Computational Screening of Tip and Stalk Cell Behavior Proposes a Role for Apelin Signaling in Sprout Progression

Angiogenesis involves the formation of new blood vessels by sprouting or splitting of existing blood vessels. During sprouting, a highly motile type of endothelial cell, called the tip cell, migrates from the blood vessels followed by stalk cells, an endothelial cell type that forms the body of the sprout. To get more insight into how tip cells contribute to angiogenesis, we extended an existing computational model of vascular network formation based on the cellular Potts model with tip and stalk differentiation, without making a priori assumptions about the differences between tip cells and stalk cells. To predict potential differences, we looked for parameter values that make tip cells (a) move to the sprout tip, and (b) change the morphology of the angiogenic networks. The screening predicted that if tip cells respond less effectively to an endothelial chemoattractant than stalk cells, they move to the tips of the sprouts, which impacts the morphology of the networks. A comparison of this model prediction with genes expressed differentially in tip and stalk cells revealed that the endothelial chemoattractant Apelin and its receptor APJ may match the model prediction. To test the model prediction we inhibited Apelin signaling in our model and in an in vitro model of angiogenic sprouting, and found that in both cases inhibition of Apelin or of its receptor APJ reduces sprouting. Based on the prediction of the computational model, we propose that the differential expression of Apelin and APJ yields a “self-generated” gradient mechanisms that accelerates the extension of the sprout.     

EphB4 controls blood vascular morphogenesis during postnatal angiogenesis

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin-1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non-neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non-neoplastic angiogenesis in general. This implies that both neoplastic and non-neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.     

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background

Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.

Methods and Findings

To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.

Conclusions

These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.     

A low-Mr chemoattractant for vascular endothelial cells

The formation of new blood vessels occurs by sprouting from previously existing microvasculature. The process involved directed migration of the vascular endothelial cells towards chemical signals released from the target tissue. We have used the Boyden chemotaxis chamber method to identify chemotactic signals for fetal bovine vascular endothelial cells. Human placenta organ cultures produce a high-Mr chemoattractant for the endothelial cells from which a low-Mr factor can be liberated with trichloroacetic acid treatment and ethanol extraction. This activity was isolated from extracts of human placenta using Sephadex LH-20, Amberlite XAD-2, and silica gel thin-layer chromatography. The Mr of the factor is less than 400, it is lipophilic and resistant to proteolytic enzymes. The factor induces chemotactic migration of both aortic endothelial cells and capillary endothelial cells from the retina, but has no effect on fibroblasts or leukocytes suggesting a specific function of the compound for the vascular endothelial cells.     

Incomplete and transitory decrease of glycolysis

During vessel sprouting, a migratory endothelial tip cell guides the sprout, while proliferating stalk cells elongate the branch. Tip and stalk cell phenotypes are not genetically predetermined fates, but are dynamically interchangeable to ensure that the fittest endothelial cell (EC) leads the vessel sprout. ECs increase glycolysis when forming new blood vessels. Genetic deficiency of the glycolytic activator PFKFB3 in ECs reduces vascular sprouting by impairing migration of tip cells and proliferation of stalk cells. PFKFB3-driven glycolysis promotes the tip cell phenotype during vessel sprouting, since PFKFB3 overexpression overrules the pro-stalk activity of Notch signaling. Furthermore, PFKFB3-deficient ECs cannot compete with wild-type neighbors to form new blood vessels in chimeric mosaic mice. In addition, pharmacological PFKFB3 blockade reduces pathological angiogenesis with modest systemic effects, likely because it decreases glycolysis only partially and transiently.     

