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1.  Computational Model of Vascular Endothelial Growth Factor Spatial Distribution in Muscle and Pro-Angiogenic Cell Therapy 
PLoS Computational Biology  2006;2(9):e127.
Members of the vascular endothelial growth factor (VEGF) family of proteins are critical regulators of angiogenesis. VEGF concentration gradients are important for activation and chemotactic guidance of capillary sprouting, but measurement of these gradients in vivo is not currently possible. We have constructed a biophysically and molecularly detailed computational model to study microenvironmental transport of two isoforms of VEGF in rat extensor digitorum longus skeletal muscle under in vivo conditions. Using parameters based on experimental measurements, the model includes: VEGF secretion from muscle fibers; binding to the extracellular matrix; binding to and activation of endothelial cell surface VEGF receptors; and internalization. For 2-D cross sections of tissue, we analyzed predicted VEGF distributions, gradients, and receptor binding. Significant VEGF gradients (up to 12% change in VEGF concentration over 10 μm) were predicted in resting skeletal muscle with uniform VEGF secretion, due to non-uniform capillary distribution. These relative VEGF gradients were not sensitive to extracellular matrix composition, or to the overall VEGF expression level, but were dependent on VEGF receptor density and affinity, and internalization rate parameters. VEGF upregulation in a subset of fibers increased VEGF gradients, simulating transplantation of pro-angiogenic myoblasts, a possible therapy for ischemic diseases. The number and relative position of overexpressing fibers determined the VEGF gradients and distribution of VEGF receptor activation. With total VEGF expression level in the tissue unchanged, concentrating overexpression into a small number of adjacent fibers can increase the number of capillaries activated. The VEGF concentration gradients predicted for resting muscle (average 3% VEGF/10 μm) is sufficient for cellular sensing; the tip cell of a vessel sprout is approximately 50 μm long. The VEGF gradients also result in heterogeneity in the activation of blood vessel VEGF receptors. This first model of VEGF tissue transport and heterogeneity provides a platform for the design and evaluation of therapeutic approaches.
Synopsis
It is not currently possible to experimentally quantify the gradients of protein concentration in the extracellular space in vivo. However, the concentration gradients of vascular endothelial growth factor (VEGF) are essential for both initiation and directed guidance of new blood vessels. The authors develop a computational model of VEGF transport in tissue in vivo (skeletal muscle, though the method is applicable to other tissues and other proteins) with realistic geometry and including biophysical interactions of VEGF, its receptors, and the extracellular matrix. Using this model, the authors predict for the first time the distribution of VEGF concentration and VEGF receptor activation throughout the tissue. VEGF concentration gradients are significant, up to 12% change in VEGF concentration over 10 μm in resting muscle. Transplanting VEGF-overexpressing myocytes (for therapeutic induction of blood vessel growth) increases the gradients significantly. Endothelial cells in sprouting vessels are approximately 50 μm long, and therefore the predicted gradients across the cell are high and sufficient for chemotactic guidance of the new vessels. The VEGF concentration gradients also result in significant heterogeneity in the activation of VEGF receptors on blood vessels throughout the tissue, a possible reason for the sporadic nature of sprout initiation.
doi:10.1371/journal.pcbi.0020127
PMCID: PMC1570371  PMID: 17002494
2.  Decreased Cell Adhesion Promotes Angiogenesis in a Pyk2-dependent Manner 
Experimental cell research  2011;317(13):1860-1871.
Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.
doi:10.1016/j.yexcr.2011.05.006
PMCID: PMC3123418  PMID: 21640103
Angiogenesis; cell adhesion; gene expression; Pyk2; VEGF
3.  Physicochemical regulation of endothelial sproutingin a 3-D microfluidic angiogenesis model 
Both physiological and pathological tissue remodeling (e.g., during wound healing and cancer, respectively) require new blood vessel formation via angiogenesis, but the underlying microenvironmental mechanisms remain poorly defined due in part to the lack of biologically relevant in vitro models. Here, we present a biomaterials-based microfluidic 3-D platform for analysis of endothelial sprouting in response to morphogen gradients. This system consists of three lithographically defined channels embedded in type I collagen hydrogels. A central channel is coated with endothelial cells, and two parallel side channels serve as a source and a sink for the steady-state generation of biochemical gradients. Gradients of vascular endothelial growth factor (VEGF) promoted sprouting, whereby endothelial cell responsiveness was markedly dependent on cell density and vessel geometry regardless of treatment conditions. These results point toward mechanical and/or autocrine mechanisms that may overwhelm pro-angiogenic paracrine signaling under certain conditions. To date, neither geometrical effects nor cell density have been considered critical determinants of angiogenesis in health and disease. This biomimetic vessel platform demonstrated utility for delineating hitherto underappreciated contributors of angiogenesis, and future studies may enable important new mechanistic insights that will inform anti-angiogenic cancer therapy.
