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Angiogenesis

Angiogenesis
this is the preferred term for processes leading to the generation of new blood vessels through sprouting (see below) from already existing blood vessels in a process involving the migration and proliferation of endothelial cells from preexisting vessels. Blood vessel growth occurs in the embryo and rarely in the adult with exceptions such as the female reproductive system, wound healing, and pathological processes such as cancer (Carmeliet, 2000; Carmeliet and Jain, 2000; Patan, 2004). Some authors have referred to this process as hemangiogenesis (see, for example, Parsons-Wingerter et al, 2006) to distinguish it from Lymphangiogenesis (see below).

Vasculogenesis
this is the term used for the de novo generation of blood vessels occurring during embryogenesis (for a process occurring later in life see also: postnatal vasculogenesis). Vasculogenesis involves the in situ differentiation into endothelial cells of circulating endothelial progenitor cells. In vasculogenesis, a primitive vascular network is formed de novo through the assembly of multipotential mesenchymal progenitor cells (angioblasts) (Carmeliet, 2000; Carmeliet and Jain, 2000; Patan, 2004).

Vascular expansion
this term refers to the enlargement of small or occluded vessels, which is observed frequently during the generation of collateral blood vessels (Carmeliet, 2000; Carmeliet and Jain, 2000).

postnatal vasculogenesis
this term pertains to the formation of new blood vessels at sites of physiological and pathological neovascularization by circulating bone marrow-derived endothelial progenitor cells (see also: angioblasts) mobilized by tumor tissue-derived cytokines and differentiating into endothelial cells at such sites (Asahara and Kawamoto, 2004). The process is promoted by tumor cell-derived VEGF and PDGF-CC (Asahara and Kawamoto, 2004), whereas endothelial progenitor cells mobilization is reduced by angiopoietin-1 (Garmy-Susini and Varner, 2005). Angiogenesis factors released by endothelial progenitor cells may further support neovascularization through paracrine actions (Urbich et al, 2005; Krenning et al, 2009).

Lyden et al (2001) have reported that hematopoietic progenitor cells that express the VEGF receptor VEGFR1 mobilize to the peripheral blood along with endothelial progenitor cells (expressing VEGFR2), and incorporate into pericapillary connective tissue, thus stabilizing tumor vasculature and possibly also providing growth factors and cytokines to support angiogenesis (Takakura, 2006; Döme et al, 2009). These cells may promote metastatic growth as they are capable of homing in before the tumor cells arrive and thus may form niches where cancer cells can locate and proliferate (Kaplan et al, 2005).

Rüger et al (2008) have reported that adult human bone marrow progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices, suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.

Lymphangiogenesis
this term refers to the growth and formation of new lymphatic vessels, which occurs in normally developing tissues and in pathological processes (e.g., inflammation, wound healing, lymphoedema, cancer) (Al-Rawi et al, 2005).

Under normal conditions all processes involving the formation of new blood vessels or the remodeling of existing blood vessels are self-limiting and require the controlled and concerted growth of various specific cell types in order to avoid the unwanted overgrowth.

endothelial cell sprouting
also: directed capillary ingrowth, endothelial sprouting, sprouting angiogenesis, or capillary sprouting. This general term refers to directed capillary ingrowth by which new capillary buds are induced from pre-existing host tissue capillaries (Ausprunk and Folkman, 1977; Paku and Paweletz, 1991). Parsons-Wingerter et al (2006) have defined sprouting as the de novo growth of a new blood or lymphatic vessel (either alone or by budding off from another vessel) and subsequent lengthening of this vessel sprout.

This formation of new tubular structures from a quiescent endothelial cell lining is the main mechanism of blood vessel growth in postnatal life and is critical for tissue remodeling and wound healing. Sprouting of new capillaries from pre-existing blood vessels is a hallmark of angiogenesis during embryonic development and also occurs during tumour growth (see also: angiogenic switch).

