Single-Cell Transcriptome Atlas of Murine Endothelial Cells.

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

A high-resolution view of the heterogeneous aging endothelium.

Vascular endothelial cell (EC) aging has a strong impact on tissue perfusion and overall cardiovascular health. While studies confined to the investigation of aging-associated vascular readouts in one or a few tissues have already drastically expanded our understanding of EC aging, single-cell omics and other high-resolution profiling technologies have started to illuminate the intricate molecular changes underlying endothelial aging across diverse tissues and vascular beds at scale. In this review, we provide an overview of recent insights into the heterogeneous adaptations of the aging vascular endothelium. We address critical questions regarding tissue-specific and universal responses of the endothelium to the aging process, EC turnover dynamics throughout lifespan, and the differential susceptibility of ECs to acquiring aging-associated traits. In doing so, we underscore the transformative potential of single-cell approaches in advancing our comprehension of endothelial aging, essential to foster the development of future innovative therapeutic strategies for aging-associated vascular conditions.

Single-cell RNA sequencing of cystic fibrosis liver disease explants reveals endothelial complement activation.

BACKGROUND & AIMS: Cystic fibrosis (CF) is considered a multisystemic disorder in which CF-associated liver disease (CFLD) is the third most common cause of mortality. Currently, no effective treatment is available for CFLD because its pathophysiology is still unclear. Interestingly, CFLD exhibits identical vascular characteristics as non-cirrhotic portal hypertension, recently classified as porto-sinusoidal vascular disorders (PSVD). METHODS: Since endothelial cells (ECs) are an important component in PSVD, we performed single-cell RNA sequencing (scRNA-seq) on four explant livers from CFLD patients to identify differential endothelial characteristics which could contribute to the disease. We comprehensively characterized the endothelial compartment and compared it with publicly available scRNA-seq datasets from cirrhotic and healthy livers. Key gene signatures were validated ex vivo on patient tissues. RESULTS: We found that ECs from CF liver explants are more closely related to healthy than cirrhotic patients. In CF patients we also discovered a distinct population of liver sinusoidal ECs-coined CF LSECs-upregulating genes involved in the complement cascade and coagulation. Finally, our immunostainings further validated the predominant periportal location of CF LSECs. CONCLUSIONS: Our work showed novel aspects of human liver ECs at the single-cell level thereby supporting endothelial involvement in CFLD, and reinforcing the hypothesis that ECs could be a driver of PSVD. Therefore, considering the vascular compartment in CF and CFLD may help developing new therapeutic approaches for these diseases.

EndoDB: a database of endothelial cell transcriptomics data.

Endothelial cells (ECs) line blood vessels, regulate homeostatic processes (blood flow, immune cell trafficking), but are also involved in many prevalent diseases. The increasing use of high-throughput technologies such as gene expression microarrays and (single cell) RNA sequencing generated a wealth of data on the molecular basis of EC (dys-)function. Extracting biological insight from these datasets is challenging for scientists who are not proficient in bioinformatics. To facilitate the re-use of publicly available EC transcriptomics data, we developed the endothelial database EndoDB, a web-accessible collection of expert curated, quality assured and pre-analyzed data collected from 360 datasets comprising a total of 4741 bulk and 5847 single cell endothelial transcriptomes from six different organisms. Unlike other added-value databases, EndoDB allows to easily retrieve and explore data of specific studies, determine under which conditions genes and pathways of interest are deregulated and assess reprogramming of metabolism via principal component analysis, differential gene expression analysis, gene set enrichment analysis, heatmaps and metabolic and transcription factor analysis, while single cell data are visualized as gene expression color-coded t-SNE plots. Plots and tables in EndoDB are customizable, downloadable and interactive. EndoDB is freely available at https://vibcancer.be/software-tools/endodb, and will be updated to include new studies.

Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation.

BACKGROUND: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. METHODS: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. RESULTS: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. CONCLUSIONS: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

Post-transcriptional regulation in hematopoiesis: RNA binding proteins take control 1.

Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP-RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.

Antisense Oligonucleotide-Based Rescue of Complex Intronic Splicing Defects in ABCA4.