doi:10.1002/jbm.a.34587
PMCID: PMC3776016  PMID: 23559519
collagen; microfluidics; gradient; angiogenesis; VEGF
4.  RAIN-Droplet: A Novel 3-D in vitro Angiogenesis Model 
Angiogenesis is fundamentally required for the initialization, development and metastatic spread of cancer. A rapidly expanding number of new experimental, chemical modulators of endothelial cell function have been described for the therapeutic inhibition of angiogenesis in cancer. Despite this expansion there has been very limited parallel growth of in vitro angiogenesis models or experimental tools. Here we present the Responsive Angiogenic Implanted Network (RAIN)-Droplet model and novel angiogenesis assay using an endothelial cell culture model of microvascular endothelial cells encapsulated in a spontaneously self-assembling, toroidal hydrogel droplet uniquely yielding discrete, pre-formed, angiogenic networks that may be embedded in 3-D matrices. On embedding, radial growth of capillary-like sprouts and cell invasion was observed. The sprouts formed as both outgrowths from endothelial cells on the surface of the droplets but also, uniquely, from the pre-formed network structures within the droplet. We demonstrate proof-of-principle for the utility of the model showing significant inhibition of sprout formation (p<0.001) in the presence of bevacizumab, an anti-angiogenic antibody. Using the RAIN-Droplet assay we also demonstrate a novel dose dependent pro-angiogenic function for the characteristically anti-angiogenic multi-kinase inhibitor sorafenib. Exposure of endothelial cells in 3-D culture to low, non-lethal doses (<1 μM) of sorafenib after initiation of sprouting resulted in the formation of significantly (p<0.05) more endothelial sprouts compared to controls over a 48-hour period. Higher doses of sorafenib (5 μM) resulted in a significant (p<0.05) reduction of sprouting over the same time period. The RAIN-Droplet model is a highly versatile and simply constructed 3-D focal sprouting approach well suited for the study of vascular morphogenesis and for preclinical testing of drugs. Furthermore, the RAIN-Droplet model has facilitated the discovery of a novel pro-angiogenic capacity for sorafenib which may impact the clinical application and dosing regimen of that drug.
doi:10.1038/labinvest.2012.77
PMCID: PMC4043634  PMID: 22565576
Endothelial; microvascular; puramatrix; 3-D cell-culture; drug screen; capillary formation assay; sorafenib; bevacizumab
5.  Computational Modeling of Interacting VEGF and Soluble VEGF Receptor Concentration Gradients 
Experimental data indicates that soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) modulates the guidance cues provided to sprouting blood vessels by VEGF-A. To better delineate the role of sFlt-1 in VEGF signaling, we have developed an experimentally based computational model. This model describes dynamic spatial transport of VEGF, and its binding to receptors Flt-1 and Flk-1, in a mouse embryonic stem cell model of vessel morphogenesis. The model represents the local environment of a single blood vessel. Our simulations predict that blood vessel secretion of sFlt-1 and increased local sFlt-1 sequestration of VEGF results in decreased VEGF–Flk-1 levels on the sprout surface. In addition, the model predicts that sFlt-1 secretion increases the relative gradient of VEGF–Flk-1 along the sprout surface, which could alter endothelial cell perception of directionality cues. We also show that the proximity of neighboring sprouts may alter VEGF gradients, VEGF receptor binding, and the directionality of sprout growth. As sprout distances decrease, the probability that the sprouts will move in divergent directions increases. This model is a useful tool for determining how local sFlt-1 and VEGF gradients contribute to the spatial distribution of VEGF receptor binding, and can be used in conjunction with experimental data to explore how multi-cellular interactions and relationships between local growth factor gradients drive angiogenesis.
doi:10.3389/fphys.2011.00062
PMCID: PMC3185289  PMID: 22007175
angiogenesis; vascular development; computational model; mathematical model; sFlt-1; VEGF; capillary sprouting
6.  Contact-Inhibited Chemotaxis in De Novo and Sprouting Blood-Vessel Growth 
PLoS Computational Biology  2008;4(9):e1000163.
Blood vessels form either when dispersed endothelial cells (the cells lining the inner walls of fully formed blood vessels) organize into a vessel network (vasculogenesis), or by sprouting or splitting of existing blood vessels (angiogenesis). Although they are closely related biologically, no current model explains both phenomena with a single biophysical mechanism. Most computational models describe sprouting at the level of the blood vessel, ignoring how cell behavior drives branch splitting during sprouting. We present a cell-based, Glazier–Graner–Hogeweg model (also called Cellular Potts Model) simulation of the initial patterning before the vascular cords form lumens, based on plausible behaviors of endothelial cells. The endothelial cells secrete a chemoattractant, which attracts other endothelial cells. As in the classic Keller–Segel model, chemotaxis by itself causes cells to aggregate into isolated clusters. However, including experimentally observed VE-cadherin–mediated contact inhibition of chemotaxis in the simulation causes randomly distributed cells to organize into networks and cell aggregates to sprout, reproducing aspects of both de novo and sprouting blood-vessel growth. We discuss two branching instabilities responsible for our results. Cells at the surfaces of cell clusters attempting to migrate to the centers of the clusters produce a buckling instability. In a model variant that eliminates the surface–normal force, a dissipative mechanism drives sprouting, with the secreted chemical acting both as a chemoattractant and as an inhibitor of pseudopod extension. Both mechanisms would also apply if force transmission through the extracellular matrix rather than chemical signaling mediated cell–cell interactions. The branching instabilities responsible for our results, which result from contact inhibition of chemotaxis, are both generic developmental mechanisms and interesting examples of unusual patterning instabilities.
