Distinct cellular mechanisms of blood vessel fusion in the zebrafish embryo

Although many of the cellular and molecular mechanisms of angiogenesis have been intensely studied [1], little is known about the processes that underlie vascular anastomosis. We have generated transgenic fish lines expressing an EGFP-tagged version of the junctional protein zona occludens 1 (ZO1) to visualize individual cell behaviors that occur during vessel fusion and lumen formation in vivo. These life observations show that endothelial cells (ECs) use two distinct morphogenetic mechanisms, cell membrane invagination and cord hollowing to generate different types of vascular tubes. During initial steps of anastomosis, cell junctions that have formed at the initial site of cell contacts expand into rings, generating a cellular interface of apical membrane compartments, as defined by the localization of the apical marker podocalyxin-2 (Pdxl2). During the cord hollowing process, these apical membrane compartments are brought together via cell rearrangements and extensive junctional remodeling, resulting in lumen coalescence and formation of a multicellular tube. Vessel fusion by membrane invagination occurs adjacent to a preexisting lumen in a proximal to distal direction and is blood-flow dependent. Here, the invaginating inner cell membrane undergoes concomitant apicobasal polarization and the vascular lumen is formed by the extension of a transcellular lumen through the EC, which forms a unicellular or seamless tube.     

Patterning of angiogenesis in the zebrafish embryo

Little is known about how vascular patterns are generated in the embryo. The vasculature of the zebrafish trunk has an extremely regular pattern. One intersegmental vessel (ISV) sprouts from the aorta, runs between each pair of somites, and connects to the dorsal longitudinal anastomotic vessel (DLAV). We now define the cellular origins, migratory paths and cell fates that generate these metameric vessels of the trunk. Additionally, by a genetic screen we define one gene, out of bounds (obd), that constrains this angiogenic growth to a specific path. We have performed lineage analysis, using laser activation of a caged dye and mosaic construction to determine the origin of cells that constitute the ISV. Individual angioblasts destined for the ISVs arise from the lateral posterior mesoderm (LPM), and migrate to the dorsal aorta, from where they migrate between somites to their final position in the ISVs and dorsal longitudinal anastomotic vessel (DLAV). Cells of each ISV leave the aorta only between the ventral regions of two adjacent somites, and migrate dorsally to assume one of three ISV cell fates. Most dorsal is a T-shaped cell, based in the DLAV and branching ventrally; the second constitutes a connecting cell; and the third an inverted T-shaped cell, based in the aorta and branching dorsally. The ISV remains between somites during its ventral course, but changes to run mid-somite dorsally. This suggests that the pattern of ISV growth ventrally and dorsally is guided by different cues. We have also performed an ENU mutagenesis screen of 750 mutagenized genomes and identified one mutation, obd that disrupts this pattern. In obd mutant embryos, ISVs sprout precociously at abnormal sites and migrate anomalously in the vicinity of ventral somite. The dorsal extent of the ISV is less perturbed. Precocious sprouting can be inhibited in a VEGF morphant, but the anomalous site of origin of obd ISVs remains. In mosaic embryos, obd somite causes adjacent wild-type endothelial cells to assume the anomalous ISV pattern of obd embryos. Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth.     

Essential and overlapping roles for laminin alpha chains in notochord and blood vessel formation

Laminins are major constituents of basement membranes and have wide ranging functions during development and in the adult. They are a family of heterotrimeric molecules created through association of an alpha, beta and gamma chain. We previously reported that two zebrafish loci, grumpy (gup) and sleepy (sly), encode laminin beta1 and gamma1, which are important both for notochord differentiation and for proper intersegmental blood vessel (ISV) formation. In this study we show that bashful (bal) encodes laminin alpha1 (lama1). Although the strongest allele, bal(m190), is fully penetrant, when compared to gup or sly mutant embryos, bal mutants are not as severely affected, as only anterior notochord fails to differentiate and ISVs are unaffected. This suggests that other alpha chains, and hence other isoforms, act redundantly to laminin 1 in posterior notochord and ISV development. We identified cDNA sequences for lama2, lama4 and lama5 and disrupted the expression of each alone or in mutant embryos also lacking laminin alpha1. When expression of laminin alpha4 and laminin alpha1 are simultaneously disrupted, notochord differentiation and ISVs are as severely affected as sly or gup mutants. Moreover, live imaging of transgenic embryos expressing enhanced green fluorescent protein in forming ISVs reveals that the vascular defects in these embryos are due to an inability of ISV sprouts to migrate correctly along the intersegmental, normally laminin-rich regions.     

