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2.  Endothelial Alpha-Parvin Controls Integrity of Developing Vasculature and Is Required for Maintenance of Cell–Cell Junctions 
Circulation Research  2015;117(1):29-40.
Supplemental Digital Content is available in the text.
Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known.
To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms.
Methods and Results:
We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv–deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell–extracellular matrix adhesion structures and an altered actin cytoskeleton.
Endothelial α-pv is essential for vessel sprouting and for vessel stability.
PMCID: PMC4470528  PMID: 25925587
adherens junctions; angiogenesis; blood vessels; cell adhesion molecules; growth and development
4.  Dynamic Endothelial Cell Rearrangements Drive Developmental Vessel Regression 
PLoS Biology  2015;13(4):e1002125.
Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.
A study of mouse retina and zebrafish vasculature shows how differential blood flow patterns direct the orientation and migration of endothelial cells as a mechanism for stabilizing or pruning individual blood vessel segments.
Author Summary
The question of how blood vessel networks achieve their branching patterns is key to our understanding of organ formation as well as diseases that involve vascular anomalies. Regression (or pruning) of blood vessel segments is required for functional vascular branching patterns; however, the molecular basis for this is poorly understood. Here we investigate remodeling of vascular networks in the mouse retina and in zebrafish and focus on the cellular components of the endothelium—the cell layer that lines blood vessels. We use high-resolution imaging to map and analyze endothelial cell orientation in relation to blood flow direction during vascular remodeling. We identify sequential steps that characterize blood vessel regression through endothelial cell migration, finding no evidence for predicted endothelial cell death in the retina. Combining endothelial cell mapping with computational modeling of flow-induced shear forces allows a systems-level prediction of endothelial cell migration patterns that drive vascular remodeling. Our work establishes how local differences in blood flow drive endothelial cells to orientate and migrate against the direction of flow. We show that the dynamic and polarized migration of endothelial cells leads to the regression of segments under low flow and the stabilization of segments under high flow. We propose that strong flow functions as an “attractor” for endothelial cells, while poorly perfused vessels are less “attractive,” thereby promoting regression of non-functional vessel segments.
PMCID: PMC4401640  PMID: 25884288
5.  VEGF and Notch in Tip and Stalk Cell Selection 
Sprouting angiogenesis is a dynamic process in which endothelial cells collectively migrate, shape new lumenized tubes, make new connections, and remodel the nascent network into a hierarchically branched and functionally perfused vascular bed. Endothelial cells in the nascent sprout adopt two distinct cellular phenotypes—known as tip and stalk cells—with specialized functions and gene expression patterns. VEGF and Notch signaling engage in an intricate cross talk to balance tip and stalk cell formation and to regulate directed tip cell migration and stalk cell proliferation. In this article, we summarize the current knowledge and implications of the tip/stalk cell concepts and the quantitative and dynamic integration of VEGF and Notch signaling in tip and stalk cell selection.
During angiogenesis, tip and stalk endothelial cells expand existing blood vessel networks. This is controlled by the VEGF and Notch signaling pathways, which induce distinct gene expression profiles in these cell types.
PMCID: PMC3530037  PMID: 23085847
6.  Computer simulations reveal complex distribution of haemodynamic forces in a mouse retina model of angiogenesis 
There is currently limited understanding of the role played by haemodynamic forces on the processes governing vascular development. One of many obstacles to be overcome is being able to measure those forces, at the required resolution level, on vessels only a few micrometres thick. In this paper, we present an in silico method for the computation of the haemodynamic forces experienced by murine retinal vasculature (a widely used vascular development animal model) beyond what is measurable experimentally. Our results show that it is possible to reconstruct high-resolution three-dimensional geometrical models directly from samples of retinal vasculature and that the lattice-Boltzmann algorithm can be used to obtain accurate estimates of the haemodynamics in these domains. We generate flow models from samples obtained at postnatal days (P) 5 and 6. Our simulations show important differences between the flow patterns recovered in both cases, including observations of regression occurring in areas where wall shear stress (WSS) gradients exist. We propose two possible mechanisms to account for the observed increase in velocity and WSS between P5 and P6: (i) the measured reduction in typical vessel diameter between both time points and (ii) the reduction in network density triggered by the pruning process. The methodology developed herein is applicable to other biomedical domains where microvasculature can be imaged but experimental flow measurements are unavailable or difficult to obtain.
