Enhancer-associated long non-coding RNA LEENE regulates endothelial nitric oxide synthase and endothelial function

Miao, Yifei; Ajami, Nassim E.; Huang, Tse‐Shun; Lin, Feng‐Mao; Lou, Chih-Hong; Wang, Yunting; Li, Shuai; Kang, Jian; Munkacsi, Hannah; Maurya, Mano Ram; Gupta, Shakti; Chien, Shu; Subramaniam, Shankar; Chen, Zhen

doi:10.1038/s41467-017-02113-y

Cited by 142 publications

(120 citation statements)

References 65 publications

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“…The delivery of the CRISPR/Cas9 components into primary endothelial and other hard-to-transfect cells has been a major obstacle for cardiovascular research. Plasmids, lenti-, adeno-or adeno-associated viral vectors have most often been used to achieve sufficient gene knockout rates in ECs (Abrahimi et al, 2015;Cullere, Plovie, Bennett, MacRae, & Mayadas, 2015;Gong et al, 2017;Miao et al, 2018;Wu et al, 2017). Just recently, we have demonstrated that gene knockouts in human ECs can be established with a crR-NA:tracrRNA:Cas9 RNP approach (Schwefel et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Precise CCM1 gene correction and inactivation in patient‐derived endothelial cells: Modeling Knudson's two‐hit hypothesis in vitro

Spiegler

Rath

Much

et al. 2019

Molec Gen & Gen Med

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Background The CRISPR/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard‐to‐transfect cells remains challenging. Methods In a proof‐of‐principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a CCM1 mutation carrier with multiple CCMs. In a CRISPR/Cas9 gene correction approach, a high‐fidelity Cas9 variant was then transfected into patient‐derived BOECs using a ribonucleoprotein complex and a single‐strand DNA oligonucleotide. In addition, patient‐specific CCM1 knockout clones were expanded after CRISPR/Cas9 gene inactivation. Results Deep sequencing demonstrated correction of the mutant allele in nearly 33% of all cells whereas no CRISPR/Cas9‐induced mutations in predicted off‐target loci were identified. Corrected BOECs could be cultured in cell mixtures but demonstrated impaired clonal survival. In contrast, CCM1 ‐deficient BOECs displayed increased resistance to stress‐induced apoptotic cell death and could be clonally expanded to high passages. When cultured together, CCM1 ‐deficient BOECs largely replaced corrected as well as heterozygous BOECs. Conclusion We here demonstrate that a non‐viral CRISPR/Cas9 approach can not only be used for gene knockout but also for precise gene correction in hard‐to‐transfect endothelial cells (ECs). Comparing patient‐derived isogenic CCM1 +/+ , CCM1 +/− , and CCM1 −/− ECs, we show that the inactivation of the second allele results in clonal evolution of ECs lacking CCM1 which likely reflects the initiation phase of CCM genesis.

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Section: Discussionmentioning

confidence: 99%

Precise CCM1 gene correction and inactivation in patient‐derived endothelial cells: Modeling Knudson's two‐hit hypothesis in vitro

Spiegler

Rath

Much

et al. 2019

Molec Gen & Gen Med

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“…Our studies demonstrate that the latest human genetics approaches such as caQTL mapping (23), allelic imbalance assay (24), and CRISPR-based assays (20,21) are powerful tools to investigate possible genetic contributions to cellular mechanotransduction. Miao et al recently applied CRISPR-Cas9 to achieve high efficiency of a 10 kb deletion of an enhancer region in bulk HUVEC (40). In this study we expanded the applications of CRISPR-based techniques to investigate key vascular functions.…”