The impact of exclusion processes on angiogenesis models

Angiogenesis is the process by which new blood vessels form from existing vessels. During angiogenesis, tip cells migrate via diffusion and chemotaxis, new tip cells are introduced through branching, loops form via tip-to-tip and tip-to-sprout anastomosis, and a vessel network forms as endothelial cells, known as stalk cells, follow the paths of tip cells (a process known as the snail-trail). Using a mean-field approximation, we systematically derive one-dimensional non-linear continuum models from a lattice-based cellular automaton model of angiogenesis in the corneal assay, explicitly accounting for cell volume. We compare our continuum models and a well-known phenomenological snail-trail model that is linear in the diffusive, chemotactic and branching terms, with averaged cellular automaton simulation results to distinguish macroscale volume exclusion effects and determine whether linear models can capture them. We conclude that, in general, both linear and non-linear models can be used at low cell densities when single or multi-species exclusion effects are negligible at the macroscale. When cell densities increase, our non-linear model should be used to capture non-linear tip cell behavior that occurs when single-species exclusion effects are pronounced, and alternative models should be derived for non-negligible multi-species exclusion effects.

     

Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.     

VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.     

Angiogenesis and vascular remodelling in normal and cancerous tissues

Vascular development and homeostasis are underpinned by two fundamental features: the generation of new vessels to meet the metabolic demands of under-perfused regions and the elimination of vessels that do not sustain flow. In this paper we develop the first multiscale model of vascular tissue growth that combines blood flow, angiogenesis, vascular remodelling and the subcellular and tissue scale dynamics of multiple cell populations. Simulations show that vessel pruning, due to low wall shear stress, is highly sensitive to the pressure drop across a vascular network, the degree of pruning increasing as the pressure drop increases. In the model, low tissue oxygen levels alter the internal dynamics of normal cells, causing them to release vascular endothelial growth factor (VEGF), which stimulates angiogenic sprouting. Consequently, the level of blood oxygenation regulates the extent of angiogenesis, with higher oxygenation leading to fewer vessels. Simulations show that network remodelling (and de novo network formation) is best achieved via an appropriate balance between pruning and angiogenesis. An important factor is the strength of endothelial tip cell chemotaxis in response to VEGF. When a cluster of tumour cells is introduced into normal tissue, as the tumour grows hypoxic regions form, producing high levels of VEGF that stimulate angiogenesis and cause the vascular density to exceed that for normal tissue. If the original vessel network is sufficiently sparse then the tumour may remain localised near its parent vessel until new vessels bridge the gap to an adjacent vessel. This can lead to metastable periods, during which the tumour burden is approximately constant, followed by periods of rapid growth.     

A Cell-Based Model of Extracellular-Matrix-Guided Endothelial Cell Migration During Angiogenesis

Angiogenesis, the formation of new blood vessels sprouting from existing ones, occurs in several situations like wound healing, tissue remodeling, and near growing tumors. Under hypoxic conditions, tumor cells secrete growth factors, including VEGF. VEGF activates endothelial cells (ECs) in nearby vessels, leading to the migration of ECs out of the vessel and the formation of growing sprouts. A key process in angiogenesis is cellular self-organization, and previous modeling studies have identified mechanisms for producing networks and sprouts. Most theoretical studies of cellular self-organization during angiogenesis have ignored the interactions of ECs with the extra-cellular matrix (ECM), the jelly or hard materials that cells live in. Apart from providing structural support to cells, the ECM may play a key role in the coordination of cellular motility during angiogenesis. For example, by modifying the ECM, ECs can affect the motility of other ECs, long after they have left. Here, we present an explorative study of the cellular self-organization resulting from such ECM-coordinated cell migration. We show that a set of biologically-motivated, cell behavioral rules, including chemotaxis, haptotaxis, haptokinesis, and ECM-guided proliferation suffice for forming sprouts and branching vascular trees.     

VEGF and endothelial guidance in angiogenic sprouting

The cellular actions of VEGF need to be coordinated to guide vascular patterning during sprouting angiogenesis. Individual endothelial tip cells lead and guide the blood vessel sprout, while neighboring stalk cells proliferate and form the vascular lumen. Recent studies illustrate how endothelial DLL4/NOTCH signalling, stimulated by VEGF, regulates the sprouting response by limiting tip cell formation in the stalk. The spatial distribution of VEGF, in turn, regulates the shape of the ensuing sprout by directing tip cell migration and determining stalk cell proliferation.     