PMCID: PMC4233731  PMID: 25079871
angiogenesis; mouse; retina; blood flow; shear stress; lattice-Boltzmann
7.  Tissue guidance without filopodia 
Filopodia are highly dynamic, rod-like protrusions that are found in abundance at the leading edge of migrating cells such as endothelial tip cells and at axonal growth cones of developing neurons. One proposed function of filopodia is that of an environmental probe, which serves to sense guidance cues during neuronal pathfinding and blood vessel patterning. However, recent studies show that tissue guidance occurs unhindered in the absence of filopodia, suggesting a dispensability of filopodia in this process. Here, we discuss evidence that support as well as dispute the role of filopodia in guiding the formation of stereotypic neuronal and blood vessel patterns.
PMCID: PMC4203535  PMID: 25346793
cell guidance; cell migration; endothelial tip cell; filopodia; neuronal pathfinding; vascular patterning
8.  Inhibition of the p110α isoform of PI 3-kinase stimulates nonfunctional tumor angiogenesis 
The Journal of Experimental Medicine  2013;210(10):1937-1945.
Inactivation of the stromal p110α isoform of PI3K increases vascular density, reduces vessel size, and alters pericyte coverage, which results in enhanced tumor hypoxia and necrosis to reduce tumor growth.
Understanding the direct, tumor cell–intrinsic effects of PI 3-kinase (PI3K) has been a key focus of research to date. Here, we report that cancer cell–extrinsic PI3K activity, mediated by the p110α isoform of PI3K, contributes in an unexpected way to tumor angiogenesis. In syngeneic mouse models, inactivation of stromal p110α led to increased vascular density, reduced vessel size, and altered pericyte coverage. This increased vascularity lacked functionality, correlating with enhanced tumor hypoxia and necrosis, and reduced tumor growth. The role of p110α in tumor angiogenesis is multifactorial, and includes regulation of proliferation and DLL4 expression in endothelial cells. p110α in the tumor stroma is thus a regulator of vessel formation, with p110α inactivation giving rise to nonfunctional angiogenesis, which can stunt tumor growth. This type of vascular aberration differs from vascular endothelial growth factor–centered antiangiogenesis therapies, which mainly lead to vascular pruning. Inhibition of p110α may thus offer a new antiangiogenic therapeutic opportunity in cancer.
PMCID: PMC3782054  PMID: 24043760
9.  Cyclic Nrarp mRNA Expression Is Regulated by the Somitic Oscillator but Nrarp Protein Levels Do Not Oscillate 
Somites are formed progressively from the presomitic mesoderm (PSM) in a highly regulated process according to a strict periodicity driven by an oscillatory mechanism. The Notch and Wnt pathways are key components in the regulation of this somitic oscillator and data from Xenopus and zebrafish embryos indicate that the Notch-downstream target Nrarp participates in the regulation of both activities. We have analyzed Nrarp/nrarp-a expression in the PSM of chick, mouse and zebrafish embryos, and we show that it cycles in synchrony with other Notch regulated cyclic genes. In the mouse its transcription is both Wnt- and Notch-dependent, whereas in the chick and fish embryo it is simply Notch-dependent. Despite oscillating mRNA levels, Nrarp protein does not oscillate in the PSM. Finally, neither gain nor loss of Nrarp function interferes with the normal expression of Notch-related cyclic genes.
PMCID: PMC3928721  PMID: 19882724
Nrarp; Notch pathway; embryo; somitic oscillator; PSM; cyclic gene
10.  Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling 
Development (Cambridge, England)  2014;141(2):448-459.
Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2.
PMCID: PMC3879820  PMID: 24353059
Crim1; Vegfa; Endothelial cell; Angiogenesis
11.  Music-evoked incidental happiness modulates probability weighting during risky lottery choices 
We often make decisions with uncertain consequences. The outcomes of the choices we make are usually not perfectly predictable but probabilistic, and the probabilities can be known or unknown. Probability judgments, i.e., the assessment of unknown probabilities, can be influenced by evoked emotional states. This suggests that also the weighting of known probabilities in decision making under risk might be influenced by incidental emotions, i.e., emotions unrelated to the judgments and decisions at issue. Probability weighting describes the transformation of probabilities into subjective decision weights for outcomes and is one of the central components of cumulative prospect theory (CPT) that determine risk attitudes. We hypothesized that music-evoked emotions would modulate risk attitudes in the gain domain and in particular probability weighting. Our experiment featured a within-subject design consisting of four conditions in separate sessions. In each condition, the 41 participants listened to a different kind of music—happy, sad, or no music, or sequences of random tones—and performed a repeated pairwise lottery choice task. We found that participants chose the riskier lotteries significantly more often in the “happy” than in the “sad” and “random tones” conditions. Via structural regressions based on CPT, we found that the observed changes in participants' choices can be attributed to changes in the elevation parameter of the probability weighting function: in the “happy” condition, participants showed significantly higher decision weights associated with the larger payoffs than in the “sad” and “random tones” conditions. Moreover, elevation correlated positively with self-reported music-evoked happiness. Thus, our experimental results provide evidence in favor of a causal effect of incidental happiness on risk attitudes that can be explained by changes in probability weighting.
