Srividya Velagapudi1, Mustafa Yalcinkaya1, Antonio Piemontese1, Roger Meier1, Simon Flyvbjerg Nørrelykke1, Damir Perisa1, Andrzej Rzepiela1, Michael Stebler1, Szymon Stoma1, Paolo Zanoni1, Lucia Rohrer2, Arnold von Eckardstein2. 1. From the Institute of Clinical Chemistry, University and University Hospital of Zurich, Schlieren, Switzerland (S.V., M.Y., A.P., D.P., P.Z., L.R., A.v.E.); Competence Center for Integrated Human Physiology, University of Zurich, Switzerland (S.V., M.Y., D.P., P.Z., L.R., A.v.E.); Department of Pharmacy, University of Parma, Italy (A.P.); and Scientific Center for Optical and Electron Microscopy, ETH Zurich, Switzerland (R.M., S.F.N., A.R., M.S., S.S.). 2. From the Institute of Clinical Chemistry, University and University Hospital of Zurich, Schlieren, Switzerland (S.V., M.Y., A.P., D.P., P.Z., L.R., A.v.E.); Competence Center for Integrated Human Physiology, University of Zurich, Switzerland (S.V., M.Y., D.P., P.Z., L.R., A.v.E.); Department of Pharmacy, University of Parma, Italy (A.P.); and Scientific Center for Optical and Electron Microscopy, ETH Zurich, Switzerland (R.M., S.F.N., A.R., M.S., S.S.). arnold.voneckardstein@usz.ch lucia.rohrer@usz.ch.
Abstract
OBJECTIVE: Low- and high-density lipoproteins (LDL and HDL) must pass the endothelial layer to exert pro- and antiatherogenic activities, respectively, within the vascular wall. However, the rate-limiting factors that mediate transendothelial transport of lipoproteins are yet little known. Therefore, we performed a high-throughput screen with kinase drug inhibitors to identify modulators of transendothelial LDL and HDL transport. APPROACH AND RESULTS: Microscopy-based high-content screening was performed by incubating human aortic endothelial cells with 141 kinase-inhibiting drugs and fluorescent-labeled LDL or HDL. Inhibitors of vascular endothelial growth factor (VEGF) receptors (VEGFR) significantly decreased the uptake of HDL but not LDL. Silencing of VEGF receptor 2 significantly decreased cellular binding, association, and transendothelial transport of 125I-HDL but not 125I-LDL. RNA interference with VEGF receptor 1 or VEGF receptor 3 had no effect. Binding, uptake, and transport of HDL but not LDL were strongly reduced in the absence of VEGF-A from the cell culture medium and were restored by the addition of VEGF-A. The restoring effect of VEGF-A on endothelial binding, uptake, and transport of HDL was abrogated by pharmacological inhibition of phosphatidyl-inositol 3 kinase/protein kinase B or p38 mitogen-activated protein kinase, as well as silencing of scavenger receptor BI. Moreover, the presence of VEGF-A was found to be a prerequisite for the localization of scavenger receptor BI in the plasma membrane of endothelial cells. CONCLUSIONS: The identification of VEGF as a regulatory factor of transendothelial transport of HDL but not LDL supports the concept that the endothelium is a specific and, hence, druggable barrier for the entry of lipoproteins into the vascular wall.
OBJECTIVE: Low- and high-density lipoproteins (LDL and HDL) must pass the endothelial layer to exert pro- and antiatherogenic activities, respectively, within the vascular wall. However, the rate-limiting factors that mediate transendothelial transport of lipoproteins are yet little known. Therefore, we performed a high-throughput screen with kinase drug inhibitors to identify modulators of transendothelial LDL and HDL transport. APPROACH AND RESULTS: Microscopy-based high-content screening was performed by incubating human aortic endothelial cells with 141 kinase-inhibiting drugs and fluorescent-labeled LDL or HDL. Inhibitors of vascular endothelial growth factor (VEGF) receptors (VEGFR) significantly decreased the uptake of HDL but not LDL. Silencing of VEGF receptor 2 significantly decreased cellular binding, association, and transendothelial transport of 125I-HDL but not 125I-LDL. RNA interference with VEGF receptor 1 or VEGF receptor 3 had no effect. Binding, uptake, and transport of HDL but not LDL were strongly reduced in the absence of VEGF-A from the cell culture medium and were restored by the addition of VEGF-A. The restoring effect of VEGF-A on endothelial binding, uptake, and transport of HDL was abrogated by pharmacological inhibition of phosphatidyl-inositol 3 kinase/protein kinase B or p38 mitogen-activated protein kinase, as well as silencing of scavenger receptor BI. Moreover, the presence of VEGF-A was found to be a prerequisite for the localization of scavenger receptor BI in the plasma membrane of endothelial cells. CONCLUSIONS: The identification of VEGF as a regulatory factor of transendothelial transport of HDL but not LDL supports the concept that the endothelium is a specific and, hence, druggable barrier for the entry of lipoproteins into the vascular wall.
Authors: Srividya Velagapudi; Lucia Rohrer; Francesco Poti; Jerzy-Roch Nofer; Arnold von Eckardstein; Renate Feuerborn; Damir Perisa; Dongdong Wang; Grigorios Panteloglou; Anton Potapenko; Mustafa Yalcinkaya; Andreas J Hülsmeier; Bettina Hesse; Alexander Lukasz; Mingxia Liu; John S Parks; Christina Christoffersen; Markus Stoffel; Manuela Simoni Journal: Arterioscler Thromb Vasc Biol Date: 2021-08-19 Impact factor: 10.514
Authors: Hong S Lu; Ann Marie Schmidt; Robert A Hegele; Nigel Mackman; Daniel J Rader; Christian Weber; Alan Daugherty Journal: Arterioscler Thromb Vasc Biol Date: 2018-10 Impact factor: 8.311
Authors: Srividya Velagapudi; Peter Schraml; Mustafa Yalcinkaya; Hella A Bolck; Lucia Rohrer; Holger Moch; Arnold von Eckardstein Journal: J Lipid Res Date: 2018-09-01 Impact factor: 5.922
Authors: Katharina Kinslechner; David Schörghofer; Birgit Schütz; Maria Vallianou; Bettina Wingelhofer; Wolfgang Mikulits; Clemens Röhrl; Markus Hengstschläger; Richard Moriggl; Herbert Stangl; Mario Mikula Journal: Mol Cancer Res Date: 2017-10-03 Impact factor: 5.852