Abstract 15691: Endothelial ADAR1-Mediated RNA Editing Controls Cathepsin S mRNA Stability and Angiogenesis
Background: Adenosine deaminase acting on RNA-1 (ADAR1) enzyme catalyze adenosine-to-inosine (A-to-I) RNA editing in double-stranded RNA. However, the functional significance of this posttranscriptional RNA regulatory mechanism has not been reported so far in vascular system.
Methods and Results: ADAR1 and ADAR2 are expressed on mRNA and protein level in human venous umbilical endothelial cells (HUVECs). Upon ADAR1 knockdown with short-interfering RNA, a significant reduction of angiogenic sprouting and tube formation, but not of cell viability or proliferation, was observed. Endothelial-specific inducible genetic ablation of ADAR1 in mice leads to impaired postnatal retinal angiogenesis, as revealed by significantly decreased vascular outgrowth and reduced vessel branching. Endothelial cell transcriptome sequencing revealed that ADAR1 is the main RNA editor inducing A-to-I RNA editing events. Cathepsin S (CTSS), which has an important function in angiogenesis and vascular diseases, was among the highest ADAR1 edited targets. Silencing of ADAR1 reduced CTSS mRNA and protein expression. Supplementation of spheroids with recombinant human CTSS partially rescued the ADAR1 knockdown-induced impairment of endothelial cell sprouting. In order to identify the mechanism of underlying CTSS regulation by ADAR1, we first tested the impact of RNA editing on CTSS mRNA secondary structure. With the help of NMR spectroscopy, we observed that RNA editing alters mRNA base-pairing and folding. RNA-immunoprecipitation experiments showed that silencing of ADAR1 reduced the binding of the stabilizing RNA-binding protein HuR to CTSS 3’UTR by about 80%. Luciferase reporter gene expression assays further confirmed our findings suggesting that RNA editing regulates HuR binding to AU-rich elements at CTSS 3’UTR.
Conclusions: These results demonstrate a novel mechanism of endothelial ADAR1 and RNA editing in gene expression and vascular function.
Author Disclosures: K. Stellos: None. A. Gatsiou: None. R.A. Boon: None. D. John: None. B. Fürtig: None. Y. Manavski: None. A. Doddaballapur: None. F.F. Lunella: None. C. Amrhein: None. X. You: None. S. Uchida: None. J. Boeckel: None. W. Chen: None. H. Schwalbe: None. A. Zeiher: None. S. Dimmeler: None.
- © 2014 by American Heart Association, Inc.