Shizuka Uchida

Long Noncoding RNA MALAT1 Regulates Endothelial Cell Function and Vessel Growth

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.

Efficient Homing of Multipotent Adult Mesenchymal Stem Cells Depends on FROUNT-Mediated Clustering of CCR2

Circulating stem cells of different origin have been demonstrated to improve repair of various organs both after systemic and local application, although the mechanisms that cause these effects are still not fully understood. We have used a combination of DNA microarray analysis and in vitro migration assays to screen for molecules that mediate homing of long-term renewing adult bone marrow-derived multipotent mesenchymal stem cells (BM-MASCs). We show that the cytokine receptor CCR2 is necessary for organ-specific homing of bone marrow-derived MASCs to the heart in a transgenic mouse model and into hearts damaged by ischemia/reperfusion. Homing and migration of stem cells was dependent on the intracellular adaptor molecule FROUNT, which interacts with CCR2. FROUNT was required for polarization of MASCs, resulting in clustering of CCR2 and reorganization of the cytoskeleton. Recruited MASCs summoned by the CCR2 ligand MCP-1/CCL2 expressed SDF1, which might trap additional bone marrow-derived circulating cells to contribute to the complex process of homing and retention of circulating stem and progenitor cells to remodel diseased organs.

Long Noncoding RNAs in Cardiovascular Diseases

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.

Identification and Characterization of Hypoxia-Regulated Endothelial Circular RNA

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.

Sca1-Derived Cells Are a Source of Myocardial Renewal in the Murine Adult Heart

Summary Although the mammalian heart is one of the least regenerative organs in the body, recent evidence indicates that the myocardium undergoes a certain degree of renewal to maintain homeostasis during normal aging. However, the cellular origin of cardiomyocyte renewal has remained elusive due to lack of lineage tracing experiments focusing on putative adult cardiac precursor cells. We have generated triple-transgenic mice based on the tet-cre system to identify descendants of cells that have expressed the stem cell marker Sca1. We found a significant and lasting contribution of Sca1-derived cells to cardiomyocytes during normal aging. Ischemic damage and pressure overload resulted in increased differentiation of Sca1-derived cells to the different cell types present in the heart. Our results reveal a source of cells for cardiomyocyte renewal and provide a possible explanation for the limited contribution of Sca1-derived cells to myocardial repair under pathological conditions.

Adenosine-to-inosine RNA editing controls cathepsin S expression in atherosclerosis by enabling HuR-mediated post-transcriptional regulation

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.

Noncoder: a web interface for exon array-based detection of long non-coding RNAs

Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called ‘noncoder’. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de.

Identification of right heart-enriched genes in a murine model of chronic outflow tract obstruction

The right ventricle (RV) differs in several aspects from the left ventricle (LV) including its embryonic origin, physiological role and anatomical design. In contrast to LV hypertrophy, little is known about the molecular circuits, which are activated upon RV hypertrophy (RVH). We established a highly reproducible model of RVH in mice using pulmonary artery clipping (PAC), which avoids detrimental RV pressure overload and thus allows long-term survival of operated mice. Magnetic resonance imaging revealed pathognomonic changes with striking similarities to human congenital heart disease- or pulmonary arterial hypertension-patients. Comparative, microarray based transcriptome analysis of right- and left-ventricular remodeling identified distinct transcriptional responses to pressure-induced hypertrophy of either ventricle, which were mainly characterized by stronger transcriptional responses of the RV compared to the LV myocardium. Hierarchic cluster analysis revealed a RV- and LV-specific pattern of gene activity after induction of hypertrophy, however, we did not find evidence for qualitatively distinct regulatory pathways in RV compared to LV. Data mining of nearly three thousand RV-enriched genes under PAC disclosed novel potential (co)-regulators of long-term RV remodeling and hypertrophy. We reason that specific inhibitory mechanisms in RV restrict excessive myocardial hypertrophy and thereby contribute to its vulnerability to pressure overload.

Long Noncoding RNA MANTIS Facilitates Endothelial Angiogenic Function

Background: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs. Methods: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression. Results: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding. Conclusion: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.

Exon Array Analyzer

SUMMARY The Exon Array Analyzer (EAA) is a web server, which provides a user-friendly interface to identify alternative splicing events analyzed with Affymetrix Exon Arrays. The EAA implements the Splice Index algorithm to identify differential expressed exons. The use of various filters allows reduction of the number of false positive hits. Results are presented with detailed annotation information and graphics to identify splice events and to facilitate biological validations. To demonstrate the versatility of the EAA, we analyzed exon arrays of 11 different murine tissues using sample data provided by Affymetrix (http://www.affymetrix.com). Data from the heart were compared with other tissues to identify exons that undergo heart-specific alternatively splicing, resulting in the identification of 885 differentially expressed probe sets in 649 genes. AVAILABILITY The web interface is available at http://EAA.mpi-bn.mpg.de/. Detailed documentation is available on the EAA web site (http://EAA.mpi-bn.mpg.de/supp.php) including screen shots, example analyzes and step by step instructions. CONTACT thomas.braun@mpi-bn.mpg.de SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.