MINIREVIEW Brain angiogenesis in developmental and pathological processes: regulation, molecular and cellular communication at the neurovascular interface Hye Shin Lee 1,2 , Jiyeon Han 1,2 , Hyun-Jeong Bai 1,2 and Kyu-Won Kim 1,2,3 1 Neurovascular Coordination Research Center, College of Pharmacy, Seoul National University, Korea 2 Research Institute of Pharmaceutical Science, Seoul National University, Korea 3 Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Korea Development of the brain vasculature Blood vessels form via two distinct processes: vasculo- genesis and angiogenesis. Vasculogenesis involves the proliferation and differentiation of mesoderm-derived angioblasts into endothelial cells [1]. Before the heart even begins to beat, the primary vascular plexus is formed throughout the body by vasculogenesis [2]. The extracerebral vascular plexus is established by vasculo- genesis within the brain vasculature [2]. Early in embryogenesis, angioblasts invade the head region and form the perineural vascular plexus, which ultimately covers the entire neural tube [3]. After the primary vas- cular plexus is formed by vasculogenesis, a more com- plex vascular network is established via angiogenesis (i.e. the production of vessel branches from pre-exist- ing vessels). Indeed, the vascular network of the brain is predominantly formed by angiogenesis. During this Keywords astrocyte; barriergenesis; blood–brain barrier; brain angiogenesis; endothelial cell; neuron; neurovascular interface; pericyte; perivascular macrophage; smooth muscle cell Correspondence K W. Kim, Neurovascular Coordination Research Center, College of Pharmacy, Seoul National University, Seoul 151-742, Korea Fax: +82 2 885 1827 Tel: +82 2 880 6988 E-mail: qwonkim@plaza.snu.ac.kr (Received 19 February 2009, revised 6 May 2009, accepted 10 June 2009) doi:10.1111/j.1742-4658.2009.07174.x The vascular network of the brain is formed by the invasion of vascular sprouts from the pia mater toward the ventricles. Following angiogenesis of the primary vascular network, brain vessels experience a maturation pro- cess known as barriergenesis, in which the blood–brain barrier is formed. In this minireview, we discuss the processes of brain angiogenesis and bar- riergenesis, as well as the molecular and cellular mechanisms underlying brain vessel formation. At the molecular level, angiogenesis and barriergen- esis occur via the coordinated action of oxygen-responsive molecules (e.g. hypoxia-inducible factor and Src-suppressed C kinase substrate ⁄ AKAP12) and soluble factors (e.g. vascular endothelial growth factor and angiopoie- tin-1), as well as axon guidance molecules and neurotrophic factors. At the cellular level, we focus on neurovascular cells, such as pericytes, astrocytes, vascular smooth muscle cells, neurons and brain macrophages. Each cell type plays a unique role, and works with other types to maintain environ- mental homeostasis and to respond to certain stimuli. Taken together, this minireview emphasizes the importance of the coordinated action of mole- cules and cells at the neurovascular interface, with regards to the regulation of angiogenesis and barriergenesis. Abbreviations Ang-1, angiopoietin-1; AQP4, aquaporin4; BBB, blood–brain barrier; BDNF, brain-derived neurotrophic factor; CNS, central nervous system; HIF, hypoxia-inducible factor; NGF, nerve growth factor; NT, neurotrophins; SEMA, semaphorin; SSeCKS, Src-suppressed C kinase substrate; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; vSMC, vascular smooth muscle cell. 4622 FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS process, vascular sprouts from the pia mater invade the brain and extend toward the ventricles [4]. Like other vascular networks, brain vessels undergo forma- tion, stabilization, branching, pruning and specializa- tion. In brief, the nascent vasculatures formed by vasculogenesis and angiogenesis are stabilized via the recruitment of mural cells and generation of the extra- cellular matrix. The nascent vasculatures are then fine- tuned in response to environmental cues from neighboring cells [5]. Finally, vessels acquire features suitable for the function of each respective organ. Brain vessels have extremely specialized characteris- tics that allow them to form the blood–brain barrier (BBB). The concept of the BBB was first suggested more than 100 years ago when Paul Ehrlich discovered that dyes injected into the vascular system did not pen- etrate brain tissues but were easily absorbed by periph- eral tissues [3]. The BBB consists of interendothelial junctions and a specialized transporter system. The impermeability of the BBB results from the physical barrier between adjacent endothelial cells lining the mi- crovessel wall. Brain endothelial cells acquire their property as a physical barrier from three different junctions (tight, adherens and possibly gap junctions) [6,7]. Tight junctions consist of three integral mem- brane proteins known as occludin, claudins and junc- tional adhesion molecules. The extracellular domains of these proteins form homophilic adhesions with those of neighboring cells, and their cytoplasmic com- ponents are linked to accessory proteins (e.g. zonula occludens proteins and cingulin), forging a connection to the actin cytoskeletons of endothelial cells [8]. Occlu- din is an  65 kDa phosphoprotein that regulates paracellular permeability [9]. Claudins are  22 kDa phosphoproteins that are thought to help maintain high transendothelial electrical resistance. Three types of claudins (claudin1 ⁄ 3, claudin5 and claudin12) are found in the BBB [10]. In addition, junctional adhesion molecule-1, -2 and -3 are present in the BBB and are thought to form part of the tight junction structure. However, the