Sprouting angiogenesis is a multistep process. It involves structural alterations of the basement membrane, partial and regulated degradation of the basement membrane, cell activation of endothelial cells, endothelial cell proliferation, migration of endothelial cells, cell invasion, lumen formation, sprout stabilization, synthesis of the new basement membrane, and the recruitment of pericytes and mural cells. During sprouting angiogenesis, groups of endothelial cells migrate together in units called sprouts. Specialized endothelial tip cells with long filopodia lead the outgrowth of blood vessel sprouts towards gradients of angiogenic growth factors such as VEGF. Stalk cells in the sprout stalks proliferate and form a vascular lumen.

The entire process of vessel formation, vessel elongation, branching, anastomosis, vessel diameter growth is subject to a tightly regulated balance of positive and negative regulators and their respective receptors and involves autocrine and paracrine control mechanisms (Carmeliet, 2005). For alternative mechanisms that do not involve sprouting (usually referred to as non-sprouting angiogenesis) see also: vasculogenic mimicry, vessel co-option, intussusceptive microvascular growth, glomeruloid angiogenesis, postnatal vasculogenesis, vascular splitting.

Tumor angiogenesis
This term pertains to angiogenic processes observed in cancer. Vascularization of malignant tumors has long been considered to be the exclusive result of directed capillary ingrowth (endothelial sprouting). Generally, tumor cells undergo what is known angiogenic switch. Having changed their phenotype to one that is more conducive to angiogenesis, one observes the directed sprouting of new blood vessels into the direction of the solid tumor mass (Carmeliet, 2000; Carmeliet and Jain, 2000; Tang and Conti, 2004; Patan, 2004). This process, together with the production of anti-angiogenic substances (see: Endostatin, Angiostatin), guarantees the blood supply of the tumor and in its absence tumor cells would die by necrosis (see also: acute phase response, Apoptosis, inflammation, wound healing). Several other mechanisms of tumor angiogenesis have been described. See: vasculogenic mimicry, vessel co-option, intussusceptive microvascular growth, glomeruloid angiogenesis, postnatal vasculogenesis.

Angiogenic factors and Angiogenic inhibitors
A plethora of experiments have suggested that tissues secrete angiogenic factors that promote angiogenesis under conditions of poor blood supply during normal and pathological angiogenesis processes. Angiogenic molecules are generated by tumor, inflammatory, and connective tissue cells in response to hypoxia and other as yet ill-defined stimuli. Susceptibility of an organism to individual angiogenesis factors has been shown to be influenced by the genetic background and thus may be one of the causes responsible for susceptibility to diseases involving the formation of new blood vessels (see: AngFq1: [angiogenesis due to FGF-2]. In addition, several miRNAs (collectively termed AngiomiRs) have been shown to regulate angiogenesis in vivo.

The first indication of the existence of such diffusible substances was gleaned from filtration experiments demonstrating that tumor cells separated from underlying tissues by filters that do not allow passage of cells are nevertheless capable of supporting vessel growth in these tissues. The formation of blood vessels is initiated and maintained by a variety of factors secreted either by the tumor cells themselves or by accessory cells. The formation of blood vessels is thought to depend on a precise balance of positive and negative regulation, involving both stimulating and inhibiting factors (Iruela-Arispe and Dvorak, 1997; Distler et al, 2003).

Many different growth factors and cytokines have been shown to exert chemotactic, mitogenic, modulatory or inhibitory activities on endothelial cells, smooth muscle cell and fibroblasts and can, therefore, be expected to participate in angiogenic processes in one way or another. The process involves the concerted action of proteolytic enzymes, extracellular matrix components, cell adhesion molecules, and vasoactive factors. Proteases, among other things, degrade the vascular basement membrane and participate in the remodelling of the extracellular matrix to facilitate cell migration and invasion. In addition, these enzymes are known to release angiogenic growth factors bound to the extracellular matrix, to generate chemotactically active fragments derived from extracellular matrix components, and to liberate endogenous angiogenesis inhibitors from inactive parent molecules (see, for example, Arresten, Canstatin, Endostatin, Restin, Tumstatin). Proteases can also modulate receptor signaling by facilitating receptor shedding. Furthermore, proteolytic enzymes can detach pericytes from vessels at sites of active angiogenesis and cleave adhesions between endothelial cells (Rundhaug et al, 2005).

