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1.  Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding 
eLife  2014;3:e03720.
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1VEGF−). Nrp1VEGF− mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.
DOI: http://dx.doi.org/10.7554/eLife.03720.001
eLife digest
Blood flows through blood vessels to carry oxygen and nutrients towards, and waste away from, the cells of the body. New blood vessels are formed not only during development but also throughout life as part of normal tissue growth and repair. However, blood vessels may also form as a consequence of diseases, such as cancer. For example, tumors often stimulate the growth of new blood vessels to ensure a good supply of blood carrying nutrients and oxygen. As such, some anti-cancer therapies try to stop blood vessels from developing in an attempt to slow down or prevent tumor growth.
New blood vessels often form by branching off from existing vessels. One molecule that stimulates this branching process is called vascular endothelial growth factor (or VEGF for short). Three ‘receptor’ proteins found on the outside of cells can bind to the VEGF molecule and then trigger a response inside the cell that guides the development of new blood vessels. VEGF and its receptor proteins—including one called NRP1—are being investigated as a possible target for drugs that could treat cancer and other diseases affecting blood vessels. However, the exact mechanisms that control the formation of new blood vessels are not fully understood, which makes it difficult to develop these treatments.
Now Gelfand et al. have created mice whose NRP1 receptors cannot bind VEGF. These mice unexpectedly survive to adulthood and develop normal blood vessels. This outcome is in contrast to mice that lack NRP1, which normally die as embryos and have severe defects with their nerves and blood vessels. Gelfand et al. instead found that mice that only lack NRP1 in the cells of their blood vessels had less of another receptor protein called VEGFR2 on the surface of these cells. This result suggests that NRP1 controls blood vessel development, not by binding to VEGF but by affecting how much of the VEGFR2 receptor is available to interact with VEGF.
These findings challenge the long-held view of how NRP1 functions and lead Gelfand et al. to suggest a new mechanism: NRP1 interacts with VEGFR2, rather than with VEGF, to control the formation of new blood vessels. Future work will aim to uncover how these interactions regulate the normal development of blood vessels, and if other molecules that bind to NRP1 are involved in this process. Furthermore, these findings may help to guide the on-going efforts to develop drugs that target NRP1 into treatments that are effective against diseases that involve problems with blood vessels—including diabetes, immune disorders, and cancer.
DOI: http://dx.doi.org/10.7554/eLife.03720.002
doi:10.7554/eLife.03720
PMCID: PMC4197402  PMID: 25244320
Neuropilin-1; developmental angiogenesis; VEGFR2; VEGF; mouse genetics; mouse
2.  Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression 
PLoS Genetics  2015;11(7):e1005324.
Vascular branching morphogenesis is activated and maintained by several signaling pathways. Among them, vascular endothelial growth factor receptor 2 (VEGFR2) signaling is largely presented in arteries, and VEGFR3 signaling is in veins and capillaries. Recent reports have documented that Snail, a well-known epithelial-to-mesenchymal transition protein, is expressed in endothelial cells, where it regulates sprouting angiogenesis and embryonic vascular development. Here, we identified Snail as a regulator of VEGFR3 expression during capillary branching morphogenesis. Snail was dramatically upregulated in sprouting vessels in the developing retinal vasculature, including the leading-edged vessels and vertical sprouting vessels for capillary extension toward the deep retina. Results from in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 promoter via cooperating with early growth response protein-1. Snail knockdown in postnatal mice attenuated the formation of the deep capillary plexus, not only by impairing vertical sprouting vessels but also by downregulating VEGFR3 expression. Collectively, these data suggest that the Snail-VEGFR3 axis controls capillary extension, especially in vessels expressing VEGFR2 at low levels.
