Abstract
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.
Highlights
Endothelial Progenitor Cells (EPCs) represent a population of stem cell circulating in small fractions in human peripheral blood with the ability to proliferate, migrate and differentiate into mature endothelial cells (ECs)
These EPCs can be identified by their uptake of Dil (3,3’ dioctadecylindocarbocyanine)-labeled acetylated LDL (DIL-ac-LDL) and by cell surface staining with Ulex europaeus agglutinin 1 (UEA-1)
The cells were removed from the flasks using trypsin-EDTA (PAA Laboratories, Pasching, Austria) seeded on FN-coated round glass coverslips and plated at the bottom of 8-well plates (Merck, Darmstadt, Germany), maintained in medium 199 supplemented with 20% fetal calf serum (FCS), 2 mM L-glutamine and antibiotics. 1 day before starting the experiment, cells were washed twice with phosphate buffered saline (PBS) and fed with medium 199 containing 20% calf lipoprotein deficient serum
Summary
Endothelial Progenitor Cells (EPCs) represent a population of stem cell circulating in small fractions in human peripheral blood with the ability to proliferate, migrate and differentiate into mature endothelial cells (ECs). Asahara et al [1] published the first detailed description of an isolation method for putative EPCs from human peripheral blood This unique cell fraction among peripheral blood mononuclear cells (PBMNCs) derived from bone marrow was shown to be incorporated into ischemic vessels that provided tissue recovery and improvement. Since this discovery, the studies on EPCs have increasingly initiated interest of scientists working in the field of vascular biology, focused on atherosclerosis and cardiovascular diseases. After two days new clusters containing EPCs appeared which were plated again to evaluate and quantify the emergence of the colony–forming EPCs designated as CFU-Hill and characterized by a central core of ‘‘round’’ cells, with rather elongated ‘‘sprouting’’ cells at the periphery with endothelial-like morphology. The number of colonies correlated negatively with the Framingham cardiovascular risk score and positively with sufficient vascular function
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