© 2020. Published by The Company of Biologists Ltd | Development (2020) 147, dev146621. doi:10.1242/dev.146621 REVIEW Understanding angiodiversity: insights from single cell biology Moritz Jakab1,2,3 and Hellmut G. Augustin1,3,4,* ABSTRACT directly exposed to the circulating blood, they are highly responsive Blood vessels have long been considered as passive conduits for and sensitive to changes in the physical and biochemical properties delivering blood. However, in recent years, cells of the vessel wall of the blood. This not only determines the genetic, transcriptomic (endothelial cells, smooth muscle cells and pericytes) have emerged and proteomic profiles of distinct types of vessels in an organ- as active, highly dynamic components that orchestrate crosstalk specific manner (Augustin and Koh, 2017), but also impacts the between the circulation and organs. Encompassing the whole body signalling of individual vascular cells within vessels. We refer to ‘ ’ and being specialized to the needs of distinct organs, it is not surprising this heterogeneity as angiodiversity (Fig. 2). that vessel lining cells come in different flavours. There is calibre- A prototypic example of intravascular angiodiversity is seen in specific specialization (arteries, arterioles, capillaries, venules, veins), the sinusoidal capillaries of the liver. Oxygen-rich blood from the but also organ-specific heterogeneity in different microvascular beds hepatic artery and nutrient-rich blood from the portal vein converge (continuous, discontinuous, sinusoidal). Recent technical advances in in the hepatic sinusoidal capillaries and exit the liver via the central the field of single cell biology have enabled the profiling of thousands of vein, leading to a strong gradient of available nutrients and oxygen single cells and, hence, have allowed for the molecular dissection of along the vessel. Conversely, hepatocytes are molecularly and such angiodiversity, yielding a hitherto unparalleled level of spatial and functionally zonated along the axis of the liver lobule. Indeed, functional resolution. Here, we review how these approaches have recent elegant work has established that EC-derived angiocrine contributed to our understanding of angiodiversity. signals exert instructive functions to orchestrate liver zonation (Wang et al., 2015; Halpern et al., 2017). Central vein ECs and KEY WORDS: Angiodiversity, Endothelial cell, Smooth muscle cell, sinusoidal ECs near the central vein express the short-range signals Single cell biology Wnt2 and Wnt9b, which are essential for creating a niche in which self-renewing Lgr5-positive hepatocytes are harboured. Loss of Introduction Wnt expression consequently leads to a loss of self-renewing The limited diffusion distance of oxygen in tissues (100-150 µm) hepatocytes, with subsequent perturbation of liver zonation and implies that all cells of the body – with few exceptions (e.g. impaired regenerative capacity of the liver (Leibing et al., 2018; cartilage) – are in close proximity to the nearest capillary. The Preziosi et al., 2018; Wang et al., 2015). vascular system can thereby be considered as a systemically This example of liver zonation is merely a prototypic stage- disseminated organ. In fact, the vascular endothelium forms one of setting example. It highlights, on the one hand, the need and, on the the body’s largest surfaces and it is estimated that an 80 kg adult other hand, the enormous scientific opportunities of deconvoluting harbours about 1 kg of endothelial cells (ECs), making them one of angiodiversity at the highest possible resolution. Indeed, recent the most abundant cell populations of the body. technological advances in the field of single cell biology have Structurally, blood vessels come in different flavours and show allowed for the transcriptional, epigenetic and genetic profiling of distinct morphological features that reflect their functional thousands of individual cells, thereby providing insights into specialization as arteries, arterioles, capillaries, venules and veins angiodiversity in various other contexts. These studies have enabled (Fig. 1). These different types of blood vessels are exposed to the molecular dissection of single cells in individual vessels and the varying physical and biochemical milieus, which are organ- and identification of crucial subsets of vascular cells as well as rare situation-specific and affect the molecular properties of their subpopulations, which may not be captured in bulk approaches. In cellular building blocks. Conceptually, blood vessel-forming cells this Review, we discuss how single cell RNA-sequencing (scRNA- have long been viewed as passive bystander cell populations that seq; see Boxes 1 and 2) approaches have