Chick Embryo Chorioallantoic Membrane Assay Research Articles

The Combined Therapeutic Effects of Bortezomib and Fenretinide on Neuroblastoma Cells Involve Endoplasmic Reticulum Stress Response

The proteasome inhibitor bortezomib inhibited cell growth and angiogenesis in neuroblastoma. Bortezomib has been shown to induce synergistic activity when combined with other antineoplastic agents. Here we have investigated the antitumor activity of bortezomib in combination with fenretinide, a synthetic retinoid, against neuroblastoma cells. Different neuroblastoma cell lines were tested for sensitivity to bortezomib and fenretinide, given alone or in different dose-dependent and time-dependent combination schedules. Cell proliferation, cell viability, and apoptosis were evaluated by measuring 3H-thymidine incorporation, trypan blue staining, DNA fragmentation, and western blot analysis. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay. An orthotopic neuroblastoma mouse model was used to examine in vivo sensitivity. Each compound alone was able to induce a dose-dependent inhibition of cell proliferation, with a significant enhanced antiproliferative effect for the drugs used in combination. This inhibition was characterized by marked G2-M and G1 cell cycle arrest with nearly complete depletion of S phase. Bortezomib and fenretinide in association triggered an increased apoptosis through activation of specific genes of the endoplasmic reticulum stress compared with either drug tested alone. Tumor-bearing mice treated with bortezomib plus fenretinide lived statistically significantly longer than mice treated with each drug alone. Histologic evaluation and chorioallantoic membrane analysis of primary tumors showed that the combined therapeutic activity of bortezomib and fenretinide rested upon antitumor and antiangiogenic mechanisms. These findings provide the rationale for the development of a new therapeutic strategy for neuroblastoma based on this pharmacologic combination.

A Paradox of Cadmium: A Carcinogen That Impairs the Capability of Human Breast Cancer Cells To Induce Angiogenesis

Cadmium, a highly persistent heavy metal, has been categorized as a human carcinogen. Even though it is known that cadmium acts as estrogens in breast cancer cells, several studies failed to demonstrate whether cadmium is a causal factor for breast cancer. The lack of a strong association between cadmium and breast cancer could be found in the antiangiogenic properties of this heavy metal, which might counteract its carcinogenic properties in the progression of breast cancer. In this study, we exposed estrogen-responsive breast cancer cells to subtoxic levels of cadmium, and we evaluated their angiogenic potential using the chick embryo chorioallantoic membrane assay. Exposure of breast cancer cells to subtoxic levels of cadmium significantly inhibited the angiogenic potential of the breast cancer cell line, suggesting the possibility that cadmium might negatively regulate the production of proangiogenic factors in breast cancer cells. Our results suggest that cadmium might exert a paradoxical effect in breast cancer: on the one hand, it could promote carcinogenesis, and, on the other hand, it could delay the onset of tumors by inhibiting breast cancer cell-induced angiogenesis.

Aprotinin stimulates angiogenesis and human endothelial cell migration through the growth factor pleiotrophin and its receptor protein tyrosine phosphatase β/ζ

Pleiotrophin is an 18 kDa secreted polypeptide growth factor with direct pro-angiogenic and tumorigenic properties. Pleiotrophin is a substrate for proteolytic enzymes, such as plasmin, leading to proteolytic fragments with distinct activities on endothelial cell activation in vitro or angiogenesis in vivo. Aprotinin is a naturally occurring broad spectrum protease inhibitor, used widely in cardiac surgery due to its ability to inhibit plasmin and reduce perioperative bleeding. Since we have seen that aprotinin inhibits proteolysis of pleiotrophin by plasmin, the aim of the present study was to evaluate the possible role of pleiotrophin in the effects of aprotinin on angiogenesis and human endothelial cell migration. Our data demonstrate that aprotinin, in a concentration-dependent manner, is angiogenic in the chicken embryo chorioallantoic membrane assay in vivo and induces human endothelial cell migration in vitro. Aprotinin inhibits pleiotrophin proteolysis and induces expression and secretion of pleiotrophin through an AP-1-dependent transcriptional activation of the pleiotrophin gene, and pleiotrophin seems to mediate the stimulatory effects of aprotinin on cell migration through its receptor protein tyrosine phosphatase β/ζ. The stimulatory effect of aprotinin on pleiotrophin expression and cell migration may explain, at least partly, the problems observed with the clinical use of aprotinin.