function of these proteins with regards to the BBB remains unclear. Adherens junctions are formed at the intersections of membrane protein cad- herins. Cadherins form a complex with the beta- and gamma-catenins in their cytoplamic tails. As with tight junctions, adherens junctions are linked to the actin cytoskeleton via the binding of beta- and gamma-cate- nin to alpha-catenin [8]. Gap junctions have been iden- tified as BBB components [7]; however, their role in the function of the BBB remains unclear. The physical barrier resulting from tight, adherens and gap junctions enhances transcellular, rather than paracellular, transport when the brain parenchyma and blood exchange factors across the vessel wall. Because the physical barrier primarily functions to protect the brain from toxins in the blood, a special- ized transport system is needed to absorb essential molecules and release substances from the brain. Nutrients are typically transported from the blood to the brain via a carrier-mediated transport system. Because glucose is one of the brain’s primary energy sources, the Glut-1 transporter is of principal impor- tance to the BBB [11]. The Glut-1 transporter is asym- metrically distributed, with a greater abundance found at the abluminal side than at the luminal membrane. This distribution ensures that the proper level of glu- cose is supplied to the brain by preventing the accumu- lation of glucose in the interstitial fluid [12,13]. Essential amino acids, nucleosides and vitamins also use carrier systems. For example, the L1 system trans- ports large neutral amino acids, whereas the y+ sys- tem transports cationic amino acids and the CNT2 adenosine transporter serves as a carrier for nucleo- sides [13]. In addition to carrier-mediated transporters, the BBB endothelium has a receptor-mediated trans- porter system used by proteins, such as insulin, trans- ferrin and leptin, to cross the BBB [13]. Molecular basis of brain angiogenesis and barriergenesis Hypoxia and the hypoxia-inducible factor system As an embryo develops and its structure increases in complexity, the simple diffusion of oxygen and nutri- ents is no longer adequate for survival [14] and a hyp- oxic gradient forms inside the body to signal for the formation of new vessels. In particular, hypoxia-induc- ible factor-1 (HIF-1) regulates the transcription of various angiogenic factors [e.g. vascular endothelial growth factor (VEGF) and erythropoietin) and plays an important role in vascular development [15]. The HIF-1 protein is a dimeric transcription factor com- posed of a and b subunits. The HIF-1a subunit is induced by low levels of oxygen, whereas the HIF-1 b (ARNT) subunit remains stable. Previous studies have shown that HIF-1b deficiency results in embryonic lethality at around days E9.5 and E10.5, with severe defects in vessel formation, especially in the yolk sac [16]. Similarly, HIF-1a deficiency results in vascular defects at a similar stage of development and neuronal defects (e.g. failure of neural tube closing and abnor- mal ventricle formation) have also been detected [17]. Moreover, the selective mutation of HIF-1a in neuro- nal cells decreases vessel density in the brain, because of enhanced apoptosis [18]. These findings highlight H. S. Lee et al. Regulation of angiogenesis and barriergenesis FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS 4623 the importance of HIF-1a in the development of brain vasculature and other tissues. Reoxygenation and the SSeCKS/AKAP12 system during barriergenesis Hypoxic signals are no longer needed once they induce new vessel formation in areas lacking oxygen and nutrients. New vessels then undergo maturation steps suitable for their environment. Vessel maturation in the brain involves the acquisition of specialized fea- tures, including the BBB. In an attempt to connect the missing link between angiogenesis and barriergenesis, Lee et al. [19] identified the Src-suppressed C kinase substrate (SSeCKS) protein (also known as AKAP12 or gravin in humans), which is upregulated by changes in oxygen tension during reoxygenation after hypoxic insult. In cultured primary astrocytes, overexpression of SSeCKS reduced VEGF expression and induced angiopoietin-1 (Ang-1), thereby promoting the expression of tight junction proteins and strengthening the bonds between brain endothelial cells [19]. Recent studies indicate that SSeCKS ⁄ AKAP12 downregulates HIF-1a expression by enhancing interac- tions with von Hippel-Lindau tumor suppressor protein (pVHL) and prolyl hydroxylase domain 2 (PHD2) [20]. These findings strongly suggest that SSeCKS may trig- ger the transition from angiogenesis to barriergenesis. Extracellular factors regulating angiogenesis and barriergenesis VEGF Within the brain, formation of the primary vascular plexus is largely dependent on VEGF signaling. The interaction between VEGF and the vascular endothe- lial growth factor receptor (VEGFR) is thought to promote the differentiation of angioblasts into endo- thelial cells. Previous studies have shown that null mutations in VEGFR2 (Flk-1) lead to defects in hemangioblast and endothelial cells, resulting in embryonic lethality at around day E9 [21]. Further- more, VEGF transcripts have been detected at the periventricular matrix zone and the VEGFR has been identified in migrating endothelial cells, suggesting that VEGF signaling contributes to the migration of vessels from the pia mater to the periventricular region [22,23]. In the healthy brain, VEGF is downregulated to maintain the balance between pro- and anti-angio- genesis. However, during the course of pathological conditions such as ischemia and tumor growth, VEGF contributes to break the BBB and promote endothelial permeability and