contributed to our facilitate barrier-function, demarcating blood and solid tissues. understanding of cellular angiodiversity. We first review how However, recent work has shown that vascular and perivascular scRNA-seq studies of individual organs have uncovered a high cells do not just respond to exogenous cytokines, but rather perform degree of heterogeneity in vascular cells. We then highlight how active gatekeeper roles and secrete growth factors and signals into such studies have provided insights into spatial aspects of their microenvironment. Such vascular cell-derived paracrine- angiodiversity. Finally, we discuss how angiodiversity contributes acting growth factors have been designated as ‘angiocrine’ factors to vascular function and responsiveness in various contexts. (reviewed by Rafii et al., 2016). Moreover, as vascular cells are Assessing vascular heterogeneity and building cellular atlases 1European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, 68167 Mannheim, Germany. 2Division of Vascular Oncology Comparison of vascular cells in adult mice at single cell resolution and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH An initial effort to assemble a single cell atlas exclusively of Alliance), 69120 Heidelberg, Germany. 3Faculty of Biosciences, Heidelberg University, 69120 Heidelberg, Germany. 4German Cancer Consortium, 69120 vascular cells was made by the group of Christer Betsholtz Heidelberg, Germany. (Vanlandewijck et al., 2018). In this study, ECs, mural cells and fibroblast-like cells were isolated from the brain and the lungs of *Author for correspondence ([email protected]) transgenic mouse reporter lines. As a result, 3436 single cell H.G.A., 0000-0002-7173-4242 transcriptomes from the adult mouse brain and 1504 single cell DEVELOPMENT 1 REVIEW Development (2020) 147, dev146621. doi:10.1242/dev.146621 Artery ArterioleCapillary Venule Vein Smooth muscle cell Endothelial cell Tunica intima Tunica media Tunica externa Continuous capillary Fenestrated capillary Sinusoidal capillary Lumen Endothelial cell Basal lamina Tight junction Pores with diaphragm Gap Capillary Artery Arteriole Venule Vein Continuous Fenestrated Sinusoidal Pressure ↑↑↑↑ ↑↑ ↑ ~ ↑↑ Oxygen ↑↑↑ ↑↑↑ ↑ ~~ Velocity ↑↑↑ ↑ ~ ↑↑↑ Diameter/ lumen (in µm) Human ~4000/2000 ~30/10 ~8/6 ~20/16 ~5000/4000 Mouse ~150/50 ~18/9 ~5/4 ~14/12 ~250/150 Demarcation, Accelerated Free fluid Primary Supply of Major site of passive Collection of fluid and and Reabsorption of rheological oxygenated vascular water and deoxygenated solute solute interstitial fluid function blood resistance solute blood exchange exchange exchange Fig. 1. Schematic of the key structural and rheological features of blood vessels. Arteries (except for pulmonary and umbilical arteries) carry oxygenated blood away from the heart. Arterioles consist of only tunica media and intima, and represent the primary site of vascular resistance. Capillaries are lined by a single layer of ECs. Typically, three major types of capillaries can be distinguished: continuous, fenestrated, and sinusoidal. Continuous capillaries are present in most organs and perform distinct barrier functions permitting diffusion of water, small solutes and lipid-soluble material. Fenestrated and sinusoidal capillaries both display discontinuity in their EC lining. Whereas fenestrated capillaries have intracellular pores that are spanned by radially oriented fibrils forming a diaphragm, sinusoidal endothelia are characterized by flattened, irregular shapes and are devoid of a basement membrane. Fenestrated ECs are found in organs with intense exchange (e.g. endocrine organs for the secretion of macromolecules) and in organs with filtration and absorption functions (e.g. the kidney). Sinusoidal ECs form the endothelium in haematopoietic organs including the bone marrow, spleen and liver. Blood from capillaries is collected in venules and drained into veins. Compared with arterioles, venules have a tunica externa and a media, although their tunica media is poorly developed so that venules have thinner walls and a larger lumen compared with arterioles. Compared with arteries, veins have a thick tunica externa, but generally a much thinner media. transcriptomes from adult mouse lungs were profiled. A substantial found to be highly heterogeneous, showing strong tissue diversity of vascular cell transcriptomes could be identified in both specialization (Table 2). The metabolic needs and general organs. The brain dataset formed 15 clusters corresponding to functions of a given tissue were also reflected in the distinct cell types and cell subtypes by means of coherent marker transcriptomes of capillary ECs, and ECs from organs with expression, whereas the lung dataset formed 17 cell