Involvement of vessels and PDGFB in muscle splitting during chick limb development

Muscle formation and vascular assembly during embryonic development are usually considered separately. In this paper, we investigate the relationship between the vasculature and muscles during limb bud development. We show that endothelial cells are detected in limb regions before muscle cells and can organize themselves in space in the absence of muscles. In chick limbs, endothelial cells are detected in the future zones of muscle cleavage, delineating the cleavage pattern of muscle masses. We therefore perturbed vascular assembly in chick limbs by overexpressing VEGFA and demonstrated that ectopic blood vessels inhibit muscle formation, while promoting connective tissue. Conversely, local inhibition of vessel formation using a soluble form of VEGFR1 leads to muscle fusion. The endogenous location of endothelial cells in the future muscle cleavage zones and the inverse correlation between blood vessels and muscle suggests that vessels are involved in the muscle splitting process. We also identify the secreted factor PDGFB (expressed in endothelial cells) as a putative molecular candidate mediating the muscle-inhibiting and connective tissue-promoting functions of blood vessels. Finally, we propose that PDGFB promotes the production of extracellular matrix and attracts connective tissue cells to the future splitting site, allowing separation of the muscle masses during the splitting process.     

Selective expression of eGFP in mouse perivascular astrocytes by modification of the Mlc1 gene using T2A‐based ribosome skipping

Perivascular astrocyte end feet closely juxtapose cerebral blood vessels to regulate important developmental and physiological processes including endothelial cell proliferation and sprouting as well as the formation of the blood‐brain barrier (BBB). The mechanisms underlying these events remain largely unknown due to a lack of experimental models for identifying perivascular astrocytes and distinguishing these cell types from other astroglial populations. Megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) is a transmembrane protein that is expressed in perivascular astrocyte end feet where it controls BBB development and homeostasis. On the basis of this knowledge, we used T2A peptide‐skipping strategies to engineer a knock‐in mouse model in which the endogenous Mlc1 gene drives expression of enhanced green fluorescent protein (eGFP), without impacting expression of Mlc1 protein. Analysis of fetal, neonatal and adult Mlc1‐eGFP knock‐in mice revealed a dynamic spatiotemporal expression pattern of eGFP in glial cells, including nestin‐expressing neuroepithelial cells during development and glial fibrillary acidic protein (GFAP)‐expressing perivascular astrocytes in the postnatal brain. EGFP was not expressed in neurons, microglia, oligodendroglia, or cerebral vascular cells. Analysis of angiogenesis in the neonatal retina also revealed enriched Mlc1‐driven eGFP expression in perivascular astrocytes that contact sprouting blood vessels and regulate blood‐retinal barrier permeability. A cortical injury model revealed that Mlc1‐eGFP expression is progressively induced in reactive astrocytes that form a glial scar. Hence, Mlc1‐eGFP knock‐in mice are a new and powerful tool to identify perivascular astrocytes in the brain and retina and characterize how these cell types regulate cerebral blood vessel functions in health and disease.     

VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.     

Sphingosine 1-phosphate released from platelets during clotting accounts for the potent endothelial cell chemotactic activity of blood serum and provides a novel link between hemostasis and angiogenesis.

