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1.  Glycolytic regulation of cell rearrangement in angiogenesis 
Nature Communications  2016;7:12240.
During vessel sprouting, endothelial cells (ECs) dynamically rearrange positions in the sprout to compete for the tip position. We recently identified a key role for the glycolytic activator PFKFB3 in vessel sprouting by regulating cytoskeleton remodelling, migration and tip cell competitiveness. It is, however, unknown how glycolysis regulates EC rearrangement during vessel sprouting. Here we report that computational simulations, validated by experimentation, predict that glycolytic production of ATP drives EC rearrangement by promoting filopodia formation and reducing intercellular adhesion. Notably, the simulations correctly predicted that blocking PFKFB3 normalizes the disturbed EC rearrangement in high VEGF conditions, as occurs during pathological angiogenesis. This interdisciplinary study integrates EC metabolism in vessel sprouting, yielding mechanistic insight in the control of vessel sprouting by glycolysis, and suggesting anti-glycolytic therapy for vessel normalization in cancer and non-malignant diseases.
Glycolytic regulator PFKFB3 is a key player in vessel sprouting. Here the authors develop a computational model predicting that PFKFB3 drives endothelial cell rearrangement during vessel sprouting by promoting filopodia formation and reducing intercellular adhesion, and empirically validate this prediction.
doi:10.1038/ncomms12240
PMCID: PMC4961802  PMID: 27436424
2.  Integrin signalling regulates YAP and TAZ to control skin homeostasis 
Development (Cambridge, England)  2016;143(10):1674-1687.
ABSTRACT
The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib.
Summary: In the squamous epithelium of the mouse skin, the transcription factors YAP and TAZ are regulated by integrin-Src signalling, and are required for proliferation of skin stem/progenitor cells.
doi:10.1242/dev.133728
PMCID: PMC4874484  PMID: 26989177
Hippo pathway; Integrin; Yes-associated protein; TAZ; Stratified squamous epithelium
3.  Synchronization of endothelial Dll4-Notch dynamics switch blood vessels from branching to expansion 
eLife  null;5:e12167.
Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning.
DOI: http://dx.doi.org/10.7554/eLife.12167.001
eLife digest
Throughout life, blood vessels are constantly remodelled to ensure that oxygen and nutrients reach every part of the body where they are needed. If a tissue is not receiving an adequate blood flow, existing blood vessels may widen or new blood vessels may sprout from their walls. In certain diseases, such as cancer, blood vessels may grow excessively to form disorganized networks, and preventing this growth may help to treat these conditions. However, we do not fully understand how the body controls the size, shape and branching pattern of blood vessels.
For a new blood vessel to sprout out of an existing vessel, the tip of the new branch must first develop. The tip forms when the endothelial cells that line the blood vessel are activated by a protein called vascular endothelial growth factor A (Vegfa), which is produced by the surrounding tissue. The activated endothelial cells respond to Vegfa stimulation by producing the protein Dll4, which talks to neighboring endothelial cells to prevent them from also forming new tips. In a way, this process bears all the signs of a competition between cells, as they fight for which one is allowed to take the lead. The losers of this competition, when forced into subordination by the tips, also serve an important function, as they will help to form and elongate the base of the new sprout.
Although it is known that changes in the levels of Vegfa in tissues can cause blood vessel branching to alter dramatically, the mechanisms that enable this to occur are not well understood. Computer simulations of the process predicted that an unexpected synchronization of Dll4 dynamics would be triggered when Vegfa levels increased; however, this remained to be observed in real cells.
Ubezio, Blanco et al. have now used fluorescent markers to observe the Dll4 production in lab-grown mouse endothelial cells as they formed new vessel sprouts in response to Vegfa. This revealed that the levels of Dll4 fluctuate widely in individual cells. Time-lapse movies of the cells showed that as a new sprout forms, the levels of Dll4 in neighbouring cells fluctuate in an uncoordinated manner. However, increasing the amount of Vegfa in the cells indeed synchronizes these fluctuations. This causes the new sprout to retract and allows the original blood vessel to widen. Increasing the levels of Dll4 had the same effect.