vascular sprouting. Six homologs of VEGF have been identified to date (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta growth factor). These homologs each play dis- tinct roles in angiogenesis. For example, VEGF-C is mainly involved in lymphangiogenesis, whereas VEGF- D may contribute to tumor angiogenesis [2,24]. Interest- ingly, VEGF-B, which works with VEGF-A to respond to brain injury, probably helps maintain the BBB [25]. A number of growth factors and cytokines are known to regulate VEGF expression. For example, epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor and tumor necrosis factor-a upregulate VEGF expression in glioma cells [26,27]. Angiopoietin Angiopoietin has also been identified as a potent angiogenic factor during embryonic vessel develop- ment. Ang-1 deficiency leads to embryonic vascular defects in the central nervous system (CNS) and many other parts of the body, because of an inappropriate association of the extracellular matrix and supporting cells [28]. Knockout mice deficient in Ang-1, tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie)-1 and Tie-2 receptors experienced vascu- lar defects at a relatively later stage than did VEGF null mutant mice [29,30]. These findings indicate that the Ang-1–Tie system may function during vessel mat- uration and stabilization, rather than during vessel sprouting. Although Ang-1-overexpressing transgenic mice experienced increased vascularization, Ang-1 also increases the tightness of BBB endothelial cells and reduces vessel permeability [19]. Despite the contro- versy surrounding the role of Ang-1 as either an angio- genic factor or a maturation factor, recent studies clearly show that Ang-1 is a prominent regulator of vascular development. In addition to its role in vascu- lar maturation, angiopoietin seems to play an impor- tant role in the maintenance of BBB homeostasis. Previous studies have shown that Ang-1 mRNA levels decrease in conditions that induce BBB breakdown, such as middle cerebral artery occlusion, whereas the expression of Ang-2, an endogenous antagonist of Ang-1, increases [31]. Moreover, when mice with ische- mic lesions that had been induced by middle cerebral artery occlusion or VEGF application were treated with Ang-1, they experienced reduced cerebral vessel leakage and smaller ischemic lesions [32,33]. Although Ang-1 is known to play a major role in BBB formation and homeostasis, it is still unclear which cell type is the major source of Ang-1. Indeed, astrocytes-condi- tioned medium contains Ang-1 and has a role in BBB tightness [19]. Furthermore, another report suggests Regulation of angiogenesis and barriergenesis H. S. Lee et al. 4624 FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS that Ang-1 secreted from pericytes mediates the expres- sion of tight junction proteins [34]. Transforming growth factor-b Transforming growth factor (TGF)-b is a prototypic member of the large TGF-b superfamily, which con- tains  30 different factors, including TGF-b, acti- vin, nodal and bone morphogenic proteins. TGF-bs are involved in a wide range of biological functions, including cell growth, differentiation, embryogenesis and morphogenesis [35]. TGF-b has five isoforms (TGF-b1, 2, 3, 4 and 5), however, only TGF-b1is known to alter BBB integrity. In an in vitro model of the BBB, TGF-b1 treatment reduced permeability [36]. As with Ang-1, TGF-b1 is thought to contri- bute to BBB permeability by mediating cellular com- munication between the endothelium and pericytes or astrocytes. Pericyte- and astrocyte-derived soluble factors seem to contribute to BBB organization, and TGF-b1 may function as a major mediator of this communication [37,38]. Wnts In addition to the classical angiogenic regulators dis- cussed above, Wnt family growth factors have recently been highlighted as key molecules for CNS angiogenesis and barriergenesis. Wnts are a large family of growth factors crucial for a variety of bio- logical processes; in particular, their functions are well established in CNS development, for example, they control dorsal–ventral, anterior–posterial pattern- ing of CNS tissues, dendrite morphogenesis and synaptogenesis (for a review, see reference [39]). According to recent reports, b-catenin, an effecter molecule of canonical Wnt pathway, is expressed in the developing CNS vasculature and has critical roles in embryonic vascular development [40–42]. Interest- ingly, Wnt ⁄ b-catenin signaling is not only responsible for angiogenesis, but also regulates barriergenesis. Conditional knockout of b-catenin in endothelial cells results in a reduction in CNS vessels, vascular hemor- rhage and malformation [40]. At the same time, it impairs Glut-1 expression and claudin-3-mediated endothelial tightness, reflecting the importance of this pathway for BBB induction [40,41]. Various types of Wnt ligands exist in neural tissues to transmit signals to the perineural endothelium. Wnt7a and Wnt7b are expressed in ventral–lateral spinal cord, whereas Wnt1, Wnt3 and Wnt3a are located in dorsal part of the spinal cord [40,42]. Neurogenic factors involved in angiogenesis and barriergenesis Vessels and nerves are located in close proximity to each other and not only share anatomical similarity, but also constantly coordinate to form a proper net- work. Neurovascular coordination requires the sharing of major signaling pathways involved in pathfinding, growth, migration and differentiation. In fact, a num- ber of factors known in CNS development, including axon guidance molecules and neurotrophins, also func- tion as regulators of vascular systems (Table 1). Con- versely, many well-known pro- and anti-angiogenic factors, including VEGF and Ang-1, are also responsi- ble for the development and function