Factors modulating growth, chemotactic behavior and/or functional activities of smooth muscle cells include Activin A, Adrenomedullin, aFGF (see also: BDGF), ANF, Angiogenin, Angiotensin-2, Betacellulin, bFGF (see also: BDGF), CLAF, ECDGF (endothelial cell-derived growth factor), ET (Endothelins), Factor X, Factor Xa, HB-EGF, Heart derived inhibitor of vascular cell proliferation, IFN-gamma, IL1, LDGF (Leiomyoma-derived growth factor), MCP-1 (see also: SMC-CF), MDGF (macrophage-derived growth factor, monocyte-derived growth factor), NPY, Oncostatin M, PD-ECGF, PDGF, Prolactin, Protein S, SDGF (smooth muscle cell-derived growth factor), SDMF (Smooth muscle cell-derived migration factor), Tachykinins, TGF-beta, Thrombospondin.

Factors modulating growth, chemotactic behavior and/or functional activities of vascular endothelial cells include AcSDKP, aFGF, ANF, Angiogenin, angiomodulin, Angiotropin, AtT20-ECGF, B61, bFGF, bFGF inducing activity, CAM-RF, ChDI, CLAF, ECGF, ECI, EDMF, EGF, EMAP-2, Neurothelin (see: EMMPRIN), Endostatin, Endothelial cell growth inhibitor, Endothelial cell-viability maintaining factor, Epo, FGF-5, IGF-2 (see: Growth-promoting activity for vascular endothelial cells), HBNF, HGF, HUAF, IFN-gamma, IL1, K-FGF, LIF, MD-ECI, MECIF, NPY, Oncostatin M, PD-ECGF, PDGF, PF4, PlGF, Prolactin, TNF-alpha, TNF-beta, Transferrin, VEGF. Some of these factors are protein factors detected initially due to some other biological activities and later shown to promote angiogenesis. The list of protein factors angiogenically active in vivo includes fibroblast growth factors (see: FGF), Angiogenin, Angiopoietin-1, EGF, HGF, NPY, VEGF, TNF-alpha, TGF-beta, PD-ECGF, PDGF, IGF, IL8, Growth hormone. Fibrin fragment E has been shown also to have angiogenic activity. In addition there are factors such as Angiopoietin-1 which do not behave as classical growth factors for endothelial cells but play a prominent role in vasculogenic and angiogenic processes. PF4 and a 16 kDa fragment of Prolactin are inhibitory in vivo.

Angiogenic activities also include a number of other compounds such as prostaglandins E1 and E2, steroids, heparin, 1-butyryl glycerol (monobutyrin) secreted by adipocytes, and many undefined derivatives of the arachidonic acid metabolism. The biologically active principle extracted from some carcinoma cells and identified as nicotinic amide is also a potent angiogenic compound in several bioassays although its mechanism of action remains to be elucidated.

These factors and compounds differ in cell specificity and also in the mechanisms by which they induce the growth of new blood vessels. Not all compounds that are active in vivo show the same spectrum of activities for endothelial cells in vitro. Many of these factors are pleiotropic and, among other things, may induce the migration and proliferation of endothelial cells, the production of collagenase and plasminogen activator. Some of these factors, however, are neither mitogenic nor chemotactic for endothelial cells (see also: Chemotaxis; Motogenic cytokines). They elicit their effects probably by attracting other cells to the site of growth, by causing cell activation, and by inducing them subsequently to secrete angiogenic factors. Localized proteolytic modification of the extracellular matrix not only allows cell migration but may be responsible also for the release of stored signaling molecules affecting angiogenesis.

