.e. the cataloguing of proteins and protein derivatives in a cell along with quantitation continues to be a major problem in biology. While mass spectrometric methods have come of age, there continues to be the bottleneck of separation of proteins that is not a high throughput process. Our objective is more modest where we will carry out context-specific proteomics and obtain a list of proteins involved in well-defined pathways. The major quest of our project is to map all the lipids that change during a given cellular function and are implicated in cellular response to input. As stated in the proposal, we will use mass spectrometry coupled with other biophysical methods to obtain a parts list of lipid and lipid derivatives involved in cellular metabolism and signaling networks. Reconstruction of biochemical pathways and networks has relied to a large extent on the knowledge of protein function and protein-protein interactions. Traditional pathway definitions (e.g. glycolysis, pentose phosphate pathway, TCA cycle) have served as conceptual frameworks for research and teaching. As useful as they have been, these traditional pathway definitions may not provide for quantitative, systemic evaluations of biological reaction networks as they focus on a subset of a network without consideration of the network-wide interactions. Only recently efforts have been made to characterize metabolism at the systems level, where genes, proteins and lipids and other substrates are mapped. In this proposal we plan to extend these approaches to construction of cellular networks incorporating lipid, along with genes and gene products.Toward this end, we will build the information infrastructure that will enable reconstruciton of the network from its component parts and subsequent modeling using quantitative data emerging from our experiments. |
University of California, San Diego San Diego Supercomputing Center 9500 Gilman Drive, m/c 0505 La Jolla, CA 92037
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Transcriptional Regulation Bridge B University of California San Diego, California Transcriptional Regulation in Macrophages Bridge will combine mammalian two-hybrid analysis and genome-wide location analysis (ChIP on Chip) with data generated by the LIPID MAPS Consortium to identify, characterize and model PPAR-dependent transcriptional networks that sense and regulate lipid metabolism in the macrophage. The mammalian two-hybrid assay will be used to detect interactions between PPARs and specific co-activators and co-repressors that depend on the production of lipid metabolites within living cells. Genome-wide location analysis is a recently developed technology that allows the detection of specific transcription factors on promoter and enhancer elements throughout the genome. The information provided by this analysis thus permits the identification of putative target genes for these transcription factors and their regulatory lipids. Two Specific Aims are proposed. Specific Aim 1 will be to assess the functional activity states of PPARg and PPARd in RAW macrophages using mammalian two hybrid assays to record interactions with specific co-activator or co-repressor interaction domains. These experiments will be performed using the standard assay conditions developed for characterization of patterns of gene expression and lipid composition by LIPID MAPS Cores. Specific Aim 2 will be to perform genome-wide location analysis of PPARg, PPARd and specific coactivators in RAW macrophages under these same conditions. By linking protein-protein interaction and protein-DNA interaction data with conventional mRNA expression profiling and lipid analysis, the temporal relationships between synthesis of bioactive lipids, DNA binding and target gene expression can be established. In principle, it should be possible to use this collective data set to construct models of regulatory networks that control lipid metabolism. |
 Chris Glass University of California, San Diego George Palade Laboratories for Cellular and Molecular Medicine Room 217 9500 Gilman Drive La Jolla, CA 92093-0651
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Lipid Imaging Bridge C University of Colorado Health Sciences Center Denver, Colorado Studies are proposed in the Bridge Unit C to develop matrix assisted laser desorption ionization (MALDI) as the primary technique of both qualitative and quantitative analysis of lipids extracted from the macrophage. The overall hypothesis centers around the ability to capture HPLC effluents on MALDI plates or similar suitable surfaces such as porous silicon and thereby convert the time domain of HPLC separation to spacial location in a 2-dimensional array. In this way, HPLC separation of lipids required for optimal analysis by both qualitative and quantitative means can be carried out in a parallel step offline from the mass spectrometric analysis. Rapid scanning mass spectrometers with MALDI ion sources can examine the special array for elution of lipids separated by HPLC. In this way sample throughput and lipid analysis would be greatly enhanced. Removing time constraints from the analysis of separated HPLC components should permit both positive and negative ion analysis to be carried out under computer control as well as a detailed analysis of a collision induced decomposition spectra of even minor ions present in complex mixtures of lipids. A second major focus of investigation will be the implementation of qualitative and quantitative assays using MALDI ionization on a high energy TOF/TOF instrument. The inherent rapid scanning of this mass spectrometer as a true tandem instrument should significantly decrease the time required for qualitative analysis of complex mixtures of lipids as well as quantitative analysis of major and minor molecular species. An additional important facet of the TOF/TOF machine is the ability to carry out high energy collision of lipid ions which are proposed to generate new structural information not attainable with low energy collisions with a quadrupole based collision cell. A new dimension of lipid analysis should result. Finally, methods will be developed to automate both qualitative and quantitative aspects of lipid analysis through the generation of specific 3-dimensional arrays of mass spectral and HPLC data. The data array will include retention time, positive or negative molecular ion species, and the complete set of collision induced decomposition ions. These data arrays should be important components of the information available from LIPID MAPS. |
