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.

In recent year, increasing evidence suggests that noncoding RNAs play important roles in the regulation of tissue homeostasis and pathophysiological conditions. Besides small noncoding RNAs (eg, microRNAs), >200-nucleotide long transcripts, namely long noncoding RNAs (lncRNAs), can interfere with gene expressions and signaling pathways at various stages. In the cardiovascular system, studies have detected and characterized the expression of lncRNAs under normal physiological condition and in disease states. Several lncRNAs are regulated during acute myocardial infarction (eg, Novlnc6 ) and heart failure (eg, Mhrt ), whereas others control hypertrophy, mitochondrial function and apoptosis of cardiomyocytes. In the vascular system, the endothelial-expressed lncRNAs (eg, MALAT1 and Tie-1-AS) can regulate vessel growth and function, whereas the smooth-muscle–expressed lncRNA smooth muscle and endothelial cell–enriched migration/differentiation-associated long noncoding RNA was recently shown to control the contractile phenotype of smooth muscle cells. This review article summarizes the data on lncRNA expressions in mouse and human and highlights identified cardiovascular lncRNAs that might play a role in cardiovascular diseases. Although our understanding of lncRNAs is still in its infancy, these examples may provide helpful insights how lncRNAs interfere with cardiovascular 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.

Intensive research in past two decades has uncovered the presence and importance of noncoding RNAs (ncRNAs), which includes microRNAs (miRs) and long ncRNAs (lncRNAs). These two classes of ncRNAs interact to a certain extent, as some lncRNAs bind to miRs to sequester them. Such lncRNAs are collectively called ‘competing endogenous RNAs’ or ‘miRNA sponges’. In this study, we screened for lncRNAs that may act as miRNA sponges using the publicly available data sets and databases. To uncover the roles of miRNA sponges, loss-of-function experiments were conducted, which revealed the biological roles as miRNA sponges. LINC00324 is important for the cell survival by binding to miR-615-5p leading to the de-repression of its target BTG2. LOC400043 controls several biological functions via sequestering miR-28-3p and miR-96-5p, thereby changing the expressions of transcriptional regulators. Finally, we also screened for circular RNAs (circRNAs) that may function as miRNA sponges. The results were negative at least for the selected circRNAs in this study. In conclusion, miRNA sponges can be identified by applying a series of bioinformatics techniques and validated with biological experiments.