miR-10 Regulates the Angiogenic Behavior of Zebrafish and Human Endothelial Cells by Promoting VEGF Signaling.
ABSTRACT Rationale: Formation and remodeling of the vasculature during development and disease involves a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their post-transcriptional dose-titration by miRNAs is poorly understood. Objective: To identify miRNAs that regulate angiogenesis. Methods and Results: We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating pro-angiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth (ISV) in the trunk of zebrafish larvae, while overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells (HUVECs). We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters VEGF, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in HUVECs inhibited the angiogenic behavior of endothelial cells largely by antagonizing VEGF receptor-2 signaling. Conclusions: Our study provides insights into how FLT1 and VEGF receptor-2 signaling is titrated in a miRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis.
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ABSTRACT: Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.01/2015; 2:14064. DOI:10.1038/mtm.2014.64
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ABSTRACT: The two principal cell types of importance for normal vessel wall physiology are smooth muscle cells and endothelial cells. Much progress has been made over the past 20 years in the discovery and function of transcription factors that coordinate proper differentiation of these cells and the maintenance of vascular homeostasis. More recently, the converging fields of bioinformatics, genomics, and next generation sequencing have accelerated discoveries in a number of classes of noncoding sequences, including transcription factor binding sites (TFBS), microRNA genes, and long noncoding RNA genes, each of which mediates vascular cell differentiation through a variety of mechanisms. Alterations in the nucleotide sequence of key TFBS or deviations in transcription of noncoding RNA genes likely have adverse effects on normal vascular cell phenotype and function. Here, the subject of noncoding sequences that influence smooth muscle cell or endothelial cell phenotype will be summarized as will future directions to further advance our understanding of the increasingly complex molecular circuitry governing normal vascular cell differentiation and how such information might be harnessed to combat vascular diseases.Cellular and Molecular Life Sciences CMLS 05/2015; DOI:10.1007/s00018-015-1936-9 · 5.86 Impact Factor
- Circulation Research 12/2014; 115(12):e79-82. DOI:10.1161/CIRCRESAHA.114.305639 · 11.09 Impact Factor