Differential apicobasal VEGF signaling at vascular blood-neural barriers.
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ABSTRACT:
SUBMITTER: Hudson N
PROVIDER: S-EPMC4160345 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Neurovascular development requires communication between two developing organs, the neuroepithelium and embryonic blood vessels. We investigated the role of VEGF-A signaling in the embryonic crosstalk required for ingression of angiogenic vessel sprouts into the developing neural tube. As the neural tube develops, blood vessels enter at specific points medially and ventrally from the surrounding perineural vascular plexus. Localized ectopic expression of heparin-binding VEGF165 or VEGF189 from the developing avian neural tube resulted in supernumerary blood vessel ingression points and disrupted vessel patterning. By contrast, localized ectopic neural expression of non-heparin-binding VEGF121 did not produce supernumerary blood vessel ingression points, although the vessels that entered the neural tube became dysmorphogenic. Localized loss of endogenous VEGF-A signaling in the developing neural tube via ectopic expression of the VEGF inhibitor sFlt-1 locally blocked blood vessel ingression. The VEGF pathway manipulations were temporally controlled and did not dramatically affect neural tube maturation and dorsal-ventral patterning. Thus, neural-derived VEGF-A has a direct role in the spatially localized molecular crosstalk that is required for neurovascular development and vessel patterning in the developing neural tube.
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Project description:Formation of the vascular system within organs requires the balanced action of numerous positive and negative factors secreted by stromal and epithelial cells. Here, we used a genetic approach to determine the role of SLITs in regulating the growth and organization of blood vessels in the mammary gland. We demonstrate that vascularization of the gland is not affected by loss of Slit expression in the epithelial compartment. Instead, we identify a stromal source of SLIT, mural cells encircling blood vessels, and show that loss of Slit in the stroma leads to elevated blood vessel density and complexity. We examine candidate SLIT receptors, Robo1 and Robo4, and find that increased vessel angiogenesis is phenocopied by loss of endothelial-specific Robo4, as long as it is combined with the presence of an angiogenic stimulus such as preneoplasia or pregnancy. In contrast, loss of Robo1 does not affect blood vessel growth. The enhanced growth of blood vessels in Robo4(-/-) endothelium is due to activation of vascular endothelial growth factor (VEGF)-R2 signaling through the Src and FAK kinases. Thus, our studies present a genetic dissection of SLIT/ROBO signaling during organ development. We identify a stromal, rather than epithelial, source of SLITs that inhibits blood vessel growth by signaling through endothelial ROBO4 to down-regulate VEGF/VEGFR2 signaling.
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Project description:RATIONALE:Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), whereas lymphangiogenesis signals are transduced by VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown. OBJECTIVE:To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability. METHODS AND RESULTS:Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated VEGFR2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell-cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3. CONCLUSIONS:VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.
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Project description:Vasculogenesis involves the differentiation of vascular endothelial progenitors de novo from undifferentiated mesoderm, their migration and coalescence to form the major embryonic vessels and the acquisition of arterial or venous identity. Vascular Endothelial Growth Factor (Vegf) signaling plays multiple roles during vascular development. However, its function during embryonic vasculogenesis has been controversial. Previous studies have implicated Vegf signaling in either regulating arteriovenous specification or overall vascular endothelial differentiation. To clarify the role of Vegf in embryonic vasculogenesis and identify its downstream targets, we used chemical inhibitors of Vegf receptor (Vegfr) signaling in zebrafish embryos as well as zebrafish genetic mutants. A high level of chemical inhibition of Vegfr signaling resulted in the reduction of overall vascular endothelial marker gene expression, including downregulation of both arterial and venous markers, ultimately leading to the apoptosis of vascular endothelial cells. In contrast, a low level of Vegfr inhibition specifically blocked arterial specification while the expression of venous markers appeared largely unaffected or increased. Inhibition of Vegfr signaling prior to the initiation of vasculogenesis reduced overall vascular endothelial differentiation, while inhibition of Vegfr signaling starting at mid-somitogenesis stages largely inhibited arterial specification. Conversely, Vegf overexpression resulted in the expansion of both arterial and pan-endothelial markers, while the expression of several venous-specific markers was downregulated. We further show that Vegf signaling affects overall endothelial differentiation by modulating the expression of the ETS transcription factor etv2/ etsrp. etv2 expression was downregulated in Vegfr- inhibited embryos, and expanded in Vegfaa-overexpressing embryos. Furthermore, vascular-specific overexpression of etv2 in Vegfr-inhibited embryos rescued defects in vascular endothelial differentiation. Similarly, vegfaa genetic mutants displayed a combination of the two phenotypes observed with chemical Vegfr inhibition: the expression of arterial and pan-endothelial markers including etv2 was downregulated while the expression of most venous markers was either expanded or unchanged. Based on these results we propose a revised model which explains the different phenotypes observed upon inhibition of Vegf signaling: low levels of Vegf signaling promote overall vascular endothelial differentiation and cell survival by upregulating etv2 expression, while high levels of Vegf signaling promote arterial and inhibit venous specification.
