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  • 1
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    Single-cell RNA sequencing profiling of mouse endothelial cells in response to pulmonary arterial hypertension
    Oxford University Press (OUP) ; 2022
    In:  Cardiovascular Research Vol. 118, No. 11 ( 2022-08-24), p. 2519-2534
    Details
    In: Cardiovascular Research, Oxford University Press (OUP), Vol. 118, No. 11 ( 2022-08-24), p. 2519-2534
    Abstract: Endothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution. Methods and results We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature. Conclusion This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.
    Type of Medium: Online Resource
    ISSN: 0008-6363 , 1755-3245
    URL: Article
    DOI: 10.1093/cvr/cvab296
    RVK:
    WA 15000
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2022
    detail.hit.zdb_id: 1499917-1
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  • 2
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    Single-cell transcriptome analyses reveal novel targets modulating cardiac neovascularization by resident endothelial cells following myocardial infarction
    Oxford University Press (OUP) ; 2019
    In:  European Heart Journal Vol. 40, No. 30 ( 2019-08-07), p. 2507-2520
    Details
    In: European Heart Journal, Oxford University Press (OUP), Vol. 40, No. 30 ( 2019-08-07), p. 2507-2520
    Abstract: A better understanding of the pathways that regulate regeneration of the coronary vasculature is of fundamental importance for the advancement of strategies to treat patients with heart disease. Here, we aimed to investigate the origin and clonal dynamics of endothelial cells (ECs) associated with neovascularization in the adult mouse heart following myocardial infarction (MI). Furthermore, we sought to define murine cardiac endothelial heterogeneity and to characterize the transcriptional profiles of pro-angiogenic resident ECs in the adult mouse heart, at single-cell resolution. Methods and results An EC-specific multispectral lineage-tracing mouse (Pdgfb-iCreERT2-R26R-Brainbow2.1) was used to demonstrate that structural integrity of adult cardiac endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region, without significant contributions from bone marrow cells or endothelial-to-mesenchymal transition. Ten transcriptionally discrete heterogeneous EC states, as well as the pathways through which each endothelial state is likely to enhance neovasculogenesis and tissue regeneration following ischaemic injury were defined. Plasmalemma vesicle-associated protein (Plvap) was selected for further study, which showed an endothelial-specific and increased expression in both the ischaemic mouse and human heart, and played a direct role in regulating human endothelial proliferation in vitro. Conclusion We present a single-cell gene expression atlas of cardiac specific resident ECs, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. These data provide a rich resource that could assist in the development of new therapeutic interventions to augment endogenous myocardial perfusion and enhance regeneration in the injured heart.
    Type of Medium: Online Resource
    ISSN: 0195-668X , 1522-9645
    URL: Article
    DOI: 10.1093/eurheartj/ehz305
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2019
    detail.hit.zdb_id: 2001908-7
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  • 3
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    Human and murine fibroblast single-cell transcriptomics reveals fibroblast clusters are differentially affected by ageing and serum cholesterol
    Oxford University Press (OUP) ; 2023
    In:  Cardiovascular Research Vol. 119, No. 7 ( 2023-07-04), p. 1509-1523
    Details
    In: Cardiovascular Research, Oxford University Press (OUP), Vol. 119, No. 7 ( 2023-07-04), p. 1509-1523
    Abstract: Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVDs). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolaemia and ageing. Methods and results Murine aorta single-cell RNA-sequencing analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, whereas traditional markers such as the cluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating along trajectories. Three trajectories, marked by CD55 (Cd55+), Cxcl chemokine 14 (Cxcl14+), and lysyl oxidase (Lox+), were reproduced in an independent RNA-seq dataset. Gene ontology (GO) analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation, and/or collagen fibril organization, respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in the adventitia of aged C57BL/6J mice, and mildly hypercholesterolaemic LDLR KO mice on chow by flow cytometry. The expansion of collagen-related CXCL14+ and LOX+ fibroblasts in aged and hypercholesterolaemic aortic adventitia, respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14+ and LOX+ fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets are differentially correlated with human atherosclerotic plaque traits. Conclusion We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and CVD in humans and mice. Biological relevance is evident from the regulation of fibroblast clusters by age and hypercholesterolaemia in vivo, associations with human atherosclerotic plaque traits, and enrichment of genes with a GWAS for CVD.
