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

FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42

Abstract The Rho-family small GTPase Cdc42 localizes at plasma membrane and Golgi complex and aside from protrusion and migration operates in vesicle trafficking, endo- and exocytosis as well as establishment and/or maintenance of cell polarity. The formin family members FMNL2 and -3 are actin assembly factors established to regulate cell edge protrusion during migration and invasion. Here we report these formins to additionally accumulate and function at the Golgi apparatus. As opposed to lamellipodia, Golgi targeting of these proteins required both their N-terminal myristoylation and the interaction with Cdc42. Moreover, Golgi association of FMNL2 or -3 induced a phalloidin-detectable actin meshwork around the Golgi. Importantly, functional interference with FMNL2/3 formins by RNAi or CRISPR/Cas9-mediated gene deletion invariably induced Golgi fragmentation in different cell lines. Furthermore, absence of these proteins led to enlargement of endosomes as well as defective maturation and/or sorting into late endosomes and lysosomes. In line with Cdc42 - recently established to regulate anterograde transport through the Golgi by cargo sorting and carrier formation - FMNL2/3 depletion also affected anterograde trafficking of VSV-G from the Golgi to the plasma membrane. Our data thus link FMNL2/3 formins to actin assembly-dependent functions of Cdc42 in anterograde transport through the Golgi apparatus

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

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