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。     

New vessel formation after surgical brain injury in the rat's cerebral cortex I. Formation of the blood vessels proximally to the surgical injury

Postnatal neovascularization has been previously considered synonymous with angiogenesis but it was found that circulating endothelial progenitor cells may home into sites of neovascularization and their differentiation into endothelial cells is consistent with vasculogenesis. In this study, we investigated neovascularization of the adult rat's cerebral cortex after surgical brain injury by electron microscopic ultrastructural and immunocytochemical studies. We found places with disrupted brain parenchyma. The blood vessels showed an incomplete endothelial lining. In the brain parenchyma we observed fibrin, likely derived from disrupted blood vessels. In the plasma there were cell aggregates characterized by endothelial-like features with fibrils in the cytoplasm, untypical for endothelial cells. These endothelial-like cells participated in the process of new vessel formation. We used the anti-alpha(v) beta3 integrin antibody to visualize the different morphogenic stages of newly formed blood vessels. We demonstrated the relationship between alpha(v) beta3 integrin localization and different stages of new vessel formation. Our data suggest that growth and development of new blood vessels due to neovascularization following trauma of the adult rat brain are not restricted to angiogenesis but encompass vasculogenesis as well.     

胶质瘤血管新生及其调控机制

体内外研究表明实体性肿瘤的生长是依赖血管的,其直径一旦超过2 mm,若没有新生的血管供血则肿瘤的生长就会停止。胶质瘤是人体内血管化程度最高恶性的肿瘤之一。胶质瘤的血管发生与其他实体性肿瘤血管发生一样,由血管形成及血管新生两种方式构成：前者由内皮祖细胞或者血管母细胞形成新的血管;而后者是由组织中既存的成熟血管的内皮细胞发生增殖和游走形成小血管。血管新生是肿瘤血管生成的主要形式,胶质瘤血管新生的机制研究以及抑制其血管生成是胶质瘤治疗的一个新途径,成为近年来的研究热点,本文就胶质瘤血管新生及其调控机制作一综述。     

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.     

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.     

Tie-2 and angiopoietin-2 expression at the fetal-maternal interface: a receptor ligand model for vascular remodelling

The blood vessels at the fetal-maternal interface widen dramatically during pregnancy in order to increase blood flow to nourish the developing fetus. This vessel remodelling destroys normal vessel integrity and encompasses the dissolution of vessel muscle and elastic tissue. It also includes the displacement of endothelial cells by fetal trophoblasts that invade the maternal arteries of the uterus. Interaction between the endothelial cell receptor, Tie-2, and its recently discovered antagonist ligand, angiopoietin-2 (Ang-2), has been implicated in the loosening of vessel structure. Using Northern blot hybridization and RNA in-situ hybridization analysis the expression pattern of Tie-2, and Ang-2 in the placenta throughout pregnancy, was investigated. We found Ang-2 expressed in the syncytiotrophoblast during the first trimester. In addition to the expected expression of the Tie-2 receptor in both fetal and maternal endothelial cells, we observed Tie-2 expression in endovascular invasive trophoblasts. These cells of epithelial origin invade the uterine spiral arteries and acquire endothelial cell properties. The temporal- and lineage-specific pattern of expression of Tie-2 and Ang-2 suggests that this receptor-ligand pair functions during the critical phase of development of the fetal vasculature and reworking of the maternal vessels during normal placentation.     

The role of angiopoietins during angiogenesis in gliomas

The formation of new blood vessels plays an important role in human disease development and progression. For instance, it is well established that the growth of most cancers critically depends on the supply of nutrition and oxygen by newly recruited blood vessels. Similarly, malignant gliomas, the most common primary brain tumors occurring in humans are highly dependent on angiogenesis. In recent years, there has been tremendous effort to uncover the molecular mechanisms that drive blood vessel growth in adult tissues, especially during cancer progression. Vascular endothelial growth factor (VEGF) and other morphogens, such as angiopoietins and ephrins have been shown to be critically involved in the formation of new blood vessels during both developmental and pathological angiogenesis as evidenced by genetic studies in mice. In this review, we focus on angiopoietins, a family of growth factor ligands binding to Tie tyrosine kinase receptors with emphasis on their functional consequences during the growth and progression of experimental tumors and malignant human gliomas.     

