Rationale: The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long noncoding RNAs in the vasculature are largely unknown. Objective: Here, we characterized the expression of long noncoding RNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Methods and Results: Endothelial cells of different origin express relative high levels of the conserved long noncoding RNAs MALAT1, taurine upregulated gene 1 (TUG1), maternally expressed 3 (MEG3), linc00657, and linc00493. MALAT1 was significantly increased by hypoxia and controls a phenotypic switch in endothelial cells. Silencing of MALAT1 by small interfering RNAs or GapmeRs induced a promigratory response and increased basal sprouting and migration, whereas proliferation of endothelial cells was inhibited. When angiogenesis was further stimulated by vascular endothelial growth factor, MALAT1 small interfering RNAs induced discontinuous sprouts indicative of defective proliferation of stalk cells. In vivo studies confirmed that genetic ablation of MALAT1 inhibited proliferation of endothelial cells and reduced neonatal retina vascularization. Pharmacological inhibition of MALAT1 by GapmeRs reduced blood flow recovery and capillary density after hindlimb ischemia. Gene expression profiling followed by confirmatory quantitative reverse transcriptase-polymerase chain reaction demonstrated that silencing of MALAT1 impaired the expression of various cell cycle regulators. Conclusions: Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial cell phenotype in vitro, and its genetic deletion or pharmacological inhibition reduces vascular growth in vivo.

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.

E ndothelial cells form the inner lining of all blood vessels and not only regulate transport of nutrients to the underlying tissue but also coordinate the formation of new blood vessels, a process termed angiogenesis. Therefore, endothelial cells are highly plastic cells that are capable of switching from a resting quiescent state in normal conduit blood vessels to a highly proliferative and migratory state when angiogenesis takes place. Resting quiescent endothelial cells are termed phalanx cells, 1 whereas migratory angiogenic endothelial cells are referred to as tip cells, which are followed by proliferating so-called stalk cells.2 Although the mechanisms regulating tip and stalk cell behavior have been extensively studied, relatively little is known about the control of the phalanx state.Shear stress, the force that laminar blood flow exerts on endothelial cells, is thought to be one of the factors that determine the quiescent state of endothelial cells.3 This biomechanical stimulus induces the expression of the transcription factor Krüppel-like factor 2 (KLF2), which orchestrates a network of genes that elicit a quiescent endothelial cell phenotype. 4,5 Among the factors that are upregulated by KLF2 are antiinflammatory and antithrombotic proteins, whereas proinflammatory and prothrombotic factors are downregulated by KLF2. 4 Although not all effects of shear stress on endothelial cells are mediated by KLF2, KLF2 coordinates approximately half of the gene expression programs evoked by shear stress. 5,6See accompanying editorial on page 13Recent studies have highlighted the importance of cellular metabolism for the control of endothelial cell phenotype. 7,8 Particularly, it was shown that angiogenic endothelial cells rely heavily on glycolysis for migration and proliferation. The enzyme PFKFB3 is a key regulator of glycolysis in endothelial cells that has been shown to promote angiogenic sprouting.9-11 However, how resting endothelial cells control © 2014 American Heart Association, Inc. Objective-Cellular metabolism was recently shown to regulate endothelial cell phenotype profoundly. Whether the atheroprotective biomechanical stimulus elicited by laminar shear stress modulates endothelial cell metabolism is not known. Approach and Results-Here, we show that laminar flow exposure reduced glucose uptake and mitochondrial content in endothelium. Shear stress-mediated reduction of endothelial metabolism was reversed by silencing the flow-sensitive transcription factor Krüppel-like factor 2 (KLF2). Endothelial-specific deletion of KLF2 in mice induced glucose uptake in endothelial cells of perfused hearts. KLF2 overexpression recapitulates the inhibitory effects on endothelial glycolysis elicited by laminar flow, as measured by Seahorse flux analysis and glucose uptake measurements. RNA sequencing showed that shear stress reduced the expression of key glycolytic enzymes, such as 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3), phosphofructokinase-1, and hexokinase 2 in a KLF2-dependent ma...