Angiogenic activity of human tumor plasma membrane components

Plasma membranes from the human colon adenocarcinoma cell line HT-29 have been isolated and examined for the presence of angiogenic activity. Membrane-associated macromolecules extracted with Triton X-100 were fractionated on immobilized wheat germ agglutinin. The fraction which bound specifically (about 200 ng of protein/mL packed cells) was highly angiogenic when assayed on the chick embryo chorioallantoic membrane. As little as 0.2 ng of this human tumor derived material consistently induced neovascularization. Similarly, 1-2 ng of this material implanted into the rabbit cornea induced new vessel growth (5-8 mm) within 10 days. The plasma membranes of eight other human tumor lines were examined for angiogenic activity. For each, the wheat germ agglutinin bound material induced neovascularization at the low nanogram level. In contrast, the wheat germ agglutinin bound material derived from purified plasma membranes of two normal human diploid fibroblast cell lines failed to induce an angiogenic response on the chick chorioallantoic membrane, even at microgram levels.     

Heparin potentiation of 3T3-adipocyte stimulated angiogenesis: mechanisms of action on endothelial cells

We have examined the cellular mechanisms by which heparin potentiates the ability of 3T3-adipocytes to stimulate the formation of new blood vessels. Both anticoagulant and non-anticoagulant heparin species enhanced the angiogenic activity of adipocyte-secreted products in the chick chorioallantoic membrane assay, indicating that the angiotropic effect of this glycosaminoglycan is independent of its effect on the coagulation cascade. Heparin alone was unable to produce a neovascular response. The ability of heparin to modulate three endothelial functions in vitro thought to be related to angiogenesis were examined: protease activity, motility, and mitogenesis. Heparin caused a 100% increase in the adipocyte-induced stimulation of endothelial cell plasminogen activator activity and motility, but had no effect on proliferation. The enhancement of plasminogen activator and chemoattractant activities had a similar ED50 (1-2 micrograms/ml) and optimum dose (10-30 micrograms/ml). When we examined the direct effect of heparin on the activity of two distinct plasminogen activator enzymes--urokinase and tissue-type--a dual action of heparin was observed: tissue-type enzyme activity was stimulated 100% by heparin at 10 micrograms/ml, whereas urokinase activity was inhibited by 77% at this dose. These data suggest that heparin potentiates angiogenesis in vivo by stimulating endothelial cell plasminogen activator, motility, or both. Our results further suggest that for adipocyte-induced blood vessel formation, in contrast to other angiogenesis systems, heparin does not appear to affect the mitogenic activity.     

Human peripheral blood eosinophils induce angiogenesis

Eosinophils play a crucial role in allergic reactions and asthma. They are also involved in responses against parasites, in autoimmune and neoplastic diseases, and in fibroses. There is increasing evidence that angiogenesis plays an important role in these processes. Since eosinophils are known to produce angiogenic mediators, we have hypothesized a direct contribution of these cells to angiogenesis. The effect of human peripheral blood eosinophil sonicates on rat aortic endothelial cell proliferation (in vitro), rat aorta sprouting (ex vivo) and angiogenesis in the chick embryo chorioallantoic membrane (in vivo) have been investigated. To determine whether eosinophil-derived vascular endothelial growth factor influences the eosinophil pro-angiogenic activity, eosinophil sonicates were incubated with anti-vascular endothelial growth factor antibodies and then added to the chorioallantoic membrane. Vascular endothelial growth factor mRNA expression and vascular endothelial growth factor receptor density on the endothelial cells were also evaluated. Eosinophils were found to enhance endothelial cell proliferation and to induce a strong angiogenic response both in the aorta rings and in the chorioallantoic membrane assays. Pre-incubation of eosinophil sonicates with anti-vascular endothelial growth factor antibodies partially reduced the angiogenic response of these cells in the chorioallantoic membrane. Eosinophils also increased vascular endothelial growth factor mRNA production on endothelial cells. Eosinophils are able to induce angiogenesis and this effect is partially mediated by their pre-formed vascular endothelial growth factor. This strongly suggests an important role of eosinophils in angiogenesis-associated diseases such as asthma.     