    Type of Medium: Online Resource
    ISSN: 0008-6363 , 1755-3245
    URL: Article
    DOI: 10.1093/cvr/cvad016
    RVK:
    WA 15000
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2023
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  • 4
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    Abstract 714: Mapping the Transcriptional Dynamics of Pluripotent Stem Cell-derived Endothelial Cell Differentiation by Single Cell RNA Sequencing
    Ovid Technologies (Wolters Kluwer Health) ; 2019
    In:  Circulation Research Vol. 125, No. Suppl_1 ( 2019-08-02)
    Details
    In: Circulation Research, Ovid Technologies (Wolters Kluwer Health), Vol. 125, No. Suppl_1 ( 2019-08-02)
    Abstract: Objectives: Human embryonic stem cell derived endothelial cell (hESC-EC) differentiation offers a model to study the process of endothelial development, commitment and maturation, thus potentially informing future regeneration strategies. We used high throughput single cell RNA sequencing (scRNA-seq) to characterise the cellular heterogeneity and associated transcriptional signatures throughout hESC-EC differentiation. Methods and Results: Using an established eight-day hESC-EC (H9) differentiation protocol yielding 66% CD31 + /CD144 + cells, we sampled cells for scRNA-seq (10X Chromium) at pluripotent (day 0), mesodermal (day 4), early (day 6) and late (day 8) endothelial stages (total 21,369 cells). The pluripotent and mesodermal population were largely homogeneous prior to the emergence of distinct endothelial and mesenchymal populations. Pseudotime analysis characterised the transcriptional signatures throughout differentiation, revealing a bifurcation point giving rise to both populations. Repeating the study with a second hESC line (RC11) produced equivalent populations and transcriptional signatures. In addition, scRNA-seq analysis of an alternative hESC-EC differentiation protocol revealed substantially more heterogeneity, including nephrogenic and haemogenic populations. However, pseudotime analysis revealed common expression of known and uncharacterised endothelial transcription factors across both protocols. Comparison of hESC-EC to foetal and mature EC revealed their transcriptional distinction, suggesting their non-specified nature. Conclusions: We sequenced over 105,000 cells allowing characterisation of hESC-EC differentiation across different hESC lines and protocols. We identified a common bifurcation point and remarkable concordance between the transcriptional signatures across different hESC lines and differentiation protocols. This included identifying novel transcription factors that may drive endothelial commitment and maturation.
    Type of Medium: Online Resource
    ISSN: 0009-7330 , 1524-4571
    URL: Article
    DOI: 10.1161/res.125.suppl_1.714
    RVK:
    XA 10000
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2019
    detail.hit.zdb_id: 1467838-X
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  • 5
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    Abstract 103: Single Cell Transcriptome Analyses Reveal Novel Targets for Therapeutic Neovascularisation by Resident Endothelial Cells in the Heart
    Ovid Technologies (Wolters Kluwer Health) ; 2019
    In:  Circulation Research Vol. 125, No. Suppl_1 ( 2019-08-02)
    Details
    In: Circulation Research, Ovid Technologies (Wolters Kluwer Health), Vol. 125, No. Suppl_1 ( 2019-08-02)
    Abstract: Aims: A better understanding of the pathways that regulate regeneration of the coronary vasculature is important for future strategies to treat patients with heart disease. We investigated (i) the clonal dynamics of endothelial cells (EC) associated with neovascularization in the ischemic border region (ii) transcriptional signatures of regenerative EC in the ischemic heart using single cell RNA-sequencing (iii) the functional relevance of selected targets. Methods: MI was induced in ‘EC-Confetti’ mice by coronary artery ligation. EC clonal proliferation was quantified or hearts dissociated for scRNAseq. Immunofluorescence staining for targets identified by scRNAseq was performed on cardiac tissue from patients with ischemic heart disease. EC proliferation was assessed in vitro following siRNA gene silencing. Results: EC-Confetti mice express YFP, RFP, GFP, or CFP specifically in EC. Fluorophores are inherited by EC progeny following proliferation, allowing quantitative clonal analysis. Clonal proliferation was significantly increased in the infarct border at 7 days post-MI compared to healthy hearts ( P 〈 0.0001). Ten transcriptionally discrete EC clusters were defined following scRNAseq with 3 clusters predominantly composed of cells from the MI group, indicating their gene expression profiles may be relevant to neovasculogenic pathways. We selected plasmalemma vesicle associated protein (Plvap) for further study and confirmed EC-specific increased Plvap expression in ischemic border regions of human ( P =0.002) and mouse ( P =0.002) hearts, compared to healthy myocardium. siRNA gene silencing of Plvap significantly inhibited EC proliferation ( P = 0.0038), strong evidence that Plvap can directly modulate EC function. Conclusions: Generation of new blood vessels following ischemic injury in the mouse heart is predominantly mediated by clonal proliferation of resident EC. We present a gene expression atlas of resident cardiac EC, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. This resource identifies novel targets, including Plvap, that may augment myocardial perfusion post-MI, and inform future design of strategies aimed at promoting vascular perfusion in ischemic heart disease.
