Evolution of angiogenesis

Endothelial cells, which are the main agents of the angiogenic process in vertebrates, are lacking in the vessels of invertebrates. These are limited by the basement membranes of epithelial or myoepithelial cells. This fact leads to the questions of how vessels grow in invertebrates and how vertebrate angiogenesis evolved. We herein review the knowledge available about vascular growth in invertebrates. The cases described include the ascidian Botryllus, the annelid Hirudo and the squid Idiosepius. All these processes of vascular growth in invertebrates show substantial differences with the vertebrate angiogenesis, although the signalling system mediated by VEGF and its receptor VEGFR seems to be involved in all cases. However, VEGF signalling is used by many processes of cell directional migration, and it cannot be considered as a hallmark of angiogenesis. We also describe the close similarity between the molecular control of the endothelial angiogenesis and the branching morphogenesis of the tracheal system of insects. In both cases, the process is regulated by hypoxia and activates a leading tip cell which inhibits responsiveness of the adjacent cells through a Delta/Notch signalling pathway. We suggest that endothelial angiogenesis in vertebrates arose through cooption of this hypoxia-sensing mechanism by replacing the FGF/FGFR axis of insects by a VEGF/VEGFR-mediated system, and adding a second layer of control of the endothelial state (quiescent or activated) mediated by angiopoietins and Tie receptors. This evolutionarily new control mechanism of endothelial angiogenesis establishes an endothelial/perivascular cell crosstalking which does not exist in invertebrates.     

Receptor Tyrosine Kinase-Mediated Angiogenesis

The endothelial cell is the essential cell type forming the inner layer of the vasculature. Two families of receptor tyrosine kinases (RTKs) are almost completely endothelial cell specific: the vascular endothelial growth factor (VEGF) receptors (VEGFR1-3) and the Tie receptors (Tie1 and Tie2). Both are key players governing the generation of blood and lymphatic vessels during embryonic development. Because the growth of new blood and lymphatic vessels (or the lack thereof) is a central element in many diseases, the VEGF and the Tie receptors provide attractive therapeutic targets in various diseases. Indeed, several drugs directed to these RTK signaling pathways are already on the market, whereas many are in clinical trials. Here we review the VEGFR and Tie families, their involvement in developmental and pathological angiogenesis, and the different possibilities for targeting them to either block or enhance angiogenesis and lymphangiogenesis.     

Differential Expression of Tie2 Receptor and VEGFR2 by Endothelial Clones Derived from Isolated Bovine Mononuclear Cells

The purpose of these experiments was to evaluate the expression of endothelial markers, such as Tie2 and VEGFR2 in endothelial cells derived from blood mononuclear endothelial progenitor cells. Bovine mononuclear cells were isolated using separation by centrifugation and were grown in endothelial specific media supplemented with growth factors. Isolation of the whole cell population of mononuclear cells (MNC) from bovine peripheral blood gave rise to progenitor-like cells (CD45⁻) that, although morphologically similar, have different phenotypes revealed by expression of endothelial specific markers Tie2 and VEGFR2. Plating of MNCs on collagen and fibronectin gave rise to more colonies than non-coated dishes. Occasional colonies from MNC isolations had a mural cell phenotype, negative for Tie2 and VEGFR2 but positive for smooth muscle actin and PDGFRβ. Although cells expressing high levels of VEGFR2 and low levels of Tie2, and vice versa were both able to form cords on Matrigel, cells with higher expression of Tie2 migrate faster in a scratch assay than ones with lower expression of Tie2. When these different clones of cells were introduced in mice through tail vein injections, they retained an ability to home to angiogenesis occurring in a subcutaneous Matrigel plug, regardless of their Tie2/VEGFR2 receptor expression patterns, but cells with high VEGFR2/low Tie2 were more likely to be CD31 positive. Therefore, we suggest that active sites of angiogenesis (such as wounds, tumors, etc.) can attract a variety of endothelial cell precursors that may differentially express Tie2 and VEGFR2 receptors, and thus affect our interpretation of EPCs as biomarkers or therapies for vascular disease.     

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

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

Fibroblast Growth Factor-2 (FGF-2) Induces Vascular Endothelial Growth Factor (VEGF) Expression in the Endothelial Cells of Forming Capillaries: An Autocrine Mechanism Contributing to Angiogenesis

FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2–induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-M_r FGF-2 (22–24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF.     