Author Summary
Endothelial cells have the intrinsic capacity to shuffle between tip, stalk, and phalanx cells in angiogenic processes. These transitions require the induction or repression of transcripts that are specific for their phenotypes, along with morphological changes. To gain insight into spatiotemporal induction during vascular branching morphogenesis, we used Affymetrix oligonucleotide arrays to screen for Snail. Then, we used stable, small-interfering RNA or the lentivirus-short hairpin RNA system to examine the angiogenic roles of endothelial Snail during retinal capillary morphogenesis. Knockdown of Snail in the developing retinal vasculature impaired deep capillary formation and attenuated vascular endothelial growth factor receptor 3 expression, indicating a functional link between Snail and vascular endothelial growth factor receptor 3. Moreover, we showed vascular endothelial growth factor receptor 3 as a transcriptional target of Snail in vitro. In the retinal vasculature, the deep capillary plexus is a unique vessel with only capillary. The deep capillary plays a critical role in retinal development, neuronal survival, and pathological conditions, including ischemic diseases. Our findings provide molecular insights into the role of the Snail-vascular endothelial growth factor receptor 3 axis in capillary formation under pathophysiological conditions.
doi:10.1371/journal.pgen.1005324
PMCID: PMC4493050  PMID: 26147525
3.  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
4.  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
5.  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
6.  Endothelial Cell Self-fusion during Vascular Pruning 
PLoS Biology  2015;13(4):e1002126.
During embryonic development, vascular networks remodel to meet the increasing demand of growing tissues for oxygen and nutrients. This is achieved by the pruning of redundant blood vessel segments, which then allows more efficient blood flow patterns. Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail. Here, we present the subintestinal vein (SIV) plexus of the zebrafish embryo as a novel model to study pruning at the cellular level. We show that blood vessel regression is a coordinated process of cell rearrangements involving lumen collapse and cell–cell contact resolution. Interestingly, the cellular rearrangements during pruning resemble endothelial cell behavior during vessel fusion in a reversed order. In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection. Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion. In a second step, the unicellular connection is resolved unilaterally, and the pruning cell rejoins the opposing branch. Thus, we show for the first time that various cellular activities are coordinated to achieve blood vessel pruning and define two different morphogenetic pathways, which are selected by the flow environment.
In vivo live imaging of zebrafish embryos reveals the diverse cellular activities involved in the pruning of unwanted blood vessels, including the self-fusion of endothelial cells.
Author Summary
The blood vasculature circulates gas, nutrients, hormones, and metabolites to all organs of the body. It is indispensable for survival and already functions at very early stages of embryonic development. At this point, new blood vessels form mainly through angiogenesis—the outgrowth of new vessels from existing ones. New vascular sprouts connect to each other to form functional loops with blood flow, a process termed anastomosis. Vascular plexuses formed in this way subsequently remodel to a final structure with efficient flow patterns. Remodeling often involves pruning or regression of unnecessary branches, leading to a simplification of the network. Our in vivo live imaging studies of the pruning process in the zebrafish embryo show that vessel regression occurs through cell rearrangements, wherein cells consecutively migrate out of the pruning branch. As a result, the initially multicellular vessel is reduced to a single cell connection that is eventually resolved when the last cell incorporates into the neighboring branch. If the lumen is maintained in the pruning vessel, the process involves transient formation of a unicellular tube through cell self-fusion. Thus, we show how a variety of cellular activities are coordinated to achieve vessel pruning.
doi:10.1371/journal.pbio.1002126
PMCID: PMC4401649  PMID: 25884426
7.  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.
doi:10.1371/journal.pcbi.1000549
PMCID: PMC2762315  PMID: 19876379
8.  Haemodynamics-Driven Developmental Pruning of Brain Vasculature in Zebrafish 
PLoS Biology  2012;10(8):e1001374.
This in vivo time-lapse imaging study in zebrafish reveals how changes to brain blood flow drive vessel pruning via endothelial cell migration, and how pruning leads to the simplification of the brain vasculature during development.
Author Summary
Although the brain comprises only 2% of body weight, it receives 15% of cardiac output and consumes 20% of total body oxygen delivered through its blood vasculature. The brain blood vasculature consists of a highly branched vessel network that is tailored to efficiently deliver oxygen and nutrients to each brain region. However, little is known about how the brain vasculature develops. Using in vivo long-term serial confocal imaging of zebrafish larvae, we analyze this process and find that the developing midbrain vasculature undergoes not only vessel growth but also blood flow-driven vessel pruning. We show that vessel pruning occurs preferentially at loop-shaped vessel segments via the migration of endothelial cells to adjacent unpruned segments; over time, such vessel pruning reduces the complexity of the early primitive midbrain vasculature. We also observe that pruned vessel segments exhibit a lower and more variable blood flow than do unpruned segments and that the local blocking of blood flow triggers vessel pruning. By contrast, increases in blood flow impair vessel pruning. Finally, we show that pruning events can be predicted using a haemodynamics based mathematical model of the midbrain vasculature. These findings demonstrate the existence of brain vessel pruning during development and provide novel insights into the role of haemodynamics in brain vascular refinement.
