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]

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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 of the new however, it also promotes vascular leakage. Vascular branch. Behind these tip cells, ECs (so-called “stalk cells”) hyperpermeability is induced by phosphorylation of vascular with high proliferative capacity migrate to connect with tip endothelial (VE) cadherin. VEGFR activated by VEGF on cells and facilitate extension of the new branch. Finally,

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ECs (so-called “phalanx cells”) emerge and assist in the mesenchymal stromal cells that can differentiate into ECs maturation of the new blood vessels by inducing tight in vitro has also been documented in the same region.29 adhesion between ECs and connections between ECs and However, the contribution of these vascular cells to neovas- mural cells.19,20 cular ECs in vivo has not been unequivocally demonstrated. Stimulation of ECs in pre-existing blood vessels by VEGF Under certain specific culture conditions, some cells may induces secretion of delta-like 4 (DLL4), which activates transdifferentiate into the EC lineage from other cell types. Notch expressed on neighboring ECs. This inhibits the From this point of view, identification of VESCs that are expression of Sox17, a transcriptional factor, resulting in already committed to the EC lineage (and indeed proven transcriptional suppression of VEGFRs such as VEGFR2, to contribute to ECs in a pathological and physiological VEGFR3, and neuropilin-1.21 Therefore, by this lateral angiogenesis process) has long been and still is required. inhibition, ECs strongly expressing DLL4 become tip cells and migration of neighboring ECs next to the tip cells is Endothelial SP Cells suppressed because they do not respond to VEGF. Here, the question arises in terms of the origin of stalk cells. ECs Methods to analyze the cellular phenotype of ECs by flow stimulated by DLL4 become non-responsive to VEGF cytometry are not well established. One reason for this is because of decreased VEGFR expression; however, stalk the difficulty in isolating single-cell suspensions from cells behind tip cells proliferate in response to VEGF. This primary organs. In the case of the hematopoietic system, suggests that stalk cells come from another place where based on sorting bone marrow hematopoietic cells by DLL4 cannot affect Notch expression by ECs and cannot different cell surface markers, stem cell populations defined adhere to tip cells for extension of new branches (Figure 1). by their bone marrow regenerative ability have been In the resting state, phenotypically different ECs such as isolated. Thus far, cell surface markers such as c-kit, sca-1, arterial, venous, and lymphatic ECs are present. In addition, and hematopoietic lineage markers have been generally at least 3 types of different ECs (i.e., tip, stalk, and phalanx used in this field of study.1 ECs and hematopoietic cells cells) emerge during angiogenesis; thus there is a great deal are derived from common progenitor (ancestor) cells, the of heterogeneity among ECs. Moreover, ECs need plasticity so-called hemangioblasts; however, identification of stem because they must adapt to the requirements for tissue/ cells committed to the endothelial lineage has not been organ-specific blood vessel formation. accomplished using cell surface markers. Because of this To date, tissue/organ-specific stem cell populations such lack of unequivocal stem cell markers for ECs, commonly as hematopoietic, neuronal, gut and epithelial stem cells used techniques for identification of stem cells need to be have been isolated and their contribution to tissue regenera- used (i.e., the SP method). SP cells were briefly mentioned tion and integrity of tissue and organs has been elucidated.22 before, but more detail is now provided. It is hypothesized that VESC populations may also be SP cell isolation is a method of purifying stem cell present in pre-existing blood vessels and could give rise to populations using the universal property of high stem cell endothelial progenitors (stalk cells) connecting to tip cells expression of drug efflux pumps such as ATP-binding for acute elongation of new