The immediate reaction following exposure of tissue to angiogenic factors is the dissolution of the basal membrane of pre-existing blood vessels. The next step is the migration and proliferation of endothelial cells. The immature new vessels is elongated, develops a lumen, and branches. The final stage of capillary development is the formation of a new basal membrane (see also: extracellular matrix). In some instances one observes the formation of a layer of perivascular cells (pericytes).

Since the development of new blood vessels is to be avoided under normal conditions it can be assumed that the synthesis of angiogenically active factors is subject to constitutive repression in normal tissues. Many control mechanisms have been described to be in operation. Some angiogenic factors are produced only if, during a process requiring the new formation of blood vessels cells have undergone the process of cell activation. Other factors such as TGF-beta are produced by normal cells in an inactive proform that can be activated in a controlled manner. Factors such as aFGF and bFGF lack a signal sequence and are not secreted but stored in depots of the basal membrane. TNF-alpha is capable of promoting angiogenesis only from without a blood vessels but not if it is released in the lumen.

In tumor cells these tight regulatory mechanisms have been corrupted in most cases by the ability of tumor cells to synthesize angiogenic growth factors constitutively. Some tumor cells also produce slightly altered factors that are directly mitogenic for endothelial cells or they elaborate hydrolytic enzymes capable of releasing angiogenesis factors from their membrane depots or of activating inactive angiogenic factors.

Angiogenic factors and also their inhibitors are of clinical significance because they could be used to directly interfere with angiogenic processes involved, for example, in wound healing, inflammation, ischemic heart and peripheral vascular disease, myocardial infarction. Angiogenesis inhibitors should be of value in treatment of diseases of pathogenic neovascularization such as Kaposi's sarcoma, diabetic retinopathy, rheumatoid arthritis, and malignant tumor growth (Indraccolo, 2004; Ziche et al, 2004; Carmeliet, 2004).

Inhibitors of angiogenesis (Folkman, 2003) have been grouped into class 1 angiogenic inhibitors, which inhibit proliferation and/or migration of endothelial cells only in a specific and semi-specific manner, and nonspecific class 2 angiogenic inhibitors, which are also toxic for tumor cells (Voest, 1996). For a historical overview see also: Ribatti D (2009).

One example of compounds, known also as Angioinhibins, which inhibit angiogenesis, is Fumagillin. Other factors negatively influencing angiogenesis by inhibiting the proliferation of endothelial cells or other modes of action include Angiomotin, Angiostatin, BAI-1 (brain-specific angiogenesis inhibitor-1), Endorepellin, Endostatin, PF4 (platelet factor-4), IFN-alpha and IFN-gamma, PRP (proliferin-related protein), Thrombospondin isolated from platelets, a proteinase inhibitor of cartilage known as CDI (cartilage-derived inhibitor), ChDI (chondrocyte-derived inhibitor), Chondrocyte-derived inhibitor of angiogenesis and metalloproteinase activity, and Prothrombin kringle-2 domain (see: kringle), Tumstatin. The designer peptide Anginex has been shown to be a potent inhibitor of angiogenesis in vivo.

A nonanticoagulating derivative of heparin, heparin adipic hydrazide (HAH), covalently linked by an acid-labile bond to the anti-angiogenic steroid, cortisol has been shown to have potent anti-angiogenesis and antitumour activity in a mouse model.

See also: Angiogenesis Dictionary section of this encyclopedia for other entries directly bearing on factors and processes involved in the generation of new blood vessels.

Angiogenesis assays
As many angiogenic factors are mitogenic and chemotactic for endothelial cells their biological activities can be determined in vitro by measuring the induced migration of endothelial cells or the effect of these factors on cell proliferation.

However, there are also a number of bioassays that allow direct determination of angiogenic activities and permit repeated, long-term quantitation of angiogenesis, physiological characterization of angiogenic vessels and the inhibition of vessel formation in response to tissues, cells, or soluble factors (Hasan et al, 2004; Staton et al, 2004; Taraboletti and Giavazzi, 2004).

cell proliferation and chemotactic assays
Cell proliferation of endothelial cells as the major players in angiogenesis, as well as their behaviour towards chemotactic stimuli are being employed regularly to test for substances that promote or block angiogenesis. It should be noted that such assays cannot be more than prescreening assays and that there are large differences between endothelial cells or cell lines of different origin.

