Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA (CTSS), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3' untranslated region (3' UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx regions, which form a long stem-loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1) to the 3' UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-γ and tumor-necrosis-factor-α induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases.

Rationale: 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. Methods and 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.

IntroductionMicroRNAs (miRNAs) are highly conserved, single-stranded noncoding short RNA molecules (18-24 nucleotides) that regulate gene expression at the posttranscriptional level. miRNAs silence gene expression by inhibiting the translation of proteins from mRNAs or by promoting the degradation of mRNAs. After transcription of the primary miRNA transcripts from the genome, their maturation is mediated by the 2 RNase III endonucleases Dicer and Drosha. Then, mature miRNAs are incorporated into the RNA-induced silencing complex, 1 which mediates the binding of the miRNA to the 3Ј-untranslated region (3Ј-UTR) of the target mRNA leading either to translational repression or degradation of the target mRNA. 2 Because miRNAs control specific expression patterns of target genes, miRNAs represent attractive candidates to interfere with neovascularization.Increasing evidence indicates that miRNAs are important regulators of vascular development and angiogenesis. 3,4 In this context, first studies addressed the function of the miRNAprocessing enzymes Dicer and/or Drosha to explore the general role of miRNAs for angiogenesis. Depletion of Dicer in zebrafish or mice revealed an aberrant vessel growth, and silencing of Dicer in endothelial cells reduced in vitro angiogenesis. [5][6][7] To date, several miRs that regulate endothelial cell function and angiogenesis have been identified, 8 including the pro-angiogenic miRs miR-130a, 9 miR-210, 5,10,11 and miR-378. 12 In addition, miR-126 was shown to regulate vascular integrity and angiogenesis during development and in ischemia-induced angiogenesis. [13][14][15] In contrast, miR-221 and miR-222, 7,16 miR-15 and miR-16, 17,18 and members of the miR-17-92 cluster 19,20 inhibit angiogenesis.In our previous study, we found that the members of the miR-23ϳ27ϳ24 cluster, miR-27a and miR-27b, were highly expressed in endothelial cells. 6 In addition, miR-27b was downregulated after Dicer and Drosha silencing, and inhibition of miR-27b significantly reduced endothelial cell sprouting in vitro, 6 indicating that miR-27b exerts pro-angiogenic effects. Recently, Zhou et al demonstrated that the miR-23ϳ27ϳ24 cluster regulates angiogenesis. 21 In muscle stem cells, miR-27b down-regulates Pax3 expression during myogenic differentiation. 22 Moreover, miR-27 down-regulates Runx1 expression during granulocyte differentiation 23 and the nuclear receptor peroxisome proliferatoractivated receptor-␥ (PPAR-␥) in adipocytes. 24 The myocyte enhancer factor 2C (MEF2C) is another important target of miR-27b during heart development. 25 However, the specific functions and targets of miR-27 in endothelial cells are largely unexplored. As the family members miR-27a and miR-27b differ in only one nucleotide and share the same seed sequence, we investigated the specific role of both family members for the angiogenic activity of endothelial cells and determined the effects on neovascularization. Here we identified the angiogenesis inhibitor semaphorin 6A as a The online version of this article contains a ...

JmjC domain-containing proteins play a crucial role in the control of gene expression by acting as protein hydroxylases or demethylases, thereby controlling histone methylation or splicing. Here, we demonstrate that silencing of Jumonji domain-containing protein 6 (Jmjd6) impairs angiogenic functions of endothelial cells by changing the gene expression and modulating the splicing of the VEGF-receptor 1 (Flt1). Reduction of Jmjd6 expression altered splicing of Flt1 and increased the levels of the soluble form of Flt1, which binds to VEGF and placental growth factor (PlGF) and thereby inhibits angiogenesis. Saturating VEGF or PlGF or neutralizing antibodies directed against soluble Flt1 rescued the angiogenic defects induced by Jmjd6 silencing. Jmjd6 interacts with the splicing factors U2AF65 that binds to Flt1 mRNA. In conclusion, Jmjd6 regulates the splicing of Flt1, thereby controlling angiogenic sprouting.