Tubulogenesis during blood vessel formation

The ability to form and maintain a functional system of contiguous hollow tubes is a critical feature of vascular endothelial cells (ECs). Lumen formation, or tubulogenesis, occurs in blood vessels during both vasculogenesis and angiogenesis in the embryo. Formation of vascular lumens takes place prior to the establishment of blood flow and to vascular remodeling which results in a characteristic hierarchical vessel organization. While epithelial lumen formation has received intense attention in past decades, more recent work has only just begun to elucidate the mechanisms controlling the initiation and morphogenesis of endothelial lumens. Studies using in vitro and in vivo models, including zebrafish and mammals, are beginning to paint an emerging picture of how blood vessels establish their characteristic morphology and become patent. In this article, we review and discuss the molecular and cellular mechanisms driving the formation of vascular tubes, primarily in vivo, and we compare and contrast proposed models for blood vessel lumen formation.     

Antagonistic interactions among Plexins regulate the timing of intersegmental vessel formation

The angioblast is an embryonic endothelial cell precursor that migrates long distances to reach its final position, navigating by sensing attractive and repulsive cues from the environment. Members of the semaphorin family have been implicated in controlling the behaviour of angioblast tip cells through repulsive signalling in vitro, but their in vivo roles are less clear. Here we show that zebrafish semaphorin3e (sema3e) is expressed by endothelial cells of the dorsal aorta, primary motoneurons, and endodermal cells. Further, loss of Sema3e leads to delayed exit of angioblasts from the dorsal aorta in ISV formation. Through transplant analysis, we show that Sema3e acts autonomously and non-autonomously in angioblasts to modulate interactions among themselves. The semaphorin receptors, PlexinD1 and PlexinB2, are expressed by zebrafish angioblasts. Loss of plxnB2 results in delayed ISV sprouting identical to that seen in sema3e morphants, while loss of plexinD1 in out of bounds (obd) mutants results in precocious ISV sprouting. Loss of either sema3e or plxnB2 in obd mutants generates an intermediate phenotype, suggesting that PlxnD1 and Sema3e/PlxnB2 antagonize each other to control timing of ISV sprouting. Consistent with this observation, we show that PlxnB2 acts cell autonomously in endothelial cells. This suggests a model where multiple semaphorin-plexin interactions control angioblast sprouting behaviour.     

Coordinating cell behaviour during blood vessel formation

The correct development of blood vessels is crucial for all aspects of tissue growth and physiology in vertebrates. The formation of an elaborate hierarchically branched network of endothelial tubes, through either angiogenesis or vasculogenesis, relies on a series of coordinated morphogenic events, but how individual endothelial cells adopt specific phenotypes and how they coordinate their behaviour during vascular patterning is unclear. Recent progress in our understanding of blood vessel formation has been driven by advanced imaging techniques and detailed analyses that have used a combination of powerful in vitro, in vivo and in silico model systems. Here, we summarise these models and discuss their advantages and disadvantages. We then review the different stages of blood vessel development, highlighting the cellular mechanisms and molecular players involved at each step and focusing on cell specification and coordination within the network.     

Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro

Summary The extracellular matrix, particularly basement membranes, plays an important role in angiogenesis (blood vessel formation). Previous work has demonstrated that a basement membranelike substrate (Matrigel) induces human umbilical vein endothelial cells to rapidly form vessel-like tubes (Kubota, et al., 1988; Grant et al., 1989b); however, the precise mechanism of tube formation is unclear. Using this in vitro model, we have investigated morphologic changes occurring during tube formation and the cytoskeletal and protein synthesis requirements of this process. Electron microscopy showed that endothelial cells attach to the Matrigel surface, align, and form cylindrical structures that contain a lumen and polarized cytoplasmic organelles. The cytoskeleton is reorganized into bundles of actin filaments oriented along the axis of the tubes and is located at the periphery of the cells. The addition of colchicine or cytochalasin D blocked tube formation, indicating that both microfilaments and microtubules are involved in this process. Cycloheximide blocked tube formation by 100%, indicating that the process also required protein synthesis. In particular, collagen synthesis seems to be required for tube formation because cis-hydroxyproline inhibited tube formation, whereas either the presence of ascorbic acid or the addition of exogenous collagen IV to the Matrigel increased tube formation. Our results indicate that endothelial cell attachment to Matrigel induces the reorganization of the cytoskeleton and elicits the synthesis of specific proteins required for the differentiated phenotype of the cells.     

Endothelial Cell Self-fusion during Vascular Pruning

During embryonic development, vascular networks remodel to meet the increasing demand of growing tissues for oxygen and nutrients. This is achieved by the pruning of redundant blood vessel segments, which then allows more efficient blood flow patterns. Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail. Here, we present the subintestinal vein (SIV) plexus of the zebrafish embryo as a novel model to study pruning at the cellular level. We show that blood vessel regression is a coordinated process of cell rearrangements involving lumen collapse and cell–cell contact resolution. Interestingly, the cellular rearrangements during pruning resemble endothelial cell behavior during vessel fusion in a reversed order. In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection. Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion. In a second step, the unicellular connection is resolved unilaterally, and the pruning cell rejoins the opposing branch. Thus, we show for the first time that various cellular activities are coordinated to achieve blood vessel pruning and define two different morphogenetic pathways, which are selected by the flow environment.     

Cellular and molecular analyses of vascular tube and lumen formation in zebrafish

Tube and lumen formation are essential steps in forming a functional vasculature. Despite their significance, our understanding of these processes remains limited, especially at the cellular and molecular levels. In this study, we analyze mechanisms of angioblast coalescence in the zebrafish embryonic midline and subsequent vascular tube formation. To facilitate these studies, we generated a transgenic line where EGFP expression is controlled by the zebrafish flk1 promoter. We find that angioblasts migrate as individual cells to form a vascular cord at the midline. This transient structure is stabilized by endothelial cell-cell junctions, and subsequently undergoes lumen formation to form a fully patent vessel. Downregulating the VEGF signaling pathway, while affecting the number of angioblasts, does not appear to affect their migratory behavior. Our studies also indicate that the endoderm, a tissue previously implicated in vascular development, provides a substratum for endothelial cell migration and is involved in regulating the timing of this process, but that it is not essential for the direction of migration. In addition, the endothelial cells in endodermless embryos form properly lumenized vessels, contrary to what has been previously reported in Xenopus and avian embryos. These studies provide the tools and a cellular framework for the investigation of mutations affecting vasculogenesis in zebrafish.     

Rasip1 is required for endothelial cell motility, angiogenesis and vessel formation

Ras proteins are small GTPases that regulate cellular growth and differentiation. Components of the Ras signaling pathway have been shown to be important during embryonic vasculogenesis and angiogenesis. Here, we report that Rasip1, which encodes a novel Ras-interacting protein, is strongly expressed in vascular endothelial cells throughout development, in both mouse and frog. Similar to the well-characterized vascular markers VEGFR2 and PECAM, Rasip1 is specifically expressed in angioblasts prior to vessel formation, in the initial embryonic vascular plexus, in the growing blood vessels during angiogenesis and in the endothelium of mature blood vessels into the postnatal period. Rasip1 expression is undetectable in VEGFR2 null embryos, which lack endothelial cells, suggesting that Rasip1 is endothelial specific. siRNA-mediated reduction of Rasip1 severely impairs angiogenesis and motility in endothelial cell cultures, and morpholino knockdown experiments in frog embryos demonstrate that Rasip1 is required for embryonic vessel formation in vivo. Together, these data identify Rasip1 as a novel endothelial factor that plays an essential role in vascular development.     