of the nervous system (Table 2) [43]. Here, we focus on the molecular factors specific to nerves and vessels, especially neuro- genic factors that affect vessel formation and differen- tiation. Table 1. Neurogenic factors affecting the vascular system. BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; NT, neurotro- phins; SEMA, semaphorin. Molecules Receptors Effects References Axon guidance cues Ephrin-B2 EphB4 Arterial–venous specification, Mural cell recruitment, Lymphatic vessel development, Tumor angiogenesis [45] SEMA3A Neurophilin 1 (NP1) Inhibit angiogenesis [46] SEMA3F Neurophilin 2 (NP2) Inhibit angiogenesis [46] SEMA4D Plexin B1 (PLEXB1) Induce tumor angiogenesis [46] Slit-2 Robo-4 and Robo-1 Repulsion or attraction of endothelial cell migration, tumor angiogenesis [47] Netrin-1 UNC5B, NeogeninA2b Vascular pathfinding, inhibit or induce angiogenesis [87] Neurotrophins NGF TrkA Promote endothelial cell proliferation and migration, induce angiogenesis [48] BDNF TrkB Cardiac vessel development, chemotactic for hematopoietic precursor cells [53] NT-3 TrkC Inhibit proliferation of cerebral endothelial cells [88] H. S. Lee et al. Regulation of angiogenesis and barriergenesis FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS 4625 Axon guidance cues Recently, it has become widely accepted that four major axon guidance cues (ephrins, semaphorins, slits and netrins) are responsible for vascular patterning [44]. In particular, ephrin B2 contributes to arterial– venous specification, mural cell recruitment, lymphatic vessel development and tumor angiogenesis via its receptor, EphB4 [45]. Semaphorins may perform two functions, with regards to angiogenesis. Class 3 sem- aphorins (SEMA), such as SEMA3A and SEMA3F, inhibit angiogenesis via competition with VEGF for their common receptor, neuropilin. By contrast, SEMA4D functions as a pro-angiogenic factor that induces tumor angiogenesis [46]. Slit2 and Netrin 1 also participate in vessel development and tumor angiogenesis via their receptor UNC5B, as well as the ROBO4 receptor for slit and the NeogeninA2b recep- tor for Netrin [47]. Neurotrophins Neurotrophins (NTs) are well-known trophic factors involved in neuronal proliferation, survival and path- finding. The NT family consists of four members [i.e. nerve growth factor (NGF), brain-derived neuro- trophic factor (BDNF), NT-3 and NT-4]. Besides their classical functions on neuronal cells, a growing body of evidence suggests that NTs play other roles in non-neuronal tissues, especially blood vessels. First and foremost, NGF is known to trigger endo- thelial cell proliferation and migration in vitro, and induces angiogenesis in in vivo angiogenic assays (e.g. the rat corneal assay and the chick embryo chorioa- llantoic membrane assay) [48]. Furthermore, the angio- genic activity of NGF was also implicated in a hind-limb ischemia model, in which NGF markedly increased arteriole length and density [49]. The role of NGF in angiogenesis is, in part, because of cross-talk with VEGF signaling. In neuronal and adipose tissues, NGF elevated VEGF expression and consequently stimulated angiogenesis [50,51]. Similarly, BDNF is also induced by ischemic conditions and overexpres- sion of this factor promotes the revascularization of ischemic tissues. During development, BDNF seems to play an important role in cardiac vascular formation, because BDNF deficiency impairs the survival of endo- thelial cells and contributes to vascular hemorrhage in cardiac vessels [52]. By contrast, BDNF overexpression increases capillary density within the heart [52,53]. Cellular basis of brain angiogenesis and barriergenesis As discussed earlier, angiogenesis and barriergenesis of the brain vasculature occur via the complex coordi- nation of various molecules. The molecular dynamics of this process involve several different types of cells surrounding the brain vessels. These cells shape the vascular environment, with each cell type playing a unique role in the development and function of brain vessels. Hence, we now discuss the major types of cells in the vascular environment, including pericytes, astro- cytes, vascular smooth muscle cells, neurons and brain macrophages (Fig. 1). Pericytes Pericytes are vascular mural cells belonging to the vas- cular smooth muscle cell (vSMC) lineage. Although these cells were discovered more than 100 years ago, pericytes seldom attracted interest because they were merely considered mural cells that supported endothe- lial cells. Recent studies have established that pericytes not only provide physical support to endothelial cells, but also play critical roles in vessel functioning. Most importantly, pericytes and endothelial cells share a basement membrane, enabling them to communicate directly. In fact, pericytes form focal contacts with endothelial cells at sites known as peg–socket contacts. At these contacts, pericytes are connected to endothe- lial cells through tight, gap and adherence junctions (Fig. 1) [54]. Pericyte coverage varies among different types of vessels. The pericyte ⁄ endothelial cell ratio ranges from 1 : 100 in skeletal muscle to 1 : 1 in the retina. In general, vessels in the CNS exhibit the high- est pericyte coverage, highlighting the importance of pericytes in the formation and maintenance of CNS vasculature [13,54]. During embryonic angiogenesis, pericyte recruitment is the first event to stabilize the primary vascular Table 2. Angiogenic regulatory factors affecting nervous system. Ang-1, angiopoietin-1; FGF, fibroblast growth factor; IGF, insulin-like growth factor; VEGF, vascular endothelial growth factor. Molecules Receptors Effects References VEGF Flk-1, Nrp1 Axonal growth, neuron survival, Neurogenesis [43] FGF-2 FGFR-1, FGFR-2 