Lentiviral rescue of vascular endothelial growth factor receptor-2 expression in flk1-/- embryonic stem cells shows early priming of endothelial precursors

The vascular endothelial growth factor (VEGF) family and its receptors are important for vascular development and maintenance of blood vessels, as well as for angiogenesis, the formation of new vessels. Loss of VEGF receptor-2 (VEGFR-2; designated Flk-1 in mouse) results in arrest of vascular and hematopoietic development in vivo. We used lentiviral transduction to reconstitute VEGFR-2 expression in flk1-/- embryonic stem (ES) cells. VEGF-induced vasculogenesis and sprouting angiogenesis were rescued in transduced ES cultures differentiating in vitro as EBs. Although the transgene was expressed in the pluripotent stem cells and lacked linage restriction during differentiation, the extent of endothelial recruitment was similar to that in wild-type EBs. Reconstitution of VEGFR-2 in flk1-/- ES cells allowed only precommitted precursors to differentiate into functional endothelial cells able to organize into vascular structures. Chimeric EB cultures composed of wild-type ES cells mixed with flk1-/- ES cells or reconstituted VEGFR-2-expressing ES cells were created. In the chimeric cultures, flk1-/- endothelial precursors were excluded from wild-type vessel structures, whereas reconstituted VEGFR-2-expressing precursors became integrated together with wild-type endothelial cells to form chimeric vessels. We conclude that maturation of endothelial precursors, as well as organization into vascular structures, requires expression of VEGFR-2. Disclosure of potential conflicts of interest is found at the end of this article.     

斑马鱼血管系统的发育遗传学研究进展

斑马鱼血管系统在原肠胚形成后不久便开始发育，血管系统的发育过程可分为血管发生和血管生成这两个不同的阶段，其过程受到多种信号通路的的调控，这些信号协同作用，以确保血管发育的正常进行。文中综述了主要以模式生物斑马鱼来研究的血管发育遗传的过程，并介绍调节血管发育进程的一些关键的调控。以斑马鱼为模式生物来研究血管系统的发育遗传学，为理解人类血管的发育和再生，为缺血性疾病和肿瘤等疾病的治疗提供了新的途径。     

Vasculogenesis and angiogenesis in the early human placenta

Vasculogenesis and angiogenesis are two consecutive processes during blood vessel development in the human placenta. While vasculogenesis, which is the formation of first blood vessels, is achieved by differentiation of pluripotent mesenchymal cells into haemangiogenic stem cells. The subsequent step, angiogenesis, is characterized by development of new vessels from already existing vessels. In this review, we aim to give an overview of vasculogenesis and angiogenesis during the first trimester of human placental development. Recent studies have shown that at the very early stages of placental development, cytotrophoblasts trigger vasculogenesis and angiogenesis, whereas as pregnancy progresses Hofbauer and stromal cells take over the task of triggering blood vessel development. Important growth factors in this scenario are the vascular endothelial growth factor (VEGF) family and their receptors, as well as Tie-1 and Tie-2. This review depicts the molecular and morphological steps of vasculogenesis and angiogenesis, which can give further insights into human placental development and maturation disorders.     

Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis.

