ARTICLE |
Correspondence to: Christine K. Abrass, Veteran Affairs Puget Sound Health Care System, 1660 S. Columbian Way, Seattle, WA 98108. E-mail: cabrass@u.washington.edu
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Summary |
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When rat glomerular mesangial cells (MCs) are cultured with IGF-1 they accumulate intracellular lipid and take on foam cell morphology. These changes were characterized by electron microscopy and Nile red staining. To define the mechanism responsible for IGF-1-mediated lipid uptake, MCs were evaluated for endocytosis, scavenger receptor activity, and receptor-mediated uptake by the LDL receptor. Lipid accumulation was markedly increased when MCs were cultured with IGF. The primary route of uptake was through enhanced endocytosis. Lipid-laden MCs have decreased phagocytic capacity and disrupted cytoskeletons. These data show that IGF-1 induces MC to take on a foam cell morphology and that lipid-laden MCs have impaired phagocytic function. (J Histochem Cytochem 50:395403, 2002)
Key Words: mesangial cell, IGF-1, atherosclerosis, lipids, glomerulosclerosis, phagocytosis
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Introduction |
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LIPID ACCUMULATION in the vessel wall is considered to be an early step in atherosclerosis. Lipoproteins bind to matrix proteins in vessel walls, where they become oxidized (
In the glomerulus, the mesangial cell (MC) functions as a pericyte with properties of a vascular smooth muscle cell. Like the smooth muscle cell, it similarly expresses LDL and scavenger receptors (
Considerable work has focused on the details of LDL and scavenger receptor biology (
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Materials and Methods |
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Materials
The following reagents were purchased: phagocytosis assay kit, FITC-dextran (10,000 MW), Nile red, Bodipy FL cholesteryl ester, Bodipy phalloidin, Texas Red phalloidin and Bodipy acetylated low-density lipoprotein (AcLDL) (Molecular Probes; Eugene, OR); trypan blue (Sigma; St Louis, MO); chambered tissue culture glass slides (NUNC; Naperville, IL); Fluoromount (Fisher Scientific; Pittsburgh, PA); poly/bed 812 resin (Ted Pella; Redding, CA); human recombinant IGF-1 (Collaborative Research; Waltham, MA).
Experimental Design
MCs were cultured in routine medium (20% FCSRPMI) with and without IGF-1 (100 nM) for 7 days and in growth arrest medium (2% FCSRPMI) for 24 hr. To confirm that IGF-1-treated MCs accumulate lipid, MCs were examined by routine electron microscopy and stained with Nile red. To examine the mechanisms whereby IGF-1 stimulates lipid uptake, particle uptake by fluid-phase pinocytosis, scavenger receptors, and LDL receptors were measured as described below. To examine the impact of intracellular lipid accumulation on function, MCs were tested for their ability to phagocytose particles and to reorganize their cytoskeleton.
Cell Culture
Rat glomerular MCs were prepared by modification (
Lipid Accumulation and Lipid Uptake in MCs
MCs (2 x 104/ml) were plated in glass chamber slides, grown for 7 days in 20% FCSRPMI medium with or without IGF-1 (100 nM), then placed in 2% FCSRPMI for 24 hr. For measurement of accumulated neutral lipids, 2.5 µg Nile red in glycerol was added to MC cultures for 5 min and processed for fluorescence microscopy. Other slides were incubated for 4 hr with 20 µg/ml FITCdextran to measure fluid-phase endocytosis, with fluoresceinated acetylated LDL for scavenger receptor function and fluoresceinated Bodipy cholesteryl esters for the LDL receptor-mediated and other nonspecific endocytosis. Trypan blue was used to quench particles that were not endocytosed. Each condition was measured in triplicate.
Phagocytosis
MC phagocytosis was measured using the phagocytosis assay kit in untreated cells and those grown in IGF-1 (100 nM). Phagocytosis was determined after 1 hr by uptake of fluoresceinated E. coli. Complete internalization was confirmed by quenching with trypan blue and slides were processed for fluorescence microscopy. Other slides were counterstained with Texas red phalloidin and processed for scanning confocal microscopy.
Cytoskeletal Organization
F-actin organization was determined by Bodipy phalloidin staining on permeablized MCs and examined by fluorescence microscopy.
Fluorescence Microscopy
Chamber slides of various experiments were rinsed in PBS and fixed in 2% paraformaldehye for 20 min. For Bodipy or Texas red phalloidin staining, MCs were permeabilized with 0.05% Triton X-100 for 3 min, rinsed in PBS, and incubated with primary antibody for 20 min at RT. MCs were mounted in Fluoromount and observed with a Leitz microscope equipped for epi-illumination. The fluorescent images were recorded on Kodak DX 400 black-and-white film or on Ektachrome film.
Confocal Microscopy
Phagocytosis slides counterstained with Texas red phalloidin were scanned on a Leica TCS-SP confocal microscope using absorptions of 596 nM and 488 nM. Images were collected in sequential xz-series and yz-series, as well as overlay images. Acquired images were exported to Adobe Photoshop for further processing.
