A Novel Selective Lipid Uptake Pathway Contributing to LDL-Induced Macrophage Foam Cell Formation

Atherosclerosis is a disease characterized by cholesterol-rich plaques within the intima of medium and large arteries. Cholesterol deposition is thought to occur by infiltration of low-density lipoprotein (LDL) into lesions followed by uptake into macrophages, generating lipid-loaded “foam cells.” Foam cells can also be generated in vitro by treatment of macrophages with LDL or oxidized LDL (oxLDL). The purpose of the current investigation was to determine the contribution of selective cholesteryl ester (CE) uptake versus whole-particle uptake during LDL-induced foam cell formation in cultured macrophages. Murine bone marrow-derived macrophages (BMMs) exhibited significant cholesterol accumulation when treated with LDL as indicated by quantification of cellular cholesterol and visualization of Oil Red-O-stained neutral lipid droplets. Uptake of LDL cholesterol was determined by measuring uptake of 3H and 125I into BMMs during treatment with [3H]CE/125I-LDL. [3H]CE uptake was linearly related to the LDL concentration at the concentrations used and was much larger than 125I uptake, indicating that the majority of LDL-cholesterol was acquired by nonsaturable, selective CE uptake. This pathway was demonstrated to be independent of whole-particle uptake by showing that inhibition of actin polymerization blocked LDL particle uptake but not selective CE uptake. Analysis by thin-layer chromatography (TLC) indicated that following uptake, [3H]CE was rapidly hydrolyzed into [3H]cholesterol by cells and largely effluxed into the culture medium. In contrast to LDL, studies of [3H]CE/125I-oxLDL uptake demonstrated that CE was acquired from oxLDL by whole-particle uptake with little or no selective CE uptake. Using a series of ten different [3H]CE/125I-oxLDLs oxidized for 0-24 hours, selective [3H]CE uptake was shown to be progressively impaired by LDL oxidation, while 125I-LDL particle uptake was increased as expected. Interestingly, the impairment of selective CE uptake occurred very early in LDL oxidation and this minimally oxidized LDL induced significantly less cholesterol accumulation in BMMs compared to native LDL. Together, these results demonstrate that selective CE uptake is the primary mode of cholesterol uptake from LDL but not oxidized LDL, a finding that has important implications for cholesterol metabolism in atherosclerotic lesions. Future studies seek to identify the molecular components that participate in the macrophage selective CE uptake mechanism

TRANSCRIPTIONAL ANALYSIS OF RETINAL AND FOREBRAIN PROGENITOR CELLS DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS

poster abstractEye development has been extensively studied in traditional model sys-tems but studies related to humans have been limited. The recent develop-ment of induced pluripotent stem cells (iPSCs) enabled the study of human development in culture at stages that would otherwise be inaccessible to in-vestigation. By definition, Pluripotent stem cells are cells that have the ca-pacity to generate any adult cell type, such as the muscle cell or the blood cell. A defined set of genes, known as eye field transcription factors (EFTFs) have proven to play an important role in eye development. Utilizing iPSCs as our model system, we sought to identify EFTFs that might play an essential role in the specification of the retina of the human eye. iPSCs were directed to develop into retinal cells as previously estab-lished. Since these events occur early in the developmental process, sam-ples were collected every two days over the first twenty days of differentia-tion. The development of retinal cells was determined by the characteriza-tion of gene expression patterns of six EFTFs over this timecourse in order to highlight important trends in retinal development. Retinal populations were identified by the expression of numerous EFTFs which were absent from other non-retinal cell types. Our preliminary data utilizing iPSCs highlights similar trends in the expression of these EFTFs as anticipated. However, the expression patterns of two key EFTFs varied from the others in a manner which implicated them to be critical for retinal devel-opment from an unspecified stem cell source. Thus, these candidate EFTFs were investigated further to establish their specific roles in retinal develop-ment using a combination of genetic and molecular biology approaches. The work presented in this study helps to elucidate the mechanisms by which retinal cells are specified and help establish iPSCs as a unique model system for studies of human development. 1also Indiana University Center for Regenerative Biology and Medicine, Indiana University Department of Medical and Molecular Genetics, and Stark Neurosciences Research Institute, Indianapolis IN 46202 This work was supported by a grant from the Indiana University Collaborative Research Grant fund of the Office of the Vice president for Research as well as startup funds from the School of Science at IUPUI

Immunoreactivity of Pluripotent Markers SSEA-5 and L1CAM in Human Tumors, Teratomas, and Induced Pluripotent Stem Cells

Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs), but little is known about SSEA-5 expression in other primitive tissues (e.g., human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors, cell lines, teratomas, and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors, such as embryonal carcinoma. In tumor cell lines, SSEA-5 was highly immunoreactive in Capan-1 cells, while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs, with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use

