Human endothelial progenitor cells internalize high density lipoprotein

In dieser Arbeit wurde eine Kombination von fluoreszenz- und elektronenmikroskopischen Methoden verwendet, um die Verteilung und intrazelluläre Lokalisation von High Density Lipoprotein (HDL), Cholesterol, und Cholesteryl Ester Cholesteryl-Oleat in menschlichen monozytären EPCs zu analysieren. Zytochemische Markierungen mit Meerrettich-Peroxidase (HRP) und Fluoreszenzkonjugationen mit BODIPY und Alexa Fluor® 568 wurden zum Nachweis dieser Substanzen eingesetzt. Aufnahme und intrazellulärer Transport dieser Stoffe wurden nach Internalisations-Perioden von 0,5 bis 4 Stunden nachgewiesen. Im Falle von HDL- Alexa Fluor® 568, Bodipy-Cholesterol und Bodipy-Cholesterol-Oleat wurde eine spezielle Photooxidations-Methode angewandt, um die aus Fluoreszenz- und Elektronenmikroskopie erhaltenen Ergebnisse hinsichtlich des Transports von HDL-assoziiertem Sterol vergleichen zu können. Es konnte klar gezeigt werden, dass bei allen Inkubationszeiten HDL-spezifische Reaktionsprodukte an Invaginationsstellen der Plasmamembran vorhanden waren. HDL-Partikel wurden internalisiert und fanden sich sowohl in endozytischen Vesikeln verschiedener Größe als auch in multivesikulären Körperchen (MVB) und bei längeren Inkubationsperioden auch in Lysosomen. Manche HDL-HRP enthaltende endozytische Kompartimente zeigten auffällige elektronendichte Anordnungen von Endosomen, die teilweise zu perlschnurartigen Gebilden fusionierten. Die MVBs wiesen zahlreiche dicht gepackte Mikrovesikel mit begrenzender Membranen auf, die mit elektronendichtem Reaktionsprodukt gefüllt waren sowie positiv-markierte tubuläre Anhänge. MVBs von HDL-Alexa Fluor® 568 behandelten EPCs zeigten intravacuolär multilamelläre Membranen, die, wenn sie größer waren, bei lockerer Anordnung deutlich hervortraten. Bei allen Inkubationszeiten waren in der Nähe der Zellmembran sowie perinukleär, im Bereich des Golgi-Apparates konzentriert, positiv gefärbte endozytische Kompartimente sichtbar, die HDL-Partikel enthielten. Bei Verwendung der Elektronentomographie konnten Ansammlungen von HDL-Partikel enthaltenden endozytischen Kompartimenten in unmittelbarer Nachbarschaft zum Trans-Golgi-Netzwerk (TGN) nachgewiesen werden. Bodipy-Cholesterol enthaltendes HDL waren in Endosomen, MVBs, Mitochondrien, vereinzelt im RER jedoch verbreitet innerhalb der gestapelten Golgi-Zisternen und im TGN vorhanden. Auch Lysosomen waren involviert. Entlang der endozytischen Transportwege war Bodipy-Cholesteryl-Oleat enthaltendes HDL erkennbar, das vor allem im Bereich des Golgi-Apparates, von Lipidtröpfchen, im RER aber auch in Mitochondrien akkumulierte. Schließlich konnten in Lysosomen Abbauprozesse von Bodipy-Cholesteryl-Oleat enthaltendem HDL nachgewiesen werden. Diese Untersuchungen zeigten,dass HDL-gebundenes Cholesterol, Bodipy-Cholesterol und Bodipy-Cholesteryl-Oleat in monozytären EPCs unterschiedliche Transportwege beschreiten, wobei HDL in Lysosomen abgebaut wird. Untersuchungen unter Verwendung von HRP-markiertem HDL, das mit Diaminobenzidin-Peroxidation nachgewiesen wurde, sowie von fluoreszenzmikroskopischen und fortgeschrittenen elektronenmikroskopischen Techniken, wie der Elektronentomographie, sollen zu einem besseren Verständnis der intrazellulären Transportrouten von HDL in EPCs beitragen.In this work, a combination of fluorescence and electron microscopical methods has been used to investigate the distribution and intracellular localization of internalized human serum high density lipoprotein (HDL), cholesterol, and the cholesteryl ester cholesteryl-oleate in human monocyte-derived endothelial progenitor cells (EPCs). Cytochemical labeling with horseradish peroxidase (HRP) and fluorochrome conjugation with BODIPY and Alexa Fluor® 568 were used for demonstration of these substances. Cellular uptake and intracellular trafficking of HDL-derived cholesterol were analyzed by fluorescence and electron microscopy after internalization times ranging from 0.5 to 4 hours. HRP labeling was visualized by diaminobenzidine (DAB) peroxidation. In case of Alexa Fluor® 568, BODIPY-cholesterol and BODIPY-cholesteryl oleate, a DAB photoconversion method was applied to correlate results from fluorescence and transmission electron microscopy concerning the intracellular trafficking of HDL-associated sterol. It could be shown that HDL-specific reaction products were clearly present at invagination sites of the plasma membrane at each incubation time. HDL particles were internalized and localized in endocytic vesicles of different size and in endosomal multivesicular bodies (MVBs) as well as with longer incubation periods also in lysosomes. Some HDL-HRP containing endocytic compartments showed conspicuous electron-dense vesicular endosomal alignments which partially fused, forming "strings of pearl"-like structures. MVBs contained numerous tightly packed microvesicles with limiting membranes filled with electron-dense reaction product and showing labeled tubular appendices. MVBs of HDL-Alexa Fluor® 568-treated EPCs exhibited multilamellar intra-vacuolar membranes appearing more distinct as they became larger and more loosely arranged. At all incubation periods of uptake, positively stained endocytic compartments and organelles were apparent in regions beneath the cell membrane, concentrated in perinuclear position around the Golgi apparatus. Electron tomographic reconstructions showed the accumulation of endocytic compartments containing HDL particles close to the trans-Golgi-network (TGN). Bodipy-cholesterol-containing HDL was localized in endosomes, MVBs, mitochondria, rarely in RER and widely distributed throughout the stacked Golgi cisternae and the TGN. Lysosomes were also involved. Internalized HDL-derived BODIPY-cholesteryl oleate was found along the endocytic pathway, accumulating prominently in all parts of the Golgi apparatus, in lipid droplets, in RER as well as in mitochondria. Subsequently, HDL degradation occurred in lysosomes. These studies demonstrated that HDL-derived cholesterol, BODIPY-cholesterol and BODIPY-cholesteryl oleate are transported in human monocyte-derived EPCs by different intracellular pathways and that HDL is degraded in lysosomes. Introduction of HRP-labeled HDL, demonstrated with the DAB peroxidation technique as well as investigations using fluorescence microscopy combined with advanced electron microscopic techniques such as electron tomography should contribute to a better understanding the intracellular traffic routes of HDL through EPCs.submitted by Kaemisa SrisenAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. Sprachehttp://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083189Wien, Med. Univ., Diss., 2014OeBB(VLID)171469

Human endothelial progenitor cells internalize high-density lipoprotein.

Endothelial progenitor cells (EPCs) originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL), and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate), cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal intraellular pathway and accumulated prominently in all parts of the Golgi apparatus and in lipid droplets. Subsequently, also the RER and mitochondria were involved. These studies demonstrated the different intracellular pathway of HDL-derived bodipy-cholesterol and HDL-derived bodipy-cholesteryl oleate by EPCs, with concomitant

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

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 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

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

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