The brain blood vasculature consists of a highly ramified vessel network that is tailored to meet its physiological functions. How the brain vasculature is formed has long been fascinating biologists. Here we report that the developing vasculature in the zebrafish midbrain undergoes not only angiogenesis but also extensive vessel pruning, which is driven by changes in blood flow. This pruning process shapes the initial exuberant interconnected meshwork into a simplified architecture. Using in vivo long-term serial confocal imaging of the same zebrafish larvae during 1.5–7.5 d post-fertilization, we found that the early formed midbrain vasculature consisted of many vessel loops and higher order segments. Vessel pruning occurred preferentially at loop-forming segments via a process mainly involving lateral migration of endothelial cells (ECs) from pruned to unpruned segments rather than EC apoptosis, leading to gradual reduction in the vasculature complexity with development. Compared to unpruned ones, pruned segments exhibited a low and variable blood flow, which further decreased irreversibly prior to the onset of pruning. Local blockade of blood flow with micro-bead obstruction led to vessel pruning, whereas increasing blood flow by noradrenergic elevation of heartbeat impeded the pruning process. Furthermore, the occurrence of vessel pruning could be largely predicted by haemodynamics-based numerical simulation of vasculature refinement. Thus, changes of blood flow drive vessel pruning via lateral migration of ECs, leading to the simplification of the vasculature and possibly efficient routing of blood flow in the developing brain.
doi:10.1371/journal.pbio.1001374
PMCID: PMC3419171  PMID: 22904685
9.  Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-β–TAK1–Dependent Mechanism 
PLoS Biology  2013;11(4):e1001527.
During angiogenesis, endothelial cells in nascent blood vessels use the transcriptional cofactors HIPK1 and HIPK2 to transduce TGF-β signals and control cell proliferation and adhesion.
Several critical events dictate the successful establishment of nascent vasculature in yolk sac and in the developing embryos. These include aggregation of angioblasts to form the primitive vascular plexus, followed by the proliferation, differentiation, migration, and coalescence of endothelial cells. Although transforming growth factor–β (TGF-β) is known to regulate various aspects of vascular development, the signaling mechanism of TGF-β remains unclear. Here we show that homeodomain interacting protein kinases, HIPK1 and HIPK2, are transcriptional corepressors that regulate TGF-β–dependent angiogenesis during embryonic development. Loss of HIPK1 and HIPK2 leads to marked up-regulations of several potent angiogenic genes, including Mmp10 and Vegf, which result in excessive endothelial proliferation and poor adherens junction formation. This robust phenotype can be recapitulated by siRNA knockdown of Hipk1 and Hipk2 in human umbilical vein endothelial cells, as well as in endothelial cell-specific TGF-β type II receptor (TβRII) conditional mutants. The effects of HIPK proteins are mediated through its interaction with MEF2C, and this interaction can be further enhanced by TGF-β in a TAK1-dependent manner. Remarkably, TGF-β-TAK1 signaling activates HIPK2 by phosphorylating a highly conserved tyrosine residue Y-361 within the kinase domain. Point mutation in this tyrosine completely eliminates the effect of HIPK2 as a transcriptional corepressor in luciferase assays. Our results reveal a previously unrecognized role of HIPK proteins in connecting TGF-β signaling pathway with the transcriptional programs critical for angiogenesis in early embryonic development.
