Dorothee Hartmann1, Jan Fiedler1, Kristina Sonnenschein1, Annette Just1, Angelika Pfanne1, Karina Zimmer1, Janet Remke1, Ariana Foinquinos1, Malte Butzlaff1, Katharina Schimmel1, Lars Maegdefessel1, Denise Hilfiker-Kleiner1, Nico Lachmann1, Andreas Schober1, Natali Froese1, Jörg Heineke1, Johann Bauersachs1, Sandor Batkai1, Thomas Thum2. 1. From Institute of Molecular and Translational Therapeutic Strategies (IMTTS), IFB-Tx, Hannover Medical School, Germany (D.H., J.F., K.S., A.J., A.P., K.Z., J.R., A.F., K.S., S.B., T.T.); Department of Cardiology and Angiology, Hannover Medical School, Germany (K.S., D.H.-K., N.F., J.H., J.B.); Cellular Neurophysiology, Center of Physiology, Hannover Medical School, Germany (M.B.); Department of Vascular and Endovascular Surgery, Technical University Munich, Germany (L.M.); Cluster of Excellence REBIRTH, Hannover Medical School, Germany (D.H.-K., N.F., J.H., J.B., T.T.); JRG Translational Hematology of Congenital Disease, Cluster of Excellence REBIRTH, Institute of Experimental Hematology, Hannover Medical School, Germany (N.L.); Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Germany (A.S.); DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Germany (A.S.); and National Heart and Lung Institute, Imperial College London, UK (T.T.). 2. From Institute of Molecular and Translational Therapeutic Strategies (IMTTS), IFB-Tx, Hannover Medical School, Germany (D.H., J.F., K.S., A.J., A.P., K.Z., J.R., A.F., K.S., S.B., T.T.); Department of Cardiology and Angiology, Hannover Medical School, Germany (K.S., D.H.-K., N.F., J.H., J.B.); Cellular Neurophysiology, Center of Physiology, Hannover Medical School, Germany (M.B.); Department of Vascular and Endovascular Surgery, Technical University Munich, Germany (L.M.); Cluster of Excellence REBIRTH, Hannover Medical School, Germany (D.H.-K., N.F., J.H., J.B., T.T.); JRG Translational Hematology of Congenital Disease, Cluster of Excellence REBIRTH, Institute of Experimental Hematology, Hannover Medical School, Germany (N.L.); Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Germany (A.S.); DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Germany (A.S.); and National Heart and Lung Institute, Imperial College London, UK (T.T.). thum.thomas@mh-hannover.de.
Abstract
BACKGROUND: The transcription factor GATA2 orchestrates the expression of many endothelial-specific genes, illustrating its crucial importance for endothelial cell function. The capacity of this transcription factor in orchestrating endothelial-important microRNAs (miRNAs/miR) is unknown. METHODS: Endothelial GATA2 was functionally analyzed in human endothelial cells in vitro. Endogenous short interfering RNA-mediated knockdown and lentiviral-based overexpression were applied to decipher the capacity of GATA2 in regulating cell viability and capillary formation. Next, the GATA2-dependent miR transcriptome was identified by using a profiling approach on the basis of quantitative real-time polymerase chain reaction. Transcriptional control of miR promoters was assessed via chromatin immunoprecipitation, luciferase promoter assays, and bisulfite sequencing analysis of sites in proximity. Selected miRs were modulated in combination with GATA2 to identify signaling pathways at the angiogenic cytokine level via proteome profiler and enzyme-linked immunosorbent assays. Downstream miR targets were identified via bioinformatic target prediction and luciferase reporter gene assays. In vitro findings were translated to a mouse model of carotid injury in an endothelial GATA2 knockout background. Nanoparticle-mediated delivery of proangiogenic miR-126 was tested in the reendothelialization model. RESULTS: GATA2 gain- and loss-of-function experiments in human umbilical vein endothelial cells identified a key role of GATA2 as master regulator of multiple endothelial functions via miRNA-dependent mechanisms. Global miRNAnome-screening identified several GATA2-regulated miRNAs including miR-126 and miR-221. Specifically, proangiogenic miR-126 was regulated by GATA2 transcriptionally and targeted antiangiogenic SPRED1 and FOXO3a contributing to GATA2-mediated formation of normal vascular structures, whereas GATA2 deficiency led to vascular abnormalities. In contrast to GATA2 deficiency, supplementation with miR-126 normalized vascular function and expression profiles of cytokines contributing to proangiogenic paracrine effects. GATA2 silencing resulted in endothelial DNA hypomethylation leading to induced expression of antiangiogenic miR-221 by GATA2-dependent demethylation of a putative CpG island in the miR-221 promoter. Mechanistically, a reverted GATA2 phenotype by endogenous suppression of miR-221 was mediated through direct proangiogenic miR-221 target genes ICAM1 and ETS1. In a mouse model of carotid injury, GATA2 was reduced, and systemic supplementation of miR-126-coupled nanoparticles enhanced miR-126 availability in the carotid artery and improved reendothelialization of injured carotid arteries in vivo. CONCLUSIONS: GATA2-mediated regulation of miR-126 and miR-221 has an important impact on endothelial biology. Hence, modulation of GATA2 and its targets miR-126 and miR-221 is a promising therapeutic strategy for treatment of many vascular diseases.
