Kurzbeschreibung
(Englisch)
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Based on many epidemiological and experimental findings high-density lipoproteins (HDL) are considered as anti-atherogenic molecules and therefore attractive targets for prevention and therapy of coronary heart disease. Some of their diverse atheroprotective properties are exerted within the arterial wall so that HDL must leave the circulation. As yet it is little understood how HDL pass the endothelial barrier to reach this extravascular space. We have previously found by a series of in vitro experiments that cultivated aortic endothelial cells bind, internalize transcytose and resecrete HDL by specific processes that are modulated by the ATP binding cassette transporters ABCG1 and the scavenger receptor SR-BI. In this project we will address to questions: 1. Through which routes is HDL internalized and trafficked by endothelial cells? 2. Which steps in transendothelial HDL transport are modulated by ABCG1 and SR-BI? To answer these questions we will use both morphological and biochemical approaches. By using (immuno)fluorescence and (immuno)electron microscopy as well as sequential ultracentrifugation with subsequent ligand or immunoblotting we will characterize the organelles and and membrane fractions of endothelial cells which contain SR-BI and ABCG1 and by which HDL are endocytosed, trafficked and excreted. The functional relevance of canonical components of the various fractions will be tested by pharmacological inhibitors and/or RNA interference.
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Abstract
(Englisch)
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High density lipoproteins (HDL) and their main protein constituent and precursor, apolipoprotein A-I (apoA-I), exert several atheroprotective actions within the vascular wall, notably the removal of cholesterol from macrophage foam cells for reverse transport to the liver. However, it is little understood how HDL and apoA-I leave the plasma compartment and pass the endothelial barrier to reach macrophages in the arterial intima and how they re-enter the blood stream. We have previously found that aortic endothelial cells bind, internalize and resecrete apoA-I and HDL by processes that are modulated by the ATP binding cassette transporters ABCA1 and ABCG1, respectively, as well as the scavenger receptor BI (SR-BI), endothelial lipase (EL), and the ectopic F0F1-ATPase/P2Y12 axis. In this project we aim to characterize the trafficking of HDL within endothelial cells. Our project will answer a key question on the pathogenesis of atherosclerosis, namely how apoA-I and HDL enter the vascular wall. This process is a pre-requisite for the mediation of anti-atherogenic functions of HDL and therefore a potential target for anti-atherosclerotic drug therapy. Supported by the Swiss COST program, we established a broad scope of fluorescence and electron microscopic techniques to investigate uptake and trafficking of HDL by endothelial cells. These studies confirmed our previous notion that initially lipid-free apoA-I and mature HDL are internalized and trafficked through the same route and that uptake, transcellular transport, and excretion of HDL are very rapid. However we were not able to associate uptake with fluid uptake or classical endocytic pathways (clathrin coated pits, caveolae). Neither did we localize HDL or apoA-I in organelles or vesicles known to be involved in the intracellular trafficking or albumin, transferrin or LDL. This indirectly indicates that HDL is internalized and trafficked by endothelial cells through an as yet little investigated or unknown itinerary. We noticed that both apoA-I and HDL stimulate the uptake of HDL. Also sphingosine-1-phosphate (S1P), a quantitatively minor lipid carried by HDL, was found to enhance transendothelial transport. Vice versa HDL lacking the S1P binding protein apoM as well as pharmacological inhibition of S1P receptors and their downstream target PI3-kinase reduced the endothelial uptake of HDL. We therefore initiated screening experiments to identify signalling cascades that are activated or inhibited by the presence of HDL or apoA-I and whose pharmacological inhibition modulates the uptake of HDL into endothelial cells. In parallel we characterized the effect of endothelial ABCA1 knock-out on HDL metabolism and atherosclerosis in a mouse model. These studies provided first in vivo evidence that transendothelial as well as lymphatic
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