Activated Notch4 inhibits angiogenesis: role of beta 1-integrin activation

Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased beta1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of beta 1-integrins. Rather, we demonstrate that Notch4-expressing cells display beta1-integrin in an active, high-affinity conformation. Furthermore, using function-activating beta 1-integrin antibodies, we demonstrate that activation of beta1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting beta 1-integrin-mediated adhesion to the underlying matrix.     

Molecular control of angiopoietin signalling

The angiopoietins act through the endothelial receptor tyrosine kinase Tie2 to regulate vessel maturation in angiogenesis and control quiescence and stability of established vessels. The activating ligand, Ang1 (angiopoietin-1), is constitutively expressed by perivascular cells, and the ability of endothelial cells to respond to the ligand is controlled at the level of the Ang1 receptor. This receptor interacts with the related protein Tie1 on the cell surface, and Tie1 inhibits Ang1 signalling through Tie2. The responsiveness of endothelium to Ang1 is determined by the relative levels of Tie2 and the inhibitory co-receptor Tie1 in the cells. Tie1 undergoes regulated ectodomain cleavage which is stimulated by a range of factors including VEGF (vascular endothelial growth factor), inflammatory cytokines and changes in shear stress. Ectodomain cleavage of Tie1 relieves inhibition of Tie2 and enhances Ang1 signalling. This mechanism regulates Ang1 signalling without requiring changes in the level of the ligand and allows Ang1 signalling to be co-ordinated with other signals in the cellular environment. Regulation of signalling at the level of receptor responsiveness may be an important adaptation in systems in which an activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.     

Attenuation of retinal vascular development and neovascularization in PECAM-1-deficient mice

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) at high levels with important roles in angiogenesis and inflammation. However, the physiological role PECAM-1 plays during vascular development and angiogenesis remains largely unknown. Here we determined the role of PECAM-1 in the postnatal development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) using PECAM-1-deficient (PECAM-1−/−) mice. A significant decrease in retinal vascular density was observed in PECAM-1−/− mice compared with PECAM-1+/+ mice. This was attributed to a decreased number of EC in the retinas of PECAM-1−/− mice. An increase in the rate of apoptosis was observed in retinal vessels of PECAM-1−/− mice, which was compensated, in part, by an increase in the rate of proliferation. However, the development and regression of hyaloid vasculature were not affected in the absence of PECAM-1. We did not observe a significant defect in astrocytes, the number of endothelial tip cell filopodias, and the rate of developing retinal vasculature progression in PECAM-1−/− mice. However, we observed aberrant organization of arterioles and venules, decreased secondary branching, and dilated vessels in retinal vasculature of PECAM-1−/− mice. In addition, retinal neovascularization was attenuated in PECAM-1−/− mice during OIR despite an expression of VEGF similar to that of PECAM-1+/+ mice. Mechanistically, these changes were associated with an increase in EphB4 and ephrin B2, and a decrease in eNOS, expression in retinal vasculature of PECAM-1−/− mice. These results suggest that PECAM-1 expression and its potential interactions with EphB4/ephrin B2 and eNOS are important for survival, migration, and functional organization of EC during retinal vascular development and angiogenesis.     

Increasing Ang1/Tie2 expression by simvastatin treatment induces vascular stabilization and neuroblast migration after stroke

In this study, we tested the hypothesis that the Angiopoietin 1 (Ang1)/Tie2 pathway mediates simvastatin-induced vascular integrity and migration of neuroblasts after stroke. Rats were subjected to 2 hrs of middle cerebral artery occlusion (MCAo) and treated, starting 1 day after stroke with or without simvastatin (1 mg/kg, daily) for 7 days. Simvastatin treatment significantly decreased blood–brain barrier (BBB) leakage and concomitantly, increased Ang1, Tie2 and Occludin expression in the ischaemic border (IBZ) compared to the MCAo control group. Simvastatin also significantly increased doublecortin (DCX, a marker of migrating neuroblasts) expression in the IBZ compared to control MCAo rats. DCX was highly expressed around vessels. To further investigate the signalling pathway of simvastatin-induced vascular stabilization and angiogenesis, rat brain microvascular endothelial cell (RBMEC) culture was employed. The data show that simvastatin treatment of RBMEC increased Ang1 and Tie2 gene and protein expression and promoted phosphorylated-Tie2 activity. Simvastatin significantly increased endothelial capillary tube formation, an index of angiogenesis, compared to non-treated control. Inhibition of Ang1 or knockdown of Tie2 gene expression in endothelial cells significantly attenuated simvastatin-induced capillary tube formation. In addition, simvastatin significantly increased subventricular zone (SVZ) explant cell migration compared to non-treatment control. Inhibition of Ang1 significantly attenuated simvastatin-induced SVZ cell migration. Simvastatin treatment of stroke increases Ang1/Tie2 expression and thereby reduces BBB leakage and promotes vascular stabilization. Ang1/Tie2 expression induced by simvastatin treatment promotes neuroblast micro-vascular coupling after stroke.     