The ABCA4 gene, involved in Stargardt disease, has a high percentage of splice-altering pathogenic variants, some of which cause complex RNA defects. Although antisense oligonucleotides (AONs) have shown promising results in splicing modulation, they have not yet been used to target complex splicing defects. Here, we performed AON-based rescue studies on ABCA4 complex splicing defects. Intron 13 variants c.1938-724A>G, c.1938-621G>A, c.1938-619A>G, and c.1938-514A>G all lead to the inclusion of different pseudo-exons (PEs) with and without an upstream PE (PE1). Intron 44 variant c.6148-84A>T results in multiple PE inclusions and/or exon skipping events. Five novel AONs were designed to target these defects. AON efficacy was assessed by in vitro splice assays using midigenes containing the variants of interest. All screened complex splicing defects were effectively rescued by the AONs. Although varying levels of efficacy were observed between AONs targeting the same PEs, for all variants at least one AON restored splicing to levels comparable or better than wildtype. In conclusion, AONs are a promising approach to target complex splicing defects in ABCA4.

Transcriptomic signatures of progressive and regressive liver fibrosis and portal hypertension.

Persistent liver injury triggers a fibrogenic program that causes pathologic remodeling of the hepatic microenvironment (i.e., liver fibrosis) and portal hypertension. The dynamics of gene regulation during liver disease progression and early regression remain understudied. Here, we generated hepatic transcriptome profiles in two well-established liver disease models at peak fibrosis and during spontaneous regression after the removal of the inducing agents. We linked the dynamics of key disease readouts, such as portal pressure, collagen area, and transaminase levels, to differentially expressed genes, enabling the identification of transcriptomic signatures of progressive vs. regressive liver fibrosis and portal hypertension. These candidate biomarkers (e.g., Tcf4, Mmp7, Trem2, Spp1, Scube1, Islr) were validated in RNA sequencing datasets of patients with cirrhosis and portal hypertension, and those cured from hepatitis C infection. Finally, deconvolution identified major cell types and suggested an association of macrophage and portal hepatocyte signatures with portal hypertension and fibrosis area.

The evolving functions of the vasculature in regulating adipose tissue biology in health and obesity.

Adipose tissue is an endocrine organ and a crucial regulator of energy storage and systemic metabolic homeostasis. Additionally, adipose tissue is a pivotal regulator of cardiovascular health and disease, mediated in part by the endocrine and paracrine secretion of several bioactive products, such as adipokines. Adipose vasculature has an instrumental role in the modulation of adipose tissue expansion, homeostasis and metabolism. The role of the adipose vasculature has been extensively explored in the context of obesity, which is recognized as a global health problem. Obesity-induced accumulation of fat, in combination with vascular rarefaction, promotes adipocyte dysfunction and induces oxidative stress, hypoxia and inflammation. It is now recognized that obesity-associated endothelial dysfunction often precedes the development of cardiovascular diseases. Investigations have revealed heterogeneity within the vascular niche and dynamic reciprocity between vascular and adipose cells, which can become dysregulated in obesity. Here we provide a comprehensive overview of the evolving functions of the vasculature in regulating adipose tissue biology in health and obesity.

Neuroepithelial organoid patterning is mediated by a neighborhood watch mechanism.

During epithelial tissue patterning, morphogens operate across multiple length scales to instruct cell identities. However, how cell fate changes are coordinated over these scales to establish spatial organization remains poorly understood. Here, we use human neural tube organoids as models of epithelial patterning and develop an in silico approach to define conditions permissive to patterning. By systematically varying morphogen position, diffusivity, and fate-inducing concentration levels, we show that cells follow a "neighborhood watch" (NW) mechanism that is deterministically dictated by initial morphogen source positions, reflecting scale-invariant in vitro phenotypes. We define how the frequency and local bias of morphogen sources stabilize pattern orientation. The model predicts enhanced patterning through floor plate inhibition, and receptor-ligand interaction analysis of single-cell RNA sequencing (scRNA-seq) data identifies wingless-related integration site (WNT) and bone morphogenic protein (BMP) as inhibition modulators, which we validate in vitro. These results suggest that developing neuroepithelia employ NW-based mechanisms to organize morphogen sources, define cellular identity, and establish patterns.

Deciphering endothelial heterogeneity in health and disease at single-cell resolution: progress and perspectives.