Modulation of Angiogenesis by a Tetrameric Tripeptide That Antagonizes Vascular Endothelial Growth Factor Receptor 1

Vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is involved in complex biological processes often associated to severe pathological conditions like cancer, inflammation, and metastasis formation. Consequently, the search for antagonists of Flt-1 has recently gained a growing interest. Here we report the identification of a tetrameric tripeptide from a combinatorial peptide library built using non-natural amino acids, which binds Flt-1 and inhibits in vitro its interaction with placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) A and B (IC(50) approximately 10 microm). The peptide is stable in serum for 7 days and prevents both Flt-1 phosphorylation and the capillary-like tube formation of human primary endothelial cells stimulated by PlGF or VEGF-A. Conversely, the identified peptide does not interfere in VEGF-induced VEGFR-2 activation. In vivo, this peptide inhibits VEGF-A- and PlGF-induced neoangiogenesis in the chicken embryo chorioallantoic membrane assay. In contrast, in the cornea, where avascularity is maintained by high levels of expression of the soluble form of Flt-1 receptor (sFlt-1) that prevents the VEGF-A activity, the peptide is able to stimulate corneal mouse neovascularization in physiological condition, as reported previously for others neutralizing anti-Flt-1 molecules. This tetrameric tripeptide represents a new, promising compound for therapeutic approaches in pathologies where Flt-1 activation plays a crucial role.

Enhanced Antitumor Efficacy of Clinical-Grade Vasculature-Targeted Liposomal Doxorubicin

In vivo evaluation of good manufacturing practice-grade targeted liposomal doxorubicin (TVT-DOX), bound to a CD13 isoform expressed on the vasculature of solid tumors, in human tumor xenografts of neuroblastoma, ovarian cancer, and lung cancer. Mice were implanted with lung, ovarian, or neuroblastoma tumor cells via the pulmonary, peritoneal, or orthotopic (adrenal gland) routes, respectively, and treated, at different days post inoculation, with multiple doses of doxorubicin, administered either free or encapsulated in untargeted liposomes (Caelyx) or in TVT-DOX. The effect of TVT-DOX treatment on tumor cell proliferation, viability, apoptosis, and angiogenesis was studied by immunohistochemical analyses of neoplastic tissues and using the chick embryo chorioallantoic membrane assay. Compared with the three control groups (no doxorubicin, free doxorubicin, or Caelyx), statistically significant improvements in survival was seen in all three animal models following treatment with 5 mg/kg (maximum tolerated dose) of TVT-DOX, with long-term survivors occurring in the neuroblastoma group; increased survival was also seen at a dose of 1.7 mg/kg in mice bearing neuroblastoma or ovarian cancer. Minimal residual disease after surgical removal of neuroblastoma primary mass, and the enhanced response to TVT-DOX, was visualized and quantified by bioluminescence imaging and with magnetic resonance imaging. When treated with TVT-DOX, compared with Caelyx, all three tumor models, as assayed by immunohistochemistry and chorioallantoic membrane, showed statistically significant reductions in cell proliferation, blood vessel density, and microvessel area, showing increased cell apoptosis. TVT-DOX should be evaluated as a novel angiostatic strategy for adjuvant therapy of solid tumors.

Friend erythroleukemia cells induce angiogenesis in chick embryo chorioallantoic membrane and in human umbilical vein endothelial cells.

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias.

Anti-angiogenic action of 5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10)

Previously, we demonstrated that 5,5-diphenyl-2-thiohydantoin (DPTH) exerts an anti-proliferation effect on subcultured human umbilical vein endothelial cells (HUVEC). In the present study, we show that 2(naphthalen-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro-imidazol-4-one (DPTH-N10), a derivative compound of DPTH, exerts a 5 times stronger inhibition of [3H]thymidine incorporation into HUVEC as compared with DPTH and at very low concentrations (0–20 μM) inhibited DNA synthesis and decreased cell number in cultured HUVEC in a concentration- and time-dependent manner, but not in human fibroblasts. [3H]thymidine incorporation analysis demonstrated that treatment of HUVEC with DPTH-N10 arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 in HUVEC increased after DPTH-N10 treatment. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH-N10 treatment. Immunoprecipitation showed that the formation of the CDK2–p21 complex, but not the CDK2–p27, CDK4–p21, and CDK4–p27 complex, was increased in the DPTH-N10-treated HUVEC. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in the DPTH-N10-treated HUVEC. Pretreatment of HUVEC with a p21, but not p27, antisense oligonucleotide reversed the DPTH-N10-induced inhibition of [3H]thymidine incorporation into HUVEC. Taken together, these data suggest that DPTH-N10 inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally interrupts the cell cycle. Capillary-like tube formation, aortic ring culture, and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of DPTH-N10.