Neurogenesis, neuroprotection, NSC proliferation [43] IGF-1 IGF-1R Neurogenesis [89] Ang-1 Tie-2 Neuroprotection [90] TSP-1 ⁄ 2 CD47 ⁄ IAP? LRP1? Synaptogenesis [91] Regulation of angiogenesis and barriergenesis H. S. Lee et al. 4626 FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS plexus. During this process, PDGF signaling plays an important role. The PDGF-B protein is expressed in sprouting endothelial cells, and its receptor PDGFR-b is expressed in pericyte precursors. Genetic ablation of PDGF-B or PDGFR-b results in the loss of pericytes and severe defects in the brain and heart, leading to vascular leakage and edema [55–57]. Ligand–receptor signaling between two types of cells may mediate cellu- lar communication and, in turn, form the proper struc- ture of mature vessels. After pericytes are recruited, the contact between pericyte precursors and endothe- lial cells signals for the production of TGF-b, which consequently induces the differentiation of pericyte precursors to mature pericytes [57]. The opposite situa- tion also seems possible, in which pericytes induce and guide vessel sprouting. In the developing human brain, migrating pericytes are found in front of growing ves- sels and pericyte-driven angiogenesis participates in the organization of growing vessels [58]. Another question that arises is: why are pericytes abundant in the brain vasculature? Brain pericytes may perform specialized roles involved in the develop- ment and maintenance of brain vessels. First and fore- most, pericytes are thought to enhance BBB integrity. Generally, in vitro models of the BBB involve the co-culturing of endothelial cells and astrocytes. How- ever, when pericytes are added to the co-culture, endo- thelial cells reorganized into stable, capillary-like structures [59]. Furthermore, pericytes play a protec- tive role in hypoxia-induced disruption of the BBB [60]. Ang-1, a key factor regulating barriergenesis, also contributes to pericyte-induced BBB formation; in fact, pericyte-derived Ang-1 induces occludin expression in cultured brain endothelial cells [34]. Pericytes are sometimes confused with vSMCs, because a specific marker capable of distinguishing pericytes from vSMCs has not yet been developed. However, it seems clear that the mural cells located in the brain microvessels are pericytes. Like vSMCs in other parts of the body, pericytes are able to regulate vessel diameter and blood flow. One of the observa- tions supporting this idea is that pericytes express a contractile protein known as a-smooth muscle actin. In addition, some in vitro studies have directly demon- strated the contractile activity of pericytes. Further- more, several kinds of molecules have been identified A C N MG vSMC E C n e m uL AA A H 2 O H 2 O culGeso Synapse PC Peg- socket junctional co mpl e x PM BM AC Endf eet N Blood vessel c u lGe so A Adenosine Amino acid AQP4 Neurotransmitter Adherence junction Gap junction Tight junction AA ulGt1 1L 2T N C Fig. 1. Cellular communication at the neurovascular interface. The neurovascular unit consists of neurons (N), endothelial cells (EC) and other types of cells located in the neurovascular unit, i.e. astrocytes (AC), pericytes (PC), vascular smooth muscle cells (vSMC), microglia (MG) and perivascular macrophages (PM). Endothelial cells form a blood–brain barrier characterized by tight, adherence and gap junctions, as well as a specialized transporter system (i.e. consisting of Glut-1, L1 and CNT2). Pericytes share basement membranes with blood vessels and directly contact endothelial cells via peg–socket junction complexes. Astrocytes stretch their endfeet toward blood vessels and neuronal synapses to integrate neuronal activity with the vascular response. Note that astrocytic endfeet contain water channel AQP4 proteins to regulate water homeostasis. The immune cells of the CNS (i.e. microglia, macrophages and pericytes) participate in the brain’s immune response. H. S. Lee et al. Regulation of angiogenesis and barriergenesis FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS 4627 to induce the constriction or dilation of pericytes. In particular, a-2 adrenergic agonists, histamine, angio- tensin II and endothelin-1 contribute to vasoconstric- tion, whereas b-2 adrenergic agonists and nitric oxide contribute to vasodilation [61]. Another interesting function of brain pericytes is their macrophage-like activity. Indeed, CNS pericytes possess a number of macrophage-like features, includ- ing the capacity to absorb soluble molecules delivered into the blood or cerebrospinal fluid, the presence of macrophage-specific class III major histocompatibility complex proteins and phagocytotic activity [61]. Although it is not clear whether pericytes function as macrophages in vivo, growing evidence supports the involvement of pericytes in CNS immunity. Astrocytes Astrocytes are the most abundant cells in the brain. In the past, astrocytes were considered ‘glue’ that provided physical support for neurons. However, recent studies suggest that astrocytes play active roles in various brain functions. In particular, astrocytes function as adult neural stem cells, participate in the formation and mod- ulation of synapses, and in the process of ‘gliotransmis- sion,’ which generates and distributes excitatory chemical signals in a neuron-coordinated manner [62]. Astrocytes are also interesting with regards to brain vas- culature, because they regulate the formation and main- tenance of BBB, modulate neurovascular coupling and maintain several parts of brain homeostasis. In this minireview, we focus on the active functions of astro- cytes in regards to brain vasculature. Anatomically, most astrocytes have stellate shapes containing multiple processes. These