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Project description:The neural crest is an excellent model to study embryonic cell migration, since cell behaviors can be studied in vivo with advanced optical imaging and molecular intervention. What is unclear is how molecular signals direct neural crest cell (NCC) migration through multiple microenvironments and into specific targets. Here, we tested the hypothesis that the invasion of cranial NCCs, specifically the rhombomere 4 (r4) migratory stream into branchial arch 2 (ba2), is due to chemoattraction through neuropilin-1-vascular endothelial growth factor (VEGF) interactions. We found that the spatio-temporal expression pattern of VEGF in the ectoderm correlated with the NCC migratory front. RT-PCR analysis of the r4 migratory stream showed that ba2 tissue expressed VEGF and r4 NCCs expressed VEGF receptor 2. When soluble VEGF receptor 1 (sVEGFR1) was injected distal to the r4 migratory front, to bind up endogenous VEGF, NCCs failed to completely invade ba2. Time-lapse imaging revealed that cranial NCCs were attracted to ba2 tissue or VEGF sources in vitro. VEGF-soaked beads or VEGF-expressing cells placed adjacent to the r4 migratory stream caused NCCs to divert from stereotypical pathways and move towards an ectopic VEGF source. Our results suggest a model in which NCC entry and invasion of ba2 is dependent on chemoattractive signaling through neuropilin-1-VEGF interactions.
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Project description:VEGFR2 is the main receptor and mediator of the vasculogenic and angiogenic activity of VEGF. Activated VEGFR2 internalizes through clathrin-mediated endocytosis and macropinocytosis. As dynamin is a key regulator of the clathrin pathway, chemical inhibitors of dynamin are commonly used to assess the role of the clathrin route in receptor signaling. However, drugs may also exert off-target effects. Here, we compare the effects of three dynamin inhibitors, dynasore, dyngo 4a and dynole, on VEGFR2 internalization and signaling. Although these drugs consistently inhibit clathrin-mediated endocytosis of both transferrin (a typical cargo of this route) and VEGFR2, surprisingly, they exert contradictory effects in receptor signaling. Thus, while dynasore has no effect on phosphorylation of VEGFR2, the other two drugs are strong inhibitors. Furthermore, although dyngo does not interfere with phosphorylation of Akt, dynasore and dynole have a strong inhibitory effect. These inconsistent effects suggest that the above dynamin blockers, besides inhibiting dynamin-dependent endocytosis of VEGFR2, exert additional inhibitory effects on signaling that are independent of endocytosis; i.e., they are due to off-target effects. Using a recently developed protocol, we comparatively validate the specificity of two endocytic inhibitors, dynasore and EIPA. Our findings highlight the importance of assessing whether the effect of an endocytic drug on signaling is specifically due to its interference with endocytosis or due to off-targets.
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Project description:Vascular endothelial growth factor (VEGF) is a potent regulator of placental vascular function. Endothelial dysfunction is a key factor associated with preeclampsia. In this study, we examined expression of VEGF, endocrine gland-derived VEGF (EG-VEGF), VEGF receptors 1 and 2 (VEGFR-1 and VEGFR-2), and neuropilin-1 and -2 (NP-1 and NP-2) in human placentas from women with normal and preeclamptic (PE) pregnancies using quantitative or semiquantitative PCR. We found that total VEGF mRNA expression was increased 2.8-fold (P < 0.05), along with increases in mRNA expression of VEGF121, 165, and 189 (P < 0.05; 1.7-, 1.9-, and 1.8-fold, respectively) in PE vs. normal placentas. Expression of VEGFR-1 mRNA, but not EG-VEGF and the other three VEGF receptors studied, was elevated (P < 0.05) 2.7-fold in PE vs. normal placentas. Protein expression of VEGF and its four receptors was determined using Western blot analysis. For VEGF, two major isoforms (VEGF165 and 189) were detected. For VEGFR-1, VEGFR-2, NP-1, and NP-2, one major band was observed at 180, 235, 130, and 130 kDa, respectively. All of these bands were corresponding to their positive controls. Of these five proteins studied, only VEGFR-1 levels were increased (P < 0.05; 1.7-fold) in PE placentas. The expression of VEGF and the four VEGF receptors was confirmed using immunohistochemistry. They were primarily present in syncytiotrophoblasts and endothelial cells of villous capillaries and large vessels. Thus, together with previous reports that VEGFR-1 mediates trophoblast function and inhibits VEGF-induced angiogenesis and endothelium-dependent vasodilation, these data suggest that the increased VEGFR-1 expression may alter VEGF- mediated function on trophoblast and endothelial cells in PE placentas.
