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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 endothelialimportant 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 realtime 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 miRNAnomescreening 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. [ABSTRACT FROM AUTHOR]

Background: Aging populations show higher incidences of myocardial infarction (MI) and heart failure (HF). Cardiac remodeling post-MI leads to progressive impaired cardiac function caused by a disarray of several processes including derailed autophagy. Microribonucleic acids (miRNAs) are known to be key players in cardiovascular disease but their involvement in cardiac autophagy and aging is not well understood.Objectives: This study sought to identify new miRNA candidates that regulate cardiac autophagy and aging.Methods: We exploited a high-throughput, fluorescence-activated cell sorting-based green fluorescent protein-LC3 detection method to measure the autophagic flux in cardiomyocytes after transfection of a precursor miRNA library consisting of 380 miRNAs. This was followed by a series of molecular and in vivo studies.Results: Together with additional expression screenings, we identified miR-22 as an abundant and strong inhibitor of the cardiac autophagy process. Cardiac miR-22 expression levels increased during aging of mice as well as in aging neonatal cardiomyocytes in vitro by a P53-dependent mechanism. Inhibition of miR-22 in aging cardiomyocytes in vitro activated autophagy and inhibited cellular hypertrophy. Pharmacological inhibition of miR-22 post-MI in older mice activated cardiac autophagy, prevented post-infarction remodeling, and improved cardiac function compared with control subjects. Interestingly, similar effects were less pronounced in younger mice with significantly lower cardiac miR-22 expression levels. In addition, circulating levels of miR-22 in 154 patients with systolic HF were highly associated with early mortality.Conclusions: We concluded that miR-22 is an important regulator of cardiac autophagy and a potential therapeutic target, especially in the older myocardium. Finally, circulating miR-22 provides prognostic information for HF patients, highlighting miR-22 as a promising therapeutic and biomarker candidate for cardiovascular disorders. [ABSTRACT FROM AUTHOR]

In response to stress, the heart undergoes extensive cardiac remodeling that results in cardiac fibrosis and pathological growth of cardiomyocytes (hypertrophy), which contribute to heart failure. Alterations in microRNA (miRNA) levels are associated with dysfunctional gene expression profiles associated with many cardiovascular disease conditions; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes. Surprisingly, evaluation of the miRNA content of cardiac fibroblast-derived exosomes revealed a relatively high abundance of many miRNA passenger strands ("star" miRNAs), which normally undergo intracellular degradation. Using confocal imaging and coculture assays, we identified fibroblast exosomal-derived miR-21_3p (miR-21*) as a potent paracrineacting RNA molecule that induces cardiomyocyte hypertrophy. Proteome profiling identified sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) as miR-21* targets, and silencing SORBS2 or PDLIM5 in cardiomyocytes induced hypertrophy. Pharmacological inhibition of miR-21* in a mouse model of Ang II-induced cardiac hypertrophy attenuated pathology. These findings demonstrate that cardiac fibroblasts secrete star miRNA-enriched exosomes and identify fibroblast-derived miR-21* as a paracrine signaling mediator of cardiomyocyte hypertrophy that has potential as a therapeutic target. [ABSTRACT FROM AUTHOR]