Induction of angiogenesis by mixtures of two angiogenic proteins, angiogenin and acidic fibroblast growth factor, in the chick chorioallantoic membrane

The chick chorioallantoic membrane assay was employed to assess the angiogenic response induced by mixtures of human angiogenin with bovine heparin-binding acidic fibroblast growth factor. Statistical evaluation of data accumulated at several molar ratios of the two proteins indicate that the angiogenic activity observed is neither an additive nor a synergistic resultant of the activities of the proteins separately. The possibility exists, however, that at an approximately 1:1 mole ratio an apparent inhibitory effect can be observed. Mechanisms which could underlie such observed effects are discussed.     

Tryptase and chymase are angiogenic in vivo in the chorioallantoic membrane assay

Human mast cells (MCs) are divided in two types depending on the expression of tryptase and chymase in their granules. Literature data indicate that both tryptase and chymase are angiogenic, but there is currently no evidence of their direct angiogenic activity in vivo. In this study, we have investigated the capacity of tryptase and chymase to promote vasoproliferation in chick embryo chorioallantoic membrane (CAM), a well established in vivo assay to study angiogenesis and anti-angiogenesis. The results showed that both tryptase and chymase stimulate angiogenesis and that the response is similar to that obtained with vascular endothelial growth factor (VEGF), a well-known angiogenic cytokine, and confirm the angiogenic activity of these two proteases stored in MC granules.     

1-Butyryl-glycerol: a novel angiogenesis factor secreted by differentiating adipocytes.

Differentiation of adipocytes is accompanied by secretion of molecules stimulating angiogenesis in vivo and endothelial cell growth and motility in vitro. We demonstrate that the angiogenic and motility-stimulating activities secreted by adipocytes are separable from the endothelial cell mitogenic activity by fractionation of adipocyte-conditioned medium. The major differentiation-dependent angiogenic molecule was purified and identified by GCMS as 1-butyryl-glycerol (monobutyrin). Monobutyrin levels increase at least 200-fold during adipocyte differentiation and represent a major fraction of the total angiogenic activity. Synthetic monobutyrin shows the same spectrum of biological activities as the adipocyte-derived factor: stimulation of angiogenesis in vivo and microvascular endothelial cell motility in vitro, with no effect on endothelial cell proliferation. Angiogenesis is stimulated at doses as low as 20 pg when tested in the chick chorioallantoic membrane assay. These results strongly suggest that monobutyrin is a key regulatory molecule in an angiogenic process linked to normal cellular and tissue development.     

A small peptide derived from Flt-1 (VEGFR-1) functions as an angiogenic inhibitor

Vascular endothelial growth factor (VEGF) is an angiogenic stimulator which functions through two endothelial specific tyrosine kinase receptors, Flt-1 and Flk-1. In this work, we show that an 11-amino acid peptide derived from the second immunoglobulin-like domain of Flt-1 functions as an angiogenic inhibitor in chick chorioallantoic membrane and inhibited VEGF-induced vascular permeability in Miles' assay without binding to VEGF directly. Circular dichroism and nuclear magnetic resonance analyses indicate that this peptide forms a stable extended structure in solution, presumably beta-sheet structure and is most likely existing as a dimer. Our results suggest that this small peptide functions as an angiogenic inhibitor by inhibiting VEGF function through a non-VEGF binding mechanism.     

Molecular characterization of recombinant human acidic fibroblast growth factor produced in E. coli: comparative studies with human basic fibroblast growth factor.

Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.     

Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells

The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.     

TNF-related activation-induced cytokine (TRANCE) induces angiogenesis through the activation of Src and phospholipase C (PLC) in human endothelial cells.

Angiogenesis is an essential step for many physiological and pathological processes. Tumor necrosis factor (TNF) superfamily cytokines are increasingly recognized as key modulators of angiogenesis. In this study, we tested whether TNF-related activation-induced cytokine (TRANCE), a new member of the TNF superfamily, possesses angiogenic activity in vitro and in vivo. TRANCE stimulated DNA synthesis, chemotactic motility, and capillary-like tube formation in primary cultured human umbilical vein endothelial cells (HUVECs). Both Matrigel plug assay in mice and chick chorioallantoic membrane assay revealed that TRANCE potently induced neovascularization in vivo. TRANCE had no effect on vascular endothelial growth factor (VEGF) expression in HUVECs and TRANCE-induced angiogenic activity was not suppressed by VEGF-neutralizing antibody, implying that TRANCE-induced angiogenesis may be the result of its direct action on endothelial cells. TRANCE evoked a time- and dose-dependent activation of the mitogen-activated protein kinases ERK1/2 and focal adhesion kinase p125(FAK) in HUVECs, which are closely linked to angiogenesis. These signaling events were blocked by the Src inhibitor PP1 or the phospholipase C (PLC) inhibitor. Furthermore, these inhibitors and the Ca(2+) chelator BAPTA-AM suppressed TRANCE-induced HUVEC migration. These results indicate that the angiogenic activity of TRANCE is mediated through the Src-PLC-Ca(2+) signaling cascade upon receptor engagement in endothelial cells, suggesting the role of TRANCE in neovessel formation under physiological and pathological conditions.     