Author Summary
A better understanding of the mechanisms by which endothelial cells (the cells lining the inner walls of blood vessels) organize into blood vessels is crucial if we need to enhance or suppress blood vessel growth under pathological conditions, including diabetes, wound healing, and tumor growth. During embryonic development, endothelial cells initially self-organize into a network of solid cords via blood vessel growth. The vascular network expands by splitting of existing blood vessels and by sprouting. Using computer simulations, we have captured a small set of biologically plausible cell behaviors that can reproduce the initial self-organization of endothelial cells, the sprouting of existing vessels, and the immediately subsequent remodeling of the resulting networks. In this model, endothelial cells both secrete diffusible chemoattractants and move up gradients of those chemicals by extending and retracting small pseudopods. By itself, this behavior causes simulated cells to accumulate to aggregate into large, round clusters. We propose that endothelial cells stop extending pseudopods along a given section of cell membrane as soon as the membrane touches the membrane of another endothelial cell (contact inhibition). Adding such contact-inhibition to our simulations allows vascular cords to form sprouts under a wide range of conditions.
doi:10.1371/journal.pcbi.1000163
PMCID: PMC2528254  PMID: 18802455
7.  Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells 
Stem cell reviews  2015;11(3):511-525.
Here we describe a strategy to model blood vessel development using a well-defined iPSC-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats.
doi:10.1007/s12015-014-9549-5
PMCID: PMC4411213  PMID: 25190668
Stem Cell; Induced Pluripotent Stem Cell; Endothelial Cell; Angiogenesis; Vascular Model; Vascular Function; Tubulogenesis; Migration; Sprouting; Barrier Function
8.  RhoA Mediates Flow-induced Endothelial Sprouting in a 3-D Tissue Analogue of Angiogenesis 
Lab on a chip  2012;12(23):10.1039/c2lc40389g.
Endothelial cells (ECs) integrate signals from the local microenvironment to guide their behaviour. RhoA is involved in vascular endothelial growth factor (VEGF) - driven angiogenesis, but its role in mechanotransduction during sprouting has not been established. Using dominant negative cell transfections in a microfluidic device that recapitulates angiogenic sprouting, we show that endothelial cells respond to interstitial flow in a RhoA-dependent manner while invading into a 3-D extracellular matrix. Furthermore, RhoA regulates flow-induced, but not VEGF gradient-5 induced tip cell filopodial extensions. Thus, RhoA pathways mediate mechanically-activated but not VEGF- induced endothelial morphogenesis.
doi:10.1039/c2lc40389g
PMCID: PMC3490212  PMID: 23073300
9.  Topography of Extracellular Matrix Mediates Vascular Morphogenesis and Migration Speeds in Angiogenesis 
PLoS Computational Biology  2009;5(7):e1000445.
The extracellular matrix plays a critical role in orchestrating the events necessary for wound healing, muscle repair, morphogenesis, new blood vessel growth, and cancer invasion. In this study, we investigate the influence of extracellular matrix topography on the coordination of multi-cellular interactions in the context of angiogenesis. To do this, we validate our spatio-temporal mathematical model of angiogenesis against empirical data, and within this framework, we vary the density of the matrix fibers to simulate different tissue environments and to explore the possibility of manipulating the extracellular matrix to achieve pro- and anti-angiogenic effects. The model predicts specific ranges of matrix fiber densities that maximize sprout extension speed, induce branching, or interrupt normal angiogenesis, which are independently confirmed by experiment. We then explore matrix fiber alignment as a key factor contributing to peak sprout velocities and in mediating cell shape and orientation. We also quantify the effects of proteolytic matrix degradation by the tip cell on sprout velocity and demonstrate that degradation promotes sprout growth at high matrix densities, but has an inhibitory effect at lower densities. Our results are discussed in the context of ECM targeted pro- and anti-angiogenic therapies that can be tested empirically.
Author Summary
A cell migrating in the extracellular matrix environment has to pull on the matrix fibers to move. When the matrix is too dense, the cell secretes enzymes to degrade the matrix proteins in order to get through. And when the matrix is too sparse, the cell produces matrix proteins to locally increase the “foothold”. How cells interact with the extracellular matrix is important in many processes from wound healing to cancer invasion. We use a computational model to investigate the topography of the matrix on cell migration and coordination in the context of tumor induced new blood vessel growth. The model shows that the density of the matrix fibers can have a strong effect on the extension speed and the morphology of a new blood vessel. Further results show that matrix degradation by the cells can enhance vessel sprout extension at high matrix density, but impede sprout extension at low matrix density. These results can potentially point to new targets for pro- and anti-angiogenesis therapies.
doi:10.1371/journal.pcbi.1000445
PMCID: PMC2709079  PMID: 19629173
10.  Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity 
Vascular Cell  2011;3:2.
Background
In the vasculature, Notch signaling functions as a downstream effecter of Vascular Endothelial Growth Factor (VEGF) signaling. VEGF regulates sprouting angiogenesis in part by inducing and activating matrix metalloproteases (MMPs). This study sought to determine if VEGF regulation of MMPs was mediated via Notch signaling and to determine how Notch regulation of MMPs influenced endothelial cell morphogenesis.