Author Summary
An essential step during early embryonic development is to establish elaborate vascular networks that provide nutrients to ensure the proper growth of the embryos. This process, known as angiogenesis, requires coordinated regulation of cell proliferation, migration, and differentiation in endothelial cells, which provide the inner-most linings of blood vessels. It is well accepted that transforming growth factor–β (TGF-β) and its downstream signal pathways are required to regulate endothelial cell growth, but the exact mechanisms remain poorly characterized. Using mouse genetics and in vitro angiogenesis assays, we show that transcriptional cofactors in the homeodomain interacting protein kinase (HIPK) family are activated by TGF-β to control the expression of target genes that regulate proliferation and adherent junction formation in endothelial cells. Our study also identifies a highly conserved tyrosine residue in HIPK proteins that is required to transduce TGF-β signal. These results provide new insights into the mechanism of TGF-β signaling in angiogenesis, and how this process may be exploited to develop therapeutic targets that control angiogenesis during development and in disease conditions.
doi:10.1371/journal.pbio.1001527
PMCID: PMC3614511  PMID: 23565059
10.  Flt-1 (VEGFR-1) is Essential for the VEGF-Notch Feedback Loop during Angiogenesis 
Objective
Vascular endothelial growth factor (VEGF) signaling induces Notch signaling during angiogenesis. Flt-1/VEGF receptor-1 (VEGFR-1) negatively modulates VEGF signaling. Therefore, we tested the hypothesis that disrupted Flt-1 regulation of VEGF signaling causes Notch pathway defects that contribute to dysmorphogenesis of Flt-1 mutant vessels.
Approach and Results
Wild-type (WT) and flt-1−/− mouse embryonic stem (ES) cell-derived vessels were exposed to pharmacological and protein-based Notch inhibitors with and without added VEGF. Vessel morphology, endothelial cell proliferation, and Notch target gene expression levels were assessed. Similar pathway manipulations were performed in developing vessels of zebrafish embryos. Notch inhibition reduced flt-1−/− ES cell-derived vessel branching dysmorphogenesis and endothelial hyper-proliferation, and rescue of flt-1−/− vessels was accompanied by a reduction of elevated Notch targets. Surprisingly, WT vessel morphogenesis and proliferation were unaffected by Notch suppression, Notch targets in WT endothelium were unchanged, and Notch suppression perturbed zebrafish intersegmental vessels (ISVs) but not caudal vein plexuses (CVPs). In contrast, exogenous VEGF caused WT ES cell-derived vessel and zebrafish ISV dysmorphogenesis that was rescued by Notch blockade.
Conclusions
Elevated Notch signaling downstream of perturbed VEGF signaling contributes to aberrant flt-1−/− blood vessel formation. Notch signaling may be dispensable for blood vessel formation when VEGF signaling is below a critical threshold.
doi:10.1161/ATVBAHA.113.301805
PMCID: PMC4521230  PMID: 23744993
vessel branching; VEGF; Flt-1; Notch; angiogenesis; ES cells; zebrafish
11.  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
12.  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
13.  Novel insights into the differential functions of Notch ligands in vascular formation 
The Notch signaling pathway is a critical component of vascular formation and morphogenesis in both development and disease. Compelling evidence indicates that Notch signaling is required for the induction of arterial-cell fate during development and for the selection of endothelial tip and stalk cells during sprouting angiogenesis. In mammals, two of the four Notch receptors (Notch1 and Notch4) and three of the five Notch ligands (Jagged1, Dll1, and Dll4) are predominantly expressed in vascular endothelial cells and are important for many aspects of vascular biology. During arterial cell-fate selection and angiogenesis, the roles of Notch1 and Notch4 are thought to be similar, and the function of Dll4 is well-characterized. However, the molecular mechanisms that determine the functional similarities and differences of Notch ligands in vascular endothelial cells remain largely unknown; consequently, additional research is needed to elucidate the ligand-specific functions and mechanisms associated with Notch activation in the vascular endothelium. Results from recent studies indicate that Dll1 and Dll4 have distinct roles in the specification and maintenance of arterial cell identity, while Dll4 and Jagged1 have opposing functions in tip- and stalk-cell selection during sprouting angiogenesis. This review will focus on the newly discovered, distinct functions of several Notch ligands in the regulation of blood vessel formation and will provide perspectives for future research in the field.
doi:10.1186/2040-2384-1-8
PMCID: PMC2794854  PMID: 20016694
14.  Spatial regulation of VEGF receptor endocytosis in angiogenesis 
Nature cell biology  2013;15(3):249-260.
Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilisation of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalisation processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalisation and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGFR endocytosis and the activity of cell polarity proteins.
doi:10.1038/ncb2679
PMCID: PMC3901019  PMID: 23354168
15.  Gamma-Secretase Inhibitor Treatment Promotes VEGF-A-Driven Blood Vessel Growth and Vascular Leakage but Disrupts Neovascular Perfusion 
PLoS ONE  2011;6(4):e18709.
The Notch signaling pathway is essential for normal development due to its role in control of cell differentiation, proliferation and survival. It is also critically involved in tumorigenesis and cancer progression. A key enzyme in the activation of Notch signaling is the gamma-secretase protein complex and therefore, gamma-secretase inhibitors (GSIs)—originally developed for Alzheimer's disease—are now being evaluated in clinical trials for human malignancies. It is also clear that Notch plays an important role in angiogenesis driven by Vascular Endothelial Growth Factor A (VEGF-A)—a process instrumental for tumor growth and metastasis. The effect of GSIs on tumor vasculature has not been conclusively determined. Here we report that Compound X (CX), a GSI previously reported to potently inhibit Notch signaling in vitro and in vivo, promotes angiogenic sprouting in vitro and during developmental angiogenesis in mice. Furthermore, CX treatment suppresses tumor growth in a mouse model of renal carcinoma, leads to the formation of abnormal vessels and an increased tumor vascular density. Using a rabbit model of VEGF-A-driven angiogenesis in skeletal muscle, we demonstrate that CX treatment promotes abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage. Based on these findings, we propose a model for how GSIs and other Notch inhibitors disrupt tumor blood vessel perfusion, which might be useful for understanding this new class of anti-cancer agents.
doi:10.1371/journal.pone.0018709
PMCID: PMC3077402  PMID: 21533193
16.  The Proangiogenic Effect of Iroquois Homeobox Transcription Factor Irx3 in Human Microvascular Endothelial Cells* 
The Journal of Biological Chemistry  2014;290(10):6303-6315.
Background: Transcription regulation is essential for angiogenesis, but the role of Irx3 in this process remains to be defined.
Results: Irx3 promotes endothelial cell migration and tip cell specification through VEGF-Notch signaling.
Conclusion: Irx3 is a novel proangiogenic mediator of endothelial cell migration and cell fate.
Significance: Manipulation of Irx3 may provide novel therapeutic strategies in adult vascular pathologies.
Angiogenesis is a dynamic process required for embryonic development. However, postnatal vascular growth is characteristic of multiple disease states. Despite insights into the multistep process in which adhesion molecules, extracellular matrix proteins, growth factors, and their receptors work in concert to form new vessels from the preexisting vasculature, there remains a lack of insight of the nuclear transcriptional mechanisms that occur within endothelial cells (ECs) in response to VEGF. Iroquois homeobox gene 3 (Irx3) is a transcription factor of the Iroquois family of homeobox genes. Irx homeodomain transcription factors are involved in the patterning and development of several tissues. Irx3 is known for its role during embryogenesis in multiple organisms. However, the expression and function of Irx3 in human postnatal vasculature remains to be investigated. Here we show that Irx3 is expressed in human microvascular endothelial cells, and expression is elevated by VEGF stimulation. Genetic Irx3 gain and loss of function studies in human microvascular endothelial cells resulted in the modulation of EC migration during wound healing, chemotaxis and invasion, and tubulogenesis. Additionally, we observed increased delta-like ligand 4 (Dll4) expression, which suggests an increase in EC tip cell population. Finally, siRNA screening studies revealed that transient knockdown of Hey1, a downstream Notch signaling mediator, resulted in increased Irx3 expression in response to VEGF treatment. Strategies to pharmacologically regulate Irx3 function in adult endothelial cells may provide new therapies for angiogenesis.
doi:10.1074/jbc.M114.601146
PMCID: PMC4358267  PMID: 25512384
Angiogenesis; Cell Migration; Endothelial Cell; Notch Pathway; Transcription Factor; VEGF; Irx3
17.  Murine Notch1 is required for lymphatic vascular morphogenesis during development 
Background
The transmembrane receptor Notch1 is a critical regulator of arterial differentiation and blood vessel sprouting. Recent evidence shows that functional blockade of Notch1 and its ligand, Dll-4, leads to postnatal lymphatic defects in mice. However, the precise role of the Notch signaling pathway in lymphatic vessel development has yet to be defined. Here we show the developmental role of Notch1 in lymphatic vascular morphogenesis by analyzing lymphatic endothelial cell (LEC)-specific conditional Notch1 knockout mice crossed with an inducible Prox1CreERT2 driver.
