Molecular Etiology of Atherogenesis – In Vitro Induction of Lipidosis in Macrophages with a New LDL Model

BACKGROUND: Atherosclerosis starts by lipid accumulation in the arterial intima and progresses into a chronic vascular inflammatory disease. A major atherogenic process is the formation of lipid-loaded macrophages in which a breakdown of the endolysomal pathway results in irreversible accumulation of cargo in the late endocytic compartments with a phenotype similar to several forms of lipidosis. Macrophages exposed to oxidized LDL exihibit this phenomenon in vitro and manifest an impaired degradation of internalized lipids and enhanced inflammatory stimulation. Identification of the specific chemical component(s) causing this phenotype has been elusive because of the chemical complexity of oxidized LDL. METHODOLOGY/PRINCIPAL FINDINGS: Lipid "core aldehydes" are formed in oxidized LDL and exist in atherosclerotic plaques. These aldehydes are slowly oxidized in situ and (much faster) by intracellular aldehyde oxidizing systems to cholesteryl hemiesters. We show that a single cholesteryl hemiester incorporated into native, non-oxidized LDL induces a lipidosis phenotype with subsequent cell death in macrophages. Internalization of the cholesteryl hemiester via the native LDL vehicle induced lipid accumulation in a time- and concentration-dependent manner in "frozen" endolysosomes. Quantitative shotgun lipidomics analysis showed that internalized lipid in cholesteryl hemiester-intoxicated cells remained largely unprocessed in those lipid-rich organelles. CONCLUSIONS/SIGNIFICANCE: The principle elucidated with the present cholesteryl hemiester-containing native-LDL model, extended to other molecular components of oxidized LDL, will help in defining the molecular etiology and etiological hierarchy of atherogenic agents

Chs increases the negative charge of Nat-LDL.

<p>(A) A brief description of the oxidation of cholesteryl linoleate to cholesteryl hemiesters; (B) Agarose gel electrophoresis of Nat-LDL, and derivatives. Lane 1, Nat-LDL; Lane 2, Ac-LDL; Lane 3, Chs-LDL (250∶1); Lane 4, Chs-LDL (500∶1); and Lane 5, Chs-LDL (1000∶1).</p

Addition of Chs-LDL to RAW cells induces the formation of large Oil Red-positive organelles.

<p>RAW cells were incubated for 48 h with Ac-LDL (A), Nat-LDL (B), Chs-LDL (1000∶1) (C), all at 400 µg LDL protein/ml; or Chs-POPC liposomes (45∶55) (D) at a particle density equivalent to that of Chs-LDL. After incubation the cells were fixed with PFA and stained with Oil-Red O and DAPI as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034822#s4" target="_blank">Methods</a>. Red stain, lipid organelles. Blue stain, nuclei. The images are projections of Z-stacks. Bars, 10 µm. Quantitative estimation of the total volume of lipid-rich structures per cell as a function of the Apo-B concentration (lower X-axis scale) or Chs (upper X-axis scale) concentrations after 24 h (E) and 48 h (F) incubations are also shown.</p

Fatty acid composition of the cholesteryl esters extracted from RAW Cells and, for purposes of comparison, the fatty acid composition of Nat-LDL.

<p>Quantitative lipidomic analysis of the intracellular cholesteryl esters in lipid extracts of cells incubated for 24 or 48 h with 300 µg/ml of Ac- or Chs-LDL. Only the major cholesteryl esters are represented in the table. Changes in the content of Cholesteryl oleate (18∶1) and Cholesteryl Linoleate (18∶2) are highlighted. Lipid extracts of native LDL were used as control. The cholesteryl esters distribution is shown as percentage of total cholesteryl esters. The results are the mean ± SD of three independent experiments obtained with three different LDL preparations.</p

Lipid composition of RAW cells subjected to various treatments.

<p>Polar lipids have been defined as the sum of phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, ceramides, diacylglycerides, free cholesterol and cholesteryl hemisuccinate. All values are expressed as a mol % of the total polar lipids. The apolar lipids (cholesteryl esters and triacylglycerides) are expressed as a mol % of the total polar lipid fraction. For purposes of comparison, the bottom row shows the lipid composition of Nat-LDL. Chs-LDL are Nat-LDL loaded with Chs with a Chs/LDL particle molar ratio of 1000∶1, Chs-POPC are POPC liposomes with a Chs/POPC molar ratio of 45∶55. The values in the columns entitled “Total Polar Lipids" is a reference point by definition and, therefore, always 100%. These values have only been included to show the standard deviation of the measurement since all the other values are given relative to the “Total Polar Lipid" content.</p

LAMP-2 decorates the neutral-lipid-rich vesicles in macrophages incubated with Chs-LDL.