A pro‐inflammatory signature mediates FGF2‐induced angiogenesis

Fibroblast growth factor-2 (FGF2) is a potent angiogenic growth factor. Here, gene expression profiling of FGF2-stimulated microvascular endothelial cells revealed, together with a prominent pro-angiogenic profile, a pro-inflammatory signature characterized by the upregulation of pro-inflammatory cytokine/chemokines and their receptors, endothelial cell adhesion molecules and members of the eicosanoid pathway. Real-time quantitative PCR demonstrated early induction of most of the FGF2-induced, inflammation-related genes. Accordingly, chick embryo chorioallantoic membrane (CAM) and murine Matrigel plug angiogenesis assays demonstrated a significant monocyte/macrophage infiltrate in the areas of FGF2-driven neovascularization. Similar results were obtained when the conditioned medium (CM) of FGF2-stimulated endothelial cells was delivered onto the CAM, suggesting that FGF2-upregulated chemoattractants mediate the inflammatory response. Importantly, FGF2-triggered new blood vessel formation was significantly reduced in phosphatidylinositol 3-kinase-gamma null mice exhibiting defective leucocyte migration or in clodronate liposome-treated, macrophage-depleted mice. Furthermore, the viral pan-chemokine antagonist M3 inhibited the angiogenic and inflammatory responses induced by the CM of FGF2-stimulated endothelial cells and impaired FGF2-driven neovascularization in the CAM assay. These findings point to inflammatory chemokines as early mediators of FGF2-driven angiogenesis and indicate a non-redundant role for inflammatory cells in the neovascularization process elicited by the growth factor.

TX-2152: A conformationally rigid and electron-rich diyne analogue of FTY720 with in vivo antiangiogenic activity

We designed FTY720 analogues with conformationally rigid and electron-rich acetylenic chains as antiangiogenic agents (the monoyne 1: TX-2148, the diyne 2: TX-2152, the triyne 3: TX-2256). Molecular orbital (MO) calculations of our designed acetylenic analogues and FTY720 showed that the localization of the lowest unoccupied MO and the highest occupied MO increased from phenyl ring to acetylenic chain compared with that of FTY720. These acetylenic analogues were synthesized from p-hydroxyphenylethanol as a starting material. The construction of the acetylenic chain was carried out by an iterative strategy using a Sonogashira cross-coupling reaction and desilylative bromination in two steps. The corresponding overall yields of the monoyne 1, the diyne 2, and the triyne 3 were 27% (11 steps), 13% (13 steps), and 10% (15 steps). The in vivo antiangiogenic activities of these acetylenic analogues and FTY720 were evaluated by the chick embryo chorioallantoic membrane (CAM) assay and compared to the activities of the known antiangiogenic agent TNP-470. The diyne 2 showed more potent antiangiogenic activity (90% inhibition) than FTY720 (77% inhibition) and other acetylenic analogues (the monoyne 1: 42% inhibition, the triyne 3: 60% inhibition), and TNP-470 (82% inhibition) at a dose of 10 μg/CAM, without showing toxicity. The diyne 2 also had potent inhibitory activity at a dose of 5 and 2.5 μg/CAM. These results indicate that the flexibility of C8 alkyl chain of FTY720 is not required for its antiangiogenic activity. We suggest that the diyne 2 (TX-2152) may be a promising candidate as an antiangiogenic agent for antineoplastic drug discovery.

Angiogenic activity of multiple myeloma endothelial cells in vivo in the chick embryo chorioallantoic membrane assay is associated to a down‐regulation in the expression of endogenous endostatin

We have attempted a fine characterization of the angiogenic response induced by multiple myeloma endothelial cells (MMEC) by using the chick embryo chorioallantoic membrane (CAM) assay and by reverse transcriptase-polymerase chain reaction (RT-PCR). Results showed that in the CAM assay MMEC induced an angiogenic response comparable to that of a well-known angiogenic cytokine, namely fibroblast growth factor-2 (FGF-2), while RT-PCR demonstrated that the expression of endostatin mRNA detected in MM treated CAM was significantly lower respect to control CAM. These data suggest that angiogenic switch in MM may involve loss of an endogenous angiogenesis inhibitor, such as endostatin.

