Rspo1/Wnt signaling promotes angiogenesis via Vegfc/Vegfr3

Here, we show that a novel Rspo1-Wnt-Vegfc-Vegfr3 signaling pathway plays an essential role in developmental angiogenesis. A mutation in R-spondin1 (rspo1), a Wnt signaling regulator, was uncovered during a forward-genetic screen for angiogenesis-deficient mutants in the zebrafish. Embryos lacking rspo1 or the proposed rspo1 receptor kremen form primary vessels by vasculogenesis, but are defective in subsequent angiogenesis. Endothelial cell-autonomous inhibition of canonical Wnt signaling also blocks angiogenesis in vivo. The pro-angiogenic effects of Rspo1/Wnt signaling are mediated by Vegfc/Vegfr3(Flt4) signaling. Vegfc expression is dependent on Rspo1 and Wnt, and Vegfc and Vegfr3 are necessary to promote angiogenesis downstream from Rspo1-Wnt. As all of these molecules are expressed by the endothelium during sprouting stages, these results suggest that Rspo1-Wnt-VegfC-Vegfr3 signaling plays a crucial role as an endothelial-autonomous permissive cue for developmental angiogenesis.     

Rspo1调控斑马鱼胚胎汇聚延伸运动的研究

Rspo1 (R-spondin 1)是分泌型Rspos (R-spondins)蛋白家族的成员,在雌性发育、血管生成和癌症等多个方面具有调控作用。为了研究Rspo1在早期胚胎发育中的功能,以斑马鱼(Danio rerio)作为模式生物,利用反转录PCR及原位杂交技术检测rspo1基因的时空表达模式;通过显微注射rspo1 mRNA或rspo1反义寡核苷酸(Morpholino, MO)对rspo1进行过表达或敲降;通过形态观察及原位杂交技术检测胚胎汇聚延伸(Convergence and extension, CE)运动是否正常;利用荧光素酶活性检测实验测定Wnt/PCP信号通路活性水平;通过蛋白印迹法检测表征Wnt/PCP信号通路活性的磷酸化JNK (Jun N-terminal kinase)蛋白的水平。结果显示:rspo1为母源基因,在12hpf前胚胎中呈全身性表达, rspo1的过表达或敲降均影响胚胎的CE运动;过表达rspo1降低Wnt/PCP信号通路报告质粒的活性,而敲降rspo1则增加其活性,与之相一致, rspo1敲降的胚胎中磷酸化JNK的水平显著升高;此外, rsp...     

R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis

Herein, we demonstrate that Lrp6-mediated R-spondin 2 signaling through the canonical Wnt pathway is required for normal morphogenesis of the respiratory tract and limbs. We show that the footless insertional mutation creates a severe hypomorphic R-spondin 2 allele (Rspo2(Tg)). The predicted protein encoded by Rspo2(Tg) neither bound the cell surface nor activated the canonical Wnt signaling reporter TOPFLASH. Rspo2 activation of TOPFLASH was dependent upon the second EGF-like repeat of Lrp6. Rspo2(Tg/Tg) mice had severe malformations of laryngeal-tracheal cartilages, limbs and palate, and lung hypoplasia consistent with sites of Rspo2 expression. Rspo2(Tg/Tg) lung defects were associated with reduced branching, a reduction in TOPGAL reporter activity, and reduced expression of the downstream Wnt target Irx3. Interbreeding the Rspo2(Tg) and Lrp6(-) alleles resulted in more severe defects consisting of marked lung hypoplasia and absence of tracheal-bronchial rings, laryngeal structures and all limb skeletal elements.     

R-spondin2 expression in the apical ectodermal ridge is essential for outgrowth and patterning in mouse limb development

Mouse R-spondin2 (Rspo2) is a member of the R-spondin protein family, which is characterized by furin-like cysteine-rich domains and a thrombospondin type 1 repeat. R-spondin is a secreted molecule that activates Wnt/ β -catenin signaling. Rspo2 -deficient mice were generated to investigate the function of mouse Rspo2 during embryonic development. The homozygous mutant forelimb showed defects in distal phalanges and nail structures, and the digits were anomalous in shape. The homozygous mutant hindlimb showed more severe malformations, including lack of digits and zeugopod components. Rspo2 is expressed in the apical ectodermal ridge (AER) of the developing limb. Fgf8 expression in the AER was significantly lower in the homozygous mutant forelimb than in the wild-type forelimb and it was disturbed along the dorsoventral axis. In the homozygous mutant hindlimb, Fgf8 and Fgf4 expression in the posterior AER and Sonic hedgehog expression in the zone of polarizing activity (ZPA) were reduced. The homozygous mutant hindlimb also showed expansion of Wnt7a expression in the dorsal ectoderm toward the ventral side. This study shows that Rspo2 is critical for maintenance of the AER and for growth and patterning in limb development.     