Myeloid cells functioning in tumor vascularization as a novel therapeutic target

Angiogenesis, the sprouting of new blood vessels to sustain growth, is an important new target in solid tumor therapy. Initial studies focused on the role of the tumor cell in promoting angiogenesis; yet more recent work has demonstrated that host cells in the tumor microenvironment also play a critical role in tumor vascularization. Additionally, vasculogenesis in which new blood vessels develop from vascular progenitor cells also contributes to tumor growth. Recent studies propose a central role for cells of the myeloid lineage in triggering vessel growth by releasing angiogenic factors and perhaps by incorporating directly into nascent blood vessels. We will review studies that support a critical role for myeloid cells in neovascularization, with a focus on cells that express various monocytic/dendritic cell markers, including vascular leukocytes (VLCs), Tie2+ monocytes, and vascular endothelial growth factor receptor 2 (VEGFR2)+ monocytes, among others. The evidence that these myeloid cells represent bona fide therapeutic targets for solid tumors will be reviewed. Finally, we will address some controversies and challenges in the field with a focus on future directions.     

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.     

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.     

Identification of genes involved in VEGF-mediated vascular morphogenesis using embryonic stem cell-derived cystic embryoid bodies

The vasculature forms during development via two processes, vasculogenesis and angiogenesis, in which vessels form de novo from angioblast precursors or as sprouts from pre-existing vessels, respectively. A common and critical aspect of both processes is vascular morphogenesis, which includes branching of endothelial cell cords and lumen formation. Although ample evidence support the central role of vascular endothelial growth factor (VEGF) in both vasculogenesis and angiogenesis, the role of VEGF in vascular morphogenesis is unclear and little is known about the regulation of vascular morphogenesis, in general. We have used the in vitro vessel differentiation system of embryonic stem (ES) cell-derived cystic embryonic bodies (CEB) as a model for studying VEGF-mediated vessel formation. Whereas CEB formed from wild-type ES cells make well-formed vessel-like structures, CEB derived from VEGF-null ES cells contain PECAM-1-positive endothelial cells, but these cells do not participate in vascular morphogenesis. Using gene expression microarray analysis to compare gene expression in these two systems, we have been able to identify many genes and novel ESTs that are downstream of VEGF function, and which may be involved in VEGF-mediated vascular morphogenesis including caveolin-1 and HEY-1. These results support using the CEB model, in combination with gene knockout ES cells, for studying vascular morphogenesis.     

In utero angiopoietin-2 gene delivery remodels placental blood vessel phenotype: a murine model for studying placental angiogenesis

Angiopoietin (Ang)-2, the natural antagonist of the Ang1/Tie2 receptor is a complex regulator of blood vessel plasticity that plays a pivotal role in both vessel sprouting [in the presence of vascular endothelial growth factor (VEGF)-A] and vessel regression (in the absence of VEGF-A). Based on the spatial and temporal expression of Ang2 throughout human gestation, we recently suggested that the Ang2 may play a pivotal role in placental angiogenesis. Further, to examine this tenet we have developed a novel murine model system in which in utero Ang2 gene delivery via a non-replicating adenoviral expression vector has the potential to manipulate the blood vessel phenotype in vivo during pregnancy. Ang2 overexpression selectively and rapidly remodels the labyrinth perivascular extracellular matrix, subsequently promoting plasticity of the maternal and fetal vessels, which appear honeycombed due to a 2-fold increase in blood vessel luminal area. High levels of Ang2 impair endothelial cell adhesiveness, leading to vascular leakiness with perivascular oedema, which increases placental weight. These observations suggest that the Ang2 overexpression may play a key role in placental vascular remodelling. Furthermore, we suggest a novel new model to study the pathobiology of placental vascularization and the effect of placental blood vessels on fetal phenotype.     