Further experiments confirmed that increasing the amount of Vegfa also reduces blood vessel branching in tumours in mice by synchronizing the fluctuations in the levels of Dll4 in neighbouring endothelial cells. In the future, these results could help refine anti-cancer treatments that work by blocking the activity of Vegfa and Dll4.
DOI: http://dx.doi.org/10.7554/eLife.12167.002
doi:10.7554/eLife.12167
PMCID: PMC4894757  PMID: 27074663
angiogenesis; agent based modelling; neovascularization; signalling dynamics; Human; Mouse
4.  Non-canonical Wnt signalling modulates the endothelial shear stress flow sensor in vascular remodelling 
eLife  null;5:e07727.
Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus.
DOI: http://dx.doi.org/10.7554/eLife.07727.001
eLife digest
Blood vessels play an essential role in growth and development as they transport many important molecules that help cells to survive. Throughout life, the forces that act on the blood vessels help to remodel the vessel network to ensure that blood gets to the parts of the body that need it. For example, the movement of blood across the surface of the endothelial cells that line the inside of the blood vessels applies a force called “shear stress” to the cells. The endothelial cells respond and adapt to the stress by altering their shape, patterns of gene activity and internal organization (known as their polarity).
It was not fully understood exactly how the forces acting on endothelial cells help to remodel the blood vessel network. Franco et al. have now investigated how a signalling pathway known as non-canonical Wnt signalling affects the remodelling of blood vessels in mice, and found that this pathway stabilizes existing connections between vessels.
Disrupting non-canonical Wnt signalling, by genetically engineering mice to lack proteins called Wnt5a and Wnt11, increased the sensitivity of endothelial cells to shear stress. Franco et al. then built a computer model that simulates blood flow and endothelial cell polarity in a network of blood vessels; this enabled them to measure the endothelial cells’ response to blood flow in complex vascular networks. The model was then used to show that endothelial cells lacking non-canonical Wnt signalling are able to reorient and become polarized against the direction of blood flow at lower levels of shear stress. Thus, non-canonical Wnt signalling helps to raise the threshold of shear stress above which endothelial cells change their properties.
Further work is now needed to identify how non-canonical Wnt signalling interferes with the ability of the endothelial cells to sense shear stress levels.
DOI: http://dx.doi.org/10.7554/eLife.07727.002
doi:10.7554/eLife.07727
PMCID: PMC4798962  PMID: 26845523
angiogenesis; retinal vasculature; vessel regression; shear stress; flow sensing; Mouse
5.  Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion 
Cell Reports  2016;14(3):440-448.
Summary
In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality.
Graphical Abstract
Highlights
•PKN2, but not PKN1 or PKN3, is essential during mouse embryogenesis•PKN2 knockout causes severe cardiovascular and morphogenetic abnormalities•PKN2 is required for mesenchymal growth and neural crest migration in vivo
The Rho effector PKN kinases regulate diverse cellular functions, but their in vivo function is unexplored. By systematically targeting the PKN family, Quetier et al. reveal a unique role for PKN2 during developmental growth and morphogenesis. These findings impact on developmental disorders and the targeting of PKN in disease.
doi:10.1016/j.celrep.2015.12.049
PMCID: PMC4733087  PMID: 26774483
PKC; protein kinase C; PKN; Protein kinase N; MEF; mouse embryonic fibroblasts; ES cells; Embryonic stem cells; 4-OHT; 4-hydroxytamoxifen; NCCs; neural crest cells
6.  Lack of CCM1 induces hypersprouting and impairs response to flow 
Human Molecular Genetics  2014;23(23):6223-6234.
Cerebral cavernous malformation (CCM) is a disease of vascular malformations known to be caused by mutations in one of three genes: CCM1, CCM2 or CCM3. Despite several studies, the mechanism of CCM lesion onset remains unclear. Using a Ccm1 knockout mouse model, we studied the morphogenesis of early lesion formation in the retina in order to provide insight into potential mechanisms. We demonstrate that lesions develop in a stereotypic location and pattern, preceded by endothelial hypersprouting as confirmed in a zebrafish model of disease. The vascular defects seen with loss of Ccm1 suggest a defect in endothelial flow response. Taken together, these results suggest new mechanisms of early CCM disease pathogenesis and provide a framework for further study.
doi:10.1093/hmg/ddu342
PMCID: PMC4222362  PMID: 24990152
7.  Fatty acid carbon is essential for dNTP synthesis in endothelial cells 
Nature  2015;520(7546):192-197.