Isolation and antigen production of herpes simplex virus in the chorioallantoic membrane of duck embryos

Summary Isolation of herpes simplex virus and production of CF antigen was tried in chorioallantoic membranes of embryonated duck eggs. Duck CAM is twice as sensitive as chick CAM to natural virus and has almost the same susceptibility as suckling mice after intracerebral inoculation. Herpes simplex virus can be more rapidly adapted to duck eggs than chicken eggs. The first few subcultures result in higher virus titers if the virus is grown in duck CAM. No difference of virus titer is found, however, if the same sample of egg-adapted virus is titrated in duck and chick membranes. Herpes reactions are in the first few passages more progressive on duck CAM than on chick membranes and show continued proliferation between 3–5 days after inoculation. Herpes reactions on chick membranes tend to decrease after the 3rd day of incubation. Intranuclear inclusions of the Lipschütz type are always found in large numbers in reactions of the first passage on duck membranes, even when they are scanty in parallel membranes of chick eggs. CF antigens of herpes simplex virus were prepared from selected duck chorioallantoic membranes harvested 5 days after inoculation. CF antigens of herpes simplex virus prepared from duck eggs are reproducible and show high antigen titers averaging about 10 times those of chick egg preparations.     

Location of the angiogenic activity in the pregnant human uterus

Angiogenic activity was known to be intensely dynamic during pregnancy with a geometric orientation towards the site of implantation. Therefore this study was designed to test whether the uterine lining of pregnancy, the decidua, induces angiogenic activity on the chorioallantoic membrane (CAM). Twenty-three decidual specimens obtained during legally induced abortions were implanted into the 9-day-old chick CAM. All decidual specimens showed strong angiogenic activity as measured by 9-fold increase in radial blood vessels, with a geometrical formation of 'spokewheel' pattern extending over a range of 1.2 cm within 48 h.     

Del1 induces integrin signaling and angiogenesis by ligation of alphaVbeta3

Del1 is a novel extracellular matrix protein encoding three Notch-like epidermal growth factor repeats, an RGD motif, and two discoidin domains. Del1 is expressed in an endothelial cell-restricted pattern during early development. In studies reported here, recombinant baculovirus Del1 protein was shown to promote alphavbeta3-dependent endothelial cell attachment and migration. Attachment of endothelial cells to Del1 was associated with clustering of alphavbeta3, the formation of focal complexes, and recruitment of talin and vinculin into these complexes. These events were shown to be associated with phosphorylation of proteins in the focal complexes, including the time-dependent phosphorylation of p125(FAK), MAPK, and Shc. When recombinant Del1 was evaluated in an in ovo chick chorioallantoic membrane assay, it was found to have potent angiogenic activity. This angiogenic activity was inhibited by a monoclonal antibody directed against alphavbeta3, and an RAD mutant Del1 protein was inactive. Thus Del1 provides a unique autocrine angiogenic pathway for the embryonic endothelium, and this function is mediated in part by productive ligation of integrin alphavbeta3.     

