MicroRNA-based Therapy of Gata2-deficient Vascular Disease
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 siRNA-mediated knockdown and lentiviral-based overexpression were applied to decipher capacity of GATA2 in regulating cell viability and capillary formation. Next, GATA2-dependent miR transcriptome was identified using a qRT-PCR-based profiling approach. Transcriptional control of miR promoters was assessed via chromatin immunoprecipitation (ChIP), luciferase promoter assays and bisulfite analysis of sites in proximity. Selected miRs were modulated in combination with GATA2 to identify signaling pathways at angiogenic cytokine level via proteome profiler and ELISAs. 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 endothelial GATA2 knockout background. Nanoparticle-mediated delivery of pro-angiogenic 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, pro-angiogenic miR-126 was regulated by GATA2 transcriptionally and targeted anti-angiogenic 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 pro-angiogenic paracrine effects. GATA2 silencing resulted in endothelial DNA hypomethylation leading to induced expression of anti-angiogenic 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 pro-angiogenic 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/or its targets miR-126 and miR-221 is a promising therapeutic strategy for treatment of many vascular diseases.
- Received March 14, 2016.
- Revision received July 15, 2016.
- Accepted October 3, 2016.