BACKGROUND: The transcription factor GATA2 orchestrates the expression of many endothelial-specific genes, illustrating its crucial importance for endothelial cell function. The capacity of this transcription factor in orchestrating endothelial-important microRNAs (miRNAs/miR) is unknown. METHODS: Endothelial GATA2 was functionally analyzed in human endothelial cells in vitro. Endogenous short interfering RNA-mediated knockdown and lentiviral-based overexpression were applied to decipher the capacity of GATA2 in regulating cell viability and capillary formation. Next, the GATA2-dependent miR transcriptome was identified by using a profiling approach on the basis of quantitative real-time polymerase chain reaction. Transcriptional control of miR promoters was assessed via chromatin immunoprecipitation, luciferase promoter assays, and bisulfite sequencing analysis of sites in proximity. Selected miRs were modulated in combination with GATA2 to identify signaling pathways at the angiogenic cytokine level via proteome profiler and enzyme-linked immunosorbent assays. Downstream miR targets were identified via bioinformatic target prediction and luciferase reporter gene assays. In vitro findings were translated to a mouse model of carotid injury in an endothelial GATA2 knockout background. Nanoparticle-mediated delivery of proangiogenic miR-126 was tested in the reendothelialization model. RESULTS:GATA2 gain- and loss-of-function experiments in human umbilical vein endothelial cells identified a key role of GATA2 as master regulator of multiple endothelial functions via miRNA-dependent mechanisms. Global miRNAnome-screening identified several GATA2-regulated miRNAs including miR-126 and miR-221. Specifically, proangiogenic miR-126 was regulated by GATA2 transcriptionally and targeted antiangiogenic SPRED1 and FOXO3a contributing to GATA2-mediated formation of normal vascular structures, whereas GATA2 deficiency led to vascular abnormalities. In contrast to GATA2 deficiency, supplementation with miR-126 normalized vascular function and expression profiles of cytokines contributing to proangiogenic paracrine effects. GATA2 silencing resulted in endothelial DNA hypomethylation leading to induced expression of antiangiogenic miR-221 by GATA2-dependent demethylation of a putative CpG island in the miR-221 promoter. Mechanistically, a reverted GATA2 phenotype by endogenous suppression of miR-221 was mediated through direct proangiogenic miR-221 target genes ICAM1 and ETS1. In a mouse model of carotid injury, GATA2 was reduced, and systemic supplementation of miR-126-coupled nanoparticles enhanced miR-126 availability in the carotid artery and improved reendothelialization of injured carotid arteries in vivo. CONCLUSIONS:GATA2-mediated regulation of miR-126 and miR-221 has an important impact on endothelial biology. Hence, modulation of GATA2 and its targets miR-126 and miR-221 is a promising therapeutic strategy for treatment of many vascular diseases.
Authors: Jan Fiedler; Andrew H Baker; Stefanie Dimmeler; Stephane Heymans; Manuel Mayr; Thomas Thum Journal: Cardiovasc Res Date: 2018-08-01 Impact factor: 10.787
Authors: Emery H Bresnick; Kyle J Hewitt; Charu Mehta; Sunduz Keles; Robert F Paulson; Kirby D Johnson Journal: Development Date: 2018-01-10 Impact factor: 6.868
Authors: Anne-Kristin Schaefer; Attila Kiss; André Oszwald; Felix Nagel; Eylem Acar; Arezu Aliabadi-Zuckermann; Matthias Hackl; Andreas Zuckermann; Renate Kain; Andrzej Jakubowski; Peter Ferdinandy; Seth Hallström; Bruno K Podesser Journal: Transpl Int Date: 2022-04-13 Impact factor: 3.842