Atherosclerosis is a leading cause of death worldwide and is characterized by accumulation of lipids and lipoproteins in the arterial vessel wall. Low density lipoproteins (LDL) as well as high density lipoproteins (HDL) have to pass the endothelial layer, which is the inner cell layer of blood vessels to exert their pro- and anti-atherogenic activities respectively. The best documented anti-atherogenic effect of HDLs is their capacity to remove excess cholesterol from the peripheral tissues and transport it back to the liver for its subsequent elimination in bile. This process is called Reverse cholesterol transport (RCT). The first step of RCT is transfer of cholesterol from lipid-laden macrophages (foam cells) to HDLs. However, in order to reach the foam cells within the arterial wall, circulating HDLs have to cross the endothelial layer via transcellular route (transcytosis). Our laboratory identified ATP binding cassette transporters A1 and G1, scavenger receptor B1, endothelial lipase and ectopic beta-ATPase/P2Y as rate limiting factors. However, they are docking receptors rather than endocytic receptors. Therefore, we are now focused on identifying the endocytic receptor as well as signaling events regulating the internalization of HDL into endothelial cells. We performed a high content screening (HCS) that allows multi-parametric analysis of biological features in single cells following treatment with drugs inhibiting kinases which are critical in cell signaling, metabolism, cellular transport and many other cellular pathways. The sublime expertise of our lab supported to breeze-through all phases of HCS workflow: assay development and validation, lab automation and screening, automated microscopy, and data analysis. Widefield-fluorescence microscopy helped to track the location and intensity of fluorescent labelled HDL in drug-treated human aortic endothelial cells (HAECs). This screening identified a signaling cascade that we are currently validating. Based on our preliminary data, we are confident that our project addresses a key question in the pathogenesis of atherosclerosis namely how HDL enters the vascular wall.