Recent studies have identified factors responsible for angiogenesis within developing tumors, but mediators of vessel formation at sites of trauma, injury, and wound healing are not clearly established. Here we show that sphingosine 1-phosphate (S1P) released by platelets during blood clotting is a potent, specific, and selective endothelial cell chemoattractant that accounts for most of the strong endothelial cell chemotactic activity of blood serum, an activity that is markedly diminished in plasma. Preincubation of endothelial cells with pertussis toxin inhibited this effect of S1P, demonstrating the involvement of a Galphai-coupled receptor. After S1P-induced migration, endothelial cells proliferated avidly and differentiated forming multicellular structures suggestive of early blood vessel formation. S1P was strikingly effective in enhancing the ability of fibroblast growth factor to induce angiogenesis in the avascular mouse cornea. Our results show that blood coagulation initiates endothelial cell angiogenic responses through the release of S1P, a potent endothelial cell chemoattractant that exerts its effects by activating a receptor-dependent process.     

Endothelial progenitor cells: A source for therapeutic vasculogenesis?

Angiogenesis has been defined as sprouting of blood vessels from pre-existing vascular structures. Risau and co-workers defined the term vasculogenesis while studying the formation of new blood vessels in embryoid bodies. This process is characterized by the recruitment of endothelial progenitor cells (EPC) to sites of new vessel formation with subsequent differentiation of EPC into mature endothelial cells, extensively proliferating in situ. Data from recent years provided evidence that EPC also exist in the adult and contribute to new vessel formation, a process called post-natal vasculogenesis. The existence of EPC has been convincingly shown in both, animals and humans. They represent a perfect cellular progenitor cell population for the ex vivo generation of EC, which in turn serve as cellular source for therapeutic vasculogenesis or tumor targeting. This review provides an overview on this hot topic of cellular-based therapeutic concepts and the therapeutic potential of ex vivo generated EPC.     

Fbxw7 controls angiogenesis by regulating endothelial notch activity

Notch signaling controls fundamental aspects of angiogenic blood vessel growth including the selection of sprouting tip cells, endothelial proliferation and arterial differentiation. The E3 ubiquitin ligase Fbxw7 is part of the SCF protein complex responsible for the polyubiquitination and thereby proteasomal degradation of substrates such as Notch, c-Myc and c-Jun. Here, we show that Fbxw7 is a critical regulator of angiogenesis in the mouse retina and the zebrafish embryonic trunk, which we attribute to its role in the degradation of active Notch. Growth of retinal blood vessel was impaired and the Notch ligand Dll4, which is also a Notch target, upregulated in inducible and endothelial cell-specific Fbxw7(iECKO) mutant mice. The stability of the cleaved and active Notch intracellular domain was increased after siRNA knockdown of the E3 ligase in cultured human endothelial cells. Injection of fbxw7 morpholinos interfered with the sprouting of zebrafish intersegmental vessels (ISVs). Arguing strongly that Notch and not other Fbxw7 substrates are primarily responsible for these phenotypes, the genetic inactivation of Notch pathway components reversed the impaired ISV growth in the zebrafish embryo as well as sprouting and proliferation in the mouse retina. Our findings establish that Fbxw7 is a potent positive regulator of angiogenesis that limits the activity of Notch in the endothelium of the growing vasculature.     

Computational Modeling Reveals that a Combination of Chemotaxis and Differential Adhesion Leads to Robust Cell Sorting during Tissue Patterning

Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.     

Neuronal clues to vascular guidance

The development of the vertebrate vascular system into a highly ordered and stereotyped network requires precise control over the branching and growth of new vessels. Recent research has highlighted the important role of genetic programs in regulating vascular patterning and in particular has established a crucial role for families of molecules previously described in controlling neuronal guidance. Like neurons, new vessels are guided along the correct path by integrating attractive and repulsive cues from the external environment. This is achieved by specialised endothelial cells at the leading tip of vessel sprouts which express receptor proteins that couple extracellular guidance signals with the cytoskeletal changes necessary to alter cell direction. Here, we review the genetic and in vitro evidence implicating four families of ligand-receptor signalling systems common to both neuronal and vessel guidance: the Ephrins and Eph receptors; Semaphorins, Neuropilins and Plexin receptors; Netrin and Unc5 receptors; and Slits and Robo receptors.