cells expand toward neurons and ves- sels. The ends of the cells, so-called endfeet, contact the vessel wall and form large compartments that enclose most blood vessels of the brain (Figs 1, 2A). Thus, one astrocyte can contact several synapses, in addition to blood vessels, making it possible to integrate signals generated from both neurons and vessels. Consequently, astrocytes are believed to function as key mediators of neurovascular coordination. Roles in BBB formation and maintenance Vessel sprouting is completed before birth, whereas astrocyte differentiation occurs during the late embry- onic and early postnatal periods. Because of the dis- cordance in developmental timing, it seems difficult for astrocytes to modulate developmental angiogenesis. Rather, astrocytes may play a role in barriergenesis. The period of astrocyte differentiation coincides with that of BBB formation. Differentiating astrocytes may extend their processes to the vessel wall, thereby send- ing signals to acquire BBB properties. When cultured astrocytes were transplanted into a rat anterior eye chamber or a chick chorioallantoic membrane (where vessels are leaky), the vessels acquired BBB properties [63]. It is also clear that if brain endothelial cells are cultured in vitro, they loose certain BBB characteris- tics, such as high TEER, the membrane localization of tight junction proteins and transporter expression; however, most BBB characteristics can be regained via co-culture with astrocytes or treatment with astrocyte- conditioned medium [38,64,65]. Moreover, most mole- cules that regulate barriergenesis are astrocyte-derived factors, which include SSeCKS, angiopoietin and TGF-b [19,38]. Although this issue remains controver- sial, it seems clear that, at least in vitro, astrocytes help endothelial cells obtain BBB characteristics. Regulation of vascular tone in response to neuronal activities One of the most surprising features of the brain is its abil- ity to control blood flow in response to neuronal activi- ties, a process known as functional hyperemia. Cerebral blood flow rate and vessel diameter are not fixed in spe- cific regions, but depend on the local demand for oxygen and nutrients by synaptic transmission and neuronal fir- ing. Imaging techniques, such as functional MRIs, have determined that neuronal activity and cerebral blood flow regulation are precisely coupled [44]. However, the exact mechanisms underlying this phenomenon are not fully understood and may depend on the anatomical position of astrocytes. As previously mentioned, astro- cytes make contact with, and possibly integrate and deliver signals between, neurons and vessels. To understand the role of astrocytes in cerebral blood flow regulation, we focus on the Ca 2+ ion. Vari- ous neurotransmitters generated from synaptic activi- ties increase intracellular Ca 2+ levels in astrocytes [66]. For example, glutamate, an excitatory neurotransmit- ter, stimulates astrocytes via the metabotropic mGluR receptor, and activated mGluR consequently triggers a Ca 2+ increase [67]. Local increases in Ca 2+ concentra- tion diffuse throughout the entire body of astrocytes, including the endfeet. The Ca 2+ signals released by as- trocytes subsequently alter vascular tone and promote either vasoconstriction [68] or vasodilation [67]. In astrocytes, vasoconstriction and vasodilation both require arachidonic acid, but the next step is different. The conversion of arachidonic acid to 20-hydroxyeico- satetraenoic acid occurs during vasoconstriction, and arachidonic acid is converted to prostaglandin E 2 or Regulation of angiogenesis and barriergenesis H. S. Lee et al. 4628 FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS epoxyeicosatrienoic acid during vasodilation [66]. In the latter event, the COX enzyme might play a role in prostaglandin E 2 production, and the nitric oxide- sensitive CYP450 enzyme may contribute to epoxyei- cosatrienoic acid conversion [66,67]. The level of nitric oxide seems to influence the type of vasomotor response, because nitric oxide produced by neighboring cells diffuses to astrocytes and arterioles, then results in vessel dilation. Maintenance of water and ion homeostasis in the brain One of the classic functions of astrocytes involves scav- enging for neurotoxic metabolites, including neuro- transmitters and ions generated from neuronal activities. Astrocytes take up neurotransmitters, which, in turn, terminate signal transmission and prevent the accumulation of toxic levels of neurotransmitters [69]. In this role, glutamate is the best-known neurotransmit- ter. Indeed, astrocytes possess glutamate transporters such as GLT1 and GLAST1 at their end processes [70]. The imported glutamate is converted to glutamine and subsequently released into the extracellular space. Because glutamine is not neurotoxic, neurons can take it up again and recycle it for further neurotransmission [69]. Similar to neurotransmitters, astrocytes also regu- late ion homeostasis of the brain. For example, neuro- nal activity causes an increase in extracellular K + content, which leads to an influx of K + into astrocytes. The clearance of neurotransmitters and ions is accom- panied by the movement of water, which is buffered by astrocytes. The increased Na + concentration caused by glutamate transport, and increased intracellular Ca 2+ levels from mGluR activation, lead to water uptake and slight swelling of the astrocytes [10,67]. The astrocytic foot processes that surround blood vessels have a high density of aquaporin4 (AQP4), a water channel, which transports water bidirectionally between the blood and the brain. Astrocytes secrete water into the perivascular space via AQP4, thereafter maintaining water homeo- stasis in the