Pancreatic Duct Cells in Human Islet Cell Preparations Are a Source of Angiogenic Cytokines Interleukin-8 and Vascular Endothelial Growth Factor

OBJECTIVE—Engraftment and function of human islet cell implants is considered to be dependent on their rapid and adequate revascularization. Studies with rodent islet grafts have shown that vascular endothelial growth factor (VEGF) expression by β-cells can promote this process. The present work examines whether human islet preparations produce VEGF as well as interleukin (IL)-8, another angiogenic protein, and assesses the role of contaminating duct cells in VEGF and IL-8–mediated angiogenesis.RESEARCH DESIGN AND METHODS—Human islet and pancreatic duct cell preparations are compared for their respective expression and production of VEGF and IL-8 during culture as well as following transplantation in nonobese diabetic (NOD)/scid mice. The associated angiogenic effects are measured in an in vitro aortic ring assay and in an in vivo chick embryo chorioallantoic membrane assay.RESULTS—Cultured pancreatic duct cells expressed 3- and 10-fold more VEGF and IL-8, respectively, than cultured human islet endocrine cells and released both proteins at angiogenic levels. The angiogenic effect of purified duct cells was higher than that of purified endocrine islet cells and was completely blocked by a combination of IL-8 and VEGF antibodies. Human duct cell implants under the kidney capsule of NOD/scid mice expressed higher levels of IL-8 and VEGF than human islet cell implants and induced circulating IL-8 and VEGF levels during the first day posttransplantation.CONCLUSIONS—Human duct cell–released IL-8 and VEGF may help revascularization of currently used human islet cell grafts. Further work should examine whether and when this effect can prevail over other inflammatory and immune influences of this cell type.

Clodronate inhibits angiogenesis in vitro and in vivo

The effects of amino-bisphosphonate clodronate on endothelial cell functions involved in angiogenesis, namely proliferation and morphogenesis on matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo. This was performed by using the chick embryo chorioallantoic membrane (CAM) assay. In vitro, clodronate inhibited the endothelial cell proliferation in a dose-dependent fashion, peaking at 30 microM. At the same concentration, clodronate inhibited the fibroblast growth factor-2 (FGF-2)-induced capillary-like tube formation in the morphogenesis assay on matrigel. In vivo, when tested with the CAM assay, clodronate again displayed the capability to inhibit FGF-2-induced angiogenesis. Overall, these results suggest that antiangiogenesis by clodronate can be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases and cancer.

Dihydrotanshinone I inhibits angiogenesis both <italic>in vitro</italic> and <italic>in vivo</italic>

Dihydrotanshinone I (DI), a naturally occurring compound extracted from Salvia miltiorrhiza Bunge, has been reported to have cytotoxicity to a variety of tumor cells. In this study, we investigated its anti-angiogenic capacity in human umbilical vein endothelial cells. DI induced a potent cytotoxicity to human umbilical vein endothelial cells, with an IC(50) value of approximately 1.28 microg/ml. At 0.25-1 microg/ml, DI dose-dependently suppressed human umbilical vein endothelial cell migration, invasion, and tube formation detected by wound healing, Transwell invasion and Matrigel tube formation assays, respectively. Moreover, DI showed significant in vivo anti-angiogenic activity in chick embryo chorioallantoic membrane assay. DI induced a 61.1% inhibitory rate of microvessel density at 0.2 microg/egg. Taken together, our results showed that DI could inhibit angiogenesis through suppressing endothelial cell proliferation, migration, invasion and tube formation, indicating that DI has a potential to be developed as a novel anti-angiogenic agent.

Friend erythroleukemia cells induce angiogenesis in chick embryo chorioallantoic membrane and in human umbilical vein endothelial cells

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias.