Methods and Results
We assessed the relationship between VEGF and Notch signaling in cultured human umbilical vein endothelial cells. Overexpression of VEGF-induced Notch4 and the Notch ligand, Dll4, activated Notch signaling, and altered endothelial cell morphology in a fashion similar to that induced by Notch activation. Expression of a secreted Notch antagonist (Notch1 decoy) suppressed VEGF-mediated activation of endothelial Notch signaling and endothelial morphogenesis. We demonstrate that Notch mediates VEGF-induced matrix metalloprotease activity via induction of MMP9 and MT1-MMP expression and activation of MMP2. Introduction of a MMP inhibitor blocked Notch-mediated endothelial morphogenesis. In mice, analysis of VEGF-induced dermal angiogenesis demonstrated that the Notch1 decoy reduced perivascular MMP9 expression.
Conclusions
Taken together, our data demonstrate that Notch signaling can act downstream of VEGF signaling to regulate endothelial cell morphogenesis via induction and activation of specific MMPs. In a murine model of VEGF-induced dermal angiogenesis, Notch inhibition led to reduced MMP9 expression.
doi:10.1186/2045-824X-3-2
PMCID: PMC3039832  PMID: 21349159
11.  Determination of Endothelial Stalk versus Tip Cell Potential during Angiogenesis by H2.0-like Homeobox-1 
Current Biology  2012;22(19):1789-1794.
Summary
Tissue branching morphogenesis requires the hierarchical organization of sprouting cells into leading “tip” and trailing “stalk” cells [1, 2]. During new blood vessel branching (angiogenesis), endothelial tip cells (TCs) lead sprouting vessels, extend filopodia, and migrate in response to gradients of the secreted ligand, vascular endothelial growth factor (Vegf) [3]. In contrast, adjacent stalk cells (SCs) trail TCs, generate the trunk of new vessels, and critically maintain connectivity with parental vessels. Here, we establish that h2.0-like homeobox-1 (Hlx1) determines SC potential, which is critical for angiogenesis during zebrafish development. By combining a novel pharmacological strategy for the manipulation of angiogenic cell behavior in vivo with transcriptomic analyses of sprouting cells, we identify the uniquely sprouting-associated gene, hlx1. Expression of hlx1 is almost entirely restricted to sprouting endothelial cells and is excluded from adjacent nonangiogenic cells. Furthermore, Hlx1 knockdown reveals its essential role in angiogenesis. Importantly, mosaic analyses uncover a cell-autonomous role for Hlx1 in the maintenance of SC identity in sprouting vessels. Hence, Hlx1-mediated maintenance of SC potential regulates angiogenesis, a finding that may have novel implications for sprouting morphogenesis of other tissues.
Highlights
► Expression of hlx1 is associated with angiogenic cell behavior in vivo ► hlx1 selectively marks sprouting endothelial cells during zebrafish development ► Hlx1 is required for intersegmental vessel angiogenesis in zebrafish embryos ► Hlx1 cell-autonomously maintains endothelial stalk cell potential
doi:10.1016/j.cub.2012.07.037
PMCID: PMC3471071  PMID: 22921365
12.  A Two-Way Communication between Microglial Cells and Angiogenic Sprouts Regulates Angiogenesis in Aortic Ring Cultures 
PLoS ONE  2011;6(1):e15846.
Background
Myeloid cells have been associated with physiological and pathological angiogenesis, but their exact functions in these processes remain poorly defined. Monocyte-derived tissue macrophages of the CNS, or microglial cells, invade the mammalian retina before it becomes vascularized. Recent studies correlate the presence of microglia in the developing CNS with vascular network formation, but it is not clear whether the effect is directly caused by microglia and their contact with the endothelium.
Methodology/Principal Findings
We combined in vivo studies of the developing mouse retina with in vitro studies using the aortic ring model to address the role of microglia in developmental angiogenesis. Our in vivo analyses are consistent with previous findings that microglia are present at sites of endothelial tip-cell anastomosis, and genetic ablation of microglia caused a sparser vascular network associated with reduced number of filopodia-bearing sprouts. Addition of microglia in the aortic ring model was sufficient to stimulate vessel sprouting. The effect was independent of physical contact between microglia and endothelial cells, and could be partly mimicked using microglial cell-conditioned medium. Addition of VEGF-A promoted angiogenic sprouts of different morphology in comparison with the microglial cells, and inhibition of VEGF-A did not affect the microglia-induced angiogenic response, arguing that the proangiogenic factor(s) released by microglia is distinct from VEGF-A. Finally, microglia exhibited oriented migration towards the vessels in the aortic ring cultures.
Conclusions/Significance
Microglia stimulate vessel sprouting in the aortic ring cultures via a soluble microglial-derived product(s), rather than direct contact with endothelial cells. The observed migration of microglia towards the growing sprouts suggests that their position near endothelial tip-cells could result from attractive cues secreted by the vessels. Our data reveals a two-way communication between microglia and vessels that depends on soluble factors and should extend the understanding of how microglia promote vascular network formation.
doi:10.1371/journal.pone.0015846
PMCID: PMC3018482  PMID: 21264342
13.  Design, Fabrication and Implementation of a Novel Multi Parameter Control Microfluidic Platform for Three-Dimensional Cell Culture and Real-Time Imaging 
Lab on a chip  2008;8(9):1468-1477.