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Project description:Angiogenesis is a key aspect of the dynamic changes occurring during the normal ovarian cycle. Hyperplasia and hypervascularity of the ovarian theca interna and stroma are also prominent features of the polycystic ovary syndrome (PCOS), a leading cause of infertility. Compelling evidence indicated that vascular endothelial growth factor (VEGF) is a key mediator of the cyclical corpus luteum angiogenesis. However, the nature of the factor(s) that mediate angiogenesis in PCOS is less clearly understood. Endocrine gland-derived (EG)-VEGF has been recently identified as an endothelial cell mitogen with selectivity for the endothelium of steroidogenic glands and is expressed in normal human ovaries. In the present study, we compared the expression of EG-VEGF and VEGF mRNA in a series of 13 human PCOS and 13 normal ovary specimens by in situ hybridization. EG-VEGF expression in normal ovaries is dynamic and generally complementary to VEGF expression in both follicles and corpora lutea. A particularly high expression of EG-VEGF was detected in the Leydig-like hilus cells found in the highly vascularized ovarian hilus. In PCOS ovaries, we found strong expression of EG-VEGF mRNA in theca interna and stroma in most of the specimens examined, thus spatially related to the new blood vessels. In contrast, VEGF mRNA expression was most consistently associated with the granulosa cell layer and sometimes the theca, but rarely with the stroma. These findings indicate that both EG-VEGF and VEGF are expressed in PCOS ovaries, but in different cell types at different stages of differentiation, thus suggesting complementary functions for the two factors in angiogenesis and possibly cyst formation.
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Project description:During neural tube closure, specialized regions called hinge points (HPs) display dynamic and polarized cell behaviors necessary for converting the neural plate into a neural tube. The molecular bases of such cell behaviors (e.g. apical constriction, basal nuclear migration) are poorly understood. We have identified a two-dimensional canonical BMP activity gradient in the chick neural plate that results in low and temporally pulsed BMP activity at the ventral midline/median hinge point (MHP). Using in vivo manipulations, high-resolution imaging and biochemical analyses, we show that BMP attenuation is necessary and sufficient for MHP formation. Conversely, BMP overexpression abolishes MHP formation and prevents neural tube closure. We provide evidence that BMP modulation directs neural tube closure via the regulation of apicobasal polarity. First, BMP blockade produces partially polarized neural cells, which retain contact with the apical and basal surfaces but where basolateral proteins (LGL) become apically localized and apical junctional proteins (PAR3, ZO1) become targeted to endosomes. Second, direct LGL misexpression induces ectopic HPs identical to those produced by noggin or dominant-negative BMPR1A. Third, BMP-dependent biochemical interactions occur between the PAR3-PAR6-aPKC polarity complex and phosphorylated SMAD5 at apical junctions. Finally, partially polarized cells normally occur at the MHP, their frequencies inversely correlated with the BMP activity gradient in the neural plate. We propose that spatiotemporal modulation of the two-dimensional BMP gradient transiently alters cell polarity in targeted neuronal cells. This ensures that the neural plate is flexible enough to be focally bent and shaped into a neural tube, while retaining overall epithelial integrity.
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Project description:Physiological functions of vascular endothelial growth factor (VEGF)-B remain an enigma, and deletion of the Vegfb gene in mice lacks an overt phenotype. Here we show that knockdown of Vegfba, but not Vegfbb, in zebrafish embryos by specific morpholinos produced a lethal phenotype owing to vascular and neuronal defects in the brain. Vegfba morpholinos also markedly prevented development of hyaloid vasculatures in the retina, but had little effects on peripheral vascular development. Consistent with phenotypic defects, Vegfba, but not Vegfaa, mRNA was primarily expressed in the brain of developing zebrafish embryos. Interestingly, in situ detection of Neuropilin1 (Nrp1) mRNA showed an overlapping expression pattern with Vegfba, and knockdown of Nrp1 produced a nearly identically lethal phenotype as Vegfba knockdown. Furthermore, zebrafish VEGF-Ba protein directly bound to NRP1. Importantly, gain-of-function by exogenous delivery of mRNAs coding for NRP1-binding ligands VEGF-B or VEGF-A to the zebrafish embryos rescued the lethal phenotype by normalizing vascular development. Similarly, exposure of zebrafish embryos to hypoxia also rescued the Vegfba morpholino-induced vascular defects in the brain by increasing VEGF-A expression. Independent evidence of VEGF-A gain-of-function was provided by using a functionally defective Vhl-mutant zebrafish strain, which again rescued the Vegfba morpholino-induced vascular defects. These findings show that VEGF-B is spatiotemporally required for vascular development in zebrafish embryos and that NRP1, but not VEGFR1, mediates the essential signaling.
| S-EPMC4640767 | biostudies-literature