title: Identification and Characterization of Hypoxia-Regulated Endothelial Circular RNA.
abstract: BACKGROUND
  Circular RNAs (circRNAs) are noncoding RNAs generated by back splicing. Back splicing has been considered a rare event, but recent studies suggest that circRNAs are widely expressed. However, the expression, regulation, and function of circRNAs in vascular cells is still unknown.
  
  
  OBJECTIVE
  Here, we characterize the expression, regulation, and function of circRNAs in endothelial cells.
  
  
  RESULTS
  Endothelial circRNAs were identified by computational analysis of ribo-minus RNA generated from human umbilical venous endothelial cells cultured under normoxic or hypoxic conditions. Selected circRNAs were biochemically characterized, and we found that the majority of them lacks polyadenylation, is resistant to RNase R digestion and localized to the cytoplasm. We further validated the hypoxia-induced circRNAs cZNF292, cAFF1, and cDENND4C, as well as the downregulated cTHSD1 by reverse transcription polymerase chain reaction in cultured endothelial cells. Cloning of cZNF292 validated the predicted back splicing of exon 4 to a new alternative exon 1A. Silencing of cZNF292 inhibited cZNF292 expression and reduced tube formation and spheroid sprouting of endothelial cells in vitro. The expression of pre-mRNA or mRNA of the host gene was not affected by silencing of cZNF292. No validated microRNA-binding sites for cZNF292 were detected in Argonaute high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation data sets, suggesting that cZNF292 does not act as a microRNA sponge.
  
  
  CONCLUSIONS
  We show that the majority of the selected endothelial circRNAs fulfill all criteria of bona fide circRNAs. The circRNA cZNF292 exhibits proangiogenic activities in vitro. These data suggest that endothelial circRNAs are regulated by hypoxia and have biological functions.
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.).
authors.forename: Jes-Niels
authors.initials: JN
authors.lastname: Boeckel
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.).
authors.forename: Nicolas
authors.initials: N
authors.lastname: Jaé
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.).
authors.forename: Andreas W
authors.initials: AW
authors.lastname: Heumüller
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.).
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.).
authors.forename: Reinier A
authors.initials: RA
authors.lastname: Boon
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.).
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Stellos
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.).
authors.forename: Andreas M
authors.initials: AM
authors.lastname: Zeiher
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.).
authors.forename: David
authors.initials: D
authors.lastname: John
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.).
authors.forename: Shizuka
authors.initials: S
authors.lastname: Uchida
authors.affiliation: From the Institute for Cardiovascular Regeneration, Center for Molecular Medicine, University Frankfurt, Frankfurt, Germany (J.-N.B., N.J., A.W.H., R.A.B., K.S., D.J., S.U., S.D.); Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany (W.C.); Department of Cardiology, Internal Medicine III, Goethe-University Hospital, Frankfurt, Germany (K.S., A.M.Z.); and German Center for Cardiovascular Research (DZHK) (J.-N.B., N.J., W.C., R.A.B., K.S., D.J., A.M.Z., S.U., S.D.). Dimmeler@em.uni-frankfurt.de.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Dimmeler
mesh_terms: D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D016000:Cluster Analysis; D003198:Computer Simulation; D005091:Exons; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D018919:Neovascularization, Physiologic; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012326:RNA Splicing; D000079962:RNA, Circular; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; D000079962:RNA, Circular; C570380:ZNF292 protein, human; D012313:RNA
keywords: back-splicing; circRNAs; circular RNA; endothelial cells; hypoxia