Functional roles of angiogenic factors and receptors on non‐endothelial cells in the oropharyngeal cavity of the chukar partridge (Alectoris chukar)

The vascular endothelial growth factor (VEGF) family belong to the platelet‐derived growth factor supergene family and is involved in angiogenesis and mitogenesis. The VEGF–VEGFR system regulates endothelial cell proliferation, migration, vascular permeability, secretion and other non‐endothelial cells functions. To clarify the possible role of endothelial and non‐endothelial cells, VEGF and its receptors, vascular endothelial cell growth inhibitor (VEGI) were immunohistochemically examined in oropharyngeal organs. Ten adult partridges were used in this study and the pharynx and larynx were dissected together with the palate and tongue. VEGI, VEGF and its receptor were highly expressed in luminal epithelial and stromal cells, when compared to glandular epithelial and muscle cells (P < 0.05). Moreover, VEGF, its receptors and VEGI were expressed rather strongly in the endothelial cells of the blood capillaries and in both the endothelial and smooth muscle cells of the large and small blood vessels. In conclusion, VEGF and its receptors (flt1/fms, flk1/KDR and flt4) and VEGI were expressed by various cell groups at varying intensity in the oropharyngeal organs. This demonstrates that they play a critical role in the regulation and maintenance of the functions in cells different from endothelial ones as well as in cell proliferation, differentiation, apoptosis and angiogenesis.     

Modulation of VEGF signalling output by the Notch pathway

The formation of blood vessels within the vascular system entails a variety of cellular processes, including proliferation, migration and differentiation. In many cases, these diverse processes need to be finely coordinated among neighbouring endothelial cells in order to establish a functional vascular network. For instance, during angiogenic sprouting specialized endothelial tip cells follow guidance cues and migrate extensively into avascular tissues while trailing stalk cells must stay connected to the patent blood vessel. The vascular endothelial growth factor (VEGF) and Notch signalling pathways have emerged as the major players in governing these different cellular behaviours. In particular, recent work indicates an important role for Notch signalling in determining how an endothelial cell responds to VEGF. In this review, we provide an overview of these biochemically distinct pathways and discuss how they may interact during endothelial cell differentiation and angiogenesis.     

Constitutive notch signaling in adult transgenic mice inhibits bFGF‐induced angiogenesis and blocks ovarian follicle development

Notch signaling is important in angiogenesis during embryonic development. However, the embryonic lethal phenotypes of knock‐out and transgenic mice have precluded studies of the role of Notch post‐natally. To develop a mouse model that would bypass the embryonic lethal phenotype and investigate the possible role of Notch signaling in adult vessel growth, we developed transgenic mice with Cre‐conditional expression of the constitutively active intracellular domain of Notch1 (IC‐Notch1). Double transgenic IC‐Notch1/Tie2‐Cre embryos with endothelial specific IC‐Notch1 expression died at embryonic day 9.5. They displayed collapsed and leaky blood vessels and defects in angiogenesis development. A tetracycline‐inducible system was used to express Cre recombinase postnatally in endothelial cells. In adult mice, IC‐Notch1 expression inhibited bFGF‐induced neovascularization and female mice lacked mature ovarian follicles, which may reflect the block in bFGF‐induced angiogenesis required for follicle growth. Our results demonstrate that Notch signaling is important for both embryonic and adult angiogenesis and indicate that the Notch signaling pathway may be a useful target for angiogenic therapies. genesis 52:809–816, 2014. © 2014 Wiley Periodicals, Inc.     