PMCID: PMC3882660  PMID: 24432007
decision making; happiness; incidental emotions; music; probability weighting; prospect theory; risk; risk aversion
12.  Visualization of Endothelial Actin Cytoskeleton in the Mouse Retina 
PLoS ONE  2012;7(10):e47488.
Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.
PMCID: PMC3480364  PMID: 23115648
13.  VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling 
Nature Cell Biology  2011;13(10):1202-1213.
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.
PMCID: PMC3261765  PMID: 21909098
14.  Regulation of angiogenesis by a non-canonical Wnt-Flt1 pathway in myeloid cells 
Nature  2011;474(7352):511-515.
Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where angiogenesis occurs postnatally1, somatic deletion in RMCs of the Wnt ligand transporter Wntless2,3 results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF)4-6. These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching.
PMCID: PMC3214992  PMID: 21623369
15.  N-CAM Exhibits a Regulatory Function in Pathological Angiogenesis in Oxygen Induced Retinopathy 
PLoS ONE  2011;6(10):e26026.
Diabetic retinopathy and retinopathy of prematurity are diseases caused by pathological angiogenesis in the retina as a consequence of local hypoxia. The underlying mechanism for epiretinal neovascularization (tuft formation), which contributes to blindness, has yet to be identified. Neural cell adhesion molecule (N-CAM) is expressed by Müller cells and astrocytes, which are in close contact with the retinal vasculature, during normal developmental angiogenesis.
Methodology/Principal Findings
Notably, during oxygen induced retinopathy (OIR) N-CAM accumulated on astrocytes surrounding the epiretinal tufts. Here, we show that N-CAM ablation results in reduced vascular tuft formation due to reduced endothelial cell proliferation despite an elevation in VEGFA mRNA expression, whereas retinal developmental angiogenesis was unaffected.
We conclude that N-CAM exhibits a regulatory function in pathological angiogenesis in OIR. This is a novel finding that can be of clinical relevance in diseases associated with proliferative vasculopathy.
PMCID: PMC3197149  PMID: 22043302
16.  A Two-Way Communication between Microglial Cells and Angiogenic Sprouts Regulates Angiogenesis in Aortic Ring Cultures 
PLoS ONE  2011;6(1):e15846.
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.
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.
PMCID: PMC3018482  PMID: 21264342
17.  Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability 
Nature medicine  2008;14(4):448-453.
The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures1. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system2,3; however, their role in the mammalian vasculature remains ill defined4–8. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165–induced migration, tube formation and permeability in vitro and VEGF-165–stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.
PMCID: PMC2875252  PMID: 18345009
18.  Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy 
PLoS ONE  2009;4(11):e7716.
The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) and Serial Block Face/Scanning Electron Microscopy (SBF/SEM). The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism.
PMCID: PMC2769265  PMID: 19893745
19.  Tipping the Balance: Robustness of Tip Cell Selection, Migration and Fusion in Angiogenesis 
PLoS Computational Biology  2009;5(10):e1000549.
Vascular abnormalities contribute to many diseases such as cancer and diabetic retinopathy. In angiogenesis new blood vessels, headed by a migrating tip cell, sprout from pre-existing vessels in response to signals, e.g., vascular endothelial growth factor (VEGF). Tip cells meet and fuse (anastomosis) to form blood-flow supporting loops. Tip cell selection is achieved by Dll4-Notch mediated lateral inhibition resulting, under normal conditions, in an interleaved arrangement of tip and non-migrating stalk cells. Previously, we showed that the increased VEGF levels found in many diseases can cause the delayed negative feedback of lateral inhibition to produce abnormal oscillations of tip/stalk cell fates. Here we describe the development and implementation of a novel physics-based hierarchical agent model, tightly coupled to in vivo data, to explore the system dynamics as perpetual lateral inhibition combines with tip cell migration and fusion. We explore the tipping point between normal and abnormal sprouting as VEGF increases. A novel filopodia-adhesion driven migration mechanism is presented and validated against in vivo data. Due to the unique feature of ongoing lateral inhibition, ‘stabilised’ tip/stalk cell patterns show sensitivity to the formation of new cell-cell junctions during fusion: we predict cell fates can reverse. The fusing tip cells become inhibited and neighbouring stalk cells flip fate, recursively providing new tip cells. Junction size emerges as a key factor in establishing a stable tip/stalk pattern. Cell-cell junctions elongate as tip cells migrate, which is shown to provide positive feedback to lateral inhibition, causing it to be more susceptible to pathological oscillations. Importantly, down-regulation of the migratory pathway alone is shown to be sufficient to rescue the sprouting system from oscillation and restore stability. Thus we suggest the use of migration inhibitors as therapeutic agents for vascular normalisation in cancer.