New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigation. While the importance of the extracellular microenvironment is clear, the ability to investigate the effects of physiologically relevant biophysical and biochemical factors is restricted in traditional cell culture platforms. Moreover, the versatility for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small population is lacking. Here we introduce a microfluidic platform for 3D cell culture in biologically derived or synthetic hydrogels with the capability to monitor cellular dynamics in response to changes in their microenvironment. Direct scaffold microinjection, was employed to incorporate 3D matrices into microfluidic devices. Our system geometry permits a unique window for studying directional migration, e.g. sprouting angiogenesis, since sprouts grow predominantly in the microscopic viewing plane. In this study, we demonstrate the ability to generate gradients (non-reactive solute), surface shear, interstitial flow, and image cells in situ. Three different capillary morphogenesis assays are demonstrated. Human adult dermal microvascular endothelial cells (HMVEC-ad) were maintained in culture for up to 7 days during which they formed open lumen-like structures which was confirmed with confocal microscopy and by perfusion with fluorescent microspheres. In the sprouting assay, time-lapse movies revealed cellular mechanisms and dynamics (filopodial projection/retraction, directional migration, cell division and lumen formation) during tip-cell invasion of underlying 3D matrix and subsequent lumen formation.
doi:10.1039/b802395f
PMCID: PMC2560179  PMID: 18818801
14.  Histone deacetylase inhibitors enhance endothelial cell sprouting angiogenesis in vitro 
Surgery  2011;150(3):429-435.
Background
Treatment with histone deacetylases inhibitors (HDACi) such as valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) has been shown to improve survival following lethal insults through mechanisms that are incompletely understood. Cell survival under adverse conditions requires a healthy network of capillaries to ensure adequate oxygen delivery. Angiogenetic activation of endothelial cells to migrate and to form sprouts is associated with characteristic changes in gene expression profiles. As HDACI can modulate expression of various genes involved in angiogenic activity, we investigated the effect of these agents on capillary-like sprout-formation in this study.
Methods
Human umbilical vein endothelial cells (HUVECs) were cultured as multicellular spheroids within a type I collagen matrix, which promotes formation of sprouts resembling angiogenesis in vitro. Human umbilical vein endothelial cells (HUVECs) were cultured as multicellular spheroids within a type I collagen matrix, which promotes formation of sprouts (in-vitro angiogenesis). Cells were cultured under following conditions: Control (no growth factors); VPA (1mM); VEGF (Vascular endothelial growth factor, 10 ng/ml); VPA+ VEGF; SAHA (5 mM) and SAHA+VEGF. After 24 hours of treatment the length of spheroid sprouting and cell migration was assessed quantitatively. The levels of acetylated histone H3, phosphor-ERK1/2 and β-Catenin in HUVECs were measured by Western blotting at 6 hours after treatment.
Results
High levels of acetylated histone H3 were detected in VPA and SAHA treated-groups. Compared to VEGF alone treated group (2379 ± 147.1 μm), the spheroid sprouting was 1.7 times increased with VPA and VEGF combined treatment (3996 ± 192.5 μm) (p < 0.01). Cell migrations did not show significant difference after addition of VPA, whereas SAHA suppressed migration. Expression of β-Catenin was significantly increased by VPA and SAHA treatments. Addition of VPA greatly enhanced expression of phosphor-ERK1/2.
Conclusions
Exposure of HUVECs to VPA and SAHA increased the expression of β-catenin and enhanced spheroid sprout formation in vitro. Modulation of HDAC dependent pathways may offer a novel approach to alter angionegenic processes and provide a useful therapeutic target.
doi:10.1016/j.surg.2011.07.001
PMCID: PMC3164968  PMID: 21878227
Endothelial cells; angiogenesis; Histone deacetylase inhibitor
15.  Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/β-catenin pathway during brain angiogenesis 
eLife  null;4:e06489.
Despite the critical role of endothelial Wnt/β-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/β-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/β-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/β-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function.
DOI: http://dx.doi.org/10.7554/eLife.06489.001
eLife digest
Organs develop alongside the network of blood vessels that supply them with oxygen and nutrients. One way that new blood vessels grow is by sprouting out of the side of an existing vessel, via a process called angiogenesis. This process relies on signals that are received by the endothelial cells that line the inner wall of blood vessels, and that direct the cells to form a new ‘sprout’, consisting of tip and stalk cells.
In the developing brain, the Wnt/β-catenin signaling pathway helps direct the formation of blood vessels. In this pathway, a member of a protein family called Wnt signals to specific proteins on the surface of the cells lining the blood vessels. Much effort has gone into uncovering the identity of these proteins, with many studies looking at blood vessel development in the brain of mouse embryos.
In this study, Vanhollebeke et al. turned to zebrafish embryos to uncover new regulators of angiogenesis and define their roles during the multi-step process of blood vessel development in the brain. A variety of experimental techniques were used to alter and study the activity of different Wnt signaling pathway components. These experiments revealed that the Wnt7a and Wnt7b proteins signal to an endothelial cell membrane protein complex containing the proteins Gpr124 and Reck.
Vanhollebeke et al. then created ‘mosaic’ zebrafish embryos, which contained two genetically distinct types of cells—cells that were missing one of the components of Wnt/β-catenin signaling pathway, and wild-type cells. Visualizing the growth of the vessels showed that all the new blood vessels that sprouted had normal tip cells. However, the cells in the stalk of the sprout could be either normal or missing a signaling protein.