Rasip1 is required for endothelial cell motility, angiogenesis and vessel formation

Ras proteins are small GTPases that regulate cellular growth and differentiation. Components of the Ras signaling pathway have been shown to be important during embryonic vasculogenesis and angiogenesis. Here, we report that Rasip1, which encodes a novel Ras-interacting protein, is strongly expressed in vascular endothelial cells throughout development, in both mouse and frog. Similar to the well-characterized vascular markers VEGFR2 and PECAM, Rasip1 is specifically expressed in angioblasts prior to vessel formation, in the initial embryonic vascular plexus, in the growing blood vessels during angiogenesis and in the endothelium of mature blood vessels into the postnatal period. Rasip1 expression is undetectable in VEGFR2 null embryos, which lack endothelial cells, suggesting that Rasip1 is endothelial specific. siRNA-mediated reduction of Rasip1 severely impairs angiogenesis and motility in endothelial cell cultures, and morpholino knockdown experiments in frog embryos demonstrate that Rasip1 is required for embryonic vessel formation in vivo. Together, these data identify Rasip1 as a novel endothelial factor that plays an essential role in vascular development.     

Endoglin promotes transforming growth factor beta-mediated Smad 1/5/8 signaling and inhibits endothelial cell migration through its association with GIPC

Transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation, migration, and angiogenesis, the ALK-1/Smad 1/5/8 and ALK-5/Smad2/3 pathways. Endoglin is a co-receptor predominantly expressed in endothelial cells that participates in TGFbeta-mediated signaling with ALK-1 and ALK-5 and regulates critical aspects of cellular and biological responses. The embryonic lethal phenotype of knock-out mice because of defects in angiogenesis and disease-causing mutations resulting in human vascular diseases both support essential roles for endoglin, ALK-1, and ALK-5 in the vasculature. However, the mechanism by which endoglin mediates TGF-beta signaling through ALK-1 and ALK-5 has remained elusive. Here we describe a novel interaction between endoglin and GIPC, a scaffolding protein known to regulate cell surface receptor expression and trafficking. Co-immunoprecipitation and immunofluorescence confocal studies both demonstrate a specific interaction between endoglin and GIPC in endothelial cells, mediated by a class I PDZ binding motif in the cytoplasmic domain of endoglin. Subcellular distribution studies demonstrate that endoglin recruits GIPC to the plasma membrane and co-localizes with GIPC in a TGFbeta-independent manner, with GIPC-promoting cell surface retention of endoglin. Endoglin specifically enhanced TGF-beta1-induced phosphorylation of Smad 1/5/8, increased a Smad 1/5/8 responsive promoter, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with GIPC. These studies define a novel mechanism for the regulation of endoglin signaling and function in endothelial cells and demonstrate a new role for GIPC in TGF-beta signaling.     

Vascular Endothelial Growth Factor Receptor-2 Activates ADP-ribosylation Factor 1 to Promote Endothelial Nitric-oxide Synthase Activation and Nitric Oxide Release from Endothelial Cells

Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates endothelial function via production and release of nitric oxide (NO), an important signaling molecule. The molecular basis leading to NO production involves phosphatidylinositiol-3 kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) activation. In this study, we have examined whether small GTP-binding proteins of the ADP-ribosylation factor (ARF) family act as molecular switches to regulate signaling cascades activated by VEGF in endothelial cells. Our results show that this growth factor can promote the rapid and transient activation of ARF1. In endothelial cells, this GTPase is present on dynamic plasma membrane ruffles. Inhibition of ARF1 expression, using RNA interference, markedly impaired VEGF-dependent eNOS phosphorylation and NO production by preventing the activation of the PI3K/Akt signaling axis. Furthermore, our data indicate that phosphorylation of Tyr⁸⁰¹, on VEGF receptor 2, is essential for activating Src- and ARF1-dependent signaling events leading to NO release from endothelial cells. Lastly, this mediator is known to regulate a broad variety of endothelial cell functions. Depletion of ARF1 markedly inhibits VEGF-dependent increase of vascular permeability as well as capillary tubule formation, a process important for angiogenesis. Taken together, our data indicate that ARF1 is a novel modulator of VEGF-stimulated NO release and signaling in endothelial cells.     