Neovascular responses induced by cultured aortic endothelial cells

Neovascularization was studied in the chorioallantoic membrane of the chick embryo after implantation of bovine aortic endothelial and smooth muscle cells, Swiss and BALB/c 3T3 cells and human diploid fibroblasts cultured separately on microcarrier beads. Quantitative analysis of neovascularization indicated a 3 1/2-fold increase in the number of blood vessels responding to endothelial cells while smooth muscle cells induced a twofold increase when compared to the response of beads without cells. Skin fibroblasts and Swiss 3T3 cells did not elicit a comparable response. The marked angiogenic response induced by endothelial cells was characterized by a 137% increase in total vessel length and a 35% increase in average vessel area when compared to controls. Two of the properties required for an angiogenesis factor--stimulation of cellular migration and proliferation--can also be demonstrated using endothelial cell-conditioned medium in cell culture systems. Medium from cultured bovine aortic endothelium stimulates DNA synthesis, proliferation, and migration of smooth muscle cells. In addition, conditioned media from both endothelial cells and smooth muscle cells produced an angiogenic response in the chorioallantoic membrane assay, which was comparable to that produced by intact cells growing on microcarrier beads. Similar responses were not evident with medium conditioned by other cell types. These results indicate the potential importance of endothelial cells and endothelial cell products in regulating blood vessel growth.     

Osteocalcin is angiogenic in vivo

The exact function of osteocalcin (OC), a protein synthesized by osteoblasts during the matrix mineralization phase, is still unknown. In this study we investigated the capacity of OC to promote vasoproliferation in chick embryo chorioallantoic membrane (CAM), a well established in vivo assay for angiogenesis and anti-angiogenesis. The results showed that OC stimulates angiogenesis and that the response is similar to that obtained with FGF-2, a well-known angiogenic cytokine. It has previously been demonstrated that OC is involved in bone repair, so the angiogenic activity reported here might also play a crucial role in bone formation.     

Embryotoxic effects of doxorubicin and N-trifluoroacetyladriamycin-14-valerate (AD-32)

Anthracyclines are mutagenic, carcinogenic, and also cardiotoxic. Concern has been shown over the use of anthracycline anticancer drugs during pregnancy as these may be teratogenic to the human fetus. We have performed a series of experiments using the chick embryo to investigate and compare the toxic and teratogenic effects of doxorubicin (DX) and its new analog N-trifluoroacetyladriamycin-14-valerate (AD-32). DX and AD-32, dissolved in 1:1 emulphor/ethanol and diluted to 1:4 with water, were injected into the air sac of white leghorn chick eggs at dose levels of 1-20 micrograms (DX) and 5-100 micrograms (AD-32) per egg. Eggs received a single injection of the drugs on days 1 and 2 of incubation. Control eggs were injected with an equivalent volume of the drug vehicle (0.05 ml per egg). Both of the drugs caused embryonic death, stunted growth, and various gross morphological malformations. Surviving embryos were sacrificed when they reached 13 days of incubation. The LD50 values for days 1 and 2 were as follows: (DX, 2.5 micrograms/egg on day 1 and 0.9 microgram/egg on day 2; AD-32, 10.6 micrograms/egg on day 1 and 11.8 micrograms/egg on day 2). Stunting of growth, a common anomaly with both drugs, decreased sharply from day 1 to day 2. The studies demonstrate that both DX and AD-32 are toxic and teratogenic during the period of early organogenesis in the chick embryos. However, the toxic and teratogenic potentials of DX and AD-32 differ quantitatively. AD-32 is a more potent teratogen than DX when injected on day 1.     

CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A

The angiogenic activity of CXC-ELR(+) chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-alpha in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck(-/-)fgr(-/-)), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck(-/-)fgr(-/-) mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck(-/-)fgr(-/-) mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck(-/-)fgr(-/-) mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.     

Cutting edge: IL-1beta mediates the proangiogenic activity of osteopontin-activated human monocytes

Inflammation plays an important role in the onset of angiogenesis. In the present study, we show that osteopontin (OPN), a proinflammatory mediator involved in tissue repair, induces IL-1beta up-regulation in human monocytes. This was accompanied by the enhanced production of TNF-alpha, IL-8, and IL-6, a decreased release of IL-10, and increased p38 phosphorylation. The supernatants of OPN-treated monocytes were highly angiogenic when delivered on the chick embryo chorioallantoic membrane. The angiogenic response was completely abrogated by a neutralizing anti-IL-1 Ab, thus indicating that this cytokine represents the major proangiogenic factor expressed by OPN-activated monocytes. Accordingly, rIL-1beta mimicked the proangiogenic activity of OPN-treated monocyte supernatants, and IL-1R (type I) was found to be expressed in the chorioallantoic membrane. In conclusion, OPN-activated monocytes may contribute to the onset of angiogenesis through a mechanism mediated by IL-1beta.