blood vessels. Several lines of cassette transporters. In this approach, cell suspensions are evidence have documented the existence of immature cells treated with the DNA-binding dye Hoechst33342. This in the vascular wall that can give rise to vascular ECs and fluorescent dye is taken up by most cells (main population smooth muscle cells. [MP]), but some remain unlabeled (SP) and can be separated by FACS. Stem cells with high efflux capacity are enriched Residual Immature Vascular Cells in the SP population. Hematopoietic stem cells have been in the Vascular Wall enriched by this Hoechst efflux method using blue emission wavelength fluorescence,30 but subsequently, by using 2 Adult (somatic) stem cells are cells that differentiate into different emission wavelengths, more enriched hemato- progenitor cells with proliferative capacity and giving rise poietic stem cell populations have been identified.31 Later, to terminally-differentiated tissue-specific cells.23 Several this method was used to isolate stem cells for which no cell studies have reported that tissue-resident cells can differen- surface markers had been identified (i.e., heart, mammary tiate into vascular lineage cells such as ECs and mural gland, liver, lung, testis, epidermis and muscle stem cells.24,25 cells).32–38 The wall of a large blood vessel has 3 layers: the tunica In pre-existing blood vessels, a line of evidence previously intima, tunica media, and tunica adventitia. In mice, vas- indicated the presence of SP cells in the vascular wall and cular progenitor cells derived from adult aorta have been reported that they can differentiate into ECs. However, in identified in the tunica media. 26 In that study, single-cell that paper, the authors showed no living ECs in their suspensions were found to contain side population (SP) single-cell suspensions, thus making it impossible to identify cells, as determined by fluorescence-activated cell sorting EC lineage-committed immature cells. Indeed, the technical (FACS) analysis. These were characterized as CD31− Sca1+ aspects of manipulating blood vessels and making single-cell and could differentiate into ECs and smooth muscle cells suspensions are challenging because ECs form such tight in vitro. Other groups identified the presence of stem and junctions with each other and adhere tightly to mural cells. progenitor cells in the adventitia. In one study, isolated Different methods of preparing single-cell suspensions Sca1+ cells in the adventitia of large and medium-sized have been explored and detection of SP cells in EC lineage- arteries and veins from adult apoE-deficient mice were committed ECs of pre-existing blood vessels has been shown to be able to differentiate into smooth muscle cells, accomplished.9 Hence, the features of SP cells can be but not ECs, in vitro and in vivo.27 In another study, described in detail next. CD34+CD31−VEGFR2+ vascular wall cells were shown to CD31 is generally used as an EC marker, but it is form capillary sprouts in the region between the media and expressed on some hematopoietic cells as well. Therefore, adventitia.28 Moreover, the presence of resident angiogenic it is necessary to define ECs as CD31-positive and at the

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Figure 2. Resident endothelial stem cell population localized on the intraluminal surface of blood vessels, where EC-SP cells produce EC progenitors and contribute to newly-developed blood vessels as terminally-differentiated ECs. EC, endothelial cell; SP, side population.

same time CD45 negative (CD31+CD45−). Among such from blood vessels (Figure 2). CD31+CD45− ECs, approximately 1% are in the SP fraction Analyzing EC-SP cells in tumors, taken as an ischemic in most organs that have been investigated, including liver, tissue, an approximately 10-fold higher frequency of these heart, lung, brain and muscle. The proliferative capacity of cells relative to normal tissues (0.7% in normal lung vs. 7% primary ECs from murine tissues and organs has been in lung cancer) has been found.11 As ischemia induces evaluated using OP9 osteoblastic stromal cells derived RNA synthesis in EC-SP cells in the hindlimb ischemia from OP/OP mice.39,40 When SP or MP cells (i.e., EC-SP model, ischemia must be one of