brain environment (Fig. 1) [69]. During pathogenesis, AQP4 is likely responsible for the forma- tion and clearance of brain edema. Interestingly, AQP4 plays opposite roles in cytotoxic and vasogenic edema (Fig. 2B). Deletion of AQP4 worsens vasogenic edema and prevents water elimination, whereas AQP4 null mutants protect against cytotoxic edema by reducing the flow of water into the brain [71]. vSMCs vSMCs are myocytes that mediate vasoconstriction and vasodilation. The thickness of the vSMC layer dif- fers according to the size of the vessels. In the brain, pial arteries invade the brain parenchyma and reduce the width of arterioles, then form deep branches that become small capillaries. As vessels become smaller, the smooth muscle layer becomes thinner and, in turn, disappears and is replaced by pericytes at the capillary level [72]. Therefore, brain vSMCs may help regulate vascular tone, especially at the arteriole level. At the forefront of functional hyperemia, vSMCs receive vasoactive signals from astrocyte endfeet or perivascu- lar nerves, which in turn alter the vascular tone by reg- ulating myofilaments [66]. In addition to their roles in functional hyperemia, parenchymal arterioles exhibit vasomotion activity (i.e. a rhythmic oscillation of ves- sel diameter) in the absence of disease. This activity coincides with an oscillation in the intracellular Ca 2+ concentration of vSMCs. Interestingly, neuronal acti- vation, followed by changes in the intracellular Ca 2+ levels of astrocytes, prevents vasomotion and promotes local changes in vascular tone [72,73]. Neurons Neurons are major participants in brain function. However, with regards to blood vessels, neurons are anatomically distant from blood vessels, with the exception of neural stem cells and perivascular nerves. As we previously discussed, neurons and vessels are functionally coordinated by sharing factors for neuro- genesis and angiogenesis, as well as arising functional hyperemia by mediating astrocytes. Although the majority of mature neurons and blood vessels are not located within a proximal distance, neural stem cells lie close to blood vessels and even make direct con- tact with specialized regions [74]. It is now widely accepted that our brain contains neural stem cells throughout our entire lifespan, and that these stem cells are located at certain regions (i.e. the subgranu- lar zone of the hippocampus and the subventricular zone of the cerebral cortex) known as the stem cell niche. Anatomical analyses of the stem cell niche have suggested that the environment surrounding neu- ral stem cells, especially the vascular environment, is important for maintenance and differentiation. Inter- estingly, the stem cell niche has high angiogenic potential, with part of the proliferating cell popula- tion composed of endothelial precursor cells [75]. These findings indicate that angiogenesis and neuro- genesis share common signals and blood vessels con- tribute to neural stem cell behavior by generating environmental cues. The direct effects of endothelial cells on neural stem cells were demonstrated via an in vitro co-culture system. When neural stem cells H. S. Lee et al. Regulation of angiogenesis and barriergenesis FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS 4629 were co-cultured with endothelial cells, they exhibited greater self-renewal activity followed by extensive neu- rogenesis [76]. These findings suggest that the vascular environment of the neural stem cell may contribute to the maintenance and proper differentiation of the stem cell population under certain conditions, with the help of soluble factors. Perivascular nerves participate in functional hyper- emia. These nerves originate from the peripheral ner- vous system (or interneurons of the CNS) and extend their terminals toward cerebral blood vessels located <1lm away (Fig. 1) [77]. In general, perivascular nerves transfer neuronal activity to blood vessels by releasing vasoactive modulators [77]. Brain macrophages Brain macrophages contribute to brain immunity. Guillemin & Brew [78] defined systemic macrophages located in the CNS (e.g. microglia, perivascular macro- phages and pericytes) as ‘brain macrophages’. The ontogeny of brain macrophages is quite controversial; however, the most acceptable hypothesis supports a monocyte origin and, to a lesser extent, a mesenchymal progenitor origin [78]. According to the monocyte hypothesis, during embryogenesis, and even in adults, migrating monocytes enter the brain via blood vessels and then differentiate into certain types of brain mac- rophages depending on the environmental signals [79]. wodkaer B A B DC nB f o BB Leukocyte infiltration Vascular hemorrhage H 2 O H 2 O H 2 O ursiDpnoitfo T JMB & att eDh cm etn a fos ortc y t e A(C)e ndfeet osaVegcinedeam y Cto t o x ic e d e ma Vucsaalr rutpure Vucsaalmeh ror r he g a go r ciMilM(a G )acitvai t no &c ykotiene s aele r Le c ok u y t e( CL ) i n ifltartino E damenoitamrof Blood vessel Blood vessel Blood vessel Blood vessel BM RBC RBC IgG MG N N N AC AC CK EC BM TJ AC BM MG LC Fig. 2. Neurovascular dysfunction. A number of brain disorders can disrupt homeostasis of the neurovascular unit. (A) Degradation of junc- tions in the blood–brain barrier (BBB) disrupts neurovascular interactions. (B) Brain edema is a clinically important symptom induced by increased extracelluar fluid, which results from the increased permeability of brain capillary endothelial cells (i.e. vasogenic edema) or swell- ing of cellular elements of the brain with interstitial fluid (i.e. cytotoxic edema). (C) Autoimmune disorders and viral infection contribute to abnormal immune responses, which promote microglia activation and leukocyte infiltration. The activated immune