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Rationale: Proangiogenic hematopoietic and endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, but the mechanisms regulating differentiation to the endothelial lineage are unclear.Objective: To elucidate the epigenetic control of endothelial gene expression in proangiogenic cells and EPCs. Methods and Results: Here we demonstrate that the endothelial nitric oxide synthase (eNOS) promoter is epigenetically silenced in proangiogenic cells (early EPCs), CD34؉ cells, and mesoangioblasts by DNA methylation and prominent repressive histone H3K27me3 marks. In order to reverse epigenetic silencing to facilitate endothelial commitment, we used 3-deazaneplanocin A, which inhibits the histone methyltransferase enhancer of zest homolog 2 and, thereby, reduces H3K27me3. 3-Deazaneplanocin A was not sufficient to increase eNOS expression, but the combination of 3-deazaneplanocin A and the histone deacetylase inhibitor Trichostatin A augmented eNOS expression, indicating that the concomitant inhibition of silencing histone modification and enhancement of activating histone modification facilitates eNOS expression. In ischemic tissue, hypoxia plays a role in recruiting progenitor cells. Therefore, we examined the effect of hypoxia on epigenetic modifications. Hypoxia modulated the balance of repressive to active histone marks and increased eNOS mRNA expression. The reduction of repressive H3K27me3 was associated with an increase of the histone demethylase Jmjd3. Silencing of Jmjd3 induced apoptosis and senescence in proangiogenic cells and inhibited hypoxia-mediated up-regulation of eNOS expression in mesoangioblasts. Key Words: Jmjd3 Ⅲ eNOS Ⅲ hypoxia Ⅲ proangiogenic cells E ndothelial or hematopoietic progenitor cells contribute to vascular repair and postnatal neovascularization. Administration of progenitor cells augmented neovascularization and functional recovery after critical ischemia studies. 1 However, the mechanisms underlying the beneficial effect of progenitor cells are unclear, and it is still intensely debated whether endothelial and hematopoietic progenitor cells can undergo differentiation toward the endothelial lineage and incorporate into newly formed blood vessels. 2 The basal mechanisms for endothelial specification and endotheliumspecific gene expression are poorly understood and an endothelial-specific master regulator has yet to be identified. Obviously, many transcription factors such as members of Ets, GATA, and forkhead families play a key role in endothelial gene activation and unique combinational regulation of multiple transcription factors is required for endothelial specification and differentiation. However, many of these transcription factors implicated in vasculogenesis are also expressed in nonendothelial cells like hematopoietic stem cells but are not sufficient to activate endothelial genes. 3 Likewise, expression of the prototypical gene endothelial NO synthase (eNOS) is controlled by a variety of transcription factors such as Sp-1, Ets-1, and YY1, but these tr...

Objective-Histone deacetylases (HDACs) modulate gene expression by deacetylation of histone and nonhistone proteins. Several HDACs control angiogenesis, but the role of HDAC9 is unclear. Methods and Results-Here, we analyzed the function of HDAC9 in angiogenesis and its involvement in regulating microRNAs. In vitro, silencing of HDAC9 reduces endothelial cell tube formation and sprouting. Furthermore, HDAC9 silencing decreases vessel formation in a spheroid-based Matrigel plug assay in mice and disturbs vascular patterning in zebrafish embryos. Genetic deletion of HDAC9 reduces retinal vessel outgrowth and impairs blood flow recovery after hindlimb ischemia. Consistently, overexpression of HDAC9 increases endothelial cell sprouting, whereas mutant constructs lacking the catalytic domain, the nuclear localization sequence, or sumoylation site show no effect. To determine the mechanism underlying the proangiogenic effect of HDAC9, we measured the expression of the microRNA (miR)-17-92 cluster, which is known for its antiangiogenic activity. We demonstrate that silencing of HDAC9 in endothelial cells increases the expression of miR-17-92. Inhibition of miR-17-20a rescues the sprouting defects induced by HDAC9 silencing in vitro and blocking miR-17 expression partially reverses the disturbed vascular patterning of HDAC9 knockdown in zebrafish embryos. Conclusion-We found that HDAC9 promotes angiogenesis and transcriptionally represses the miR-17-92 cluster.(Arterioscler Thromb Vasc Biol. 2013;33:533-543.)Key Words: angiogenesis ◼ histone deacetylase 9 and histone deacetylase 5 ◼ histone deacetylases ◼ microRNAs ◼ miR-17

Objective-Jumonji C (JmjC) domain-containing proteins modify histone and nonhistone proteins thereby controlling cellular functions. However, the role of JmjC proteins in angiogenesis is largely unknown.