Ancestral Vascular Lumen Formation via Basal Cell Surfaces

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.     

Prohibitin-1 maintains the angiogenic capacity of endothelial cells by regulating mitochondrial function and senescence

Prohibitin 1 (PHB1) is a highly conserved protein that is mainly localized to the inner mitochondrial membrane and has been implicated in regulating mitochondrial function in yeast. Because mitochondria are emerging as an important regulator of vascular homeostasis, we examined PHB1 function in endothelial cells. PHB1 is highly expressed in the vascular system and knockdown of PHB1 in endothelial cells increases mitochondrial production of reactive oxygen species via inhibition of complex I, which results in cellular senescence. As a direct consequence, both Akt and Rac1 are hyperactivated, leading to cytoskeletal rearrangements and decreased endothelial cell motility, e.g., migration and tube formation. This is also reflected in an in vivo angiogenesis assay, where silencing of PHB1 blocks the formation of functional blood vessels. Collectively, our results provide evidence that PHB1 is important for mitochondrial function and prevents reactive oxygen species–induced senescence and thereby maintains the angiogenic capacity of endothelial cells.     

Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA(1/3), COX-2, and NF-kappaB activation- and EGFR transactivation-dependent mechanisms

Lysophosphatidic acid (LPA) is a lipid bioactive mediator which binds to G-protein-coupled receptors and activates a variety of cellular functions. LPA modulates multiple behaviors in endothelial cells, including cell proliferation and migration, capillary-like tube formation in vitro, activation of proteases, interactions with leukocytes, and expressions of inflammation-related genes, thereby regulating vessel formation. LPA has been reported to modulate the angiogenesis process. However, the role of LPA in the lymphangiogenesis process has not been studied. In this study, we showed that LPA upregulated vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) and subsequent endothelial cell tube formation in vitro and in vivo. These enhancement effects were LPA(1)- and LPA(3)-dependent and required cyclooxygenase-2 (COX-2), endothelial growth factor receptor (EGFR) transactivation and activation of nuclear factor kappaB (NF-kappaB). Moreover, LPA induced the protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HUVECs, and these enhancement effects were dependent on LPA(1) and LPA(3) activation and EGFR transactivation. Our results demonstrated that LPA might regulate VEGF-C and lymphatic marker expression in endothelial cells, which contributes to endothelial cell tube formation in vitro and in vivo, thus facilitating endothelial cell participation in the lymphangiogenesis process. This study clarifies the signaling mechanism of LPA-regulated VEGF-C expression and lymphatic marker expressions in endothelial cells, which suggest that LPA may be a suitable target for generating therapeutics against lymphangiogenesis and tumor metastasis.     

Mechanisms controlling human endothelial lumen formation and tube assembly in three-dimensional extracellular matrices

Recent data have revealed new mechanisms that underlie endothelial cell (EC) lumen formation during vascular morphogenic events in development, wound repair, and other disease states. It is apparent that EC interactions with extracellular matrices (ECMs) establish signaling cascades downstream of integrin ligation leading to activation of the Rho GTPases, Cdc42 and Rac1, which are required for lumen formation. In large part, this process is driven by intracellular vacuole formation and coalescence, which rapidly leads to the creation of fluid-filled matrix-free spaces that are then interconnected via EC-EC interactions to create multicellular tube structures. EC vacuoles markedly accumulate in a polarized fashion directly adjacent to the centrosome in a region that strongly accumulates Cdc42 protein as indicated by green fluorescent protein (GFP)-Cdc42 during the lumen formation process. Downstream of Cdc42-mediated signaling, key molecules that have been identified to be required for EC lumen formation include Pak2, Pak4, Par3, Par6, and the protein kinase C (PKC) isoforms zeta and epsilon. Together, these molecules coordinately regulate the critical EC lumen formation process in three-dimensional (3D) collagen matrices. These events also require cell surface proteolysis mediated through membrane type 1 matrix metalloproteinase (MT1-MMP), which is necessary to create vascular guidance tunnels within the 3D matrix environment. These tunnels represent physical spaces within the ECM that are necessary to regulate vascular morphogenic events, including the establishment of interconnected vascular tube networks as well as the recruitment of pericytes to initiate vascular tube maturation (via basement membrane matrix assembly) and stabilization. Current research continues to analyze how specific molecules integrate signaling information in concert to catalyze EC lumen formation, pericyte recruitment, and stabilization processes to control vascular morphogenesis in 3D extracellular matrices.     