The metabolism of endothelial cells (ECs) during vessel sprouting remains poorly studied. Here, we report that endothelial loss of CPT1a, a rate-limiting enzyme of fatty acid oxidation (FAO), caused vascular sprouting defects due to impaired proliferation, not migration of ECs. Reduction of FAO in ECs did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labeling studies in control ECs showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1a silencing reduced these processes and depleted EC stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1a-silenced ECs. Finally, CPT1 blockade inhibited pathological ocular angiogenesis, suggesting a novel strategy for blocking angiogenesis.
doi:10.1038/nature14362
PMCID: PMC4413024  PMID: 25830893
9.  Alk1 and Alk5 inhibition by Nrp1 controls vascular sprouting downstream of Notch 
Nature Communications  2015;6:7264.
Sprouting angiogenesis drives blood vessel growth in healthy and diseased tissues. Vegf and Dll4/Notch signalling cooperate in a negative feedback loop that specifies endothelial tip and stalk cells to ensure adequate vessel branching and function. Current concepts posit that endothelial cells default to the tip-cell phenotype when Notch is inactive. Here we identify instead that the stalk-cell phenotype needs to be actively repressed to allow tip-cell formation. We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5. Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour. Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-β/BMP signalling.
Notch signals are crucial for organization of angiogenic sprouting cells into the leading ‘tip' and trailing ‘stalk' cells. Here the authors show that endothelial neuropilin-1 quantitatively inhibits TGF-β/BMP signalling, explaining how Notch-mediated regulation of neuropilin-1 specifies endothelial tip and stalk cells.
doi:10.1038/ncomms8264
PMCID: PMC4557308  PMID: 26081042
10.  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.
Rationale:
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.
Objective:
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.
Conclusions:
Endothelial α-pv is essential for vessel sprouting and for vessel stability.
doi:10.1161/CIRCRESAHA.117.305818
PMCID: PMC4470528  PMID: 25925587
adherens junctions; angiogenesis; blood vessels; cell adhesion molecules; growth and development
12.  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.
doi:10.1371/journal.pbio.1002125
PMCID: PMC4401640  PMID: 25884288
13.  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.
doi:10.1101/cshperspect.a006569
PMCID: PMC3530037  PMID: 23085847
14.  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.
doi:10.1098/rsif.2014.0543
PMCID: PMC4233731  PMID: 25079871
angiogenesis; mouse; retina; blood flow; shear stress; lattice-Boltzmann
15.  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.
doi:10.4161/cib.28820
PMCID: PMC4203535  PMID: 25346793
cell guidance; cell migration; endothelial tip cell; filopodia; neuronal pathfinding; vascular patterning
16.  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.
doi:10.1084/jem.20121571
PMCID: PMC3782054  PMID: 24043760
17.  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.
doi:10.1002/dvdy.22139
PMCID: PMC3928721  PMID: 19882724
Nrarp; Notch pathway; embryo; somitic oscillator; PSM; cyclic gene
18.  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.
doi:10.1242/dev.097949
PMCID: PMC3879820  PMID: 24353059
Crim1; Vegfa; Endothelial cell; Angiogenesis
19.  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.
doi:10.3389/fpsyg.2013.00981
PMCID: PMC3882660  PMID: 24432007
decision making; happiness; incidental emotions; music; probability weighting; prospect theory; risk; risk aversion
20.  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.
doi:10.1371/journal.pone.0047488
PMCID: PMC3480364  PMID: 23115648
21.  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.
doi:10.1038/ncb2331
PMCID: PMC3261765  PMID: 21909098
22.  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.
doi:10.1038/nature10085
PMCID: PMC3214992  PMID: 21623369
23.  N-CAM Exhibits a Regulatory Function in Pathological Angiogenesis in Oxygen Induced Retinopathy 
PLoS ONE  2011;6(10):e26026.
Background
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.
Conclusion/Significance
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.
doi:10.1371/journal.pone.0026026
PMCID: PMC3197149  PMID: 22043302
24.  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
25.  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.
doi:10.1038/nm1742
PMCID: PMC2875252  PMID: 18345009

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