The formation of new blood vessels proceeds by both vasculogenesis and angiogenesis. The development of models, which fully recapitulate spatio-temporal events involved during these processes, are crucial to fully understand their mechanisms of regulation. In vitro differentiation of murine embryonic stem (ES) cells has been shown to be a useful tool to investigate factors and genes potentially involved in vasculogenesis (Hirashima et al, 1999; Risau et al, 1988; Vittet et al, 1996; Wang et al, 1992; Wartenberg et al, 1998). We asked here whether this model system can also recapitulate angiogenesis, which may offer new means to study mechanisms involved in this process. ES-derived embryoid bodies (EBs) obtained after 11 days of differentiation, in which a primitive vascular network had formed, were then subcultured into a type I collagen matrix. In the presence of angiogenic growth factors, EBs rapidly developed branching pseudopods. Whole mount immunostainings with a PECAM antibody revealed that more than 75% EBs displayed, within a few days, a large number of endothelial outgrowths that can give tube-like structures with concomitant differentiation of alpha-smooth muscle actin positive cells, thus evoking sprouting angiogenesis. High expression levels of flk1 (VEGFR2), flt1 (VEGFR1), tie-1, and tie-2 are also found, indicating that budding endothelial cells displayed an angiogenic phenotype. The endothelial sprouting response was specifically induced by angiogenic factors with a major contribution of vascular endothelial growth factor (VEGF). Known angiostatic agents, such as platelet factor 4 (PF4), angiostatin, and endostatin inhibited the formation of endothelial sprouts induced by angiogenic factors. Moreover, consistent with the in vivo phenotype, VE-cadherin deficient EBs failed to develop angiogenesis in this model. ES cell differentiation can then recapitulate, in addition to vasculogenesis, the early stages of sprouting angiogenesis. This model system, in which genetic modifications can be easily introduced, may be of particular interest to investigate unsolved questions and molecular mechanisms involved in blood vessel formation.     

Molecular and biological properties of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a fundamental regulator of normal and abnormal angiogenesis. Recent evidence indicates that VEGF is essential for embryonic vasculogenesis and angiogenesis. Furthermore, VEGF is required for the cyclical blood vessel proliferation in the female reproductive tract and for longitudinal bone growth and endochondral bone formation. Substantial experimental evidence also implicates VEGF in pathological angiogenesis. Anti-VEGF monoclonal antibodies or other VEGF inhibitors block the growth of many tumor cell lines in nude mice. Furthermore, the concentrations of VEGF are elevated in the aqueous and vitreous humors of patients with proliferative retinopathies such as the diabetic retinopathy. In addition, VEGF-induced angiogenesis results in a therapeutic benefit in several animal models of myocardial or limb ischemia. Currently, both therapeutic angiogenesis using recombinant VEGF or VEGF gene transfer and inhibition of VEGF-mediated pathological angiogenesis are being pursued.     

Regulation of endothelial cell differentiation and arterial specification by VEGF and Notch signaling

Analysis of molecular and cellular mechanisms underlying vascular development in vertebrates indicates that initially vasculogenesis occurs when a primary capillary plexus forms de novo from endothelial cell precursors derived from nascent mesodermal cells. Transplantation experiments in avian embryos demonstrate that embryonic endothelial cells originate from two different mesodermal lineages: splanchnic mesoderm and somites. Genetic analysis of mouse and zebrafish reveals that vascular endothelial growth factor (VEGF)/Flk1 and Notch signaling play crucial roles throughout embryonic vascular development. VEGFA plays a major role in endothelial cell proliferation, migration, survival, and regulation of vascular permeability. Flk1, the primary VEGFA receptor, is the earliest marker of the developing endothelial lineage and is essential for endothelial differentiation during vasculogenesis. Notch signaling has been demonstrated to directly induce arterial endothelial differentiation. Recent studies suggest that Notch signaling is activated downstream of VEGF signaling and negatively regulates VEGF-induced angiogenesis and suppresses aberrant vascular branching morphogenesis. In addition to altering endothelial cell fate through Notch activation, VEGFA directly guides endothelial cell migration in an isoform-dependent manner, modifying vascular patterns. Interestingly, genetic studies in mice show that many molecules involved in VEGF or Notch signaling must be tightly regulated for proper vascular formation. Taken together, VEGF and Notch signaling apparently coordinate vascular patterning by regulating each other.     

Vasculogenesis drives pulmonary vascular growth in the developing chick embryo.