A Distinct Mechanism of Vascular Lumen Formation in Xenopus Requires EGFL7

During vertebrate blood vessel development, lumen formation is the critical process by which cords of endothelial cells transition into functional tubular vessels. Here, we use Xenopus embryos to explore the cellular and molecular mechanisms underlying lumen formation of the dorsal aorta and the posterior cardinal veins, the primary major vessels that arise via vasculogenesis within the first 48 hours of life. We demonstrate that endothelial cells are initially found in close association with one another through the formation of tight junctions expressing ZO-1. The emergence of vascular lumens is characterized by elongation of endothelial cell shape, reorganization of junctions away from the cord center to the periphery of the vessel, and onset of Claudin-5 expression within tight junctions. Furthermore, unlike most vertebrate vessels that exhibit specialized apical and basal domains, we show that early Xenopus vessels are not polarized. Moreover, we demonstrate that in embryos depleted of the extracellular matrix factor Epidermal Growth Factor-Like Domain 7 (EGFL7), an evolutionarily conserved factor associated with vertebrate vessel development, vascular lumens fail to form. While Claudin-5 localizes to endothelial tight junctions of EGFL7-depleted embryos in a timely manner, endothelial cells of the aorta and veins fail to undergo appropriate cell shape changes or clear junctions from the cell-cell contact. Taken together, we demonstrate for the first time the mechanisms by which lumens are generated within the major vessels in Xenopus and implicate EGFL7 in modulating cell shape and cell-cell junctions to drive proper lumen morphogenesis.     

Electrostatic cell-surface repulsion initiates lumen formation in developing blood vessels

Blood vessels function in the uptake, transport, and delivery of gases and nutrients within the body. A key question is how the central lumen of blood vessels develops within a cord of vascular endothelial cells. Here, we demonstrate that sialic acids of apical glycoproteins localize to apposing endothelial cell surfaces and generate repelling electrostatic fields within an endothelial cell cord. Both in?vitro and in?vivo experiments show that the negative charge of sialic acids is required for the separation of endothelial cell surfaces and subsequent lumen formation. We also demonstrate that sulfate residues can substitute for sialic acids during lumen initiation. These results therefore reveal a key step in the creation of blood vessels, the most abundant conduits in the vertebrate body. Because negatively charged mucins and proteoglycans are often found on luminal cell surfaces, it is possible that electrostatic repulsion is a general principle also used to initiate lumen formation in other organs.     

Endothelial development taking shape

Blood vessel development is a vital process during embryonic development, during tissue growth, regeneration and disease processes in the adult. In the past decade researchers have begun to unravel basic molecular mechanisms that regulate the formation of vascular lumen, sprouting angiogenesis, fusion of vessels, and pruning of the vascular plexus. The understanding of the biology of these angiogenic processes is increasingly driven through studies on vascular development at the cellular resolution. Single cell analysis in vivo, advanced genetic tools and the widespread use of powerful animal models combined with improved imaging possibilities are delivering new insights into endothelial cell form, function and behavior angiogenesis. Moreover, the combination of in silico modeling and experimentation including dynamic imaging promotes insights into higher level cooperative behavior leading to functional patterning of vascular networks. Here we summarize recent concepts and advances in the field of vascular development, focusing in detail on the endothelial cell.     

