The receptor tyrosine kinase TIE is required for integrity and survival of vascular endothelial cells.

Vascular endothelial cells are critical for the development and function of the mammalian circulatory system. We have analyzed the role of the endothelial cell-specific receptor tyrosine kinase TIE in the mouse vasculature. Mouse embryos homozygous for a disrupted Tie allele developed severe edema, their microvasculature was ruptured and they died between days 13.5 and 14.5 of gestation. The major blood vessels of the homozygous embryos appeared normal. Cells lacking a functional Tie gene were unable to contribute to the adult kidney endothelium in chimeric animals, further demonstrating the intrinsic requirement for TIE in endothelial cells. We conclude that TIE is required during embryonic development for the integrity and survival of vascular endothelial cells, particularly in the regions undergoing angiogenic growth of capillaries. TIE is not essential, however, for vasculogenesis, the early differentiation of endothelial cells.     

Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse.

Integrins alpha6beta1 and alpha6beta4 are cell surface receptors for laminins. Integrin alpha6-null mice die at birth with severe skin blistering and defects in the cerebral cortex and in the retina. Integrin alpha3beta1 can associate with laminins and other ligands. Integrin alpha3-null mice also die at birth, with kidney and lung defects at late stages of development, and moderate skin blistering. To investigate possible overlapping functions between alpha3 and alpha6 integrins, we analyzed the phenotype of compound alpha3-/-/alpha6-/- mutant embryos. Double homozygous mutant embryos were growth-retarded and displayed several developmental defects not observed in the single mutant animals. First, limb abnormalities characterized by an absence of digit separation and the fusion of preskeletal elements were observed. Further analyses indicated a defect in the apical ectodermal ridge, an essential limb organizing center. In the double mutant, the ridge appeared flattened, and ridge cells did not show a columnar morphology. A strong reduction in ridge cell proliferation and alterations of the basal lamina underlying the ectoderm were observed. These results suggest that alpha3 and alpha6 integrins are required for the organization or compaction of presumptive apical ectodermal ridge cells into a distinct differentiated structure. Additional defects were present: an absence of neural tube closure, bilateral lung hypoplasia, and several abnormalities in the urogenital tract. Finally, an aggravation of brain and eye lamination defects was observed. The presence of novel phenotypes in double mutant embryos demonstrates the synergism between alpha3 and alpha6 integrins and their essential roles in multiple processes during embryogenesis.     

NgBR is essential for endothelial cell glycosylation and vascular development

NgBR is a transmembrane protein identified as a Nogo‐B‐interacting protein and recently has been shown to be a subunit required for cis‐prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial‐specific NgBR knockout embryos. Here, we show that endothelial‐specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE‐cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo‐B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.     

Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation

Notch signaling functions to regulate cell-fate decisions by modulating differentiation, proliferation, and survival of cells. Notch receptors and ligands are expressed in embryonic vasculature and are required for the remodeling of the primary embryonic vasculature of mice. Here, we characterize the expression patterns of Notch1, Notch4, and Jagged1 proteins during the process of folliculogenesis and corpus luteum formation in the mouse ovary, an organ with dynamic physiological angiogenic growth. These Notch proteins and ligand are expressed in a subset of ovarian vessels, including both mature ovarian vasculature as well as angiogenic neovessels. Their expression in the ovary was found in both endothelial and vascular associated mural cells. Our data suggest a complex regulatory role for the Notch signaling pathway during mouse oogenesis and ovarian neovascularization.     

Developmental ablation of Id1 and Id3 genes in the vasculature leads to postnatal cardiac phenotypes

The Id1 and Id3 genes play major roles during cardiac development, despite their expression being confined to non-myocardial layers (endocardium–endothelium–epicardium). We previously described that Id1Id3 double knockout (dKO) mouse embryos die at mid-gestation from multiple cardiac defects, but early lethality precluded the studies of the roles of Id in the postnatal heart. To elucidate postnatal roles of Id genes, we ablated the Id3 gene and conditionally ablated the Id1 gene in the endothelium to generate conditional KO (cKO) embryos. We observed cardiac phenotypes at birth and at 6 months of age. Half of the Id cKO mice died at birth. Postnatal demise was associated with cardiac enlargement and defects in the ventricular septum, trabeculation and vasculature. Surviving Id cKO mice exhibited fibrotic vasculature, cardiac enlargement and decreased cardiac function. An abnormal vascular response was also observed in the healing of excisional skin wounds of Id cKO mice. Expression patterns of vascular, fibrotic and hypertrophic markers were altered in the Id cKO hearts, but addition of Insulin-Like Growth Factor binding protein-3 (IGFbp3) reversed gene expression profiles of vascular and fibrotic, but not hypertrophic markers. Thus, ablation of Id genes in the vasculature leads to distinct postnatal cardiac phenotypes. These findings provide important insights into the role/s of the endocardial network of the endothelial lineage in the development of cardiac disease, and highlight IGFbp3 as a potential link between Id and its vascular effectors.     

Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis

Focal adhesion kinase (FAK) is a critical mediator of signal transduction by integrins and growth factor receptors in a variety of cells including endothelial cells (ECs). Here, we describe EC-specific knockout of FAK using a Cre-loxP approach. In contrast to the total FAK knockout, deletion of FAK specifically in ECs did not affect early embryonic development including normal vasculogenesis. However, in late embryogenesis, FAK deletion in the ECs led to defective angiogenesis in the embryos, yolk sac, and placenta, impaired vasculature and associated hemorrhage, edema, and developmental delay, and late embryonic lethal phenotype. Histologically, ECs and blood vessels in the mutant embryos present a disorganized, detached, and apoptotic appearance. Consistent with these phenotypes, deletion of FAK in ECs isolated from the floxed FAK mice led to reduced tubulogenesis, cell survival, proliferation, and migration in vitro. Together, these results strongly suggest a role of FAK in angiogenesis and vascular development due to its essential function in the regulation of multiple EC activities.     

The role of blood flow and microRNAs in blood vessel development

The circulatory system is the first organ system that develops during embryogenesis, and is essential for embryo viability and survival. Crucial for developing a functional vasculature are the specification of arterial-venous identity in vessels and the formation of a hierarchical branched vascular network. Sprouting angiogenesis, intussusception, and flow driven remodeling events collectively contribute to establishing the vascular architecture. At the molecular level, arterial-venous identity and branching are regulated by genetically hardwired mechanisms involving Notch, vascular endothelial growth factor and neural guidance molecule signaling pathways, modulated by hemodynamic factors. MicroRNAs are small, non-coding RNAs that act as silencers to fine-tune the gene expression profile. MicroRNAs are known to influence cell fate decisions, and microRNA expression can be controlled by blood flow, thus placing microRNAs potentially at the center of the genetic cascades regulating vascular differentiation. In the present review, we summarize current progress regarding microRNA functions in blood vessel development with an emphasis on studies performed in zebrafish and mouse models.     

Endoglin null endothelial cells proliferate faster and are more responsive to transforming growth factor beta1 with higher affinity receptors and an activated Alk1 pathway

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.     

A requirement for neuropilin-1 in embryonic vessel formation.

Neuropilin-1 is a membrane protein that is expressed in developing neurons and functions as a receptor or a component of the receptor complex for the class 3 semaphorins, which are inhibitory axon guidance signals. Targeted inactivation of the neuropilin-1 gene in mice induced disorganization of the pathway and projection of nerve fibers, suggesting that neuropilin-1 mediates semaphorin-elicited signals and regulates nerve fiber guidance in embryogenesis. Neuropilin-1 is also expressed in endothelial cells and shown to bind vascular endothelial growth factor (VEGF), a potent regulator for vasculogenesis and angiogenesis. However, the roles of neuropilin-1 in vascular formation have been unclear. This paper reported that the neuropilin-1 mutant mouse embryos exhibited various types of vascular defects, including impairment in neural vascularization, agenesis and transposition of great vessels, insufficient aorticopulmonary truncus (persistent truncus arteriosus), and disorganized and insufficient development of vascular networks in the yolk sac. The vascular defects induced by neuropilin-1 deficiency in mouse embryos suggest that neuropilin-1 plays roles in embryonic vessel formation, as well as nerve fiber guidance.     

Gastrulation and angiogenesis,not endothelial specification,is sensitive to partial deficiency in vascular endothelial growth factor-a in mice

Mouse embryogenesis is dose sensitive to vascular endothelial growth factor-A (VEGF-A), and mouse embryos partially deficient in VEGF-A die in utero because of severe vascular defects. In this study, we investigate the possible causes that underlie this phenomenon. Although the development of vascular defects in VEGF-A-deficient embryos seems to suggest that endothelial differentiation depends on the presence of a sufficient level of VEGF-A, we were surprised to find that endothelial differentiation per se is insensitive to a significant loss of VEGF-A activity. Instead, the development of the multipotent mesenchymal cells, from which endothelial progenitors arise in the yolk sac, is most highly dependent on VEGF-A. As a result of VEGF-A deficiency, dramatically fewer multipotent mesenchymal cells are generated in the prospective yolk sac. However, among the small number of mesenchymal cells that do enter the prospective yolk sac, endothelial differentiation occurs at a normal frequency. In the embryo proper, vasculogenesis is initiated actively in spite of a significant VEGF-A deficiency, but the subsequent steps of vascular development are defective. We conclude that a full-level VEGF-A activity is not critical for endothelial specification but is important for two distinct processes before and after endothelial specification: the development of the yolk sac mesenchyme and angiogenic sprouting of blood vessels.     

Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo

Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.     

Coagulation factor III (tissue factor) is required for vascularization in zebrafish embryos

Tissue factor (coagulation factor III) is a cell surface receptor for coagulation factor VII/VIIa; it was initially recognized as an initiator of the extrinsic coagulation pathway. Recently, the zebrafish tissue factor gene (TF) has been cloned. Paralogs encode coagulation factors IIIa and IIIb; both show remarkable sequence identity to the human and mouse coagulation factor III gene. It has been reported that TF could have additional properties that are essential for normal embryonic development, since knockout of the murine coagulation factor III gene resulted in 90% embryonic lethality. We examined the role of coagulation factor IIIb (f3b) during zebrafish embryonic development. Expression analysis revealed that endogenous f3b was chronologically expressed in the pectoral fins and in the vicinity of the pharynx. Knockout of f3b by injection of an f3b morpholino at the one-to-two cell stage caused distinctive morphological defects in embryos, including edema in the fourth brain ventricle at early embryonic stages and occasional bleeding at later stages. Furthermore, f3b morphants displayed abnormal vascular patterning. We conclude that f3b is required for brain vascular development and for development of part of the somatic vasculature during embryogenesis in the zebrafish.     

The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development

The vertebrate embryonic vasculature develops from angioblasts, which are specified from mesodermal precursors and develop in close association with blood cells. The signals that regulate embryonic vasculogenesis and angiogenesis are incompletely understood. Here, we show that R-spondin 3 (Rspo3), a member of a novel family of secreted proteins in vertebrates that activate Wnt/beta-catenin signaling, plays a key role in these processes. In Xenopus embryos, morpholino antisense knockdown of Rspo3 induces vascular defects because Rspo3 is essential for regulating the balance between angioblast and blood cell specification. In mice, targeted disruption of Rspo3 leads to embryonic lethality caused by vascular defects. Specifically in the placenta, remodeling of the vascular plexus is impaired. In human endothelial cells, R-spondin signaling promotes proliferation and sprouting angiogenesis in vitro, indicating that Rspo3 can regulate endothelial cells directly. We show that vascular endothelial growth factor is an immediate early response gene and a mediator of R-spondin signaling. The results identify Rspo3 as a novel, evolutionarily conserved angiogenic factor in embryogenesis.     

Role of vascular endothelial-cadherin in vascular morphogenesis

Vascular endothelial (VE)-cadherin is an adhesive transmembrane protein specifically expressed at interendothelial junctions. Its extracellular domain exhibits Ca2+-dependent homophilic reactivity, promoting cell-cell recognition. Mice deficient in VE-cadherin die at mid-gestation resulting from severe vascular defects. At the early phases of vascular development (E8.5) of VE-cadherin-deficient embryos, in situ differentiation of endothelial cells was delayed although their differentiation program appeared normal. Vascularization was defective in the anterior part of the embryo, while dorsal aortae and vitelline and umbilical arteries formed normally in the caudal part. At E9.25, organization of endothelial cells into large vessels was incomplete and angiogenesis was impaired in mutant embryos. Defects were more severe in extraembryonic vasculature. Blood islands of the yolk sac and clusters of angioblasts in allantois failed to establish a capillary plexus and remained isolated. This was not due to defective cell-cell recognition as endothelial cells formed intercellular junctions, as shown by electron microscopy. These data indicate that VE-cadherin is dispensable for endothelial homophilic adhesion but is required for vascular morphogenesis.     

Role of kinase-independent and -dependent functions of FAK in endothelial cell survival and barrier function during embryonic development

Focal adhesion kinase (FAK) is essential for vascular development as endothelial cell (EC)–specific knockout of FAK (conditional FAK knockout [CFKO] mice) leads to embryonic lethality. In this study, we report the differential kinase-independent and -dependent functions of FAK in vascular development by creating and analyzing an EC-specific FAK kinase-defective (KD) mutant knockin (conditional FAK knockin [CFKI]) mouse model. CFKI embryos showed apparently normal development through embryonic day (E) 13.5, whereas the majority of CFKO embryos died at the same stage. Expression of KD FAK reversed increased EC apoptosis observed with FAK deletion in embryos and in vitro through suppression of up-regulated p21. However, vessel dilation and defective angiogenesis of CFKO embryos were not rescued in CFKI embryos. ECs without FAK or expressing KD FAK showed increased permeability, abnormal distribution of vascular endothelial cadherin (VE-cadherin), and reduced VE-cadherin Y658 phosphorylation. Together, our data suggest that kinase-independent functions of FAK can support EC survival in vascular development through E13.5 but are insufficient for maintaining EC function to allow for completion of embryogenesis.