Electron Microscopy
MCs were rinsed with PBS, fixed in 2% gluteraldehyde for 2 hr at 4C, treated with 1% osmium tetroxide for 30 min at RT, and washed three times with cacodylate buffer. They were sequentially dehydrated through ascending concentrations of alcohol (35100%) to resin and polymerized. A Sorvall MT6000 ultramicrotome was used for sectioning and specimens were examined on a JEOL TEM S100.
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Results |
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IGF-1-induced Foam Cell Formation
Compared to MCs cultured for 7 days in growth medium alone, cells supplemented with IGF-1 (100 nM) displayed morphological changes consistent with a foam cell. These include a convoluted nucleus and an expansive cytoplasm with extended Golgi and endoplasmic reticulum. The cytoplasm contained multiple lipid- and myelin-filled vacuoles and distorted fat-laden mitochondria (Fig 1).
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To confirm that MCs with foam cell morphology contain increased amounts of lipid, untreated MCs or those treated with IGF-1 were stained with Nile red. As shown in Fig 2, IGF-1-treated MCs had increased neutral lipid content compared to untreated cells. FCS contains 200 mg/dl of total lipid, including cholesterol, HDL, LDL, triglycerides, and phospholipids. Therefore, medium containing 20% FCS has 40 mg/dl of lipid. Intracellular lipid accumulation may result from uptake from the medium and/or intracellular synthesis.
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IGF-1 Stimulation of Lipid Uptake by MCs
Having shown that IGF-1 treatment of MCs leads to lipid accumulation in MCs, we analyzed IGF-1-induced uptake of various test substances to determine the pathways involved. As shown in Fig 3, FITCdextran was scarcely seen within untreated MCs even after 7 days in growth medium (Fig 3A), whereas uptake was increased in IGF-1-treated cells in regular growth media (Fig 3B). This is consistent with the known effect of IGF-1 in increasing membrane internalization, turnover, and rates of general endocytosis (
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Within an hour, small amounts of labeled AcLDL were taken up by both untreated and IGF-1-treated MCs (data not shown). AcLDL initially taken up by untreated MCs is no longer present by 7 days (Fig 3C). However, IGF-1-treated MCs show slightly increased intracellular accumulation of AcLDL (Fig 3D). These results suggest that IGF-1 has no direct effect on scavenger receptor-mediated uptake of AcLDL. However, IGF-1 may affect scavenger receptors after chronic exposure or may influence the turnover of intracellular lipid such that oxidized LDL accumulates within the cell.
To investigate lipid uptake by receptor-mediated endocytosis in clathrin-coated pits, where the LDL receptor is located, as well as "selected transport of lipoprotein" (
Impact of Lipid Accumulation on Phagocytosis
Phagocytosis is the process by which cells recognize and engulf large particles. MCs are known to take up large particles, including immune complexes (
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Cytoskeletal Organization
MC phagocytosis, migration, and contraction all require an intact cytoskeleton that can rapidly reorganize in response to a variety of stimuli (
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Discussion |
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In this study we show that long-term exposure of MCs to IGF-1 induces foam cell formation, as indicated by ultrastructural characteristics and accumulation of neutral lipids. This occurs in routine medium with relatively low concentrations of lipid. Studies of particle uptake indicate that a primary effect of IGF-1 is to enhance endocytosis. Although this mechanism can lead to generalized particle uptake from the medium, lipids accumulated to a marked degree. This suggests that lipid disposal mechanisms may be impaired or may become overloaded. Once MCs become lipid-laden and assume the appearance of foam cells, they are no longer able to phagocytose particles. Mesangial foam cells have a disrupted cytoskeleton, which may in part be responsible for impaired cell function because an intact cytoskeleton that can be rapidly reorganized is required for phagocytosis.
IGF-1 and the Mechanisms of Intracellular Lipid Accumulation
IGF-1 stimulated MCs uptake and accumulation of lipid by several mechanisms. Using FITC-labeled dextran, we confirmed that IGF-1 increased fluid-phase endocytosis via caveolae or non-coated vesicle transport. Although this pathway was commonly believed to be a method of internalization only of large macromolecules, such as modified albumin and fluid, many molecules are endocytosed via caveolae. Lipids such as cholesterol, gangliosides, and fatty acids are often concentrated in caveolae (
MCs express scavenger receptors and take up modified LDL by this pathway (
Long-term treatment of MCs with IGF-1 increased cholesteryl ester uptake compared to control MCs. Bodipy-labeled cholesteryl ester has classically been used to measure LDL receptor binding, which suggests that IGF-1 modulates LDL receptor expression. Recent data suggest that uptake of labeled cholesteryl ester may also define a "selective" pathway of vesicles or intracellular membrane sheets by which lipids are imported into the cell (
Altered Phagocytosis and Cytoskeletal Reorganization
MCs are known to phagocytose both particulate matter and immune complexes (
Foam Cells and Renal Disease
Information supporting a role for lipids and progressive renal disease is emerging. Lipid deposits can be found in the glomeruli and renal interstitium of patients with focal and glomerulosclerosis, diabetic nephropathy, membranoproliferative glomerulonephritis, and hemolytic uremic syndrome (
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Acknowledgments |
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Supported by the Medical Research Service of the Department of Veterans Affairs.
Received for publication October 17, 2001; accepted October 24, 2001.
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