Endothelial cells (ECs) constitute the inner lining of vascular beds in mammals and are crucial for homeostatic regulation of blood vessel physiology, but also play a key role in pathogenesis of many diseases, thereby representing realistic therapeutic targets. However, it has become evident that ECs are heterogeneous, encompassing several subtypes with distinct functions, which makes EC targeting and modulation in diseases challenging. The rise of the new single-cell era has led to an emergence of studies aimed at interrogating transcriptome diversity along the vascular tree, and has revolutionized our understanding of EC heterogeneity from both a physiological and pathophysiological context. Here, we discuss recent landmark studies aimed at teasing apart the heterogeneous nature of ECs. We cover driving (epi)genetic, transcriptomic, and metabolic forces underlying EC heterogeneity in health and disease, as well as current strategies used to combat disease-enriched EC phenotypes, and propose strategies to transcend largely descriptive heterogeneity towards prioritization and functional validation of therapeutically targetable drivers of EC diversity. Lastly, we provide an overview of the most recent advances and hurdles in single EC OMICs.

Prioritization and functional validation of target genes from single-cell transcriptomics studies.

Translation of academic results into clinical practice is a formidable unmet medical need. Single-cell RNA-sequencing (scRNA-seq) studies generate long descriptive ranks of markers with predicted biological function, but without functional validation, it remains challenging to know which markers truly exert the putative function. Given the lengthy/costly nature of validation studies, gene prioritization is required to select candidates. We address these issues by studying tip endothelial cell (EC) marker genes because of their importance for angiogenesis. Here, by tailoring Guidelines On Target Assessment for Innovative Therapeutics, we in silico prioritize previously unreported/poorly described, high-ranking tip EC markers. Notably, functional validation reveals that four of six candidates behave as tip EC genes. We even discover a tip EC function for a gene lacking in-depth functional annotation. Thus, validating prioritized genes from scRNA-seq studies offers opportunities for identifying targets to be considered for possible translation, but not all top-ranked scRNA-seq markers exert the predicted function.

In Vivo Screening Unveils Pervasive RNA-Binding Protein Dependencies in Leukemic Stem Cells and Identifies ELAVL1 as a Therapeutic Target.

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML. SIGNIFICANCE: LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171.

The pulmonary vasculature in lethal COVID-19 and idiopathic pulmonary fibrosis at single-cell resolution.

AIMS: Severe acute respiratory syndrome coronavirus-2 infection causes COVID-19, which in severe cases evokes life-threatening acute respiratory distress syndrome (ARDS). Transcriptome signatures and the functional relevance of non-vascular cell types (e.g. immune and epithelial cells) in COVID-19 are becoming increasingly evident. However, despite its known contribution to vascular inflammation, recruitment/invasion of immune cells, vascular leakage, and perturbed haemostasis in the lungs of severe COVID-19 patients, an in-depth interrogation of the endothelial cell (EC) compartment in lethal COVID-19 is lacking. Moreover, progressive fibrotic lung disease represents one of the complications of COVID-19 pneumonia and ARDS. Analogous features between idiopathic pulmonary fibrosis (IPF) and COVID-19 suggest partial similarities in their pathophysiology, yet, a head-to-head comparison of pulmonary cell transcriptomes between both conditions has not been implemented to date. METHODS AND RESULTS: We performed single-nucleus RNA-sequencing on frozen lungs from 7 deceased COVID-19 patients, 6 IPF explant lungs, and 12 controls. The vascular fraction, comprising 38 794 nuclei, could be subclustered into 14 distinct EC subtypes. Non-vascular cell types, comprising 137 746 nuclei, were subclustered and used for EC-interactome analyses. Pulmonary ECs of deceased COVID-19 patients showed an enrichment of genes involved in cellular stress, as well as signatures suggestive of dampened immunomodulation and impaired vessel wall integrity. In addition, increased abundance of a population of systemic capillary and venous ECs was identified in COVID-19 and IPF. COVID-19 systemic ECs closely resembled their IPF counterparts, and a set of 30 genes was found congruently enriched in systemic ECs across studies. Receptor-ligand interaction analysis of ECs with non-vascular cell types in the pulmonary micro-environment revealed numerous previously unknown interactions specifically enriched/depleted in COVID-19 and/or IPF. CONCLUSIONS: This study uncovered novel insights into the abundance, expression patterns, and interactomes of EC subtypes in COVID-19 and IPF, relevant for future investigations into the progression and treatment of both lethal conditions.