Author Summary
Abnormal vasculature exacerbates many diseases such as cancer and diabetic retinopathy. In angiogenesis new blood vessels, headed by a migrating tip cell, sprout from pre-existing vessels in response to chemical signals. The signals are released from newly oxygen deficient tissue. The signals are known to be different in disease and are thought to cause the process of angiogenesis to progress abnormally, though the reasons for this remain unclear. Normalisation of angiogenesis has great potential as a therapeutic strategy; it has been shown to reduce metastasis and improve drug delivery in tumours. Here we focus on the behaviours of three inter-related initial angiogenic pathways associated with changes in tissue signal conditions, utilising both in silico and in vivo approaches. By the construction and implementation of a novel computational model for cell motility and signal processing we present a new theory on why angiogenesis exhibits such sensitivity to signal changes and show that the behaviour in disease is surprisingly more robust than normal functioning. This we attribute to the positive feedback of cell migration reinforcing abnormal oscillations in cell fate selection. We make the unique prediction that normalisation could be achieved by reducing cell migration alone.
PMCID: PMC2762315  PMID: 19876379
20.  Wnt/β-catenin signaling controls development of the blood–brain barrier 
The Journal of Cell Biology  2008;183(3):409-417.
The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.
PMCID: PMC2575783  PMID: 18955553
21.  VEGF and endothelial guidance in angiogenic sprouting 
Organogenesis  2008;4(4):241-246.
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 neighbouring 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.
PMCID: PMC2634329  PMID: 19337404
VEGF; Notch; angiogenesis; tip cells; vascular patterning
22.  Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice 
The Journal of Clinical Investigation  2008;118(11):3725-3737.
Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1–derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn–/– mice). EHC T-syn–/– mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn–/– mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn–/– lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn–/– defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn–/– mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn–/– pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.
PMCID: PMC2567837  PMID: 18924607
23.  VEGF and Notch Signaling 
Cell Adhesion & Migration  2007;1(3):133-136.
Tubular sprouting in angiogenesis relies on division of labour between endothelial tip cells, leading and guiding the sprout, and their neighboring stalk cells, which divide and form the vascular lumen. We previously learned how the graded extracellular distribution of heparin-binding vascular endothelial growth factor (VEGF)-A orchestrates and balances tip and stalk cell behavior. Recent data now provided insight into the regulation of tip cell numbers, illustrating how delta-like (Dll)4-Notch signalling functions to limit the explorative tip cell behavior induced by VEGF-A. These data also provided a first answer to the question why not all endothelial cells stimulated by VEGF-A turn into tip cells. Here we review this new model and discuss how VEGF-A and Dll4/Notch signalling may interact dynamically at the cellular level to control vascular patterning.
PMCID: PMC2634014  PMID: 19262131
Notch; angiogenesis; mouse development; VEGF-A; tip cells
24.  Pericytes limit tumor cell metastasis 
Journal of Clinical Investigation  2006;116(3):642-651.
Previously we observed that neural cell adhesion molecule (NCAM) deficiency in β tumor cells facilitates metastasis into distant organs and local lymph nodes. Here, we show that NCAM-deficient β cell tumors grew leaky blood vessels with perturbed pericyte-endothelial cell-cell interactions and deficient perivascular deposition of ECM components. Conversely, tumor cell expression of NCAM in a fibrosarcoma model (T241) improved pericyte recruitment and increased perivascular deposition of ECM molecules. Together, these findings suggest that NCAM may limit tumor cell metastasis by stabilizing the microvessel wall. To directly address whether pericyte dysfunction increases the metastatic potential of solid tumors, we studied β cell tumorigenesis in primary pericyte-deficient Pdgfbret/ret mice. This resulted in β tumor cell metastases in distant organs and local lymph nodes, demonstrating a role for pericytes in limiting tumor cell metastasis. These data support a new model for how tumor cells trigger metastasis by perturbing pericyte-endothelial cell-cell interactions.
PMCID: PMC1361347  PMID: 16470244
25.  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.
PMCID: PMC2172999  PMID: 12810700
VEGF; endothelial cell; filopodia; astrocyte; migration; proliferation

Results 1-25 (27)