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Acetoacetylated Lipoproteins Used to Distinguish Fibroblasts from Macrophages In Vitro by Fluorescence Microscopy

Acetoacetylated Lipoproteins Used to Distinguish Fibroblasts from Macrophages In Vitro by... We have developed a procedure (or labeling lipoproteins with the fluorescent probe 3,3-dioctadecylindocarbocyanlne (Dll) and have used Dil-labeled native and acetoacetylated lipoproteins to differentiate macrophages from fibroblasts In mixed cell culture. Lipoproteins labeled with this probe were suitable for the direct viewing of their binding and Internallzatlon by cells In vitro. The labeling technique has been applied to human low density lipoproteins (LDL) and to two canine cholesterolInduced lipoproteins: apo-E HDLC, which contain only the E apoprotein (apo-E), and beta-migrating, very low density lipoproteins (β-VLDL), which contain apo-B and apo-E. The Dll-labeled lipoproteins showed specific high affinity binding to human fibroblasts via the LDL (apo-B,-E) receptors. The equilibrium dissociation constant for the binding of Dll-labeled apo-E HDLCand LDL were the same as for the respective native lipoproteins. The specific binding of Dil-labeled LDL and apo-E HDLCwas further substantiated by fluorescence microscopy. When an excess of native (nonfluorescent) lipoproteins was added to the Dll-labeled llpoprotein, essentially no fluorescently labeled lipoproteins were seen associated with the cells. The Dil-labeled LDL, apo-E HDLC, and β9-VLDL, which were bound to the cells at 4° C, were associated with the cell surface and were often observed In linear arrays. Cells that were either incubated with Dil-labeled lipoproteins at 4°C and subsequently heated to 37° C or incubated with the Dil-labeled lipoproteins at 37° C showed Internalized perlnuclear fluorescence. When Dil-labeled LDL, apo-E HDLC, or β-VLDL were treated with dlketene to acetoacetylate their lysine residues, and then were incubated at 37° C with mixtures of fibroblasts and mouse peritoneal macrophages In culture, the fibroblasts did not become fluorescently labeled. The macrophages became highly fluorescent, however. The acetoacetylatlon Inhibited the interaction of these lipoproteins with the apo-B, −E receptors of fibroblasts and stimulated their uptake by macrophages. The use of fluorescently labeled native lipoproteins and chemically modified lipoproteins may allow the functional differentiation of macrophages from other cell types (e.g., fibroblasts and smooth muscle cells) In the arterial wall. This differentiation may be useful in determining the origin of the llpld-laden foam cells of atherosclerotic lesions. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Arteriosclerosis Wolters Kluwer Health

Acetoacetylated Lipoproteins Used to Distinguish Fibroblasts from Macrophages In Vitro by Fluorescence Microscopy

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Copyright
© 1981 by American Heart Association, Inc.
ISSN
0276-5047

Abstract

We have developed a procedure (or labeling lipoproteins with the fluorescent probe 3,3-dioctadecylindocarbocyanlne (Dll) and have used Dil-labeled native and acetoacetylated lipoproteins to differentiate macrophages from fibroblasts In mixed cell culture. Lipoproteins labeled with this probe were suitable for the direct viewing of their binding and Internallzatlon by cells In vitro. The labeling technique has been applied to human low density lipoproteins (LDL) and to two canine cholesterolInduced lipoproteins: apo-E HDLC, which contain only the E apoprotein (apo-E), and beta-migrating, very low density lipoproteins (β-VLDL), which contain apo-B and apo-E. The Dll-labeled lipoproteins showed specific high affinity binding to human fibroblasts via the LDL (apo-B,-E) receptors. The equilibrium dissociation constant for the binding of Dll-labeled apo-E HDLCand LDL were the same as for the respective native lipoproteins. The specific binding of Dil-labeled LDL and apo-E HDLCwas further substantiated by fluorescence microscopy. When an excess of native (nonfluorescent) lipoproteins was added to the Dll-labeled llpoprotein, essentially no fluorescently labeled lipoproteins were seen associated with the cells. The Dil-labeled LDL, apo-E HDLC, and β9-VLDL, which were bound to the cells at 4° C, were associated with the cell surface and were often observed In linear arrays. Cells that were either incubated with Dil-labeled lipoproteins at 4°C and subsequently heated to 37° C or incubated with the Dil-labeled lipoproteins at 37° C showed Internalized perlnuclear fluorescence. When Dil-labeled LDL, apo-E HDLC, or β-VLDL were treated with dlketene to acetoacetylate their lysine residues, and then were incubated at 37° C with mixtures of fibroblasts and mouse peritoneal macrophages In culture, the fibroblasts did not become fluorescently labeled. The macrophages became highly fluorescent, however. The acetoacetylatlon Inhibited the interaction of these lipoproteins with the apo-B, −E receptors of fibroblasts and stimulated their uptake by macrophages. The use of fluorescently labeled native lipoproteins and chemically modified lipoproteins may allow the functional differentiation of macrophages from other cell types (e.g., fibroblasts and smooth muscle cells) In the arterial wall. This differentiation may be useful in determining the origin of the llpld-laden foam cells of atherosclerotic lesions.

Journal

ArteriosclerosisWolters Kluwer Health

Published: May 1, 1981

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