Results
LEC-specific Notch1 mutant embryos exhibited enlarged lymphatic vessels. The phenotype of lymphatic overgrowth accords with increased LEC sprouting from the lymph sacs and increased filopodia formation. Furthermore, cell death was significantly reduced in Notch1-mutant LECs, whereas proliferation was increased. RNA-seq analysis revealed that expression of cytokine/chemokine signaling molecules was upregulated in Notch1-mutant LECs isolated from E15.5 dorsal skin, whereas VEGFR3, VEGFR2, VEGFC, and Gja4 (Connexin 37) were downregulated.
Conclusions
The lymphatic phenotype of LEC-specific conditional Notch1 mouse mutants indicates that Notch activity in LECs controls lymphatic sprouting and growth during development. These results provide evidence that similar to postnatal and pathological lymphatic vessel formation, the Notch signaling pathway plays a role in inhibiting developmental lymphangiogenesis.
doi:10.1002/dvdy.24129
PMCID: PMC4062592  PMID: 24659232
Notch; lymphatic vessel development; lymphangiogenesis; Prox1
18.  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
19.  The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis 
eLife  null;4:e06726.
A key step in the de novo formation of the embryonic vasculature is the migration of endothelial precursors, the angioblasts, to the position of the future vessels. To form the first axial vessels, angioblasts migrate towards the midline and coalesce underneath the notochord. Vascular endothelial growth factor has been proposed to serve as a chemoattractant for the angioblasts and to regulate this medial migration. Here we challenge this model and instead demonstrate that angioblasts rely on their intrinsic expression of Apelin receptors (Aplr, APJ) for their migration to the midline. We further show that during this angioblast migration Apelin receptor signaling is mainly triggered by the recently discovered ligand Elabela (Ela). As neither of the ligands Ela or Apelin (Apln) nor their receptors have previously been implicated in regulating angioblast migration, we hereby provide a novel mechanism for regulating vasculogenesis, with direct relevance to physiological and pathological angiogenesis.
DOI: http://dx.doi.org/10.7554/eLife.06726.001
eLife digest
The circulatory system enables blood to move around the body and deliver substances including nutrients and oxygen to the cells that need them. In the embryos of animals with a backbone, blood flows from the heart through the aorta into branching smaller vessels to the cells. The blood then gets collected by progressively bigger vessels and flows back to the heart via the cardinal vein. The cells that make up these blood vessels develop from cells called angioblasts—but first, during development these angioblasts must move to the place where the vessels will form.
A protein called Vascular endothelial growth factor (VEGF) had been suggested to help guide and align the angioblasts as the embryo develops. Now, Helker, Schuermann et al. have examined developing zebrafish embryos using new technologies. This revealed that VEGF is in fact not essential for the dorsal aorta and cardinal vein to develop. Instead, the angioblasts only move to the correct part of the embryo if they can produce the Apelin receptor protein, which forms part of a signaling pathway.
There are two hormones—called Apelin and Elabela—that can bind to and activate the Apelin receptor. Helker, Schuermann et al. show that Elabela alone is needed to guide the angioblasts to the right part of the embryo during blood vessel development. However, in embryos where there is not enough Elabela, the Apelin hormone can compensate for this deficiency and the first blood vessels will later develop correctly. Future research will address whether this signaling pathway not only guides angioblasts to establish a circulatory system, but also guides blood vessel growth. As blood vessel growth is very relevant to human disease, identifying the mechanisms that regulate it will have an impact on biomedical research.
DOI: http://dx.doi.org/10.7554/eLife.06726.002
doi:10.7554/eLife.06726
PMCID: PMC4468421  PMID: 26017639
angioblast migration; endothelial cell migration; Vegf; dorsal aorta; chemoattractant; notochord; zebrafish
20.  reg6 is required for branching morphogenesis during blood vessel regeneration in zebrafish caudal fins 
Developmental biology  2003;264(1):263-274.