Blood vessel tubulogenesis requires Rasip1 regulation of GTPase signaling

Cardiovascular function depends on patent blood vessel formation by endothelial cells (ECs). However, the mechanisms underlying vascular "tubulogenesis" are only beginning to be unraveled. We show that endothelial tubulogenesis requires the Ras interacting protein 1, Rasip1, and its binding partner, the RhoGAP Arhgap29. Mice lacking Rasip1 fail to form patent lumens in all blood vessels, including the early endocardial tube. Rasipl null angioblasts fail to properly localize the polarity determinant Par3 and display defective cell polarity, resulting in mislocalized junctional complexes and loss of adhesion to extracellular matrix (ECM). Similarly, depletion of either Rasip1 or Arhgap29 in cultured ECs blocks in vitro lumen formation, fundamentally alters the cytoskeleton, and reduces integrin-dependent adhesion to ECM. These defects result from increased RhoA/ROCK/myosin II activity and blockade of Cdc42 and Rac1 signaling. This study identifies Rasip1 as a unique, endothelial-specific regulator of Rho GTPase signaling, which is essential for blood vessel morphogenesis.     

Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo.

Endothelial cells are known to bind to laminin, and two peptides derived from the laminin A (CTFALRGDNP) and B1 (CDPGYIGSR) chains block the capillary-like tube formation on a laminin-rich basement membrane matrix, Matrigel. In the present study, we have used various in vitro and in vivo assays to investigate the angiogenic-biologic effects of a third active site in the laminin A chain, CSRARKQAASIKVAVSADR (designated PA22-2) on endothelial cells. The SIKVAV-containing peptide was as active as the YIGSR-containing peptide for endothelial cell attachment but was less active than either the RGD-containing peptide or intact laminin. Endothelial cells seeded on this peptide appeared fibroblastic with many extended processes, unlike the normal cobblestone morphology observed on tissue culture plastic. In addition, in contrast to normal tube formation on Matrigel, short irregular structures formed, some of which penetrated the matrix and sprouting was more apparent. Analysis of endothelial cell conditioned media of cells cultured in the presence of this peptide indicated degradation of the Matrigel and zymograms demonstrated active collagenase IV (gelatinase) at 68 and 62 Kd. A murine in vivo angiogenesis assay and the chick yolk sac/chorioallantoic membrane assays with the peptide demonstrated increased endothelial cell mobilization, capillary branching, and vessel formation. These data suggest that the -SIKVAV-site may play an important role in initiating branching and formation of new capillaries from the parent vessels, a behavior that is observed in vivo in response to tumor growth or in the normal vascular response to injury.     

Nobiletin, a polymethoxylated flavonoid from citrus, shows anti-angiogenic activity in a zebrafish in vivo model and HUVEC in vitro model

Traditional Chinese medicinal herbs are a rich source of compounds with reported anti-inflammatory and anti-carcinogenic effects. Growing evidence shows the codependence of chronic inflammation and angiogenesis, and the potential benefits of targeting angiogenesis in the treatment of chronic inflammation and targeting inflammation in the treatment of diseases with impaired angiogenesis. We hypothesized that the anti-inflammatory activity of the natural compounds may owe at least some of its efficacy to their anti-angiogenic activity and hence we investigated the anti-angiogenic activity of these compounds in vivo in zebrafish embryos and in vitro in human umbilical vein endothelial cells (HUVECs). Nobiletin, a polymethoxylated flavonoid from citrus fruits, showed anti-angiogenic activity in both assays. Nobiletin inhibited the formation of intersegmental vessels (ISVs) in live transgenic zebrafish embryos expressing green fluorescent protein (GFP) in the vasculature. Cell cycle analysis of dissociated zebrafish embryo cells showed that nobiletin induced G0/G1 phase accumulation in a dose-dependent manner in GFP-positive endothelial cells. Nobiletin also dose-dependently induced VEGF-A mRNA expression. In HUVECs, nobiletin inhibited endothelial cell proliferation and, to a greater extent, tube formation in a dose-dependent manner. As in the in vivo study, nobiletin induced G0/G1 cell cycle arrest in HUVECs. However, this arrest was not accompanied by an increase in apoptosis, indicating a cytostatic effect of nobiletin. This study, for the first time, identifies nobiletin as having potent anti-angiogenic activity and suggests that nobiletin has a great potential for future research and development as a cytostatic anti-proliferative agent.