Formation of the pulmonary vasculature has been described as occurring by outgrowth of existing vessels (angiogenesis), de novo formation of new vessels (vasculogenesis), or a combination of both processes. Uncertainty about the contribution of angiogenesis and vasculogenesis to pulmonary vascular formation is partly due to methodologic approaches. Evidence in favor of angiogenesis stems from studies that used vascular-filling methods. Such methods identify only directly continuous lumina. Evidence for vasculogenesis has been provided by the use of molecular markers of blood vessel endothelium. Use of both methods has not been combined in the same species, however. We hypothesized, based on published evidence from quail and mouse, that chick pulmonary vascular formation occurs by vasculogenesis. To test that hypothesis, we used vascular filling, serial section, and immunohistochemical methods to analyze the developing lungs of chick embryos from Hamburger and Hamilton stages 20 to 43. Vascular filling suggested that the lumen of the pulmonary arteries sprouted from the sixth pharyngeal arch arteries. However, serial sections and immunohistochemical localization of fetal liver kinase-1 protein, the receptor for vascular endothelial growth factor, showed that the pulmonary arterial tree formed from endothelial cell precursors and coalescence of isolated blood vessels in the mediastinal splanchnic mesenchyme centrally to the developing lung tissue distally. Pulmonary veins grew from the left atrium to the developing lungs. Pulmonary blood vessel formation occurred continuously throughout the embryonic period studied. Our results show that vasculogenesis is the main process by which the pulmonary vasculature forms in the developing chick embryo.     

The vascular endothelial growth factor family; proteins which guide the development of the vasculature

The development of the vascular tree during embryogenesis involves vasculogenesis, angiogenesis and tissue-specific differentiation of endothelium which gives rise to many different vessel types. These processes are physiologically complex and are therefore difficult to study in vitro. However, the discovery of endothelial cell-specific receptors and cognate ligands has led to the generation of transgenic and knockout mouse models which have shed light on the molecular mechanisms that regulate the development of blood and lymphatic vessels during embryogenesis. Such mouse models have demonstrated that members of the vascular endothelial growth factor (VEGF) family of proteins and the VEGF receptors are critical regulators of vasculogenesis, angiogenesis and endothelial cell differentiation. The availability of purified VEGF family members and of inhibitors of these growth factors may provide a means to modulate blood vessel growth for the treatment of cancer, retinopathies and diseases of ischemia.     

Signaling pathways induced by vascular endothelial growth factor (review)

Vasculogenesis and angiogenesis are the mechanisms responsible for the development of the blood vessels. Angiogenesis refers to the formation of capillaries from pre-existing vessels in the embryo and adult organism, while vasculogenesis is the development of new blood vessels from the differentiation of endothelial precursors (angioblasts) in situ. Vascular endothelial growth factor (VEGF) family members are major mediators of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to promote endothelial cell viability, mitogenesis, chemotaxis, and vascular permeability. It mediates its activity mainly via two tyrosine kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR), although other receptors, such as neuropilin-1 and -2, can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (flt-4) binds VEGF-C and VEGF-D and is more important in the development of lymphatic vessels. While the functional effects of VEGF on endothelial cells has been well studied, not as much is known about VEGF signaling. This review summarizes the different pathways known to be involved in VEGF signal transduction and the biological responses triggered by the VEGF signaling cascade.     

VEGF signaling is required for the assembly but not the maintenance of embryonic blood vessels.

Here we investigated the importance of vascular endothelial growth factor (VEGF) signaling to the de novo formation of embryonic blood vessels, vasculogenesis, as opposed to the maintenance of blood vessels. We found that antagonizing the activity of the VEGF signaling pathway by using soluble VEGF receptor 1 (sFlt1) or VEGF antibodies inhibited vasculogenesis that occurs in embryos and in cultures of 7.5 days postcoitus prevascular mesoderm. Antagonist treatment resulted in the formation of clusters of endothelial cells not normally observed during vasculogenesis. In contrast, when embryos with established vasculatures or cultures of vascularized mesoderm were treated with sFlt1 or VEGF antibodies, no discernible alterations to the preexisting blood vessels were observed. These observations indicate that, although VEGF signaling is required to promote the mesenchymal to epithelial transition by which angioblasts assemble into nascent endothelial tubes, it is not required by endothelial cells to maintain their organization as an endothelium.     