the triggers promoting cells or EC-MP cells) are cultured on OP9 cells, the latter proliferation (self-renewal) of vascular EC-SP cells. Thus, rarely generate EC colonies, and those that form are small it can be concluded that EC-SP cells may be the source of (the frequency of colony-forming units is approximately stalk cells during sprouting angiogenesis and contribute to 1/100 cells), whereas the former generate very large EC acute proliferation of ECs. colonies at a frequency of approximately 1 in 8 SP cells. Cells passaged from EC-SP cell primary cultures are able Hierarchy of ECs Marked by CD157 to generate secondary EC colonies on fresh OP9 cells but cells from EC-MP colonies are unable to do so. Detection of stem cell populations by the Hoechst method On cell-cycle analysis, EC-SP cells are dormant, but they is a promising approach, but it is context dependent. In initiate RNA synthesis when exposed to ischemia. The order to prove the stemness of ECs, their hierarchy from high drug efflux capacity of EC-SP cells is consistent with stem cells to terminally-differentiated ECs and their self- their high levels of expression of ABC transporters (i.e., renewal potential should ideally be proven. To do this ABCG2, ABCB1a, ABCB2, and ABCA5). In contrast, definitively, injection of a single cell should be shown to expression of other endothelial-related molecules is similar cause a long-term vascular EC contribution and expansion between EC-SP cells and EC-MP cells. Similar levels of as a stem cell population. The hindlimb ischemia model is VE-cadherin, VEGFR2, and CD34 suggest that EC-SP not appropriate for demonstrating single-cell transplanta- cells are indeed EC lineage-committed ECs. tion reconstitution ability. Using a liver injury model of Occlusion of the femoral artery causing hindlimb isch- monocrotaline to induce damage to liver sinusoidal ECs, it emia is a frequently used mouse model of neovasculariza- was found that a single EC-SP cell from the liver could tion. In this model, it was found that EC-MP cells could reconstitute ECs long-term. Which molecules are expressed not generate functional blood vessels but ECs derived from on EC-SP cells specifically has also been investigated and EC-SP cells were able to generate entire tubular formations 2 markers, CD200 and CD157,12 which are highly expressed of new blood vessels containing large amounts of red blood by VESCs have been found. cells and connecting with recipient blood vessels. Moreover, CD200 is an immunoglobulin superfamily membrane- blood vessels in the hindlimb were gradually replaced by anchored glycoprotein containing 2 immunoglobulin ECs from EC-SP cells over the long-term (≥6 months). domains and 1 transmembrane domain. CD200 is expressed It has been reported that EPCs are located in the bone by some leukocytes, especially B cells,41 but its function marrow and therefore it is possible that EC-SP cells origi- has not been fully clarified. CD157 is a glycosylphosphati- nate from bone marrow. However, as far as my group dylinositol-binding membrane glycoprotein with a 33% could determine, there were no EC-SP cells in the bone amino acid homology to CD38. This molecule has ADP- marrow, as shown in a mouse model of bone marrow ribosyl cyclase activity and promotes pre-B cell growth.42 transplantation in neonates and adults. Localization of ECs from the liver could be fractioned into different popu- dormant EC-SP cells reveals that most of these cells localize lations according to their expression of these 2 molecules: to the intraluminal inner surface of the vessel but not distant CD200+CD157+, CD200+CD157− and CD200−CD157−

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been investigated. However, bone marrow cells may contribute to some vascular wall cells in the liver, and significant factor VIII elevation was not observed.44 On the other hand, it has been shown that transplantation of CD200+CD157+ VESCs from controls to mice with hemophilia A effectively improved bleeding episodes in the latter. Moreover, factor VIII elevation is continuously induced over the long-term because VESCs can maintain blood vessels induced by normal ECs.12 In the future, it is possible that VESC transplantation may become a curative treatment for hemophilia A.