Man1, an inner nuclear membrane protein, regulates vascular remodeling by modulating transforming growth factor beta signaling

A growing number of integral inner nuclear membrane (INM) proteins have been implicated in diverse cellular functions. Man1, an INM protein, has recently been shown to regulate transforming growth factor (Tgf) beta superfamily signaling by interacting with receptor-associated Smads. However, the in vivo roles of Man1 have not been fully characterized. Here, we show that Man1 regulates vascular remodeling by analyzing Man1-deficient embryos lacking the Smad interacting domain. Man1-deficient embryos die at midgestation because of defects in embryonic vasculature; the primary capillary plexus forms, but subsequent remodeling is perturbed. It has been proposed that the angiogenesis process is divided into two balanced phases, the activation and resolution/maturation phases, both of which are regulated by Tgfbeta1. We have demonstrated, in Man1-deficient embryos, the expression of Tgfb1 is upregulated and Smad2/3 signaling is abnormally activated, resulting in increased extracellular matrix deposition, a hallmark of the resolution phase of angiogenesis. We have also showed that the recruitment of mural cells to the vascular wall is severely disturbed in mutants, which may lead to disruption of intercellular communication between endothelial and mural cells required for proper vascular remodeling. These results have revealed a novel role for Man1 in angiogenesis and provide the first evidence that vascular remodeling can be regulated at the INM through the interaction between Man1 and Smads.     

Inhibition of angiogenesis by a mouse sprouty protein

Sprouty negatively modulates branching morphogenesis in the Drosophila tracheal system. To address the role of mammalian Sprouty homologues in angiogenesis, another form of branching morphogenesis, a recombinant adenovirus engineered to express murine Sprouty-4 selectively in endothelial cells, was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Sprouty-4 expression inhibited branching and sprouting of small vessels, resulting in abnormal embryonic development. In vitro, Sprouty-4 inhibited fibroblast growth factor and vascular endothelial cell growth factor-mediated cell proliferation and migration and prevented basic fibroblast growth factor and vascular endothelial cell growth factor-induced MAPK phosphorylation in endothelial cells, indicating inhibition of tyrosine kinase-mediated signaling pathways. The ability of constitutively activated mutant Ras(L61) to rescue Sprouty-4 inhibition of MAPK phosphorylation suggests that Sprouty inhibits receptor tyrosine kinase signaling upstream of Ras. Thus, Sprouty may regulate angiogenesis in normal and disease processes by modulating signaling by endothelial tyrosine kinases.     

R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.     

The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis

Endothelial cells play essential roles in maintenance of vascular integrity, angiogenesis, and wound repair. We show that an endothelial cell-restricted microRNA (miR-126) mediates developmental angiogenesis in vivo. Targeted deletion of miR-126 in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, due to a loss of vascular integrity and defects in endothelial cell proliferation, migration, and angiogenesis. The subset of mutant animals that survives displays defective cardiac neovascularization following myocardial infarction. The vascular abnormalities of miR-126 mutant mice resemble the consequences of diminished signaling by angiogenic growth factors, such as VEGF and FGF. Accordingly, miR-126 enhances the proangiogenic actions of VEGF and FGF and promotes blood vessel formation by repressing the expression of Spred-1, an intracellular inhibitor of angiogenic signaling. These findings have important therapeutic implications for a variety of disorders involving abnormal angiogenesis and vascular leakage.     

Essential role for sphingosine kinases in neural and vascular development

Sphingosine-1-phosphate (S1P), an important sphingolipid metabolite, regulates diverse cellular processes, including cell survival, growth, and differentiation. Here we show that S1P signaling is critical for neural and vascular development. Sphingosine kinase-null mice exhibited a deficiency of S1P which severely disturbed neurogenesis, including neural tube closure, and angiogenesis and caused embryonic lethality. A dramatic increase in apoptosis and a decrease in mitosis were seen in the developing nervous system. S1P(1) receptor-null mice also showed severe defects in neurogenesis, indicating that the mechanism by which S1P promotes neurogenesis is, in part, signaling from the S1P(1) receptor. Thus, S1P joins a growing list of signaling molecules, such as vascular endothelial growth factor, which regulate the functionally intertwined pathways of angiogenesis and neurogenesis. Our findings also suggest that exploitation of this potent neuronal survival pathway could lead to the development of novel therapeutic approaches for neurological diseases.     

Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein–protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W¹⁵³ and W¹⁵⁶. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W¹⁵³ and W¹⁵⁶ by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W¹⁵⁶ but not W¹⁵³ based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W¹⁵⁶ and found that DPY19L3-mediated C-mannosylation of Rspo1 at W¹⁵⁶ is required for its secretion.     

A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos

Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP)(s843) transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.     

miR-126 regulates angiogenic signaling and vascular integrity

Precise regulation of the formation, maintenance, and remodeling of the vasculature is required for normal development, tissue response to injury, and tumor progression. How specific microRNAs intersect with and modulate angiogenic signaling cascades is unknown. Here, we identified microRNAs that were enriched in endothelial cells derived from mouse embryonic stem (ES) cells and in developing mouse embryos. We found that miR-126 regulated the response of endothelial cells to VEGF. Additionally, knockdown of miR-126 in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development. miR-126 functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1 and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-beta). Increased expression of Spred1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown. These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for modulating vascular formation and function.     