Dehydroepiandrosterone Stimulates Endothelial Proliferation and Angiogenesis through Extracellular Signal-Regulated Kinase 1/2-Mediated Mechanisms

Dehydroepiandrosterone (DHEA) activates a plasma membrane receptor on vascular endothelial cells and phosphorylates ERK 1/2. We hypothesize that ERK1/2-dependent vascular endothelial proliferation underlies part of the beneficial vascular effect of DHEA. DHEA (0.1-10 nm) activated ERK1/2 in bovine aortic endothelial cells (BAECs) by 15 min, causing nuclear translocation of phosphorylated ERK1/2 and phosphorylation of nuclear p90 ribosomal S6 kinase. ERK1/2 phosphorylation was dependent on plasma membrane-initiated activation of Gi/o proteins and the upstream MAPK kinase because the effect was seen with albumin-conjugated DHEA and was blocked by pertussis toxin or PD098059. A 15-min incubation of BAECs with 1 nm DHEA (or albumin-conjugated DHEA) increased endothelial proliferation by 30% at 24 h. This effect was not altered by inhibition of estrogen or androgen receptors or nitric oxide production. There was a similar effect of DHEA to increase endothelial migration. DHEA also increased the formation of primitive capillary tubes of BAECs in vitro in solubilized basement membrane. These rapid DHEA-induced effects were reversed by the inhibition of either Gi/o-proteins or ERK1/2. Additionally, DHEA enhanced angiogenesis in vivo in a chick embryo chorioallantoic membrane assay. These findings indicate that exposure to DHEA, at concentrations found in human blood, causes vascular endothelial proliferation by a plasma membrane-initiated activity that is Gi/o and ERK1/2 dependent. These data, along with previous findings, define an important vascular endothelial cell signaling pathway that is activated by DHEA and suggest that this steroid may play a role in vascular function.

Endogenous opioid peptides, endomorphin-1 and -2 and deltorphin I, stimulate angiogenesis in the CAM assay

The opioid peptides modulate extensive bioactivities, including pain, cardiovascular response, development and so on. The effects of endogenous opioid peptides on angiogenesis were evaluated in the chick embryo chorioallantoic membrane (CAM) assay for the first time in the present study. Endomorphin-1, endomorphin-2 and deltorphin I at the dosage of 1, 10, 100 nmol/embryo could stimulate angiogenesis dose-dependently, respectively. Naloxone, the nonselective opioid receptor antagonist, did not influence angiogenesis alone; but it could antagonize the stimulative effects of the opioid peptides on angiogenesis when it was administrated in combination with the opioid peptides. Taken altogether, the results suggested that endogenous opioid peptides (endomorphin-1 and -2 and deltorphin I) stimulated angiogenesis in the CAM assay, and these effects were modulated with the opioid receptors. These data are important for potential future clinical implementation.

In Vivo Angiogenesis Is Suppressed by Unsaturated Vitamin E, Tocotrienol ,

Antiangiogenic therapy using drugs and food components is a recognized strategy for the prevention of various angiogenesis-mediated disorders such as tumor growth, diabetic retinopathy, and rheumatoid arthritis. Our preliminary cell culture studies, using both bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVEC) on screening for food-derived antiangiogenic compounds, showed tocotrienol (T3), an unsaturated version of vitamin E, to be a potential angiogenic inhibitor. We therefore investigaged the in vivo antiangiogenic properties of T3 using 2 well-characterized angiogenic models [mouse dorsal air sac (DAS) assay and the chick embryo chorioallantoic membrane (CAM) assay]. In the DAS assay, the increased neovascularization (angiogenesis index, 4.8 α 0.6) in tumor cell–implanted mice was suppressed (angiogenesis index, 2.7 α 0.6) by dietary supplementation of 10 mg T3-rich oil/d (equivalent to 4.4 mg T3/d). In the CAM assay, T3 (500–1000 μg/egg) inhibited new blood vessel formation on the growing CAM and increased the frequency of avascular zone (36–50%). To evaluate the antiangiogenic mechanism, we conducted cell-culture studies and found that T3 significantly reduced fibroblast growth factor -induced proliferation, migration, and tube formation in HUVEC (P < 0.05), with δ-T3 having the highest activity. Western blot analysis revealed that δ-T3 suppressed the phosphorylation of phosophoinositide-dependent protein kinase (PDK) and Akt, and increased the phosphorylation of apoptosis signal-regulating kinase and p38 in fibroblast growth factor-treated HUVEC, indicating that the antiangiogenic effects of T3 are associated with changes in growth factor-dependent phosphatidylinositol-3 kinase /PDK/Akt signaling as well as induction of apoptosis in endothelial cells. Our findings suggest that T3 has potential as a therapeutic dietary supplement for preventing angiogenic disorders, and therefore future clinical study will be required to evaluate the efficacy and safety of T3.

