The optimal expression of endothelial nitric oxide synthase (eNOS), the hallmark of endothelial homeostasis, is vital to vascular function. Dynamically regulated by various stimuli, eNOS expression is modulated at transcriptional, post-transcriptional, and post-translational levels. However, epigenetic modulations of eNOS, particularly through long non-coding RNAs (lncRNAs) and chromatin remodeling, remain to be explored. Here we identify an enhancer-associated lncRNA that enhances eNOS expression (LEENE). Combining RNA-sequencing and chromatin conformation capture methods, we demonstrate that LEENE is co-regulated with eNOS and that its enhancer resides in proximity to eNOS promoter in endothelial cells (ECs). Gain- and Loss-of-function of LEENE differentially regulate eNOS expression and EC function. Mechanistically, LEENE facilitates the recruitment of RNA Pol II to the eNOS promoter to enhance eNOS nascent RNA transcription. Our findings unravel a new layer in eNOS regulation and provide novel insights into cardiovascular regulation involving endothelial function.

Bone marrow-derived mesenchymal stem cells (MSC) have been promoted for multiple therapeutic applications. Many beneficial effects of MSCs are paracrine, dependent on extracellular vesicles (EVs). Although MSC-derived EVs (mEVs) are beneficial for acute lung injury and pulmonary fibrosis, mechanisms of mEV uptake by lung fibroblasts and their effects on myofibroblastic differentiation have not been established. We demonstrate that mEVs, but not fibroblast EVs (fEVs), suppress TGFβ1-induced myofibroblastic differentiation of normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. MEVs display increased time- and dose-dependent cellular uptake compared to fEVs. Removal or blocking of Thy-1, or blocking Thy-1-beta integrin interactions, decreased mEV uptake and prevented suppression of myofibroblastic differentiation. MicroRNAs (miRs) 199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p are selectively packaged in mEVs. In silico analyses indicated that IPF lung fibroblasts have increased expression of genes that are targets of mEV-enriched miRs. MiR-630 mimics blocked TGFβ1 induction of CDH2 in normal and IPF fibroblasts, and antagomiR-630 abrogated the effect of mEV on CDH2 expression. These data suggest that the interaction of Thy-1 with beta integrins mediates mEV uptake by lung fibroblasts, which blocks myofibroblastic differentiation, and that mEVs are enriched for miRs that target profibrotic genes up-regulated in IPF fibroblasts.

MicroRNAs (miRNAs) are small RNAs ∼22 nt in length that are involved in the regulation of a variety of physiological and pathological processes. Advances in high-throughput small RNA sequencing (smRNA-seq), one of the next-generation sequencing applications, have reshaped the miRNA research landscape. In this study, we established an integrative database, the YM500 (http://ngs.ym.edu.tw/ym500/), containing analysis pipelines and analysis results for 609 human and mice smRNA-seq results, including public data from the Gene Expression Omnibus (GEO) and some private sources. YM500 collects analysis results for miRNA quantification, for isomiR identification (incl. RNA editing), for arm switching discovery, and, more importantly, for novel miRNA predictions. Wetlab validation on >100 miRNAs confirmed high correlation between miRNA profiling and RT-qPCR results (R = 0.84). This database allows researchers to search these four different types of analysis results via our interactive web interface. YM500 allows researchers to define the criteria of isomiRs, and also integrates the information of dbSNP to help researchers distinguish isomiRs from SNPs. A user-friendly interface is provided to integrate miRNA-related information and existing evidence from hundreds of sequencing datasets. The identified novel miRNAs and isomiRs hold the potential for both basic research and biotech applications.

Objective— The topographical distribution of atherosclerosis in vasculature underscores the importance of shear stress in regulating endothelium. With a systems approach integrating sequencing data, the current study aims to explore the link between shear stress-regulated master transcription factor and its regulation of endothelial cell (EC) function via epigenetic modifications. Approach and Results— H3K27ac (acetylation of histone 3 lysine 27)-ChIP-seq (chromatin immunoprecipitation followed by high throughput sequencing), ATAC-seq (an assay for transposase-accessible chromatin-sequencing), and RNA-seq (RNA-sequencing) were performed to investigate the genome-wide epigenetic regulations in ECs in response to atheroprotective pulsatile shear stress (PS). In silico prediction revealed that KLF4 binding motifs were enriched in the PS-enhanced H3K27ac regions. By integrating PS- and KLF4-modulated H3K27ac, we identified 18 novel PS-upregulated genes. The promoter regions of these genes showed an overlap between the KLF4-enhanced assay for transposase-accessible chromatin signals and the PS-induced H3K27ac peaks. Experiments using ECs isolated from mouse aorta, lung ECs from EC-KLF4-TG versus EC-KLF4-KO mice, and atorvastatin-treated ECs showed that ITPR3 (inositol 1,4,5-trisphosphate receptor 3) was robustly activated by KLF4 and statins. KLF4 ATAC-qPCR (quantitative polymerase chain reaction) and ChIP-qPCR further demonstrated that a specific locus in the promoter region of the ITPR3 gene was essential for KLF4 binding, H3K27ac enrichment, chromatin accessibility, RNA polymerase II recruitment, and ITPR3 transcriptional activation. Deletion of this KLF4 binding locus in ECs by using CRISPR-Cas9 resulted in blunted calcium influx, reduced expression of endothelial nitric oxide synthase, and diminished nitric oxide bioavailability. Conclusions— These results from a novel multiomics study suggest that KLF4 is crucial for PS-modulated H3K27ac that allow the transcriptional activation of ITPR3. This novel mechanism contributes to the Ca 2+ -dependent eNOS (endothelial nitric oxide synthase) activation and EC homeostasis.