A brain-specific angiogenic mechanism enabled by tip cell specialization.

Vertebrate organs require locally adapted blood vessels. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals.

Mosaic deletion of claudin-5 reveals rapid non-cell-autonomous consequences of blood-brain barrier leakage.

Claudin-5 (CLDN5) is an endothelial tight junction protein essential for blood-brain barrier (BBB) formation. Abnormal CLDN5 expression is common in brain disease, and knockdown of Cldn5 at the BBB has been proposed to facilitate drug delivery to the brain. To study the consequences of CLDN5 loss in the mature brain, we induced mosaic endothelial-specific Cldn5 gene ablation in adult mice (Cldn5).

Molecular anatomy of adult mouse leptomeninges.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence.

A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature.

Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain diseases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing.

Immune-interacting lymphatic endothelial subtype at capillary terminals drives lymphatic malformation.

Oncogenic mutations in PIK3CA, encoding p110α-PI3K, are a common cause of venous and lymphatic malformations. Vessel type-specific disease pathogenesis is poorly understood, hampering development of efficient therapies. Here, we reveal a new immune-interacting subtype of Ptx3-positive dermal lymphatic capillary endothelial cells (iLECs) that recruit pro-lymphangiogenic macrophages to promote progressive lymphatic overgrowth.

A single-cell transcriptomic inventory of murine smooth muscle cells.

Smooth muscle cells (SMCs) execute important physiological functions in numerous vital organ systems, including the vascular, gastrointestinal, respiratory, and urogenital tracts. SMC differ morphologically and functionally at these different anatomical locations, but the molecular underpinnings of the differences remain poorly understood. Here, using deep single-cell RNA sequencing combined with in situ gene and protein expression analysis in four murine organs-heart, aorta, lung, and colon-we identify a molecular basis for high-level differences among vascular, visceral, and airway SMC, as well as more subtle differences between, for example, SMC in elastic and muscular arteries and zonation of elastic artery SMC along the direction of blood flow.

ADAMTS18 villus tip telocytes maintain a polarized VEGFA signaling domain and fenestrations in nutrient-absorbing intestinal blood vessels.

The small intestinal villus tip is the first point of contact for lumen-derived substances including nutrients and microbial products. Electron microscopy studies from the early 1970s uncovered unusual spatial organization of small intestinal villus tip blood vessels: their exterior, epithelial-facing side is fenestrated, while the side facing the villus stroma is non-fenestrated, covered by pericytes and harbors endothelial nuclei. Such organization optimizes the absorption process, however the molecular mechanisms maintaining this highly specialized structure remain unclear.

CD49b identifies functionally and epigenetically distinct subsets of lineage-biased hematopoietic stem cells.

Hematopoiesis is maintained by functionally diverse lineage-biased hematopoietic stem cells (HSCs). The functional significance of HSC heterogeneity and the regulatory mechanisms underlying lineage bias are not well understood. However, absolute purification of HSC subtypes with a pre-determined behavior remains challenging, highlighting the importance of continued efforts toward prospective isolation of homogeneous HSC subsets.

The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells: Implications for COVID-19 vascular research.

Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is important to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed.

A human cell type similar to murine central nervous system perivascular fibroblasts.

The brain vasculature has several specific features, one of them being the blood-brain barrier (BBB), which supports and protects the brain by allowing for the passage of oxygen and nutrients, while at the same time preventing passage of pathogens and toxins. The BBB also prevents efficient delivery of drugs to the brain, e.g.

The infantile myofibromatosis NOTCH3 L1519P mutation leads to hyperactivated ligand-independent Notch signaling and increased PDGFRB expression.

Infantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal dominant forms of IMF are caused by mutations in the gene, but a family carrying a L1519P mutation in the gene has also recently been identified. In this report, we address the molecular consequences of the NOTCH3 mutation and the relationship between the NOTCH and PDGFRB signaling in IMF.

Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss.

Rationale: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive.

Publisher Correction: Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder.

A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature.

Single-cell transcriptomics promise to revolutionize our understanding of the vasculature. Emerging computational methods applied to high-dimensional single-cell data allow integration of results between samples and species and illuminate the diversity and underlying developmental and architectural organization of cell populations. Here, we illustrate these methods in the analysis of mouse lymph node (LN) lymphatic endothelial cells (LEC) at single-cell resolution.

Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta.

Despite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. We report the comprehensive characterization of transcriptomes (bulk and single-cell) and chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse aortic endothelial cells. First, S1PR1 regulates NFκB and nuclear glucocorticoid receptor pathways to suppress inflammation-related mRNAs.

Integrative discovery of treatments for high-risk neuroblastoma.

Despite advances in the molecular exploration of paediatric cancers, approximately 50% of children with high-risk neuroblastoma lack effective treatment. To identify therapeutic options for this group of high-risk patients, we combine predictive data mining with experimental evaluation in patient-derived xenograft cells. Our proposed algorithm, TargetTranslator, integrates data from tumour biobanks, pharmacological databases, and cellular networks to predict how targeted interventions affect mRNA signatures associated with high patient risk or disease processes.

Astrocyte-microglial association and matrix composition are common events in the natural history of primary familial brain calcification.

Primary familial brain calcification (PFBC) is an age-dependent and rare neurodegenerative disorder characterized by microvascular calcium phosphate deposits in the deep brain regions. Known genetic causes of PFBC include loss-of-function mutations in genes involved in either of three processes-platelet-derived growth factor (PDGF) signaling, phosphate homeostasis or protein glycosylation-with unclear molecular links. To provide insight into the pathogenesis of PFBC, we analyzed murine models of PFBC for the first two of these processes in Pdgfb and Slc20a2 mice with regard to the structure, molecular composition, development and distribution of perivascular calcified nodules.

Heterogeneity and plasticity in healthy and atherosclerotic vasculature explored by single-cell sequencing.

Cellular characteristics and their adjustment to a state of disease have become more evident due to recent advances in imaging, fluorescent reporter mice, and whole genome RNA sequencing. The uncovered cellular heterogeneity and/or plasticity potentially complicates experimental studies and clinical applications, as markers derived from whole tissue 'bulk' sequencing is unable to yield a subtype transcriptome and specific markers. Here, we propose definitions on heterogeneity and plasticity, discuss current knowledge thereof in the vasculature and how this may be improved by single-cell sequencing (SCS).

Publisher Correction: Claudin-3-deficient C57BL/6J mice display intact brain barriers.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Claudin-3-deficient C57BL/6J mice display intact brain barriers.

The tight junction protein claudin-3 has been identified as a transcriptional target of the Wnt/β-catenin signaling pathway regulating blood-brain barrier (BBB) maturation. In neurological disorders loss of claudin-3 immunostaining is observed at the compromised BBB and blood-cerebrospinal fluid barrier (BCSFB). Although these observations support a central role of claudin-3 in regulating brain barriers' tight junction integrity, expression of claudin-3 at the brain barriers has remained a matter of debate.

Prolonged systemic hyperglycemia does not cause pericyte loss and permeability at the mouse blood-brain barrier.

Diabetes mellitus is associated with cognitive impairment and various central nervous system pathologies such as stroke, vascular dementia, or Alzheimer's disease. The exact pathophysiology of these conditions is poorly understood. Recent reports suggest that hyperglycemia causes cerebral microcirculation pathology and blood-brain barrier (BBB) dysfunction and leakage.

Single-Cell mRNA Sequencing of the Mouse Brain Vasculature.

In this chapter, we describe a method for analyzing the vasculature of the mouse brain using single-cell transcriptomics. More specifically, we focus on the use of fluorescence-activated cell sorting (FACS) for selection of the cells of interest and sorting of these cells in a 384-well format, allowing for enrichment for the cells being studied. Furthermore, we outline the Smart-Seq2 single-cell library construction method for transforming single-cell mRNA into Illumina sequencing compatible libraries.

Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types.

Vascular diseases are major causes of death, yet our understanding of the cellular constituents of blood vessels, including how differences in their gene expression profiles create diversity in vascular structure and function, is limited. In this paper, we describe a single-cell RNA sequencing (scRNA-seq) dataset that defines vascular and vessel-associated cell types and subtypes in mouse brain and lung. The dataset contains 3,436 single cell transcriptomes from mouse brain, which formed 15 distinct clusters corresponding to cell (sub)types, and another 1,504 single cell transcriptomes from mouse lung, which formed 17 cell clusters.