Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.

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. In order to allow user-friendly access to our data, we constructed a searchable database (http://betsholtzlab.org/VascularSingleCells/database.html). Our dataset constitutes a comprehensive molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung, and as such provides a strong foundation for future studies of vascular development and diseases.

Mural cells (MCs) consisting of vascular smooth muscle cells and pericytes cover the endothelial cells (ECs) to regulate vascular stability and homeostasis. Here, we clarified the mechanism by which MCs develop and cover ECs by generating transgenic zebrafish lines that allow live imaging of MCs and by lineage tracing in vivo. To cover cranial vessels, MCs derived from either neural crest cells or mesoderm emerged around the preformed EC tubes, proliferated and migrated along EC tubes. During their migration, the MCs moved forward by extending their processes along the inter-EC junctions, suggesting a role for inter-EC junctions as a scaffold for MC migration. In the trunk vasculature, MCs derived from mesoderm covered the ventral side of the dorsal aorta (DA), but not the posterior cardinal vein. Furthermore, the MCs migrating from the DA or emerging around intersegmental vessels (ISVs) preferentially covered arterial ISVs rather than venous ISVs, indicating that MCs mostly cover arteries during vascular development. Thus, live imaging and lineage tracing enabled us to clarify precisely how MCs cover the EC tubes and to identify the origins of MCs.

Endothelial cells (ECs) line the inside of blood vessels and respond to mechanical cues generated by blood flow. Mechanical stimuli regulate the localization of YAP by reorganizing the actin cytoskeleton. Here we demonstrate blood-flow-mediated regulation of endothelial YAP in vivo. We indirectly monitored transcriptional activity of Yap1 (zebrafish YAP) and its spatiotemporal localization in living zebrafish and found that Yap1 entered the nucleus and promoted transcription in response to blood flow. In cultured human ECs, laminar shear stress induced nuclear import of YAP and its transcriptional activity in a manner independent of Hippo signaling. We uncovered a molecular mechanism by which flow induced the nuclear translocation of YAP through the regulation of filamentous actin and angiomotin. Yap1 mutant zebrafish showed a defect in vascular stability, indicating an essential role for Yap1 in blood vessels. Our data imply that endothelial Yap1 functions in response to flow to maintain blood vessels.

Pericytes, the mural cells of blood microvessels, regulate microvascular development and function and have been implicated in many brain diseases. However, due to a paucity of defining markers, pericyte identification and functional characterization remain ambiguous and data interpretation problematic. In mice carrying two transgenic reporters, Pdgfrb-eGFP and NG2-DsRed, we found that double-positive cells were vascular mural cells, while the single reporters marked additional, but non-overlapping, neuroglial cells. Double-positive cells were isolated by fluorescence-activated cell sorting (FACS) and analyzed by RNA sequencing. To reveal defining patterns of mural cell transcripts, we compared the RNA sequencing data with data from four previously published studies. The meta-analysis provided a conservative catalogue of 260 brain mural cell-enriched gene transcripts. We validated pericyte-specific expression of two novel markers, vitronectin (Vtn) and interferon-induced transmembrane protein 1 (Ifitm1), using fluorescent in situ hybridization and immunohistochemistry. We further analyzed signaling pathways and interaction networks of the pericyte-enriched genes in silico. This work provides novel insight into the molecular composition of brain mural cells. The reported gene catalogue facilitates identification of brain pericytes by providing numerous new candidate marker genes and is a rich source for new hypotheses for future studies of brain mural cell physiology and pathophysiology.

Histamine-induced vascular leakage is an integral component of many highly prevalent human diseases, including allergies, asthma, and anaphylaxis. Yet, how histamine induces the disruption of the endothelial barrier is not well defined. By using genetically modified animal models, pharmacologic inhibitors, and a synthetic biology approach, here we show that the small GTPase RhoA mediates histamine-induced vascular leakage. Histamine causes the rapid formation of focal adherens junctions, disrupting the endothelial barrier by acting on H1R Gαq-coupled receptors, which is blunted in endothelial Gαq/11 KO mice. Interfering with RhoA and ROCK function abolishes endothelial permeability, while phospholipase Cβ plays a limited role. Moreover, endothelial-specific RhoA gene deletion prevents vascular leakage and passive cutaneous anaphylaxis in vivo, and ROCK inhibitors protect from lethal systemic anaphylaxis. This study supports a key role for the RhoA signaling circuitry in vascular permeability, thereby identifying novel pharmacological targets for many human diseases characterized by aberrant vascular leakage.

Rap1 potentiates endothelial cell junctions by spatially controlling non-muscle myosin II activity through activation of the Cdc42–MRCK pathway and suppression of the Rho–ROCK pathway.

During angiogenesis in vivo, endothelial cells (ECs) at the tips of vascular sprouts actively extend filopodia that are filled with bundles of linear actin filaments. To date, signaling pathways involved in the formation of endothelial filopodia have been studied using in-vitro-cultured ECs that behave differently from those in vivo. Herein, we have delineated a signaling pathway that governs the assembly of endothelial filopodia during angiogenic sprouting of the caudal vein plexus (CVP) in zebrafish. During CVP formation, bone morphogenetic protein induces the extension of endothelial filopodia and their migration via Arhgef9b-mediated activation of Cdc42. Active Cdc42 binds to and stimulates Formin-like 3, an actin-regulatory protein of the formin family, which, in turn, promotes the extension of endothelial filopodia to facilitate angiogenic sprouting of the CVP. Thus, this study has elucidated molecular mechanisms underlying the formation of endothelial filopodia and their role in angiogenesis in vivo.