These findings demonstrate that Wnt/β-catenin signaling controls the pattern of blood vessel development in the brain by acting specifically on the invasive behaviors of the tip cells of new sprouts, a cellular mechanism that allows efficient organ-specific control of vascularization.
DOI: http://dx.doi.org/10.7554/eLife.06489.002
doi:10.7554/eLife.06489
PMCID: PMC4456509  PMID: 26051822
brain vascular development; tip cell; angiogenesis; Reck; Gpr124; Wnt7a/Wnt7b; zebrafish
16.  Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation 
PLoS ONE  2009;4(6):e5798.
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.
doi:10.1371/journal.pone.0005798
PMCID: PMC2688382  PMID: 19495422
17.  Activated Notch4 Inhibits Angiogenesis: Role of β1-Integrin Activation 
Molecular and Cellular Biology  2002;22(8):2830-2841.
Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased β1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of β1-integrins. Rather, we demonstrate that Notch4-expressing cells display β1-integrin in an active, high-affinity conformation. Furthermore, using function-activating β1-integrin antibodies, we demonstrate that activation of β1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting β1-integrin-mediated adhesion to the underlying matrix.
doi:10.1128/MCB.22.8.2830-2841.2002
PMCID: PMC133705  PMID: 11909975
18.  VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia 
The Journal of Cell Biology  2003;161(6):1163-1177.
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.
doi:10.1083/jcb.200302047
PMCID: PMC2172999  PMID: 12810700
VEGF; endothelial cell; filopodia; astrocyte; migration; proliferation
19.  Geometrical Microfeature Cues for Directing Tubulogenesis of Endothelial Cells 
PLoS ONE  2012;7(7):e41163.
Angiogenesis, the formation of new blood vessels by sprouting from pre-existing ones, is critical for the establishment and maintenance of complex tissues. Angiogenesis is usually triggered by soluble growth factors such as VEGF. However, geometrical cues also play an important role in this process. Here we report the induction of angiogenesis solely by SVVYGLR peptide micropatterning on polymer surfaces. SVVYGLR peptide stripes were micropatterned onto polymer surfaces by photolithography to study their effects on endothelial cell (EC) behavior. Our results showed that the EC behaviors (cell spreading, orientation and migration) were significantly more guided and regulated on narrower SVVYGLR micropatterns (10 and 50 µm) than on larger stripes (100 µm). Also, EC morphogenesis into tube formation was switched on onto the smaller patterns. We illustrated that the central lumen of tubular structures can be formed by only one-to-four cells due to geometrical constraints on the micropatterns which mediated cell-substrate adhesion and generated a correct maturation of adherens junctions. In addition, sprouting of ECs and vascular networks were also induced by geometrical cues on surfaces micropatterned with SVVYGLR peptides. These micropatterned surfaces provide opportunities for mimicking angiogenesis by peptide modification instead of exogenous growth factors. The organization of ECs into tubular structures and the induction of sprouting angiogenesis are important towards the fabrication of vascularized tissues, and this work has great potential applications in tissue engineering and tissue regeneration.
doi:10.1371/journal.pone.0041163
PMCID: PMC3400641  PMID: 22829923
20.  Formation of VEGF isoform-specific spatial distributions governing angiogenesis: computational analysis 
BMC Systems Biology  2011;5:59.
Background
The spatial distribution of vascular endothelial growth factor A (VEGF) is an important mediator of vascular patterning. Previous experimental studies in the mouse hindbrain and retina have suggested that VEGF alternative splicing, which controls the ability of VEGF to bind to heparan sulfate proteoglycans (HSPGs) in the extracellular matrix (ECM), plays a key role in controlling VEGF diffusion and gradients in tissues. Conversely, proteolysis notably by matrix metalloproteinases (MMPs), plays a critical role in pathological situations by releasing matrix-sequestered VEGF and modulating angiogenesis. However, computational models have predicted that HSPG binding alone does not affect VEGF localization or gradients at steady state.
Results
Using a 3D molecular-detailed reaction-diffusion model of VEGF ligand-receptor kinetics and transport, we test alternate models of VEGF transport in the extracellular environment surrounding an endothelial sprout. We show that differences in localization between VEGF isoforms, as observed experimentally in the mouse hindbrain, as well as the ability of proteases to redistribute VEGF in pathological situations, are consistent with a model where VEGF is endogenously cleared or degraded in an isoform-specific manner. We use our predictions of the VEGF distribution to quantify a tip cell's receptor binding and gradient sensing capacity. A novel prediction is that neuropilin-1, despite functioning as a coreceptor to VEGF165-VEGFR2 binding, reduces the ability of a cell to gauge the relative steepness of the VEGF distribution. Comparing our model to available in vivo vascular patterning data suggests that vascular phenotypes are most consistently predicted at short range by the soluble fraction of the VEGF distributions, or at longer range by matrix-bound VEGF detected in a filopodia-dependent manner.
Conclusions
Isoform-specific VEGF degradation provides a possible explanation for numerous examples of isoform specificity in VEGF patterning and examples of proteases relocation of VEGF upon release.
doi:10.1186/1752-0509-5-59
PMCID: PMC3113235  PMID: 21535871
21.  Mechanical Cell-Matrix Feedback Explains Pairwise and Collective Endothelial Cell Behavior In Vitro 
PLoS Computational Biology  2014;10(8):e1003774.