Combined Therapeutic Effects of Vinblastine and Rapamycin on Human Neuroblastoma Growth, Apoptosis, and Angiogenesis

Vinblastine and rapamycin displayed synergistic inhibition of human neuroblastoma-related angiogenesis. Here, we studied the antitumor activity of vinblastine and rapamycin against human neuroblastoma. Cell proliferation, cell cycle progression, and apoptosis were evaluated by measuring (3)H-thymidine incorporation, bromodeoxyuridine uptake, and phosphatidylserine exposure, respectively. The in vivo sensitivity of neuroblastoma cells to vinblastine and rapamycin was determined in orthotopic neuroblastoma-engrafted mice. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay. Each compound alone was able to induce a dose-dependent significant inhibition of cell proliferation, with a dramatically enhanced antiproliferative effect for the drugs used in combination. A marked G(2)-M cell cycle arrest with a nearly complete depletion of S phase was associated. The combined treatment triggered an increased apoptosis compared with either drug tested alone. A significant inhibition of tumor growth and microvessel area was obtained in neuroblastoma-bearing mice when treated with vinblastine or rapamycin alone, and a more dramatic effect with the combined treatment, compared with control mice. The therapeutic effectiveness, expressed as increased life span, was statistically improved by the combined therapy, compared with mice treated with either drug tested separately. Histologic evaluation of primary tumors showed that the combined treatment inhibited proliferation and angiogenesis and induced apoptosis. Combined treatment of neuroblastoma cells and neuroblastoma-bearing mice with vinblastine and rapamycin induced the down-modulation of both vascular endothelial growth factor production and vascular endothelial growth factor receptor 2 expression. In the chorioallantoic membrane assay, angiogenesis induced by human neuroblastoma biopsy specimens was significantly inhibited by vinblastine and rapamycin. These results may be relevant to design new therapeutic strategies against neuroblastoma.

β amyloid angiogenic activity in vitro and in vivo

Angiogenesis has been suggested as a direct contributor to Alzheimer's disease (AD) pathology. The major pathological hallmarks of AD are the presence of neurofibrillary tangles and, beta-amyloid plaques associated with activated microglia, astrocytes, degenerating neurons and vascular toxicity. In this study, Abeta1-40 and Abeta1-42 peptides, both components of the senile plaques in AD, were used to study their angiogenic activity in vitro, by using normal human cerebral endothelial cells (HCECs), and in vivo, by using the chick embryo chorioallantoic membrane (CAM) assay. Results showed that both peptides stimulate in vitro endothelial cell proliferation, chemotaxis and morphogenesis in Matrigel. Moreover, by using the aorta ring assay, both peptides stimulated the formation of capillary-like structures. An angiogenic response was induced in the CAM assay, similar to that induced by fibroblast growth factor-2 (FGF-2), a well-known angiogenic cytokine. Overall, these data support the hypothesis that Abeta peptides may contribute to angiogenesis occurring in AD and suggest that limiting the pro-angiogenic activity of Abeta peptides may therefore provide a useful target to control angiogenesis associated to AD and therefore limit the disease progression.

Assessment of angiogenic properties of biomaterials using the chicken embryo chorioallantoic membrane assay

The angiogenic potential of a biomaterial is a critical factor for successful graft intake in tissue engineering. We developed a modified, rapid and reproducible chicken embryo chorioallantoic membrane (CAM) assay to evaluate the ability of biomaterials in inducing blood vessel density. Five biomaterials including one-layer porcine small intestinal submucosa (SIS), two-layer SIS, four-layer vacuum pressed (VP) SIS, polyglycolic acid (PGA) and PGA modified with poly(lactic-co-glycolic acid) (PLGA) were analyzed. A circular section (1.2 mm diameter) of each biomaterial was placed near a group of blood vessels in the CAM. Blood vessels around the biomaterials were captured with black and white images at 96 h post implantation; and the images were subjected to densitometry evaluation. One-layer SIS induced a significant increase in blood vessel density as compared to the cellulose nitrate negative control, and had the greatest increase in blood vessel density as compared to four-layer VP SIS, PGA, or PLGA modified PGA. Although two-layer SIS has enhanced physical structure for surgical manipulation, its induction in blood vessel density was significantly lower than the one-layer SIS. Stripping the SIS proteins or incubating one-layer SIS with neutralizing antibodies against basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) resulted in decreased angiogenesis. Consistent with results obtained from bladder augmentation animal models, these results confirmed that angiogenic growth factors were present in SIS and affected the angiogenic potential of biomaterials. These data also demonstrated that the CAM assay can be used to ascertain methodically the angiogenic potential of biomaterials.