Postnatal neovascularization is essential for wound healing, cancer progression, and many other physiological functions. However, its genetic mechanism is largely unknown. In this report, we study neovascularization in regenerating adult zebrafish fins using transgenic fish that express EGFP in blood vessel endothelial cells. We first describe the morphogenesis of regenerating vessels in wild-type animals and then the phenotypic analysis of a genetic mutation that disrupts blood vessel regeneration. In wild-type zebrafish caudal fins, amputated blood vessels heal their ends by 24 h postamputation (hpa) and then reconnect arteries and veins via anastomosis, to resume blood flow at wound sites by 48 hpa. The truncated vessels regenerate by first growing excess vessels to form unstructured plexuses, resembling the primary capillary plexuses formed during embryonic vasculogenesis. Interestingly, this mode of vessel growth switches by 8 days postamputation (dpa) to growth without a plexus intermediate. During blood vessel regeneration, vessel remodeling begins during early plexus formation and continues until the original vasculature pattern is reestablished at ~35 dpa. Temperature-sensitive mutants for reg6 have profound defects in blood vessel regeneration. At the restrictive temperature, reg6 regenerating blood vessels first fail to make reconnections between severed arteries and veins, and then form enlarged vascular sinuses rather than branched vascular plexuses. Reciprocal temperature-shift experiments show that reg6 function is required throughout plexus formation, but not during later growth. Our results suggest that the reg6 mutation causes defects in branch formation and/or angiogenic sprouting.
PMCID: PMC3665419  PMID: 14623247
Zebrafish; Regeneration; Angiogenesis; Plexus; Branching; Anastomosis
21.  NO mediates mural cell recruitment and vessel morphogenesis in murine melanomas and tissue-engineered blood vessels 
Journal of Clinical Investigation  2005;115(7):1816-1827.
NO has been shown to mediate angiogenesis; however, its role in vessel morphogenesis and maturation is not known. Using intravital microscopy, histological analysis, α–smooth muscle actin and chondroitin sulfate proteoglycan 4 staining, microsensor NO measurements, and an NO synthase (NOS) inhibitor, we found that NO mediates mural cell coverage as well as vessel branching and longitudinal extension but not the circumferential growth of blood vessels in B16 murine melanomas. NO-sensitive fluorescent probe 4,5-diaminofluorescein imaging, NOS immunostaining, and the use of NOS-deficient mice revealed that eNOS in vascular endothelial cells is the predominant source of NO and induces these effects. To further dissect the role of NO in mural cell recruitment and vascular morphogenesis, we performed a series of independent analyses. Transwell and under-agarose migration assays demonstrated that endothelial cell–derived NO induces directional migration of mural cell precursors toward endothelial cells. An in vivo tissue-engineered blood vessel model revealed that NO mediates endothelial–mural cell interaction prior to vessel perfusion and also induces recruitment of mural cells to angiogenic vessels, vessel branching, and longitudinal extension and subsequent stabilization of the vessels. These data indicate that endothelial cell–derived NO induces mural cell recruitment as well as subsequent morphogenesis and stabilization of angiogenic vessels.
doi:10.1172/JCI24015
PMCID: PMC1143589  PMID: 15951843
22.  17β-Estradiol Enhances Signalling Mediated by VEGF-A-Delta-Like Ligand 4-Notch1 Axis in Human Endothelial Cells 
PLoS ONE  2013;8(8):e71440.
