Identification of Laminar Flow Regulated Non-Coding RNAs in Endothelial Cells

Laminar flow across endothelial cells (ECs) protects against atherosclerotic lesion formation in part via activating the transcription factor KLF2. Previously, it was shown that laminar flow regulates a number of microRNAs, including the atheroprotective miR-143/-145. As known from genome sequencing only 2% of the whole genome encodes for proteins. The vast majority is transcribed as non-protein-coding sequences that are classified in short (<200 nt) and long (>200 nt) non-coding RNAs (lncRNAs). lncRNAs have been described to play an important role in many biological processes. We hypothesize that shear stress-regulated lncRNAs are involved in physiological and pathophysiological processes in the vascular wall. Therefore, we determined the expression levels of lncRNAs by next generation sequencing in RNA isolated from static control and laminar flow-exposed ECs. Our results indicate that in HUVEC, 22% (7311) of all (polyA+) RNAs are lncRNAs. Of these lncRNAs, 19% were up-regulated and 15% down-regulated by laminar flow. Next, we confirmed the induction of one the most prominent lncRNAs MALAT1 by flow (3.39± 0.62 fold induction) by RT-qPCR. Additionally, we showed that MALAT1 is also up-regulated by the transcription factor KLF2 (2.18± 0.33 fold induction, p<0.05). MALAT1 has been shown to localize to nuclear bodies. Therefore, we analyzed the subcellular localization and identified that MALAT1 is indeed localized in the nucleus in HUVECs. To address the putative role of MALAT1, we measured endothelial cell sprouting and determined a potential anti-inflammatory property of MALAT1. MALAT1 was efficiently silenced with siRNAs and gapmeRs. Silencing of MALAT1 induced sprouting angiogenesis in vitro (2.51±0.25 fold, p<0.05). MALAT1 silencing additionally augmented the expression of the adhesion molecules VCAM1, ICAM1 and E-Selectin under baseline conditions and after TNF-α stimulation. In summary, our data identify endothelial ncRNAs. Furthermore, these data demonstrate that shear stress alters the expression of lncRNAs in ECs, in particular MALAT1. We also identified MALAT1 as an anti-angiogenic and anti-inflammatory lncRNA in ECs. The up-regulation of MALAT1 by laminar flow may contribute to the anti-atherosclerotic effects of flow.