Spatial stochasticity and non-continuum effects in gas flows

We investigate the relationship between spatial stochasticity and non-continuum effects in gas flows. A kinetic model for a dilute gas is developed using strictly a stochastic molecular model reasoning, without primarily referring to either the Liouville or the Boltzmann equations for dilute gases. The kinetic equation, a stochastic version of the well-known deterministic Boltzmann equation for dilute gas, is then associated with a set of macroscopic equations for the case of a monatomic gas. Tests based on a heat conduction configuration and sound wave dispersion show that spatial stochasticity can explain some non-continuum effects seen in gases

The Abundance of Interstellar Nitrogen

Using the HST Goddard High Resolution Spectrograph (GHRS), we have obtained high S/N echelle observations of the weak interstellar N I 1160, 1161 A absorption doublet toward the stars Gamma Cas, Lambda Ori, Iota Ori, Kappa Ori, Delta Sco, and Kappa Sco. In combination with a previous GHRS measurement of N I toward Zeta Oph, these new observations yield a mean interstellar gas phase nitrogen abundance (per 10$^6$ H atoms) of 10$^6$ N/H = 75 +/- 4. There are no statistically significant variations in the measured N abundances from sightline to sightline and no evidence of density-dependent depletion from the gas-phase. Since N is not expected to be depleted much into dust grains in these diffuse sightlines, its gas-phase abundance should reflect the total interstellar abundance. Consequently, the GHRS observations imply that the abundance of interstellar nitrogen (gas plus grains) in the local Milky Way is about 80% of the solar system value of 10$^6$ N/H = 93 +/- 16. Although this interstellar abundance deficit is somewhat less than that recently found for oxygen and krypton with GHRS, the solar N abundance and the N I oscillator strengths are too uncertain to definitively rule out either a solar ISM N abundance or a 2/3 solar ISM N abundance similar to that of O and Kr.Comment: 14 pages, LaTeX, 2 Postscript figures; ApJ Letters, in pres

Fatty acid transport protein 4 is required for incorporation of saturated ultralong-chain fatty acids into epidermal ceramides and monoacylglycerols

Fatty acid transport protein 4 (FATP4) is an acyl-CoA synthetase that is required for normal permeability barrier in mammalian skin. FATP4 (SLC27A4) mutations cause ichthyosis prematurity syndrome, a nonlethal disorder. In contrast, Fatp4-/- mice die neonatally from a defective barrier. Here we used electron microscopy and lipidomics to characterize defects in Fatp4-/- mice. Mutants showed lamellar body, corneocyte lipid envelope, and cornified envelope abnormalities. Lipidomics identified two lipids previously speculated to be present in mouse epidermis, sphingosine β-hydroxyceramide and monoacylglycerol; mutants displayed decreased proportions of these and the two ceramide classes that carry ultralong-chain, amide-linked fatty acids (FAs) thought to be critical for barrier function, unbound ω-O-acylceramide and bound ω-hydroxyceramide, the latter constituting the major component of the corneocyte lipid envelope. Other abnormalities included elevated amounts of sphingosine α-hydroxyceramide, phytosphingosine non-hydroxyceramide, and 1-O-acylceramide. Acyl chain length alterations in ceramides also suggested roles for FATP4 in esterifying saturated non-hydroxy and β-hydroxy FAs with at least 25 carbons and saturated or unsaturated ω-hydroxy FAs with at least 30 carbons to CoA. Our lipidomic analysis is the most thorough such study of the Fatp4-/- mouse skin barrier to date, providing information about how FATP4 can contribute to barrier function by regulating fatty acyl moieties in various barrier lipids

Exosomes from Human Adipose Tissue-Derived Mesenchymal Stem Cells Promote Epidermal Barrier Repair by Inducing de Novo Synthesis of Ceramides in Atopic Dermatitis.

Atopic dermatitis (AD) is a multifactorial, heterogeneous disease associated with epidermal barrier disruption and intense systemic inflammation. Previously, we showed that exosomes derived from human adipose tissue-derived mesenchymal stem cells (ASC-exosomes) attenuate AD-like symptoms by reducing multiple inflammatory cytokine levels. Here, we investigated ASC-exosomes' effects on skin barrier restoration by analyzing protein and lipid contents. We found that subcutaneous injection of ASC-exosomes in an oxazolone-induced dermatitis model remarkably reduced trans-epidermal water loss, while enhancing stratum corneum (SC) hydration and markedly decreasing the levels of inflammatory cytokines such as IL-4, IL-5, IL-13, TNF-α, IFN-γ, IL-17, and TSLP, all in a dose-dependent manner. Interestingly, ASC-exosomes induced the production of ceramides and dihydroceramides. Electron microscopic analysis revealed enhanced epidermal lamellar bodies and formation of lamellar layer at the interface of the SC and stratum granulosum with ASC-exosomes treatment. Deep RNA sequencing analysis of skin lesions demonstrated that ASC-exosomes restores the expression of genes involved in skin barrier, lipid metabolism, cell cycle, and inflammatory response in the diseased area. Collectively, our results suggest that ASC-exosomes effectively restore epidermal barrier functions in AD by facilitating the de novo synthesis of ceramides, resulting in a promising cell-free therapeutic option for treating AD