Thromboxane A2 receptor activation via Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction inhibits angiogenic sprouting of human endothelial cells
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Thromboxane A2 receptor activation via Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction inhibits angiogenic sprouting of human endothelial cells. / Eckenstaler, Robert; Ripperger, Anne; Hauke, Michael; Braun, Heike; Ergün, Süleyman; Schwedhelm, Edzard; Benndorf, Ralf A.
in: BIOCHEM PHARMACOL, Jahrgang 201, 115069, 07.2022.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Thromboxane A2 receptor activation via Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction inhibits angiogenic sprouting of human endothelial cells
AU - Eckenstaler, Robert
AU - Ripperger, Anne
AU - Hauke, Michael
AU - Braun, Heike
AU - Ergün, Süleyman
AU - Schwedhelm, Edzard
AU - Benndorf, Ralf A
N1 - Copyright © 2022 Elsevier Inc. All rights reserved.
PY - 2022/7
Y1 - 2022/7
N2 - We could previously show that thromboxane A2 receptor (TP) activation inhibits the angiogenic capacity of human endothelial cells, but the underlying mechanisms remained unclear. Therefore, the aim of this study was to elucidate TP signal transduction pathways relevant to angiogenic sprouting of human endothelial cells. To clarify this matter, we used RNAi-mediated gene silencing as well as pharmacological inhibition of potential TP downstream targets in human umbilical vein endothelial cells (HUVEC) and VEGF-induced angiogenic sprouting of HUVEC spheroids in vitro as a functional read-out. In this experimental set-up, the TP agonist U-46619 completely blocked VEGF-induced angiogenic sprouting of HUVEC spheroids. Moreover, in live-cell analyses TP activation induced endothelial cell contraction, sprout retraction as well as endothelial cell tension and focal adhesion dysregulation of HUVEC. These effects were reversed by pharmacological TP inhibition or TP knockdown. Moreover, we identified a TP-Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction pathway to be relevant for U-46619-induced inhibition of VEGF-mediated HUVEC sprouting. In line with these results, U-46619-mediated TP activation potently induced RhoA and RhoC activity in live HUVEC as measured by FRET biosensors. Interestingly, pharmacological inhibition of ROCK and LIMK2 also normalized U-46619-induced endothelial cell tension and focal adhesion dysregulation of HUVEC. In summary, our work reveals mechanisms by which the TP may disturb angiogenic endothelial function in disease states associated with sustained endothelial TP activation.
AB - We could previously show that thromboxane A2 receptor (TP) activation inhibits the angiogenic capacity of human endothelial cells, but the underlying mechanisms remained unclear. Therefore, the aim of this study was to elucidate TP signal transduction pathways relevant to angiogenic sprouting of human endothelial cells. To clarify this matter, we used RNAi-mediated gene silencing as well as pharmacological inhibition of potential TP downstream targets in human umbilical vein endothelial cells (HUVEC) and VEGF-induced angiogenic sprouting of HUVEC spheroids in vitro as a functional read-out. In this experimental set-up, the TP agonist U-46619 completely blocked VEGF-induced angiogenic sprouting of HUVEC spheroids. Moreover, in live-cell analyses TP activation induced endothelial cell contraction, sprout retraction as well as endothelial cell tension and focal adhesion dysregulation of HUVEC. These effects were reversed by pharmacological TP inhibition or TP knockdown. Moreover, we identified a TP-Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction pathway to be relevant for U-46619-induced inhibition of VEGF-mediated HUVEC sprouting. In line with these results, U-46619-mediated TP activation potently induced RhoA and RhoC activity in live HUVEC as measured by FRET biosensors. Interestingly, pharmacological inhibition of ROCK and LIMK2 also normalized U-46619-induced endothelial cell tension and focal adhesion dysregulation of HUVEC. In summary, our work reveals mechanisms by which the TP may disturb angiogenic endothelial function in disease states associated with sustained endothelial TP activation.
KW - 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology
KW - GTP-Binding Protein alpha Subunits, G12-G13/metabolism
KW - Human Umbilical Vein Endothelial Cells/cytology
KW - Humans
KW - Lim Kinases/metabolism
KW - Neovascularization, Physiologic
KW - Receptors, Thromboxane A2, Prostaglandin H2/metabolism
KW - Signal Transduction
KW - Vascular Endothelial Growth Factor A/metabolism
KW - rho-Associated Kinases
KW - rhoA GTP-Binding Protein/genetics
KW - rhoC GTP-Binding Protein
U2 - 10.1016/j.bcp.2022.115069
DO - 10.1016/j.bcp.2022.115069
M3 - SCORING: Journal article
C2 - 35525325
VL - 201
JO - BIOCHEM PHARMACOL
JF - BIOCHEM PHARMACOL
SN - 0006-2952
M1 - 115069
ER -