In vitro cultures of endothelial cells are a widely used model system of the collective behavior of endothelial cells during vasculogenesis and angiogenesis. When seeded in an extracellular matrix, endothelial cells can form blood vessel-like structures, including vascular networks and sprouts. Endothelial morphogenesis depends on a large number of chemical and mechanical factors, including the compliancy of the extracellular matrix, the available growth factors, the adhesion of cells to the extracellular matrix, cell-cell signaling, etc. Although various computational models have been proposed to explain the role of each of these biochemical and biomechanical effects, the understanding of the mechanisms underlying in vitro angiogenesis is still incomplete. Most explanations focus on predicting the whole vascular network or sprout from the underlying cell behavior, and do not check if the same model also correctly captures the intermediate scale: the pairwise cell-cell interactions or single cell responses to ECM mechanics. Here we show, using a hybrid cellular Potts and finite element computational model, that a single set of biologically plausible rules describing (a) the contractile forces that endothelial cells exert on the ECM, (b) the resulting strains in the extracellular matrix, and (c) the cellular response to the strains, suffices for reproducing the behavior of individual endothelial cells and the interactions of endothelial cell pairs in compliant matrices. With the same set of rules, the model also reproduces network formation from scattered cells, and sprouting from endothelial spheroids. Combining the present mechanical model with aspects of previously proposed mechanical and chemical models may lead to a more complete understanding of in vitro angiogenesis.
Author Summary
During the embryonic development of multicellular organisms, millions of cells cooperatively build structured tissues, organs and whole organisms, a process called morphogenesis. How the behavior of so many cells is coordinated to produce complex structures is still incompletely understood. Most biomedical research focuses on the molecular signals that cells exchange with one another. It has now become clear that cells also communicate biomechanically during morphogenesis. In cell cultures, endothelial cells—the building blocks of blood vessels—can organize into structures resembling networks of capillaries. Experimental work has shown that the endothelial cells pull onto the protein gel that they live in, called the extracellular matrix. On sufficiently compliant matrices, the strains resulting from these cellular pulling forces slow down and reorient adjacent cells. Here we propose a new computational model to show that this simple form of mechanical cell-cell communication suffices for reproducing the formation of blood vessel-like structures in cell cultures. These findings advance our understanding of biomechanical signaling during morphogenesis, and introduce a new set of computational tools for modeling mechanical interactions between cells and the extracellular matrix.
doi:10.1371/journal.pcbi.1003774
PMCID: PMC4133044  PMID: 25121971
22.  MMP-Sensitive PEG Diacrylate Hydrogels with Spatial Variations in Matrix Properties Stimulate Directional Vascular Sprout Formation 
PLoS ONE  2013;8(3):e58897.
The spatial presentation of immobilized extracellular matrix (ECM) cues and matrix mechanical properties play an important role in directed and guided cell behavior and neovascularization. The goal of this work was to explore whether gradients of elastic modulus, immobilized matrix metalloproteinase (MMP)-sensitivity, and YRGDS cell adhesion ligands are capable of directing 3D vascular sprout formation in tissue engineered scaffolds. PEGDA hydrogels were engineered with mechanical and biofunctional gradients using perfusion-based frontal photopolymerization (PBFP). Bulk photopolymerized hydrogels with uniform mechanical properties, degradation, and immobilized biofunctionality served as controls. Gradient hydrogels exhibited an 80.4% decrease in elastic modulus and a 56.2% decrease in immobilized YRGDS. PBFP hydrogels also demonstrated gradients in hydrogel degradation with degradation times ranging from 10–12 hours in the more crosslinked regions to 4–6 hours in less crosslinked regions. An in vitro model of neovascularization, composed of co-culture aggregates of endothelial and smooth muscle cells, was used to evaluate the effect of these gradients on vascular sprout formation. Aggregate invasion in gradient hydrogels occurred bi-directionally with sprout alignment observed in the direction parallel to the gradient while control hydrogels with homogeneous properties resulted in uniform invasion. In PBFP gradient hydrogels, aggregate sprout length was found to be twice as long in the direction parallel to the gradient as compared to the perpendicular direction after three weeks in culture. This directionality was found to be more prominent in gradient regions of increased stiffness, crosslinked MMP-sensitive peptide presentation, and immobilized YRGDS concentration.
doi:10.1371/journal.pone.0058897
PMCID: PMC3595229  PMID: 23554954
23.  Microfluidics embedded within extracellular matrix to define vascular architectures and pattern diffusive gradients 
Lab on a chip  2013;13(16):3246-3252.