cells release self-targeted antibodies and enzymes that cause cellular damage at the neurovascular interface. (D) Hemorrhage, induced by events such as brain trauma and stroke, is one of the most common abnormalities of brain vessels. N, neuron; MG, microglia; AC, astrocyte; EC, endothelial cell; TJ, tight junction; BM, basement membrane; LC, leukocyte; Ck, cytokine; RBC, red blood cell. Regulation of angiogenesis and barriergenesis H. S. Lee et al. 4630 FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS Perivascular macrophages are located within astrocyte endfeet (Fig. 1) and may belong to a similar lineage of blood-derived macrophages, rather than to resident microglia [78]. It has also been suggested that blood- derived macrophages enhance the tightness of the BBB, based on studies of an in vitro co-culture system [80]. As we discussed previously, pericytes play a role in the brain immune response and are thus included in brain macrophages. Microglia are the major type of immune cell found in brain parenchyma. Because brain macrophages fundamentally mediate the immune response, these cells play crucial roles in pathological, rather than physiological, conditions. However, some experiments have demonstrated the involvement of these cells in BBB integrity. During the resting state, microglia acquire different shapes and functions, and continuously survey their microenvironment [81]. When they sense brain damage, microglia begin to modify their behavior and acquire an ameboid form. This, in turn, alters their antigen presentation and stimulates the release of cytokines, thereby instigating subsequent immune responses by recruiting leukocytes from blood to the brain parenchyma (Fig. 2C) [13]. The transmigration of leukocytes through the BBB occurs via both the paracellular and transcellular path- ways. Leukocyte and endothelial cell interactions are necessary for extravasation, a process during which rolling leukocytes dock to the luminal membrane of endothelial cells via interactions between selectins, chemokines and integrins. After docking to the vessel wall, leukocytes extend their processes toward interen- dothelial junctions to search for abluminal chemokine cues. Chemokine–chemokine receptor interactions encourage leukocytes to migrate to the perivascular space. Some leukocytes are retained at the perivascular space, whereas others keep migrating toward brain parenchyma across the glia limitance [82]. In this pro- cess, leukocytes migrate through the extracellular matrix with the help of matrix metalloproteinase [82]. The consequent event of brain macrophages is one of the most important defense mechanisms used by the brain. However, this phenomenon sometimes leads to neuroinflammatory disorders, such as multiple sclerosis [83] and neuro-AIDS [84]. The inflammatory response resulting from the activation of microglia and leuko- cyte infiltration affects normal cells, which, in turn, causes neuronal dysfunction. Cell–cell interaction in the neurovascular unit As discussed, all types of cells in the brain have unique roles and coordinate with each other to main- tain brain homeostasis and enable proper reactions to environmental stimuli. From this point of view, neuro- vascular research has been moving toward an inte- grated theory that defines the entire cellular and molecular population, anatomically and functionally, as a single ‘unit’. In particular, the concept of a ‘neurovascular unit’ that encompasses neurons, vessels and other types of cells located at their interface has become one of the main themes of brain science [43,44]. Researchers targeting the neurovascular unit have uncovered clues that may help treat several kinds of brain disease. Indeed, a number of brain disorders (e.g. neurodegenerative diseases, such as stroke, Alzheimer’s disease and Parkinson’s disease; neuroimmune diseases, such as multiple sclerosis and neuro-AIDS; and many types of brain tumors) are accompanied by vascular dysfunction, which presents as BBB disruption, edema formation, leukocyte infil- tration and vascular hemorrhage (Fig. 2). Under cer- tain pathological conditions, cells at the neurovascular interface have protective roles while also accelerating pathologic progression. For example, astrocytes pro- tect the brain from toxic materials via their high buf- fering capacity; however, if astrocytes reach the state of reactive gliosis, they are also capable of releasing cytokines that disrupt the BBB and elicit inflammatory responses [85]. In addition, the slight edema of astro- cytes helps maintain the water balance in the brain, but pathologic cytotoxic edema is a main cause of increased intracranial pressure [86]. To overcome such neurodiseases, it may be necessary to understand the precise mechanisms underlying cellular communication within the neurovascular unit. However, the in vitro systems used for studying cellular communication (e.g. the co-culture system or treatment with conditioned medium) have inevitable limitations and cannot entirely reproduce the in vivo environment. For exam- ple, co-cultures of astrocytes and endothelial cells elim- inate the effects of the basement membrane and differences in the luminal–abluminal polarity of the endothelium. To bridge the gap between in vitro condi- tions and the actual environment, experiments should incorporate improved in vivo imaging techniques, con- struct clear marker systems and develop proper animal models for certain brain diseases. Acknowledgements This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Ministry of Education, Science & Technology (MEST) through the Creative Research Initiatives Program (Grant R16-2004-001-01001-0, 2008). H. S. Lee et al. 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