Physiological relevance of HDL uptake and resecretion

Cholesterol transport via the high density lipoprotein (HDL) is an essential regulatory mechanism to remove excess cholesterol from peripheral tissue and to deliver it back to the liver for disposal. In contrast to the low density lipoprotein (LDL) receptor pathway, in which the entire LDL particle is degraded by the cell, HDL delivers only its core lipids to the cell. The scavenger receptor class B, type I (SR-BI) was described to mediate the selective uptake of cholesteryl ester from HDL. Thus, SR-BI is the "HDL-receptor" involved in the reverse cholesterol transport and thereby exerting its anti-atherogenic role. Furthermore, SR-BI is also described to mediate cholesterol efflux. In the present project we propose to study the role of SR-BI in holo-HDL particle uptake and resecretion and its physiological relevance. 3-D Electron Microscopy and fluorescence imaging techniques will be applied to follow the binding of individual HDL particles and their subsequent internalization. Based on our novel findings, we will characterize the mechanism leading to the uptake of the holo-HDL particle. Such a characterization has to comprise the subcellular location of its transport routes. This will be performed by dual color imaging of individual HDL particles within the cell in correlation with intracellular markers and by using fluorescence analysis. The exact localization will be inferred from electron microscopy studies. The compartments involved in intracellular HDL-traffic and their relationships to defined membranes and organelles will be analyzed by using electron tomography, and details will be three dimensionally reconstructed. The possible point(s) where the HDL particle transfers its cholesterol during resecretion will be identified by using cholesterol surrogates like NBP-cholesterol. We will extend our studies by using polarized liver cells, WifB9 cells, to assess the role of HDL particle uptake in hepatic cholesterol homeostasis. Finally, fibroblast cell lines defective in a part of the cholesterol metabolism like Tangier disease will help to clarify the role of HDL retroendocytosis for cholesterol efflux. Influence of the proposed work on the development of the field This project will help to merge two distinct scientific fields in life science: lipidology and cell biology. The combined application of potent novel methodologies - fluorescence microscopy, high pressure freezing and 3D- electron microscopy, single organelle fluorescence analysis - will allow us to directly image and characterize in detail each step in the uptake and resecretion pathways of HDL and the purpose of its uptake. The physiological relevance of HDL resecretion for cholesterol efflux will be assessed.

Cholesterol transport via the high density lipoprotein (HDL) is an essential regulatory mechanism to remove excess cholesterol from peripheral tissue and to deliver it back to the liver for disposal. In contrast to the low density lipoprotein (LDL) receptor pathway, in which the entire LDL particle is degraded by the cell, HDL delivers only its core lipids to the cell. The scavenger receptor class B, type I (SR-BI) was described to mediate the selective uptake of cholesteryl ester from HDL. Thus, SR-BI is the "HDL-receptor" involved in the reverse cholesterol transport and thereby exerting its anti-atherogenic role. Furthermore, SR-BI is also described to mediate cholesterol efflux. In the present project we propose to study the role of SR-BI in holo-HDL particle uptake and resecretion and its physiological relevance. 3-D Electron Microscopy and fluorescence imaging techniques will be applied to follow the binding of individual HDL particles and their subsequent internalization. Based on our novel findings, we will characterize the mechanism leading to the uptake of the holo-HDL particle. Such a characterization has to comprise the subcellular location of its transport routes. This will be performed by dual color imaging of individual HDL particles within the cell in correlation with intracellular markers and by using fluorescence analysis. The exact localization will be inferred from electron microscopy studies. The compartments involved in intracellular HDL- traffic and their relationships to defined membranes and organelles will be analyzed by using electron tomography, and details will be three dimensionally reconstructed. The possible point(s) where the HDL particle transfers its cholesterol during resecretion will be identified by using cholesterol surrogates like NBP-cholesterol. We will extend our studies by using polarized liver cells, WifB9 cells, to assess the role of HDL particle uptake in hepatic cholesterol homeostasis. Finally, fibroblast cell lines defective in a part of the cholesterol metabolism like Tangier disease will help to clarify the role of HDL retroendocytosis for cholesterol efflux. Influence of the proposed work on the development of the field This project will help to merge two distinct scientific fields in life science: lipidology and cell biology. The combined application of potent novel methodologies - fluorescence microscopy, high pressure freezing and 3D- electron microscopy, single organelle fluorescence analysis - will allow us to directly image and characterize in detail each step in the uptake and resecretion pathways of HDL and the purpose of its uptake. The physiological relevance of HDL resecretion for cholesterol efflux will be assessed.