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.     

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.     

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.     

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.     

Angiogenesis: now and then

Angiogenesis or new blood vessel formation plays an essential role during embryogenesis, adult vascular remodeling and in several pathological disorders, as in tumor development. Although sprouting of blood vessels is the principal angiogenic mechanism, additional ones, such as the recruitment of bone marrow-derived cells, have recently been described. These processes are controlled by several molecules, although members of the VEGF family of angiogenic factors and its receptors seem to be the main mediators. Initially, VEGF receptors were described as endothelial specific; however, further studies have reported their presence in several types of cells of non-endothelial origin, such as tumor cells. This VEGF receptor altered expression has suggested an angiogenesis-independent growth advantage mechanism on certain types of cancers by the generation of autocrine loops. A possible role in tumorigenesis and a potential novel target in cancer therapy have been hypothesized. Detection of other receptors and molecules considered to be angiogenic players has also been observed on tumor cells. Currently, their clinical significance as well as their potential as therapeutic targets for the treatment of certain cancers is being evaluated, having in mind the future development of promising mechanism-based therapies. The aspects mentioned above are the main focus of this review, which aims to throw light on recent findings respecting angiogenesis and novel therapeutic approaches.     

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.     

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.     

TGFbeta1 induces vasculogenesis and inhibits angiogenic sprouting in an embryonic stem cell differentiation model: respective contribution of ALK1 and ALK5

Transforming growth factor-beta1 (TGFbeta1) is a multipotent cytokine that is involved in the regulation of vasculogenesis and angiogenesis. However, the actions of TGFbeta1 on vascular cells in vitro and in vivo are extremely complex and still incompletely understood. The aim of the present study was to investigate the role of TGFbeta1 and its two type I receptors, activin receptor-like kinase-1 (ALK1) and ALK5, in an embryonic stem cell (ESC) differentiation model that recapitulates the developmental steps of vasculogenesis and sprouting angiogenesis. We show that TGFbeta1 increases endothelial cell differentiation in a vascular endothelial growth factor (VEGF)-independent manner and inhibits endothelial tube formation. Furthermore, we demonstrate that undifferentiated ESCs express ALK5 but do not express ALK1, with ALK1 being expressed only after day 5 of differentiation. Finally, we demonstrate that constitutively active forms of ALK1 and ALK5 both inhibit growth factor-induced endothelial sprouting from embryoid bodies. In conclusion, the use of this ESC differentiation model allowed us to propose the following model: at early stages of development, TGFbeta1, through the ALK5 receptor, is provasculogenic in a VEGF-independent manner. Later, in differentiated endothelial cells in which both ALK1 and ALK5 are expressed, both receptors are implicated in inhibition of sprouting angiogenesis.     

Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome

Human placental development combines elements of tumorigenesis and vasculogenesis. The organ's specialized epithelial cells, termed cytotrophoblasts, invade the uterus where they reside in the interstitial compartment. They also line uterine arteries and veins. During invasion, ectodermally derived cytotrophoblasts undergo pseudovasculogenesis, switching their adhesion molecule repertoire to mimic that of vascular cells. Failures in this transformation accompany the pregnancy complication preeclampsia. Here, we used a combination of in situ and in vitro analyses to characterize the cell's expression of vascular endothelial growth factor (VEGF) family ligands and receptors, key regulators of conventional vasculogenesis and angiogenesis. Cytotrophoblast differentiation and invasion during the first and second trimesters of pregnancy were associated with down-regulation of VEGF receptor (VEGFR)-2. Invasive cytotrophoblasts in early gestation expressed VEGF-A, VEGF-C, placental growth factor (PlGF), VEGFR-1, and VEGFR-3 and, at term, VEGF-A, PlGF, and VEGFR-1. In vitro the cells incorporated VEGF-A into the surrounding extracellular matrix; PlGF was secreted. We also found that cytotrophoblasts responded to the VEGF ligands they produced. Blocking ligand binding significantly decreased their expression of integrin alpha1, an adhesion molecule highly expressed by endovascular cytotrophoblasts, and increased apoptosis. In severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome, immunolocalization on tissue sections showed that cytotrophoblast VEGF-A and VEGFR-1 staining decreased; staining for PlGF was unaffected. Cytotrophoblast secretion of the soluble form of VEGFR-1 in vitro also increased. Together, the results of this study showed that VEGF family members regulate cytotrophoblast survival and that expression of a subset of family members is dysregulated in severe forms of preeclampsia.     

Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat

Vascular endothelial growth factor (VEGF), a key regulator of vasculogenesis and embryonic angiogenesis, was recently found to be up-regulated in an animal model of stroke. Unlike VEGF, angiopoietin (Ang)-1 and -2, their receptor tie-2, and the associated receptor tie-1 exert their functions at later stages of vascular development, i.e., during vascular remodeling and maturation. To assess the role of the angiopoietin/tie family in ischemia-triggered angiogenesis we analyzed their temporal and spatial expression pattern after middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells with no apparent change in expression after MCAO. Ang-2 mRNA was up-regulated 6 hours after MCAO and was mainly observed in endothelial cell (EC) cord tips in the peri-infarct and infarct area. Up-regulation of both Ang-2 and VEGF coincided with EC proliferation. Interestingly, EC proliferation was preceded by a transient period of EC apoptosis, correlating with a change in VEGF/Ang-2 balance. Our observation of specific stages of vascular regression and growth after MCAO are in agreement with recent findings suggesting a dual role of Ang-2 in blood vessel formation, depending on the availability of VEGF.     

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.     

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 microvascular permeability by vascular endothelial growth factors

Generation of new blood vessels from pre-existing vasculature (angiogenesis) is accompanied in almost all states by increased vascular permeability. This is true in physiological as well as pathological angiogenesis, but is more marked during disease states. Physiological angiogenesis occurs during tissue growth and repair in adult tissues, as well as during development. Pathological angiogenesis is seen in a wide variety of diseases, which include all the major causes of mortality in the west: heart disease, cancer, stroke, vascular disease and diabetes. Angiogenesis is regulated by vascular growth factors, particularly the vascular endothelial growth factor family of proteins (VEGF). These act on two specific receptors in the vascular system (VEGF-R1 and 2) to stimulate new vessel growth. VEGFs also directly stimulate increased vascular permeability to water and large-molecular-weight proteins. We have shown that VEGFs increase vascular permeability in mesenteric microvessels by stimulation of tyrosine auto-phosphorylation of VEGF-R2 on endothelial cells, and subsequent activation of phospholipase C (PLC). This in turn causes increased production of diacylglycerol (DAG) that results in influx of calcium across the plasma membrane through store-independent cation channels. We have proposed that this influx is through DAG-mediated TRP channels. It is not known how this results in increased vascular permeability in endothelial cells in vivo. It has been shown, however, that VEGF can stimulate formation of a variety of pathways through the endothelial cell, including transcellular gaps, vesiculovacuolar organelle formation, and fenestrations. A hypothesis is outlined that suggests that these all may be part of the same process.     

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.     

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.     

Emerging roles of the Angiopoietin-Tie and the ephrin-Eph systems as regulators of cell trafficking

Vascular receptor tyrosine kinases (RTK) have been identified as critical regulatory signaling molecules of developmental and adult vascular morphogenic processes [vascular endothelial growth factor (VEGF) receptors=sprouting; EphB receptors=assembly; Tie2 receptor=maturation and quiescence]. It is intriguing that the same molecules that control the growth of blood and lymphatic vessels play critical roles in the adult to regulate maintenance functions related to vascular homeostasis. VEGF is among the most potent inducers of vascular permeability. The second vascular RTK system, the interaction of paracrine-acting Angiopoietin-1 with its cognate receptor Tie2, acts as an endothelial maintenance and survival-mediating molecular system, which stabilizes the vessel wall and controls endothelial cell quiescence. The third vascular RTK system, the interaction of Eph receptors with their Eph family receptor-interacting protein (ephrin) ligands, transduces positional guidance cues on outgrowing vascular sprouts, which are critical for proper arteriovenous assembly and establishment of blood flow. As such, Eph-ephrin interactions act as an important regulator of cell-cell interactions, exerting propulsive and repulsive functions on neighboring cells and mediating adhesive functions. This review summarizes recent findings related to the roles of the Angiopoietin-Tie and the Eph-ephrin systems as regulators of cell trafficking in the vascular system. The recognition of vascular homeostatic functions of vascular RTKs marks an important change of paradigm in the field of angiogenesis research as it relates angiogenesis-inducing molecules to vascular maintenance functions in the adult. This may also broaden the scope of vascular RTK-targeted therapies.     

Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells

One of the fundamental principles that underlies tissue-engineering strategies using cell transplantation is that a newly formed tissue must acquire and maintain sufficient vascularization in order to support its growth. Enhancing angiogenesis through delivery of growth factors is one approach to establishing a vascular network to these tissues. In this study, we tested the potential of bone marrow stromal cells (BMSCs) to modulate the growth and differentiation activities of blood vessel precursors, endothelial cells (ECs), by their secretion of soluble angiogenic factors. The growth and differentiation of cultured ECs were enhanced in response to exposure to BMSC conditioned medium (CM). Enzyme-linked immunosorbent assays demonstrated that both mouse and human BMSCs secreted significant quantities of vascular endothelial growth factor (VEGF) (2.4-3.1 ng/10(6) cells per day). Furthermore, eliminating the activity of BMSC-secreted VEGF with blocking antibodies completely blocked the CM effects on cultured ECs. These data demonstrate that human BMSCs secrete sufficient quantities of VEGF to enhance survival and differentiation of endothelial cells in vitro, and suggest they may be capable of directly orchestrating angiogenesis in vivo.