The microRNA Lifecycle in Health and Cancer.

MicroRNAs (miRNAs) are small non-coding RNAs of ~22 nucleotides that regulate gene expression at the post-transcriptional level. They can bind to around 60% of all protein-coding genes with an average of 200 targets per miRNA, indicating their important function within physiological and pathological cellular processes. miRNAs can be quickly produced in high amounts through canonical and non-canonical pathways that involve a multitude of steps and proteins. In cancer, miRNA biogenesis, availability and regulation of target expression can be altered to promote tumour progression. This can be due to genetic causes, such as single nucleotide polymorphisms, epigenetic changes, differences in host gene expression, or chromosomal remodelling. Alternatively, post-transcriptional changes in miRNA stability, and defective or absent components and mediators of the miRNA-induced silencing complex can lead to altered miRNA function. This review provides an overview of the current knowledge on the lifecycle of miRNAs in health and cancer. Understanding miRNA function and regulation is fundamental prior to potential future application of miRNAs as cancer biomarkers.

Generation of vessel co-option lung metastases mouse models for single-cell isolation of metastases-derived cells and endothelial cells.

Tumor vessel co-option, a process in which cancer cells "hijack" pre-existing blood vessels to grow and invade healthy tissue, is poorly understood but is a proposed resistance mechanism against anti-angiogenic therapy (AAT). Here, we describe protocols for establishing murine renal (RENCA) and breast (4T1) cancer lung vessel co-option metastases models. Moreover, we outline a reproducible protocol for single-cell isolation from murine lung metastases using magnetic-activated cell sorting as well as immunohistochemical stainings to distinguish vessel co-option from angiogenesis. For complete details on the use and execution of this protocol, please refer to Teuwen et al. (2021).

Single-cell RNA sequencing profiling of mouse endothelial cells in response to pulmonary arterial hypertension.

AIMS: 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.

Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast.

Since a detailed inventory of endothelial cell (EC) heterogeneity in breast cancer (BC) is lacking, here we perform single cell RNA-sequencing of 26,515 cells (including 8433 ECs) from 9 BC patients and compare them to published EC taxonomies from lung tumors. Angiogenic ECs are phenotypically similar, while other EC subtypes are different. Predictive interactome analysis reveals known but also previously unreported receptor-ligand interactions between ECs and immune cells, suggesting an involvement of breast EC subtypes in immune responses. We also identify a capillary EC subtype (LIPEC (Lipid Processing EC)), which expresses genes involved in lipid processing that are regulated by PPAR-γ and is more abundant in peri-tumoral breast tissue. Retrospective analysis of 4648 BC patients reveals that treatment with metformin (an indirect PPAR-γ signaling activator) provides long-lasting clinical benefit and is positively associated with LIPEC abundance. Our findings warrant further exploration of this LIPEC/PPAR-γ link for BC treatment.

Endothelial cell heterogeneity and microglia regulons revealed by a pig cell landscape at single-cell level.

Pigs are valuable large animal models for biomedical and genetic research, but insights into the tissue- and cell-type-specific transcriptome and heterogeneity remain limited. By leveraging single-cell RNA sequencing, we generate a multiple-organ single-cell transcriptomic map containing over 200,000 pig cells from 20 tissues/organs. We comprehensively characterize the heterogeneity of cells in tissues and identify 234 cell clusters, representing 58 major cell types. In-depth integrative analysis of endothelial cells reveals a high degree of heterogeneity. We identify several functionally distinct endothelial cell phenotypes, including an endothelial to mesenchymal transition subtype in adipose tissues. Intercellular communication analysis predicts tissue- and cell type-specific crosstalk between endothelial cells and other cell types through the VEGF, PDGF, TGF-ß, and BMP pathways. Regulon analysis of single-cell transcriptome of microglia in pig and 12 other species further identifies MEF2C as an evolutionally conserved regulon in the microglia. Our work describes the landscape of single-cell transcriptomes within diverse pig organs and identifies the heterogeneity of endothelial cells and evolutionally conserved regulon in microglia.