Abstract 565: Vascular Endothelial Growth Factor - An Important Factor in HDL Binding and Transcytosis Through Aortic Endothelial Cells

Abstract

In the reverse cholesterol transport pathway cholesterol acceptors like high density lipoproteins (HDL) and apolipoprotein A-I (apoA-I), must cross the endothelium to get access to the donor cells in the arterial initima. However, it is unknown how they get translocated from the blood stream into the vascular wall. Previously, we showed that transendothelial HDL transport is modulated by scavenger receptor B I (SR-BI), ATP binding cassette transporter G1, endothelial lipase (EL), and the ectopic beta-ATPase/purinergic receptor axis. None of these proteins is a good candidate to directly mediate holoparticle uptake. They appear rather to indirectly modulate transendothelial transport of HDL by signalling or altering the structure of HDL. To unravel HDL transcytosis, a high-throughput screening was performed, incubating aortic endothelial cells (ECs) with kinase inhibiting drugs and fluorescent labelled HDL. Fixed cells were imaged using fluorescence wide-field microscope. Processing of microscopy acquired data using cell profiler and principle component analysis yielded, only inhibitors of the vascular endothelial growth factor receptor (VEGFR) and its down-stream signaling kinases. To verify and further characterize the screening hits towards their impact on binding, internalization and transport of 125I-HDL, we applied RNA interference and pharmacological means to ECs. Binding, internalization and transport of 125I-HDL through ECs were increased by VEGF treatment but decreased by inhibitors of VEGFR2 as well as knock-down of VEGFR2. RNA interference with VEGFR1 or VEGFR3 had no effect. The stimulatory effect of VEGF on 125I-HDL binding and internalization was decreased by knocking-down SR-BI or PDZK1. The identification of VEGF as a regulatory factor of transendothelial transport supports the concept that the endothelium is a specific and hence druggable barrier for the entry of HDL into the vascular wall.