Conclusions and Perspective The reason why bone marrow replacement therapy is useful Figure 3. Hierarchy of endothelial cell (ECs). CD157+CD200+ in leukemia patients is because hematopoietic stem cells in ECs are present as a stem cell population with self-renewal the graft maintain an immature status with the ability to capacity, as demonstrated by the finding that a single cell can differentiate into all hematopoietic lineage cells. As observed construct blood vessels that are sustainable long-term. in the hematopoietic system, in every tissue regeneration + + − − CD157 CD200 ECs differentiate into CD157 CD200 ECs therapy, use of a stem cell population is critical for the through a CD157−CD200+ stage, possibly endothelial pro- genitor cells in the liver. continuous repopulation of newly-developed tissues. The vascular system is no exception and long-term sustainable blood vessel formation needs to be induced and maintained for therapeutic angiogenesis. In this context, the discovery of VESCs will significantly contribute to future therapeutic ECs. It was found that 70% of SP cells express both CD200 angiogenesis therapy. and CD157. When cultured on an OP9 stromal layer, Although the usefulness of VESCs in tissue regeneration CD200+CD157+ ECs generate higher number of EC colo- is clear, the number of VESCs in pre-existing blood vessels nies than CD200+CD157− ECs. The latter do also generate is very low. For these cells to be used effectively, their in EC colonies but fewer in number and smaller in size. In vitro or ex vivo expansion or induction of VESCs from ES contrast, CD200−CD157− ECs were completely unable to cells or induced pluripotent stem cells may be required. form any EC colonies. The CD200+CD157+ ECs contrib- The study of VESCs is still in its infancy. After the uted long-term to ECs after massive monocrotaline-induced identification of CD157+ VESCs in mice, many researchers liver injury. CD200+CD157− ECs also formed blood vessels are now focusing on vascular stem cell populations in in the damaged liver, but at much lower efficiency. As pre-existing blood vessels. Recently, it was reported that observed in vitro, CD200−CD157− ECs were completely existing EC stem cell-like cells in the aorta facilitate recovery unable to contribute to new blood vessels in the liver. from massive intraluminal vascular injury.45 Moreover, as When a single CD200+CD157+ EC was transplanted into reported, liver-resident EC lineage cells alone, but not mice with liver injury, it generated CD200+CD157+ ECs bone marrow cells, can rescue vascular damage occurring and many differentiated CD200−CD157− ECs via a stage in the liver.46 In addition to the heterogeneity of ECs where cells were CD200+CD157−. Therefore, it can be during angiogenesis (i.e., tip, stalk, and phalanx cells), the concluded that CD200+CD157+ ECs are at the top of the concept of VESC populations needs to be considered when EC hierarchy of self-renewing stem cells, and that they seeking to clarify the precise molecular mechanisms of maintain the vasculature by supplying terminally differen- angiogenesis. tiating ECs through a stage of CD200+CD157− endothelial progenitors (Figure 3). Acknowledgments I thank all people involved in the isolation of endothelial stem cell populations in pre-existing blood vessels. This work was partly Therapeutic Applications of ESCs supported by the Japan Agency for Medical Research and Devel- + + opment (AMED) under Grant number (18 cm0106508 h0002, Based on the fact that CD200 CD157 ECs constantly 18 gm5010002s110), Japan Society for the Promotion of Science supply terminally-differentiated ECs and contribute to (JSPS) Grants-in-Aid for Scientific Research (A) (16H02470). This blood vessel formation long-term, it can be proposed that research was supported by Integrated Frontier Research for Medical this type of VESC can be exploited for vascular regeneration Science Division, Institute for Open and Transdisciplinary Research therapy in ischemic patients. Moreover, when disease is Initiatives, Osaka University. caused by a lack of secretion of certain factors by ECs, transplantation of normal VESCs can restore the function References of the blood vessels. It is well known that liver is the major 1. Mendelson A, Frenette PS. Hematopoietic stem cell niche source of blood coagulation factor VIII, a blood-clotting maintenance during homeostasis and regeneration. Nat Med 2014; 20: 833 – 846. protein. Defects in the factor VIII-encoding gene (F8 gene) 2. Ding BS, Cao Z, Lis R, Nolan DJ, Guo P, Simons M, et al. cause hemophilia A, a recessive X-linked coagulation dis- Divergent angiocrine signals from vascular niche balance liver ease. 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Circulation Journal Vol.83, January 2019