The properties of endothelial cells derived from large vessels differ from those derived of microvascular origin. In addition, endothelial cells obtained from different organ sites and even within single organs (Arap et al, 1998; Gumkowski et al, 1987; Auerbach, 1991) noticeably differ from one another. Other important consideration are possible species differences and the use of cell lines (proliferative) versus quiescent endothelial cells in the vasculature.

Chicken chorioallantoic membrane assay (CAM assay)
This is one of the most frequently used angiogenesis assays (Ribatti, 2004; Richardson and Singh, 2003). The extraembryonic chicken embryo chorioallantoic membrane is formed by fusion of the chorion and the allantois. It is in direct contact with the shell and contains a very thick capillary network (Ausprunk et al, 1974). The membrane can be used in ovo, which utilizes a sealed window cut into the shell (Ribatti et al, 1996), or in vitro, which involves the transfer of the chicken embryo together with its extraembryonic membranes into a Petri dish (Auerbach et al, 1974). Test substances are applied to the membrane by using biologically inert polymers that allow controlled and sustained release. Widely used polymers are Elvax 40 (ethylene-vinyl acetate copolymer) and hydron (a poly-2-hydroxyethyl-methacrylate polymer) (Langer and Folkman, 1976). Nguyen et al (1994) have used collagen gels impregnated with the test substances for the same purpose. Ribatti et al (1997) have described a modification of this assay, which used implantation of gelatin sponges on the top of growing membrane. The use of the chicken chorioallantoic membrane in angiogenesis assays is inexpensive but more problematic because of the expertise required to differentiate pre-existing from newly formed blood vessels (Ribatti et al, 2000; Peek et al, 1988; Splawinsky et al, 1988). Miller et al (2004) have modified the assay for quantifying changes in vascular density, endothelial cell proliferation and protein expression in response to modulators of angiogenesis. Adaptations of the CAM assays for the use with quail eggs (quail chorioallantoic membrane assay) have been described (Parsons-Wingerter et al, 2002; Gonzalez-Iriarte et al, 2003).

Cornea pocket assay
This assay employs the use of the nonvascularized rabbit eye. The assay employs slow release polymer pellets containing angiogenic substances. These pellets are implanted into the corneal stroma (Hartwell, 1998; Presta et al, 1999; Rogers et al, 2007). This assay has the advantage that new blood vessels are easily detected and essentially must be newly formed blood vessels (Morbidelli and Ziche, 2004; Ribatti and Vacca, 1999). Micromethods for the use in the rat eye have been described by Fournier et al (1981).

Co-culture angiogenesis assay
This assay uses primary endothelial cells and primary ECM producing fibroblasts. Distinguishable patterns of vascular structures develop under the influence of single factors or combination of factors (Beilmann et al, 2004).

