Ultrastructural appearance of EPCs after 30 minute of HDL-HRP internalization.

<p><b>A</b>) HDL-HRP particles can be seen at the cell surface (black arrow), the various sizes of HDL-HRP positive endosomal vesicles (E) are present beneath the cell membrane and close to the Golgi apparatus (G). <b>B</b>) HDL-positive and -negative MVBs (MVBs, asterisk) are visible. The white arrowhead indicates HDL-negative MVBs representing an early stage of lipid internalization; lipid droplets (Li) appear unstained. <b>C</b>) Lysosomes (L) shows intraluminal HDL-HRP particles. <b>D</b>) The RER is HDL-HRP negative while two invaginations of the plasma membrane (black arrowheads) show a strong reaction. (Scale bar: A, C = 1 µm; B, D = 0.5 µm).</p

Appearance of EPCs after 3 hour of HDL-HRP internalization.

<p><b>A</b>) The population of positively stained endosomal vesicles increase and could be observed throughout the cytoplasm. Positive MVBs (MVB) in various sizes and shapes with tubular membranous extensions as well as prominent Golgi stacks (G) are visible. <b>B</b>) This figure shows the appearance of a tubular endosome. <b>C</b>) Arrows point to an endosome alignment forming “strings of pearl-like structures” (arrowheads). <b>D</b>) A large secondary lysosome shows intraluminal HDL-HRP particles. <b>E</b>) Numerous autophagosomes (white thick arrows) with included endosomal vesicles are noted. (Scale bar: A, D, E = 1 µm; B, C = 0.5 µm).</p

Flow chart summarizing the intracellular traffic routes of HDL, HDL cholesterol and HDL cholesteryl oleate.

<p>Flow chart summarizing the intracellular traffic routes of HDL, HDL cholesterol and HDL cholesteryl oleate.</p

Micrographs of EPCs incubated with HDL labeld bodipy-cholesterol between 30 and 240 min.

<p><b>A</b>) Small cytoplasmic vesicles are stained (black thin arrows), the large 2 positive MVBs exhibit tightly-packed intraluminal microvesicles with reaction products. <b>B</b>), <b>C</b>) Reaction products are widely dispersed in all parts of the Golgi apparatus and the TGN. <b>D</b>) RER (white arrows) and mitochondria (M and in inset) are weakly stained. <b>E</b>) Demonstration of close association between unlabeled lipid droplets and RER. (Scale bar: A = 0.5 µm; B = 1 µm; C, D, D inset, E = 0.25 µm).</p

ET indicates HDL-containing endosomes.

<p><b>A</b>) shows a virtual slice, <b>B</b>) a corresponding model with endosomes (bright blue color) are accumulated in close proximity to the Golgi apparatus (orange color). (Scale bar: A, B = 200 µm).</p

Time course of HDL-Alexa Fluor® 568 internalization after interval from 30 to 240 min shows the fluorescence-marked structures in bright signals increasing in size and number of intracellular endocytic compartments.

<p>(Scale bar: A, B, C, D = 50 µm)</p

Ultrastructural detection of the internalization of EPCs with HDL-bodipy-cholesteryl oleate between 30 and 240 min.

<p><b>A</b>) MVBs at the cell periphery are filled with numerous tightly packed positively stained microvesicles; the membrane invagination are full filled with the reaction products also present on the other side of cell (inset). <b>B</b>), <b>D</b>) The TGN and stacked Golgi cisternae are positively stained. <b>C</b>) Mitochondria (M) and lipid droplet (Li) are labeled. <b>E</b>), <b>F</b>) A close association between labeled lipid droplets and RER could be demonstrated. (Scale bar: A = 1 µm; A inset = 0.5 µm; B, C, D, E, F = 0.25 µm).</p

Differentiation of PBMNCs derived EPCs after isolation and seeding onto FN-coated flasks with medium 199 shown in phase contrast.

<p><b>A</b>) PBMNCs, 30 min after seeding form many large and small clusters (C). <b>B</b>) They are able to differentiate into spindle cells 48 hours after culture (white arrows). <b>C</b>) Most of the attached cells become spindle shape on day 5 in culture; cell clusters (C). <b>D</b>) Spindle shaped morphology form tubular like structure (black arrows) after 7 days in culture. (Scale bar: A and B = 50 µm; C and D = 100 µm).</p

Micrographs of EPCs at 1 hour of incubation with HDL-HRP.

<p><b>A</b>) Numerous positive endosomal vesicles of varying sizes are found throughout cytoplasm, membrane invagination are indicated by arrowheads, positive MVBs are present. <b>B</b>) Positive MVBs with irregular shape and tubular appendices (white thin arrows), an endosome alignment (black thin arrows) as well as membrane invagination (black arrowhead) are demonstrated. <b>C</b>) Showing positively and negatively stained MVBs (asterisk). <b>D</b>) The large structures represent autophagosomes (white thick arrows) containing endosomes, are enclosed by a multilamellar membrane. <b>E</b>) The HDL-HRP particles are apparent in lysosomes (L, black thick arrow) (Scale bar: A, B, E = 1 µm; C, D = 0.5 µm).</p

Ultrastructural appearance of EPCs after 4 hour of incubation with HDL-HRP.

<p><b>A</b>) Autophagosomes (white thick arrows) with engulfed endosomal vesicles are noted. <b>B</b>) Positive MVBs with the characteristic tubular membranous extensions are visible. <b>C</b>) High magnification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083189#pone-0083189-g006" target="_blank">figure 6a</a>, shows a large spherical secondary lysosomes (L) close to Golgi apparatus (G). <b>D</b>) Demonstration of “strings of pearls-like structures”. (Scale bar: A = 1 µm; B, C, D = 0.5 µm).</p