<p>Macrophages incubated for 48 h with Ac-LDL (A-<b>A^IV</b>) or Chs-LDL (B-<b>B^IV</b>) were fixed and double labelled with anti-LAMP-2 antibodies and Bodipy. Merged images (panels (A-<b>A^I</b>) and (B-<b>B^I</b>)) show LAMP-2 in red and Bodipy in green. Panels (A<b>^I</b>) and (B<b>^I</b>) are the zoomed regions outlined by the rectangles in panels (A) and (B), respectively. Panels (A<b>^II</b>) and (B<b>^II</b>) show LAMP-2 staining. Panels (A<b>^III</b>) and (B<b>^III</b>) show lipid droplet staining. All panels show confocal single slice images. Asterisks point to LAMP-2-positive lipid droplets. Bars, 10 µm. The graphs (A<b>^IV</b>) and (B<b>^IV</b>) show relative intensity scans along the arrows in panels (A<b>^I</b>) and (B<b>^I</b>), respectively, of the LAMP-2 (red line) and Bodipy (green line) stains.</p

Chs is internalized mainly by passive diffusion.

<p>Panel (A) shows the incorporation of intracellular radioactivity as a function of time when RAW cells were exposed to Chs-LDL (×); Chs-(Ac-LDL) (□); and Chs-POPC liposomes (○), with similar ³H-Chs levels. After incubation the cells were acid washed and then scraped, and finally the radioactivity and protein were quantified as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034822#s4" target="_blank">Methods</a>. Panel (B) shows the effect of specifically blocking the scavenger receptors CD36, SRBI and CD204 by first exposing the RAW cells to specific inhibitory antibodies for 15 min on ice followed by raising the temperature to 37°C and exposing the cells to ³H-Chs-LDL, ³H-Chs-(Ac-LDL), both at 300 ug/ml Apo-B, or ³H-Chs-POPC liposomes. The Chs concentration and the ³H radioactivity was similar in all Chs-containing particles. Results are expressed as percentages relatively to the control cells (treated similarly but with non-inhibitory antibodies). After incubation with the Chs-containing particles the cells were processed as described above. Chs uptake results were expressed as percentages relative to the control. The results are the mean ± SD of three independent experiments. **, p<0.01; *, p<0.05. Panel (C-<b>C^II</b>) shows that RAW cells pre-incubated with Chs-POPC liposomes during 16 h and subsequently exposed to Nat-LDL accumulate neutral lipids in the lysosomal compartment. RAW cells were incubated with Chs-POPC liposomes during 16 h and then with Nat-LDL (300 µg/ml) for 48 h in absence of liposomes. The cells were then fixed and double labeled with Bodipy (C<b>^I</b>) for neutral lipid staining and anti-LAMP-2 antibody (C<b>^II</b>). The merged image (C) shows Bodipy staining in green and LAMP-2 staining in red. Arrows point to LAMP-2-positive cytosolic structures that contain neutral lipids. Bar, 10 µm. Panels (D–<b>G</b>) show that the lysosomal accumulation of neutral lipids induced by pre-treatment of RAW cells with Chs-LDL is irreversible. Raw cells were pulsed with 300 µg/ml of Ac-LDL (panel <b>D</b>) or Chs-LDL (panel <b>F</b>) for 48 h and then chased for 96 h. Cells pulsed with Ac-LDL (D) and then chased (E). Cells pulsed with Chs-LDL (F) and then chased (G). Lipid-rich structures visualized by Oil-red staining (red), DAPI staining (blue). All are merged images. Bars, 10 µm. The total volume of lipid structures per cell, quantified after the 96 h chase, is shown in panel (H). The results are the mean ± SD of three independent experiments. In every experiment 20 individual cells were analyzed. ***, p<0.0001; *, p<0.05; ns, not significant.</p

ADRP, a marker of lipid droplets, decorates the intracellular lipid structures induced by Chs-LDL.

<p>RAW cells were incubated for 48 h with Ac-LDL (A-<b>A^III</b>) or Chs-LDL (B-<b>B^III</b>). Panels (A) and (B) are confocal single-slice images and show the neutral lipids and the ADRP distribution. Neutral lipid-rich structures, in green, were stained with Bodipy 493/503. ADRP, in red, was visualized by immuno-staining with polyclonal antibodies as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034822#s4" target="_blank">Methods</a>. Panels (A) and (B) are merged images. In panels (A<b>^I</b>) and (B<b>^I</b>) the regions outlined with the rectangles in panels (A) and (B), respectively, are enlarged. Panels (A<b>^II</b>) and (B<b>^II</b>) show Bodipy staining 493/503, panels (A<b>^III</b>) and (B<b>^III</b>) show the distribution of ADRP. Bars, 10 µm.</p

Chs-LDL induces the formation of bilayer vesicles full of electron dense material.

<p>Transmission electron microscopy of cells treated for 24 h with Ac-LDL (A) or Chs-LDL (B). Cytoplasmic lipid droplets (organelles with a single monolayer) and vesicular structures with a bilayer and with electron dense material are visible in cells treated with Ac-LDL and Chs-LDL, respectively. The asterisk (*) indicates cytoplasmic lipid droplets in (A). Arrows point to bilayer vesicles with electron dense material inside in (B). Bars, 500 nm.</p