Section: Discussionmentioning

confidence: 99%

A genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics

Krause

Huang

et al. 2018

Preprint

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Biomechanical cues dynamically control major cellular processes but whether genetic variants actively participate in mechano-sensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies have identified chromosome 1p32.2 as one of the most strongly associated loci with CAD/IS; however, the causal mechanism related to this locus remains unknown. Employing statistical analyses, ATAC-seq, and H3K27ac/H3K4me2 ChIP-Seq in human aortic endothelium (HAEC), our results demonstrate that rs17114036, a common noncoding polymorphism at the 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR/Cas9-based genome editing shows that rs17114036containing region promotes endothelial quiescence under unidirectional flow by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays further demonstrate that CAD/IS protective allele at rs17114036 in PLPP3 intron 5 confers an increased endothelial enhancer activity. ChIP-PCR and luciferase assays show that CAD/IS protective allele at rs17114036 creates a binding site for transcription factor Krüppel-like factor 2, which increases the enhancer activity under unidirectional flow. These results demonstrate for the first time that a human single-nucleotide polymorphism contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cisregulatory elements provide a previously unappreciated layer of regulatory control in cellular mechano-sensing mechanisms. Significance StatementBiomechanical stimuli control major cellular functions and play critical roles in the pathogenesis of diverse human diseases. Although recent studies have implicated genetic variation in regulating key biological processes, whether human genetic variants contribute to the cellular mechano-sensing mechanisms remains unclear. This study provides the first line of evidence supporting an underappreciated role of genetic 2 predisposition in cellular mechanotransduction mechanisms. Employing epigenomic profiling, genome-editing, and latest human genetics approaches, our data demonstrate that rs17114036, a common noncoding polymorphism implicated in coronary artery disease and ischemic stroke by genome-wide association studies, dynamically regulates endothelial responses to blood flow (hemodynamics) related to atherosclerosis via regulation of an intronic enhancer. The results provide new molecular insights linking disease-associated genetic variants to cellular mechanobiology.

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“…ChIP assays were performed as previously described (Miao et al, 2018). Briefly, 1×10 7 human fetal endocardial cells or iPSC-ECs were treated with 0.75% formaldehyde for 20 minutes at room temperature.…”

Section: Chromatin Immunoprecipitationmentioning

confidence: 99%

Single-Cell RNA-Seq Reveals Endocardial Defect in Hypoplastic Left Heart Syndrome

Miao

Tian

Martin

et al. 2019

Preprint

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Hypoplastic left heart syndrome (HLHS) is one of the most challenging forms of congenital heart diseases. Previous studies were mainly focused on intrinsic defects in myocardium. However, this does not sufficiently explain the abnormal development of the cardiac valve, septum, and vasculature, known to originate from the endocardium. Here, using single-cell RNA profiling, induced pluripotent stem cells, and human fetal heart tissue with an underdeveloped left ventricle, we identified a developmentally impaired endocardial population in HLHS. The intrinsic endocardial deficits contributed to abnormal endothelial to mesenchymal transition, NOTCH signaling, and extracellular matrix organization, all of which are key factors in valve formation.Consequently, endocardial abnormalities conferred reduced proliferation and maturation of cardiomyocytes through a disrupted fibronectin-integrin interaction. Several recently described HLHS de novo mutations were associated with abnormal endocardial gene and FN1 regulation and expression. Our studies provide a rationale for considering endocardial function in future regenerative strategies for HLHS.Page 5 together, our findings have uncovered a mechanism whereby a developmentally impaired endocardium can underlie the ventricular and valvular hypoplasia in HLHS. Results HLHS patient de novo mutations were highly enriched in the endocardial and endothelial populations in a developing human heart.It is well appreciated that the genetics of HLHS is complex. Previously, a set of congenital heart disease (CHD) related de novo mutations (DNMs) were reported (Jin et al., 2017), some of which were strongly associated with HLHS. To further understand the role of these HLHS-associated DNMs in the development of human heart, we first examined their expression level at single cell resolution. A normal human fetal heart at a gestational age of 83 days was micro-dissected into the left-and right-side of the heart. Each side was enzymatically dispersed as single cells, enriched with endocardial/endothelial cells using a CD144 antibody and processed for single-cell RNA-sequencing (scRNA-seq) ( Figure 1A). The majority of the cell types expected to be present in the heart were successfully retrieved by scRNA-seq analysis as shown in the UMAP projection, including a certain amount of CMs (TNNI3 + ) in addition to pan-ECs (CDH5 +

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Enhancer-associated long non-coding RNA LEENE regulates endothelial nitric oxide synthase and endothelial function

Cited by 142 publications

References 65 publications

Precise CCM1 gene correction and inactivation in patient‐derived endothelial cells: Modeling Knudson's two‐hit hypothesis in vitro

Precise CCM1 gene correction and inactivation in patient‐derived endothelial cells: Modeling Knudson's two‐hit hypothesis in vitro

A genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics

Single-Cell RNA-Seq Reveals Endocardial Defect in Hypoplastic Left Heart Syndrome

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