Paracrine and autocrine mechanisms of apelin signaling govern embryonic and tumor angiogenesis

Apelin and its G protein-coupled receptor APJ play important roles in blood pressure regulation, body fluid homeostasis, and possibly the modulation of immune responses. Here, we report that apelin-APJ signaling is essential for embryonic angiogenesis and upregulated during tumor angiogenesis. A detailed expression analysis demonstrates that both paracrine and autocrine mechanisms mark areas of embryonic and tumor angiogenesis. Knockdown studies in Xenopus reveal that apelin-APJ signaling is required for intersomitic vessel angiogenesis. Moreover, ectopic expression of apelin but not vascular endothelial growth factor A (VEGFA) is sufficient to trigger premature angiogenesis. In vitro, apelin is non-mitogenic for primary human endothelial cells but promotes chemotaxis. Epistasis studies in Xenopus embryos suggest that apelin-APJ signaling functions downstream of VEGFA. Finally, we show that apelin and APJ expression is highly upregulated in microvascular proliferations of brain tumors such as malignant gliomas. Thus, our results define apelin and APJ as genes of potential diagnostic value and promising targets for the development of a new generation of anti-tumor angiogenic drugs.     

A vascular gene trap screen defines RasGRP3 as an angiogenesis-regulated gene required for the endothelial response to phorbol esters

We identified Ras guanine-releasing protein 3 (RasGRP3) as a guanine exchange factor expressed in blood vessels via an embryonic stem (ES) cell-based gene trap screen to identify novel vascular genes. RasGRP3 is expressed in embryonic blood vessels, down-regulated in mature adult vessels, and reexpressed in newly formed vessels during pregnancy and tumorigenesis. This expression pattern is consistent with an angiogenic function for RasGRP3. Although a loss-of-function mutation in RasGRP3 did not affect viability, RasGRP3 was up-regulated in response to vascular endothelial growth factor (VEGF) stimulation of human umbilical vein endothelial cells, placing RasGRP3 regulation downstream of VEGF signaling. Phorbol esters mimic the second messenger diacylglycerol (DAG) in activating both protein kinase C (PKC) and non-PKC phorbol ester receptors such as RasGRP3. ES cell-derived wild-type blood vessels exposed to phorbol myristate acetate (PMA) underwent extensive aberrant morphogenesis that resulted in the formation of large endothelial sheets rather than properly branched vessels. This response to PMA was completely dependent on the presence of RasGRP3, as mutant vessels were refractory to the treatment. Taken together, these findings show that endothelial RasGRP3 is up-regulated in response to VEGF stimulation and that RasGRP3 functions as an endothelial cell phorbol ester receptor in a pathway whose stimulation perturbs normal angiogenesis. This suggests that RasGRP3 activity may exacerbate vascular complications in diseases characterized by excess DAG, such as diabetes.     

The interaction of endoglin with beta-arrestin2 regulates transforming growth factor-beta-mediated ERK activation and migration in endothelial cells

In endothelial cells, transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation and migration, the ALK-1/Smads 1/5/8 pathway and the ALK-5/Smads 2/3 pathway. TGF-beta signaling through these pathways is further regulated in endothelial cells by the endothelial specific TGF-beta superfamily co-receptor, endoglin. The importance of endoglin, ALK-1, and ALK-5 in endothelial biology is underscored by the embryonic lethal phenotypes of knock-outs in mice due to defects in angiogenesis, and by the presence of disease-causing mutations in these genes in human vascular diseases. However, the mechanism of action of endoglin is not well defined. Here we define a novel interaction between endoglin and the scaffolding protein beta-arrestin2. Both co-immunoprecipitation and fluorescence confocal studies demonstrate the specific interaction between endoglin and beta-arrestin2 in endothelial cells, enhanced by ALK-1 and to a lesser extent by the type II TGF-beta receptor. The endoglin/beta-arrestin2 interaction results in endoglin internalization and co-accumulation of endoglin and beta-arrestin2 in endocytic vesicles. Whereas endoglin did not have a direct impact on either Smad 2/3 or Smad 1/5/8 activation, endoglin antagonized TGF-beta-mediated ERK signaling, altered the subcellular distribution of activated ERK, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with beta-arrestin2. Reciprocally, small interfering RNA-mediated silencing of endogenous beta-arrestin2 expression restored TGF-beta-mediated ERK activation and increased endothelial cell migration in an endoglin-dependent manner. These studies define a novel function for endoglin, and further expand the roles mediated by the ubiquitous scaffolding protein beta-arrestin2.     

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis

Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.