12 TAKAKURA N Circulation Journal REVIEW Circ J 2019; 83: 12 – 17 doi: 10.1253/circj.CJ-18-1180 Discovery of a Vascular Endothelial Stem Cell (VESC) Population Required for Vascular Regeneration and Tissue Maintenance Nobuyuki Takakura, MD, PhD The roles that blood vessels play in the maintenance of organs and tissues in addition to the delivery of oxygen and nutrients are being gradually clarified. The maintenance of tissue-specific organ stem cells, such as hematopoietic and neuronal stem cells, is supported by endothelial cells (ECs), which represent an important component of the stem cell niche. The maintenance of organogenesis, for example, osteogenesis and liver generation/regeneration, is supported by molecules referred to as “angiocrine signals” secreted by EC. The mechanisms responsible for the well-known functions of blood vessels, such as thermoregulation and metabolism, especially removal of local metabolites, have now been determined at the molecular level. Following the development of single-cell genetic analysis, blood cell heterogeneity, especially of mural cell populations, has been established and tissue-specific blood vessel formation and function are now also understood at the molecular level. Among the heterogeneous populations of ECs, it seems that a stem cell population with the ability to maintain the production of ECs long-term is present in pre-existing blood vessels. Neovascularization by therapeutic angiogenesis yields benefits in many diseases, not only ischemic disease but also metabolic disease and other vascular diseases. Therefore, vascular endothelial stem cells should be considered to use in vascular regeneration therapy. Key Words: Angiogenesis; Endothelial cells; Stem cells he fundamental role of blood vessels is clearly to related to the improvement of therapies, the development maintain constant supplies of oxygen and nutrients of agents targeting blood vessels is being vigorously pursued T to cells in tissues and organs, as well as the rapid by many pharmacological companies. In order to develop deployment of immune cells to regions of infection or drugs to induce or suppress new blood vessel formation, it damage. In addition to these crucial roles, blood vessels is necessary to understand the cellular and molecular are also involved in the formation of stem cell niches to mechanisms of neovascularization. In the adult, it is well support and maintain the immature phenotype and self- known that new blood vessel formation is mainly induced renewal capacity of organ-specific stem cells.1 Moreover, by sprouting angiogenesis from pre-existing blood vessels. not only for stem cells, vascular endothelial cells (ECs) Two decades ago, endothelial progenitor cells (EPCs) were secrete several growth factors known as “angiocrine signals” identified in the bone marrow and found to promote to maintain the integrity of and assist in the regeneration neovascularization in ischemic regions.5 Although a direct of tissues/organs. One example of this angiocrine signaling contribution of EPCs to ECs is still controversial,6,7 injection is secretion of hepatocyte growth factor and Wnt2 from of bone marrow cells into ischemic regions has been sug- liver sinusoidal ECs to maintain hepatocytes.2 Another gested to yield clinical benefit.8 My group recently discov- example is the function of Noggin from ECs in the bone ered a vascular endothelial stem cell (VESC) population in marrow to support the turnover of osteocytes.3 pre-existing blood vessels and have documented its direct In addition to the function of blood vessels for extravasa- contribution to ECs in ischemia and tissue injury.9–12 By tion of oxygen and nutrients into tissues/organs, capillaries means of cell surface marker studies, a hierarchy of ECs drain materials from organs into intraluminal cavities. traversing a differentiation pathway from immature VESCs Regulation of permeability is an important function of to terminally-differentiated ECs has been identified,12 and blood vessels throughout the body. Particularly in terms of here, I will review blood vessel formation and the role of their tissue-specific functions, control of intravasation and these newly-discovered VESCs. extravasation is extremely important in the brain, liver, lung and kidney. In the brain, removal of waste products Structure of Blood Vessels and Overview of such as amyloid β through the blood-brain barrier is critical Blood Vessel Formation for the maintenance of neuronal function and survival.4 Because regulation of blood vessel formation is directly Blood pumped from the heart into the aorta flows through Received November 1, 2018; accepted November 1, 2018; J-STAGE Advance Publication released online November 28, 2018 