Angiomotin regulates endothelial cell migration during embryonic angiogenesis

The development of the embryonic vascular system into a highly ordered network requires precise control over the migration and branching of endothelial cells (ECs). We have previously identified angiomotin (Amot) as a receptor for the angiogenesis inhibitor angiostatin. Furthermore, DNA vaccination targeting Amot inhibits angiogenesis and tumor growth. However, little is known regarding the role of Amot in physiological angiogenesis. We therefore investigated the role of Amot in embryonic neovascularization during zebrafish and mouse embryogenesis. Here we report that knockdown of Amot in zebrafish reduced the number of filopodia of endothelial tip cells and severely impaired the migration of intersegmental vessels. We further show that 75% of Amot knockout mice die between embryonic day 11 (E11) and E11.5 and exhibit severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain. Furthermore, using ECs differentiated from embryonic stem (ES) cells, we demonstrate that Amot-deficient cells have intact response to vascular endothelial growth factor (VEGF) in regard to differentiation and proliferation. However, the chemotactic response to VEGF was abolished in Amot-deficient cells. We provide evidence that Amot is important for endothelial polarization during migration and that Amot controls Rac1 activity in endothelial and epithelial cells. Our data demonstrate a critical role for Amot during vascular patterning and endothelial polarization.     

The role of FGF and VEGF in angioblast induction and migration during vascular development.

The embryonic vasculature forms by the processes of vasculogenesis and angiogenesis. Angioblasts (endothelial cell precursors) appear to be induced by fibroblast growth factor 2 (FGF-2). The angioblasts contributing to the dorsal aortae arise by an epithelial to mesenchymal transformation of cells originating from the splanchnic mesoderm. QH-l and vascular endothelial growth factor receptor 2 (VEGFR-2) both appear to label these cells as they adopt a mesenchymal morphology. Since VEGFR-2 is the earliest known VEGF receptor this suggests that VEGF is not involved in angioblast induction. VEGF does appear to be critical, however, for growth and morphogenesis of angioblasts into the initial vascular pattern. Controlled delivery of FGF-2 from beads and aggregates of cells transfected with quail VEGF have been used in our laboratory to study the role of these growth factors in angioblast induction and migration. We have induced cells from the epithelial quail somite to differentiate into angioblasts with FGF-2 both in the embryo and in culture. This is a useful model system to study the origins of endothelial cells that are normally more diffusely induced during gastrulation by an obscure process probably involving signals from the embryonic endoderm. The origins of arterial versus venous endothelial cells is also poorly understood but recent findings on the distribution of ephrins and Eph receptors suggest that molecular differences exist prior to the onset of circulation. Finally, studies on the role of growth factors in such diverse phenomena as stem cell biology, angiogenesis, and molecular medicine in addition to vascular development suggest multiple roles for FGF-2 and VEGF in vascular development.     

