Experiments are proposed to study intracellular processing of lipoproteins by monocyte-derived macrophages cultured from White Carneau pigeons. The hypothesis is that modified LDLs and high capacity low affinity binding of betaVLDL stimulates macropinocytosis. Uptake via the stimulated macropinocytosis rather than internalization by receptor-mediated endocytosis is associated with foam cell formation. Modified LDL stimulated macropinocytosis also causes uptake of co-internalized lipoproteins via fluid-phase, or high capacity, low-affinity binding, or nonspecific binding to the membrane resulting in foam cell formation. These studies will be done by a combination of ultrastructural and biochemical approaches. Ultrastructurally receptor-mediated coated-pit endocytosis will be differentiated from macropinocytosis by localization of colloidal gold conjugated ligands in pits and vesicles (diameter less than or equal to 0.05) and endosomes (diameter less than or equal to 0.2microm) for receptor-mediated endocytosis versus entry into macropinosomes (diameter greater than or equal to 0.5 microm under membrane ruffled surface) for macropinocytosis. Phase-contrast light microscopy will identify the stimulation of membrane ruffling and phase-bright macropinosomes (greater than or equal to 0.5microm). Fluorescent microscopy with co-localization with fluid-phase markers or endocytic markers will be used to study trafficking of lipoproteins in either macropinosomes or endosomes, respectively. Stimulation of the fluid-phase uptake will be measured with 14C-sucrose, lucifer yellow (LY), horseradish peroxidase (HRP), and dextrans Additionally macropinocytosis and endocytosis will be functionally differentiated by wortmannin, nocodazole, hypertonic medium, and cytoplasmic acidification. The mechanism of stimulated uptake and degradation of LDL in the presence of modified LDLs will be examined using the biochemical parameters of binding, degradation, acyl-CoA; cholesterol acyltransferase (ACAT) stimulation and cholesterol loading. The role of receptors and non-specific binding will be determined by competition studies. The proposed studies will provide potential mechanism(s) for native LDL to cause in vitro foam cell formation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL041990-07A2
Application #
2903239
Study Section
Pathology A Study Section (PTHA)
Project Start
1988-12-01
Project End
2003-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
7
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Pathology
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
Zhao, Bin; Li, Yifu; Buono, Chiara et al. (2006) Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF). J Biol Chem 281:15757-62
Kruth, Howard S; Jones, Nancy L; Huang, Wei et al. (2005) Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein. J Biol Chem 280:2352-60
Jones, N L; Saunders, J A; Mallory, R R (2000) Intracellular trafficking of pigeon beta-very low density lipoprotein and low density lipoprotein at low and high concentrations in pigeon macrophages. J Lipid Res 41:1823-31
Jones, N L; Reagan, J W; Willingham, M C (2000) The pathogenesis of foam cell formation: modified LDL stimulates uptake of co-incubated LDL via macropinocytosis. Arterioscler Thromb Vasc Biol 20:773-81
Jones, N L; Willingham, M C (1999) Modified LDLs are internalized by macrophages in part via macropinocytosis. Anat Rec 255:57-68
Jones, N L; Allen, N S; Willingham, M C et al. (1999) Modified LDLs induce and bind to membrane ruffles on macrophages. Anat Rec 255:44-56
Jones, N L (1997) Simultaneous labeling of lipoprotein intracellular trafficking in pigeon monocyte-derived macrophages. Am J Pathol 150:1113-24
Zha, X; Tabas, I; Leopold, P L et al. (1997) Evidence for prolonged cell-surface contact of acetyl-LDL before entry into macrophages. Arterioscler Thromb Vasc Biol 17:1421-31
Jones, N L; Gupta, M; Lewis, J C (1995) The LDL receptor and LRP are receptors for beta VLDL on pigeon monocyte-derived macrophages. Virchows Arch 426:189-98
Lewis, J C; Jones, N L; Hermanns, M I et al. (1995) Tissue factor expression during coculture of endothelial cells and monocytes. Exp Mol Pathol 62:207-18

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