Department of Signal Transduction, Research Institute for Microbial Deseases, Osaka University, Suita, Japan Mailing address: Nobuyuki Takakura, MD, PhD, Department of Signal Transduction, Research Institute for Microbial Deseases, Osaka University, 3-1 Yamada-oka, Suita 565-0871, Japan. E-mail: [email protected] ISSN-1346-9843 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Circulation Journal Vol.83, January 2019 Endothelial Stem Cell Population 13 Figure 1. What is the origin of stalk cells? After stimulation with vascular endothelial growth factor (VEGF), tip cell candidates start to secrete DLL4 and stimulate neighboring endothelial cells (ECs). Notch activated in ECs by DLL4 reduces expression of Sox17, resulting in the suppression of VEGFR2, VEGFR3, and neuropilin-1 (NRP1) expression. Tip cell candidates alone respond to VEGF and become tip cells to guide the migration of new branches from pre-existing blood vessels. Behind tip cells, highly proliferating ECs emerge and adhere to tip cells. Neighboring ECs around tip cells may be non-responders to VEGF, which otherwise induces EC proliferation. In this case, where do stalk cells come from? arteries to supply oxygen and nutrients through the capil- ECs phosphorylates VE-cadherin through several signaling laries, returning to the heart through venules and veins. pathways initiated by src phosphorylation.17 Activated Although according to the caliber of the vessel, the wall VE-cadherin is internalized into the cytoplasm of ECs, and may be thinner or thicker, the structure is basically the results in weakening of the junctions between the cells. same, consisting of 2 types of vascular cells. Most of the During physiological blood vessel formation, vascular inner surface is lined with vascular ECs and mural cells leakage is inhibited by mural cell attachment to ECs. When adhering at the basal side of the ECs. In capillaries and these cells have weak junctions, they secrete platelet-derived some areas of venules, pericytes adhere to ECs, and vascular growth factor (PDGF), mainly the PDGF-BB isoform, smooth muscle cells adhere to ECs in arteries and veins. and recruit mural cells expressing the PDGF receptor β.16 Pericytes and vascular smooth muscle cells are collectively During embryogenesis, mural cell populations produce designated “mural” cells. angiopoietin-1, a ligand for Tie2 expressed on ECs. This In capillaries, pericytes adhere to ECs sparsely in order promotes tight EC–EC junctions by cross-linking Tie2 to control permeability and allow ease of diffusion of with Tie2 on neighboring ECs and causing membrane materials through EC–EC junctions. On the other hand, localization of VE-cadherin by inhibiting activation of venules act as gates for extravasation of leukocytes for src.17,18 Finally, mural cells adhere to ECs for final matura- immune responses to invasive challenges. In the venules, tion of blood vessels. The molecular mechanisms of mural pericytes adhere densely to ECs, such that the ratio of ECs cell adhesion to ECs have not been clearly identified; to pericytes is 1:1. It has been recently documented that however, tight adhesion between ECs must be a trigger for leukocytes first migrate between ECs and subsequently this process. enter the parenchyma through gaps between pericytes.13,14 In contrast to embryonic angiogenesis, new blood vessel Blood vessel formation is induced by 2 different processes. formation after birth is usually induced by sprouting and During embryogenesis, ECs developing from the mesoderm extension of vascular branches from pre-existing blood form tubes in situ where blood vessels are required by the vessels, so-called sprouting angiogenesis.16 This process is body plan, and then mural cells are gradually recruited involved in the progression of many vascular diseases near the ECs. When the mural cells adhere to ECs, EC–EC such as cancer, retinopathy and chronic inflammation. By junctions and EC–mural connections are formed and controlling this process, therapeutic interventions in structurally stable blood vessels are created. This de novo angiogenesis have been developed and are in clinical use. vascular formation normally observed in embryos is called 15 vasculogenesis. Functional Diversity of ECs During Angiogenesis ECs developing from the mesoderm form vascular tubes through activation of vascular endothelial growth factor Sprouting angiogenesis is the process by which ECs sprout receptors (VEGFRs).16 Activation of VEGFRs on ECs from pre-existing blood vessels and extend to new plays an important role in proliferation, movement and branches.16 Initially, tip cells emerge from intraluminal matrix reconstitution during blood vessel formation; lining ECs to guide the direction of migration