Ovarian steroids in endometrial angiogenesis

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is fundamental for human endometrial development and differentiation, which are necessary for implantation. This vascular process is supposed to be mainly mediated by the vascular endothelial growth factor (VEGF), also named vascular permeability factor (VPF). We report here the expression and modulation of VEGF and its receptors, Flk-1/KDR and Flt-1, in the functionalis throughout the menstrual cycle. Using immunocytochemistry, VEGF is localized in glandular epithelial cells and in the surrounding stroma, as well as in capillaries and spiral arterioles. The localization of VEGF on the endothelium correlates with the presence of Flt-1 and Flk-1/KDR receptors on vascular structures, including capillary strands that have not yet formed a lumen and that have been previously described in tumors as angiogenic capillaries. The strongest immunoreactivity for both VEGF and Flk-1/KDR receptor on endothelial cells is detected in the proliferative and midsecretory phases. Enhanced expression of VEGF and its Flk-1 receptors on narrow capillary strands during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration from the residual basal layer following menstrual shedding of the functionalis. The vascular expression of Flt-1 is more important in the secretory than in the proliferative phase, associated with a high microvascular density and an increase in vascular permeability in the implantation period. Consistently with these in vivo observations, the treatment of isolated endometrial stromal cells with estradiol (E(2)), or E(2) + progesterone, significantly increased VEGF mRNA over the control value in a dose-dependent manner. These results demonstrate that the expression of VEGF and its receptors is cyclically modulated by ovarian steroids, and that this endothelial growth factor acts on the endothelium in a paracrine fashion to control endometrial angiogenesis and permeability.     

Macrophages overexpressing VEGF,transdifferentiate into endothelial-like cells in vitro and in vivo

Vascular endothelial growth factor (VEGF) induces endothelial cell differentiation. To investigate the role of VEGF overexpression in regulating the phenotype of macrophages, we transfected mouse macrophages with human VEGF₁₆₅ and examined the expression of the genes and proteins for various endothelial markers. Macrophages overexpressing VEGF significantly expressed fetal liver kinase 1 (FLK-1), vascular endothelial cadherin (VE-cadherin), CD31, Von Willebrand factor (vWF), endothelial nitric oxide synthase (eNOS) and CD105. Furthermore, in a model of myocardial infarction (MI), macrophages overexpressing VEGF incorporated into blood vessels. Thus macrophages overexpressing VEGF were transdifferentiate into endothelial-like cells (ELCs) both in vitro and in vivo.     

Mechanisms of angiogenesis

Tissue activity of angiogenesis depends on the balance of many stimulating or inhibiting factors. The key signaling system that regulates proliferation and migration of endothelial cells forming the basis of any vessel are vascular endothelium growth factors (VEGF) and their receptors. The VEGF-dependent signaling system is necessary for formation of the embryonic vascular system. Neoangiogenesis during tumor growth is also associated with activation of this signaling system. The biological significance of the effect of such system on the cells depends on the content in tissue of various factors of the VEGF family and their receptors, while in the case of VEGFA it is defined by the ratio of different isoforms of this growth factor. A number of other signaling systems are also involved in regulation of the main steps of vessel formation. The signaling system Dll4/Notch regulates selection of endothelial cells for beginning of angiogenic expansion by endowing particular properties to endothelial cells leading in this process. An important step in vessel stabilization and maturation is vascular wall formation. Signaling system PDGFB/PDGFRbeta as well as angiopoietins Ang1, Ang2, and their receptor Tie2 are involved in recruiting mural cells (pericytes and smooth muscle cells). Identification of key molecules involved in the regulation of angiogenesis may provide new possibilities for development of drugs suitable for inhibition of angiogenesis or its stimulation in various pathologies.     

Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation

Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin’s actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin’s actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin’s effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.     

Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling

The vasculature of the embryo requires vascular endothelial growth factor (VEGF) during development, but most adult blood vessels lose VEGF dependence. However, some capillaries in the respiratory tract and selected other organs of adult mice regress after VEGF inhibition. The present study sought to identify the sequence of events and the fate of endothelial cells, pericytes, and vascular basement membrane during capillary regression in mouse tracheas after VEGF signaling was blocked with a VEGF-receptor tyrosine kinase inhibitor AG-013736 or soluble receptor construct (VEGF Trap or soluble adenoviral VEGFR-1). Within 1 day, patency was lost and fibrin accumulated in some tracheal capillaries. Apoptotic endothelial cells marked by activated caspase-3 were present in capillaries without blood flow. VEGF inhibition was accompanied by a 19% decrease in tracheal capillaries over 7 days and 30% over 21 days. During this period, desmin/NG2-immunoreactive pericytes moved away from regressing capillaries onto surviving vessels. Empty sleeves of basement membrane, left behind by regressing endothelial cells, persisted for about 2 wk and served as a scaffold for vascular regrowth after treatment ended. The amount of regrowth was limited by the number of surviving basement membrane sleeves. These findings demonstrate that, after inhibition of VEGF signaling, some normal capillaries regress in a systematic sequence of events initiated by a cessation of blood flow and followed by apoptosis of endothelial cells, migration of pericytes away from regressing vessels, and formation of empty basement membrane sleeves that can facilitate capillary regrowth.