    Type of Medium: Online Resource
    ISSN: 0009-7330 , 1524-4571
    URL: Article
    DOI: 10.1161/res.125.suppl_1.103
    RVK:
    XA 10000
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2019
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  • 6
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    Transcriptional dynamics of pluripotent stem cell-derived endothelial cell differentiation revealed by single-cell RNA sequencing
    Oxford University Press (OUP) ; 2020
    In:  European Heart Journal Vol. 41, No. 9 ( 2020-03-01), p. 1024-1036
    Details
    In: European Heart Journal, Oxford University Press (OUP), Vol. 41, No. 9 ( 2020-03-01), p. 1024-1036
    Abstract: Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. Methods and results A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. Conclusion A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.
    Type of Medium: Online Resource
    ISSN: 0195-668X , 1522-9645
    URL: Article
    DOI: 10.1093/eurheartj/ehz351
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2020
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  • 7
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    Mapping the developing human cardiac endothelium at single-cell resolution identifies MECOM as a regulator of arteriovenous gene expression
    Oxford University Press (OUP) ; 2022
    In:  Cardiovascular Research Vol. 118, No. 14 ( 2022-11-10), p. 2960-2972
    Details
    In: Cardiovascular Research, Oxford University Press (OUP), Vol. 118, No. 14 ( 2022-11-10), p. 2960-2972
    Abstract: Coronary vasculature formation is a critical event during cardiac development, essential for heart function throughout perinatal and adult life. However, current understanding of coronary vascular development has largely been derived from transgenic mouse models. The aim of this study was to characterize the transcriptome of the human foetal cardiac endothelium using single-cell RNA sequencing (scRNA-seq) to provide critical new insights into the cellular heterogeneity and transcriptional dynamics that underpin endothelial specification within the vasculature of the developing heart. Methods and results We acquired scRNA-seq data of over 10 000 foetal cardiac endothelial cells (ECs), revealing divergent EC subtypes including endocardial, capillary, venous, arterial, and lymphatic populations. Gene regulatory network analyses predicted roles for SMAD1 and MECOM in determining the identity of capillary and arterial populations, respectively. Trajectory inference analysis suggested an endocardial contribution to the coronary vasculature and subsequent arterialization of capillary endothelium accompanied by increasing MECOM expression. Comparative analysis of equivalent data from murine cardiac development demonstrated that transcriptional signatures defining endothelial subpopulations are largely conserved between human and mouse. Comprehensive characterization of the transcriptional response to MECOM knockdown in human embryonic stem cell-derived EC (hESC-EC) demonstrated an increase in the expression of non-arterial markers, including those enriched in venous EC. Conclusions scRNA-seq of the human foetal cardiac endothelium identified distinct EC populations. A predicted endocardial contribution to the developing coronary vasculature was identified, as well as subsequent arterial specification of capillary EC. Loss of MECOM in hESC-EC increased expression of non-arterial markers, suggesting a role in maintaining arterial EC identity.
    Type of Medium: Online Resource
    ISSN: 0008-6363 , 1755-3245
    URL: Article
    DOI: 10.1093/cvr/cvac023
    RVK:
    WA 15000
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2022
    detail.hit.zdb_id: 1499917-1
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