Rat aortic ring model
This assay provides another reproducible assay for discovering angiogenic agonists and antagonists (Nissanov et al, 1995; Go and Owen, 2003; Zhu and Nicosia, 2002; Nicosia and Ottinetti, 1990; Blacher et al, 2001). In this ex vivo model of angiogenesis differential migration of endothelial cells and/or smooth muscle cell populations produces radial outgrowths of microvessels. They arise from the cut edge of an aorta that has been cut into small disks and cultured in a fibrin matrix with growth media. The technique has been used also with mouse aorta (Mouse aortic ring model) (Masson V Ve et al, 2002; Berger et al, 2004). The chick aortic arch assay employs aortic arches from day 12–14 chick embryos cut into rings similar to those of the rat aorta and can be carried out in serum-free medium (Muthukkaruppan VR et al, 2000).
Stiffey-Wilusz et al (2001) have adapted the assay to the use of commercial porcine carotid artery. The automated quantitative analysis of angiogenesis in the rat aorta model has been described (Blatt et al, 2004). Zhu et al (2003) have reported that, unlike rat aorta explants, unstimulated mouse aortic rings are unable to spontaneously produce an angiogenic response under serum-free conditions. Also, there are differences in angiogenic responses in different mouse strains of different ages and these effects have to be taken into account when these assays are used to evaluate the biological activities of factors in knock-out mice or mice overexpressing certain genes influencing growth factors, their receptors, or signaling cascades. Zogakis et al (2002) have described the use of microarray gene expression profiling to evaluate the modulation of gene expression in angiogenesis using the rat aortic ring assay. An in vitro serum-free three-dimensional rat aortic model has been developed that overcomes the limitations of two-dimensional in vitro angiogenesis assays that usually measure only one facet of this process. This assay closely approximates the complexities of angiogenesis in vivo, from endothelial activation to pericyte acquisition and remodelling, and most of these can be quantified by image analysis, immunohistochemistry, and biochemical analysis (West and Burbridge, 2009).

MRSG assay [miniature ring-supported gel assay]
This assay has been described by Reed et al, 2007). It is a modification of the classic aortic explant model of angiogenesis. Aortic segments from mice are placed within small (5.6 mm diameter, 30 microl volume) lenticular hydrogels of collagen-1 supported at the edge by nylon mesh rings. Advantages of the MRSG assay are low volumes and microwell format, which optimize handling, cytological staining, and conventional imaging.

Rodent mesenteric window angiogenesis assay (mesenteric angiogenesis assay)
This assay employs the virtually avascular membranous rat mesentery to measure angiogenic activities and the kinetics of angiogenesis in the intact animal (Norrby, 1992; Jakobsson et al, 1994). Benest and Bates (2009) have employed adenoviruses expressing growth factors (VEGF and angiopoietin-1) injected into the mesenteric fat pad of adult male Wistar rats to study the angiogenic phenotype.

Placenta fragment assay
Brown et al (1995) have described the use of human placenta fragments to establish a physiologically relevant in vitro model for human angiogenesis that can be used to screen for enhancers and inhibitors of human angiogenesis and allow further investigation of this process. For this purpose they embedded a fragment of human placental blood vessel in a fibrin gel in microculture plates. This was found to give rise to a complex network of microvessels during a period of 7 to 21 days in culture without the addition of exogenous growth factors. The placenta fragment assay thus provides a convenient system for testing substances for their ability to stimulate or inhibit a human in vitro angiogenic response.

Tube formation angiogenesis assay
Endothelial cells have the ability to form three-dimensional structures (tube formation) (Madri et al, 1988), and this behaviour has been used as a basis of angiogenesis tests. An assay that is based on the differentiation of endothelial cells and the formation of tube-like structures on Matrigel as extracellular matrix has been detailed by Ponce (2009). Tubulogenesis has been assessed also by using co-cultures of differentiated endothelial cells with monolayers of human fibroblasts (Sieveking et al, 2008).

Matrigel plug assay
This assay employs reconstituted basement membrane material and has been used as an in vitro and in vivo angiogenesis model to study the activity of angiogenic and anti-angiogenic cytokines and other substances. Test compounds are introduced into cold liquid Matrigel which, after subcutaneous injection, solidifies and permits penetration by host cells and the formation of new blood vessels (Akhtar et al, 2002; Kragh et al, 2003, 2004; Malinda, 2009).

microcarrier-based angiogenesis assay s
The alginate bead assay assay makes use of cells entrapped in alginate beads. Several authors have reported the use of micro-encapsulated cells (agarose beads, gelatine-coated microcarrier beads) transplanted into experimental animals to study angiogenic processes (Okada et al, 1995; Nehls and Drenckhahn, 1995). Crabtree B and Subramanian (2007) have described an assay that combines cells grown on Cytodex-3 microcarrier beads with Matrigel for evaluating and measuring angiogenic activity. Dietrich and Lelkes (2006) have reported a three-dimensional microcarrier-based angiogenesis assay for the analysis of co-cultures of endothelial cells and mesenchymal cells in fibrin and collagen gels.