Estrogens play a protective role in coronary artery disease. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. In several cell types, estrogens modulate the Notch pathway, which is involved in controlling angiogenesis downstream of vascular endothelial growth factor A (VEGF-A). The goal of our study was to establish whether estrogens modulate Notch activity in endothelial cells and the possible consequences on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were treated with 17β-estradiol (E2) and the effects on Notch signalling were evaluated. E2 increased Notch1 processing as indicated by i) decreased levels of Notch1 transmembrane subunit ii) increased amount of Notch1 in nuclei iii) unaffected level of mRNA. Similarly, E2 increased the levels of the active form of Notch4 without altering Notch4 mRNA. Conversely, protein and mRNA levels of Notch2 were both reduced suggesting transcriptional repression of Notch2 by E2. Under conditions where Notch was activated by upregulation of Delta-like ligand 4 (Dll4) following VEGF-A treatment, E2 caused a further increase of the active form of Notch1, of the number of cells with nuclear Notch1 and of Hey2 mRNA. Estrogen receptor antagonist ICI 182.780 antagonized these effects suggesting that E2 modulation of Notch1 is mediated by estrogen receptors. E2 treatment abolished the increase in endothelial cells sprouting caused by Notch inhibition in a tube formation assay on 3D Matrigel and in mouse aortic ring explants. In conclusion, E2 affects several Notch pathway components in HUVECs, leading to an activation of the VEGF-A-Dll4-Notch1 axis and to a modulation of vascular branching when Notch signalling is inhibited. These results contribute to our understanding of the molecular mechanisms of cardiovascular protection exerted by estrogens by uncovering a novel role of E2 in the Notch signalling-mediated modulation of angiogenesis.
doi:10.1371/journal.pone.0071440
PMCID: PMC3742772  PMID: 23967210
23.  Axon Guidance Molecules in Vascular Patterning 
Endothelial cells (ECs) form extensive, highly branched and hierarchically organized tubular networks in vertebrates to ensure the proper distribution of molecular and cellular cargo in the vertebrate body. The growth of this vascular system during development, tissue repair or in disease conditions involves the sprouting, migration and proliferation of endothelial cells in a process termed angiogenesis. Surprisingly, specialized ECs, so-called tip cells, which lead and guide endothelial sprouts, share many feature with another guidance structure, the axonal growth cone. Tip cells are motile, invasive and extend numerous filopodial protrusions sensing growth factors, extracellular matrix and other attractive or repulsive cues in their tissue environment. Axonal growth cones and endothelial tip cells also respond to signals belonging to the same molecular families, such as Slits and Roundabouts, Netrins and UNC5 receptors, Semaphorins, Plexins and Neuropilins, and Eph receptors and ephrin ligands. Here we summarize fundamental principles of angiogenic growth, the selection and function of tip cells and the underlying regulation by guidance cues, the Notch pathway and vascular endothelial growth factor signaling.
“Tip cells” guide branching of blood vessels during development and tissue repair. These are surprisingly similar to axon growth cones, using many of the same guidance molecules.
doi:10.1101/cshperspect.a001875
PMCID: PMC2857165  PMID: 20452960
24.  Tie1 deletion inhibits tumor growth and improves angiopoietin antagonist therapy 
The endothelial Tie1 receptor is ligand-less, but interacts with the Tie2 receptor for angiopoietins (Angpt). Angpt2 is expressed in tumor blood vessels, and its blockade inhibits tumor angiogenesis. Here we found that Tie1 deletion from the endothelium of adult mice inhibits tumor angiogenesis and growth by decreasing endothelial cell survival in tumor vessels, without affecting normal vasculature. Treatment with VEGF or VEGFR-2 blocking antibodies similarly reduced tumor angiogenesis and growth; however, no additive inhibition was obtained by targeting both Tie1 and VEGF/VEGFR-2. In contrast, treatment of Tie1-deficient mice with a soluble form of the extracellular domain of Tie2, which blocks Angpt activity, resulted in additive inhibition of tumor growth. Notably, Tie1 deletion decreased sprouting angiogenesis and increased Notch pathway activity in the postnatal retinal vasculature, while pharmacological Notch suppression in the absence of Tie1 promoted retinal hypervasularization. Moreover, substantial additive inhibition of the retinal vascular front migration was observed when Angpt2 blocking antibodies were administered to Tie1-deficient pups. Thus, Tie1 regulates tumor angiogenesis, postnatal sprouting angiogenesis, and endothelial cell survival, which are controlled by VEGF, Angpt, and Notch signals. Our results suggest that targeting Tie1 in combination with Angpt/Tie2 has the potential to improve antiangiogenic therapy.
doi:10.1172/JCI68897
PMCID: PMC3904604  PMID: 24430181
25.  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

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