Genomic Landscapes of Noncoding RNAs Regulating VEGFA and VEGFC Expression in Endothelial Cells

Abstract

Vascular endothelial growth factors (VEGFs) are best known as key regulators of angiogenesis and lymphangiogenesis. Although VEGFs have been promising therapeutic targets for various cardiovascular related diseases, their regulatory landscape in endothelial cells remains elusive. Several studies have highlighted the involvement of non-coding RNAs (ncRNAs) in the modulation of VEGF expression. In this study, we investigated the role of two classes of non-coding RNAs, long ncRNA (lncRNA) and enhancer RNAs (eRNAs), in the transcriptional regulation of VEGFA and VEGFC. By integrating genome-wide global run-on (GRO-seq) and Hi-C data, we identified putative lncRNAs and eRNAs associated with VEGFA and VEGFC genes in endothelial cells. A subset of the identified putative enhancers demonstrated regulatory activity in a reporter assay. Importantly, we demonstrate that deletion of enhancers and lncRNAs by CRISPR/Cas9 promoted significant changes in VEGFA and VEGFC expression. RNA-seq data from lncRNA deletions showed downstream factors implicated in VEGFA and VEGFC linked pathways, such as angiogenesis and lymphangiogenesis, suggesting functional roles for these lncRNAs. Our study uncovers novel lncRNAs and eRNAs regulating VEGFA and VEGFC that can be targeted to modulate the expression of these important molecules in endothelial cells.

Characterization of a functional endothelial super-enhancer that regulates ADAMTS18 and angiogenesis

“…However, super-enhancers hierarchical organization does not exclude contribution of some or all enhancers within a SE, and as such it was demonstrated that combinatorial deletion of enhancers within a SE led to a greater effect on gene expression ( 66 , 69 ). This collaborative activity of enhancers within a SE is also reflected in our data from the enhancer deletions, whereby deletion of either enhancer resulted in similar gene regulation of approximately 2000 genes, in the range reported in previous enhancer deletion studies ( 70 , 71 ). Thus, the close overlap of differentially regulated genes would indicate that most of the enhancer activity is related to the SE activity as a whole, and perturbation of any enhancer within the cluster may negatively affect the SE activity.…”

“…We have recently observed a similar discrepancy between in vitro reporter assays and genomic deletion of enhancers. However, in this case enhancers showed activity in vitro but not following CRISPR/Cas9 deletion ( 71 ). What is clear is that in vitro assays alone are insufficient to analyse enhancer activity, and multiple approaches are required to adequately investigate these regulatory regions.…”

Inflammatory Immune-Associated eRNA: Mechanisms, Functions and Therapeutic Prospects

“…The recent studies showed that non-immune inflammatory cells have inflammatory changes and altered cellular states under eRNA regulation ( 135 – 138 ). Isidore et al ( 136 ) found that VEGFA-eRNA5 and VEGFC-eRNA3 in endothelial cells affect angiogenesis and lymphangiogenesis by regulating endogenous transcription and VEGFA and VEGFC expression. Additionally, eRNAs were found to be well correlated with VEGF expression across cell types and in response to hypoxic stimuli using GRO-Seq.…”

Gene Therapy for Cardiovascular Disease: Basic Research and Clinical Prospects

“…Local transferring of certain genes has been verified to be effective in inhibiting neointimal hyperplasia and neoatherosclerosis. These genes include Inducible NOS (iNOS) (157), vascular endothelial growth factor (VEGF) (158), tissue inhibitor of metalloproteinase 1 (TIMP-1) (159), monocyte chemoattractant protein-1(MCP-1) (160), Ras mutation (161), TGF-β1 receptor Type II (TβRII) (162). iNOS is rarely expressed under physiological conditions but highly expressed under inflammatory stimuli.…”