DIVAA [directed in vivo angiogenesis assay]
This assay employs semiclosed small silicone cylinders (termed angioreactors by the authors) that are implantated subcutaneously in nude mice. The cylinders are filled with 18 microliters of extracellular matrix, premixed with or without angiogenic factors. Vascularization within the cylinders is quantified by intravenous injection of FITC-labelled dextran before their recovery, followed by spectrofluorimetry.

Cells and invading angiogenic vessels at different developmental stages are visualised by immunofluorescence. With bFGF or VEGF, the minimally detectable angiogenic response requires an implantation period of 9 days and approximately 50 ng/mL of the angiogenesis factor.

The assay system allows accurate dose-response analysis and identification of effective doses of factors modulating angiogenesis in vivo. The angiogenesis inhibitor TNP-470 potently inhibits angiogenesis (88 pmol/L) induced by 500 ng/mL of bFGF. This inhibition correlates with decreased endothelial cell invasion. The assay has been used to detect differences in anti-angiogenic potencies of thrombospondin peptides (25 micro mol/L). It has been used also to demonstrate a partial inhibition of angiogenesis in knock-out mice lacking expression of the matrix metalloprotease MMP-2 compared with that in wild-type animals and to reveal quantitative changes in MMP expression by zymography of the cylinder contents.

Subcutaneous Air Sac model
This assay has been described by Lichtenberg et al (1999). An air sac is induced by injection of air subcutaneously on the back of an experimental animal. The air sac gives rise to an almost transparent avascular membrane after 10-14 days that can be used to study the formation or disappearance of new vessels following implantation of materials (cells or slow-release pellets) containing angiogenic factors or angiogenic inhibitors or systemic application of bioactive compounds such as the anti-angiogenic compound TNP-470.

Leech angiogenesis assay
de Eguileor et al (2004) have reviewed the utility of the leech Hirudo medicinalis for studies of the angiogenic and anti-angiogenic compounds. The leech possesses a virtually avascular muscular body wall. Leech angiogenic growth factor receptors have been shown to respond to human/mammalian recombinant growth factors and cytokines. Endogenous angiogenic growth factors and receptors of the leech also react with antibodies against human/mammalian factors.

three-dimensional human tumor angiogenesis assay
This assay has been described by Gulec and Woltering (2004). It employs fragments of tumor tissue embedded in fibrin gels to study the angiogenic potential of human tumors. Simultaneous evaluation of antitumor compounds and their antiangiogenic effects is possible because angiogenic vessels grow into the fibrin gel matrix, which is separate from the tumor stroma.

embryoid body angiogenesis assay
This assay is based on the use of embryonic stem cells under conditions leading to angiogenic sprouting of embryoid bodies during ES cell differentiation. Embryoid bodies contain embryonic stem cells and non-embryonic stem cells and thus provide a three-dimensional environment for vascular development that can occur in a context of continuous interactions between these two cell types. Various techniques have been described (Jacobsson et al, 2006, 2007). Hermant et al (2007) have demonstrated that endothelial sprouting is stimulated specifically in the presence of VEGF and bFGF and that endothelial sprouting induced by angiogenic activators is inhibited by angiogenesis inhibitors such as angiostatin, TGF-beta and PF4.

spheroidal angiogenesis assay
This assay makes use of aggregates of endothelial cells formed under suitable conditions in suspension cultures. These endothelial cell spheroids usually consist of a surface monolayer of differentiated cells and a center of unorganized cells that will die by apoptosis if they are not rescued by survival factors (Oudar, 2000; Korff and Augustin, 1998). Such spheroids have been used to assay the activities of angiogenesis factors and their inhibitors.

ZFYM assay
See: zebrafish yolk membrane angiogenesis assay

LAST MODIFIED: August 2009

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