Gradients of diffusive molecules within 3D extracellular matrix (ECM) are essential in guiding many processes such as development, angiogenesis, and cancer. The spatial distribution of factors that guide these processes is complex, dictated by the distribution and architecture of vasculature and presence of surrounding cells, which can serve as sources or sinks of factors. To generate temporally and spatially defined soluble gradients within a 3D cell culture environment, we developed an approach to patterning microfluidically ported microchannels that pass through a 3D ECM. Micromolded networks of sacrificial conduits ensconced within an ECM gel precursor solution are dissolved following ECM gelation to yield functional microfluidic channels. The dimensions and spatial layout of channels are readily dictated using photolithographic methods, and channels are connected to external flow via a gasket that also serves to house the 3D ECM. We demonstrated sustained spatial patterning of diffusive gradients dependent on the architecture of the microfluidic network, as well as the ability to independently populate cells in either the channels or surrounding ECM, enabling the study of 3D morphogenetic processes. To highlight the utility of this approach, we generated model vascular networks by lining the channels with endothelial cells and examined how channel architecture, through its effects on diffusion patterns, can guide the location and morphology of endothelial sprouting out from the channels. We show that locations of strongest gradients define positions of angiogenic sprouting, suggesting a mechanism by which angiogenesis is regulated in vivo and a potential means to spatially defining vasculature in tissue engineering applications. This flexible 3D microfluidic approach should have utility in modeling simple tissues and will aid in the screening and identification of soluble factor conditions that drive morphogenetic events such as angiogenesis.
doi:10.1039/c3lc50493j
PMCID: PMC4082768  PMID: 23787488
microfluidics; tissue engineering; cell migration; chemotaxis; angiogenesis
24.  MOSAIC: A Multiscale Model of Osteogenesis and Sprouting Angiogenesis with Lateral Inhibition of Endothelial Cells 
PLoS Computational Biology  2012;8(10):e1002724.
The healing of a fracture depends largely on the development of a new blood vessel network (angiogenesis) in the callus. During angiogenesis tip cells lead the developing sprout in response to extracellular signals, amongst which vascular endothelial growth factor (VEGF) is critical. In order to ensure a correct development of the vasculature, the balance between stalk and tip cell phenotypes must be tightly controlled, which is primarily achieved by the Dll4-Notch1 signaling pathway. This study presents a novel multiscale model of osteogenesis and sprouting angiogenesis, incorporating lateral inhibition of endothelial cells (further denoted MOSAIC model) through Dll4-Notch1 signaling, and applies it to fracture healing. The MOSAIC model correctly predicted the bone regeneration process and recapitulated many experimentally observed aspects of tip cell selection: the salt and pepper pattern seen for cell fates, an increased tip cell density due to the loss of Dll4 and an excessive number of tip cells in high VEGF environments. When VEGF concentration was even further increased, the MOSAIC model predicted the absence of a vascular network and fracture healing, thereby leading to a non-union, which is a direct consequence of the mutual inhibition of neighboring cells through Dll4-Notch1 signaling. This result was not retrieved for a more phenomenological model that only considers extracellular signals for tip cell migration, which illustrates the importance of implementing the actual signaling pathway rather than phenomenological rules. Finally, the MOSAIC model demonstrated the importance of a proper criterion for tip cell selection and the need for experimental data to further explore this. In conclusion, this study demonstrates that the MOSAIC model creates enhanced capabilities for investigating the influence of molecular mechanisms on angiogenesis and its relation to bone formation in a more mechanistic way and across different time and spatial scales.
Author Summary
The healing of a fracture largely depends on the development of a new blood vessel network (angiogenesis), which can be investigated and simulated with mathematical models. The current mathematical models of angiogenesis during fracture healing do not, however, implement all relevant biological scales (e.g. a tissue, cellular and intracellular level) rigorously in a multiscale framework. This study established a novel multiscale platform of angiogenesis during fracture healing (called MOSAIC) which allowed us to investigate the interactions of several influential factors across the different biological scales. We focused on the biological process of tip cell selection, during which a specific cell of a blood vessel, the “tip cell”, is selected to migrate away from the original vessel and lead the new branch. After showing that the MOSAIC model is able to correctly predict the bone regeneration process as well as many experimentally observed aspects of tip cell selection, we have used the model to investigate the influence of stimulating signals on the development of the vasculature and the progression of healing. These results raised an important biological question concerning the criterion for tip cell selection. This study demonstrates the potential of multiscale modeling to contribute to the understanding of biological processes like angiogenesis.
doi:10.1371/journal.pcbi.1002724
PMCID: PMC3469420  PMID: 23071433
25.  Local Guidance of Emerging Vessel Sprouts Requires Soluble Flt-1 (VEGFR-1) 
Developmental cell  2009;17(3):377-386.
Summary
Blood vessel networks form via sprouting of endothelial cells from parent vessels. Extrinsic cues guide sprouts once they leave the initiation site, but these cues are likely insufficient to regulate initial outward movement, and many embryonic vessel networks form in the absence of a strong extrinsic gradient. We hypothesized that nascent sprouts are guided by spatial cues produced along their own vessels, and that soluble Flt-1 (sFlt-1) participates in this guidance. Analysis of developing vessels with perturbed flt-1 function revealed mis-guided emerging sprouts, and transgenic sFlt-1 rescued sprout guidance parameters. sFlt-1 activity in endothelial cells immediately adjacent to the emerging sprout significantly improved local sprout guidance. Thus we propose that a vessel-intrinsic system initially guides emerging sprouts away from the parent vessel, utilizing spatially regulated expression of sFlt-1 in conjunction with exogenous VEGF. Local sprout guidance defects are predicted to contribute to vessel dysmorphogenesis during perturbed development and disease.
doi:10.1016/j.devcel.2009.07.011
PMCID: PMC2747120  PMID: 19758562
vessel branching; VEGF; soluble Flt-1; sprout guidance; angiogenesis; ES cells; retinal vessels

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