Angiogenesis: basic and clinical aspects

The cardiovascular system is the first functional organ system to develop in the vertebrate embryo. A widely accepted view is that blood vessels arise through two mechanisms during development, vasculogenesis and angiogenesis. New vessels in the adult arise mainly through angiogenesis, although vasculogenesis also may occur. The existence of a postnatal vasculogenesis is also supported by the evidence that both endothelial cells and endothelial precursor cells co-exist in the circulation. Angiogenesis is a biological process by which new capillaries are formed and it occurs in many physiological and pathological conditions. It is controlled by the net balance between molecules that have positive and negative regulatory activity and this concept had led to the notion of the "angiogenic switch", depending on an increased production of one or more of the positive regulators of angiogenesis. Considerable benefit can be derived in the clinical setting from manipulating angiogenesis, either positively or negatively. There is a variety of important clinical situations in which it would be desiderable to promote angiogenic processes, such as situations in which it would be desiderable to promote angiogenic processes, such as for the induction of collateral vascularization in an ischemic heart or limb. Conversely, there are pathologic conditions in which preventing angiogenic processes could be useful in the treatment of a growing tumor or a chronic inflammatory process.     

Semaphoring vascular morphogenesis.

In vertebrate embryos, development of an architecturally optimized blood vessel network allows the efficient transport of oxygen and nutrients to all other tissues. The final shape of the vascular system results from vasculogenesis and angiogenesis, during which motile endothelial cells (ECs) modify their integrin-mediated interactions with the extracellular matrix (ECM) in response to pro- and anti-angiogenic factors. There is mounting evidence that different members of the semaphorin (SEMA) family of neural guidance cues participate in developmental and postnatal vessel formation and patterning as well. It turns out that paracrine secretion of class 3 SEMA (SEMA3) by nonendothelial tissues cooperates with vascular endothelial growth factor in regulating EC precursor migration and assembly during vasculogenesis and funnels navigating blood vessel through tissue boundaries during sprouting angiogenesis. Autocrine loops of endothelial SEMA3 instead appears to regulate vascular remodeling, which occurs through blood vessel intussusception and fusion. SEMA3 activity both on the vascular and nervous systems relies upon their ability to hamper the affinity of integrin receptors towards ECM ligands. Indeed, signaling from SEMA-activated plexin receptors negatively regulates cell-ECM adhesive interactions by inhibiting two key integrin activators, such as the small GTPase R-Ras and the focal adhesion protein talin.     

Vasodilator-stimulated phosphoprotein expression and its cytokine-mediated regulation in vasculogenesis during human placental development

Vasculogenesis and the subsequent step, angiogenesis, are the most important stages for the continuity of placental development. Vasodilator-stimulated phosphoprotein (VASP) has a widespread role in the control of cell motility and participates in filamentous actin formation. We hypothesized that VASP participates in vasculogenesis and angiogenesis, by regulating endothelial cell migration. We therefore studied VASP expression in vasculogenic sites in placenta throughout pregnancy and the effect of vascular endothelial growth factor (VEGF) and interleukin (IL)-8 on the regulation of VASP expression in placental explant cultures. We found that VASP is expressed in a spatially and temporally regulated manner by various cells of the villi. In the villous stroma, the most intense immunoreactivity was observed in vasculogenic areas and in endothelial cells. In the second and third trimesters, endothelial cells demonstrated weaker immunoreactivity for VASP compared to samples from first trimester. Ultrastructural analysis of corresponding sites for VASP showed that this protein was increased in pre-endothelial cells. Areas of the strongest VEGF and IL-8 expression by villous trophoblasts corresponded to the areas of strongest VASP expression by endothelial cells, and VEGF and IL-8 showed a stimulatory effect on VASP expression in placental explants (P < 0.05). These results suggest that VASP may participate in vasculogenesis and endothelial sprouting during placental vasculogenesis. In addition, one of the effects of VEGF and IL-8 in angiogenesis may be to induce VASP expression in a paracrine manner.     

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.