Qualitative and quantitative analysis of embryonic pulmonary vessel formation

Vessel formation in the lung has been described as occurring by two mechanisms: proximal, or branch, pulmonary arteries develop via angiogenesis; and distal, smaller vessels form by vasculogenesis. Connections between the proximal and distal vessels establish the final vascular network. The preponderance of vessel formation has been suspected to occur during the canalicular stage of lung development. To test these hypotheses, reporter gene expression under control of the regulatory domain of fetal liver kinase-1 (flk), an early endothelial cell-specific marker, was used to evaluate mouse lungs from embryonic day 10.5 (E10.5) through 2 wk postnatal age. Morphologic assessment was performed after histochemical staining, and quantification of vessel development by a chemiluminescent assay was compared with overall embryonic lung growth. LacZ expression under flk promoter control allowed: (1) early identification of differentiating endothelial cells of the branch pulmonary arteries; (2) visualization of distal vessels forming in the lung mesenchyme (primary capillary network) with subsequent remodeling; (3) recognition of early continuity between proximal and distal vessels, occurring by E10.5; and (4) assessment of developing pulmonary veins and venous confluence. Quantitative analysis revealed increased flk regulated beta-galactosidase (beta-gal) activity of 12 ng beta-gal/lung at E12.5 to 3,215 ng beta-gal/lung at 2 wk, which corresponded to overall lung growth during this period as shown by an increase in total protein content per lung from 35 microg at E12.5 to 6,456 microg at 2 wk after birth. We identified endothelial cell precursors of the developing pulmonary vasculature before vessel lumen formation. Continuity between the proximal pulmonary artery and vessels forming in the distal mesenchyme was present even at the earliest stage evaluated, suggesting endothelial cell differentiation at the site of vessel formation (i.e., vasculogenesis) as occurs with development of the aorta. Finally, we demonstrated that lung vessel development was not accentuated during the canalicular stage, but occurred at all stages and directly corresponded to overall lung growth.     

Vascular update: morphogenesis, tumors, malformations, and molecular dimensions

This vascular review is organized under the following headings: vasculogenesis and angiogenesis; vascular endothelial growth factors, their receptors, TIE receptors, and angiopoietins; other factors in blood vessel formation; parallel patterning in blood vessels and nerves; physiological and pathological neovascularization; the role of VEGF receptors in metastasis; anti-angiogenic therapy for tumors; association of blood vessels with fat; vascular malformations and vascular tumors; infantile hemangiomas; congenital hemangiomas; lymphatic malformations; molecular characteristics of some disorders with vascular malformations; Kasabach-Merritt phenomenon; Sturge-Weber syndrome, Klippel-Trenaunay syndrome, and Parkes Weber syndrome; diagnostic and laboratory studies; and future perspectives.     

Human embryonic stem cells as an in vitro model for human vascular development and the induction of vascular differentiation

Early embryonic blood vessels are typically composed of fragile tubes of endothelial cells encircled by vascular smooth muscle cells. Early human vasculogenesis was explored in spontaneous and directed differentiation models derived from human embryonic stem (HES) cells. In a 3-dimensional (3D) model, HES cells were studied for their potential for vascular differentiation during the spontaneous formation of embryoid bodies. Directed differentiation was investigated by means of a 2-dimensional (2D) differentiation method to promote vascular differentiation from HES cells (without the formation of embryoid bodies). Using this latter approach, up-regulation of early lineage markers of endothelial progenitors were induced. Additional culture under strict conditions and exposure to angiogenic growth factors resulted in a prolonged differentiation pathway into mature endothelial cells and up-regulation of vascular smooth muscle cell markers. The use of 3D collagen gels and Matrigel assays for the induction and inhibition of human vascular sprouting in vitro further established the vascular potential of the cells generated by the 2D differentiation system. Our study shows that HES cells can provide useful models to study early differentiation and development of blood vessels. Moreover, the 2D differentiation model facilitates both the production of vascular lineage cells from HES cells for various potential therapeutic applications and also provides a model for studying the mechanisms involved in early human embryonic blood vessel development.     

Endothelial and Hematopoietic Progenitor Cells (EPCs and HPCs): Hand in Hand Fate Determining Partners for Cancer Cells

Tumor growth and metastasis need new vessel formation by angiogenesis provided by mature endothelial cells and postnatal vasculogenesis provided by endothelial progenitor cells (EPCs). Emerging data suggest a coordinated interaction between EPCs and hematopoietic progenitor cells (HPCs) in these processes. The complexity of the mechanisms governing the new vessel formation by postnatal vasculogenesis has increased by new evidence that not only bone marrow derived EPCs and HPCs seem to be involved in this process but also local progenitors residing within the vascular wall are mobilized and activated to new vessel formation by tumor cells. This review attempts to bring these systemic and local players of postnatal vasculogenesis together and to highlight their role in tumor growth and mestastasis.