Robert C. Murphy, Ph.D. Department of Pharmacology University of Colorado Health Sciences Center Mail Stop 8303 P.O. Box 6511 Aurora, CO 80045-0511
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Oxidized Lipids in Macrophages Bridge D University of California San Diego, California Oxidized LDL (OxLDL) is believed to play a major role in atherogenesis because of its major proinflammatory and proatherogenic properties. For example, profoundly OxLDL as well as minimally oxidized LDL (mmLDL) can effect profound changes in gene expression in cells such as macrophages, resulting in adverse effects. Undoubtedly, many of these effects are mediated by the oxidized lipid moieties generated when LDL undergoes oxidation.. Although there is a vast literature now on the biological properties of OxLDL, most of these studies have utilized LDL oxidized in vitro by exposure to copper, a situation that may not occur in vivo, where most likely OxLDL is generated by exposure to cells, such as macrophages. Yet little is known about the oxidized lipid moieties generated when LDL is incubated with macrophages, nor is there information on the biological effects on macrophages of the specific oxidized moieties present in such cell-mediated OxLDL. In an analogous situation, we and others have shown that when cells undergo apoptosis, a condition known to induce oxidative stress, oxidized phospholipids are generated on the surface of the apoptotic cells, as well as on apoptotic blebs that are released from the dying cell. These apoptotic blebs too have been shown to have many proinflammatory properties similar to OxLDL. Again, little is known of the oxidized lipid moieties present in such apoptotic bodies. In this proposal we will incubate LDL with macrophages in culture, under a variety of conditions to simulate resting and activated states known to initiate oxidation of LDL. The lipids of the oxidized LDL will be analyzed to determine the oxidized moieties formed, with an initial emphasis on oxidized phospholipids and cholesterol and cholesteryl esters. Prominent identified oxidized moieties will then be tested for their ability to affect macrophage function, including their ability to alter in turn the lipid moieties of the macrophage. We will also determine the effects on macrophage gene expression, by testing "candidate" gene expression and functions known to be altered by exposure to "OxLDL", as well as by more global techniques such as examination of gene expression by microarray techniques. In a similar manner, apoptotic bodies will be isolated and purified from cells undergoing apoptosis and subjected to a similar analysis. The oxidized moieties will be compared to those of cell-mediated OxLDL, and the biological consequences of unique identified moieties will be determined. Knowledge of the actual oxidized lipid moieties generated when LDL undergoes cell-mediated oxidation, or when apoptotic bodies are released from a dying cell could provide novel insight into pathogenic moieties that may not only give specific insights into mechanisms of atherogenesis, but into proinflammatory mechanisms in general. |
 Joseph Witztum University of California, San Diego Dept of Medicine-0682 La Jolla, CA 92093 Delivery Address for samples: Univ of Calif, San Diego Basic Science Building-Room 1080 9500 Gilman Drive La Jolla, CA 92093
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Subcellular Localization Bridge E University of California San Diego, California The goals of this bridge proposal focus on the qualitative and quantitative analyses of lipid components in the various subcompartments of macrophages with the intent of furthering our knowledge of how these lipids are involved in the regulation of macrophage-initiated disorders. Macrophages are the hallmark of chronic inflammation. They are recognized as major contributors to the initiation and progress of atherosclerosis, rheumatoid arthritis, and fibrosis. Through major investments in research and development of sophisticated technology to analyze genes and proteins our understanding of the details of how such molecules are involved in macrophage-related disorders has occurred. Unfortunately, this has not been the case for the lipid details and it is the details that are the sights of pharmaceutical intervention. A major objective of this bridge proposal is to build on the advances made by the Lipidomics Cores to develop standardized procedures for lipid analysis and extend the LIPID MAPS database to include the location of lipids in the organelles isolated from macrophages. This will be done with both resting and macrophages activated in vitro by factors involved in macrophage-initiated diseases. In order to accomplish the above goals and objectives, three Specific Aims will be evaluated: (1) Adapt or develop routine procedures for the isolation of macrophage subcellular fractions for lipid analysis by the Lipidomic Cores. The subcellular fractions will include endoplasmic reticulum, Golgi, endosomes, lysosomes, peroxisomes, plasma membrane and its apical and basolateral domains for cells in monolayers, and the inner and outer membranes of mitochondria and nuclei. (2) Determine the changes in lipid composition of macrophage organelles activated by lipopolysaccharide (LPS) and during endocytosis of polystyrene beads in vitro. Both LPS activation and endocytosis albeit of microorganisms occur in inflammation. (3) Determine the changes in lipid composition of macrophage organelles that occur during the different stages of macrophage adhesion to a collagen matrix. The stages are cell attachment, spreading, and migration. Such stages are analogous to those used by macrophages during normal and activated functions in tissues. Accomplishing these aims and making the LIPID MAPS database generated from the results immediately available to others should expedite our understanding of how lipids initiate, regulate and propagate disease. |
Edward A. Dennis University of California, San Diego Department of Chemistry and Biochemistry Basic Science Building 9500 Gilman Drive m/c 0601 La Jolla, CA 92093-0601 (For deliveries, please include delivery code -- e.g. "BSB 4080")
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