Directed In Vivo Angiogenesis Assay Research Articles

Investigating the Role of Von Willebrand Factor (VWF) in Modulating In Vivo angiogenesis in VWF-Deficient Mice

Background: Previous studies have demonstrated the negative regulatory role of von Willebrand Factor (VWF) in angiogenesis involving extracellular and intracellular pathways that promote vascular endothelial growth factor (VEGF) signaling. These findings have provided direct evidence for the longstanding association between inherited and acquired abnormalities of VWF and the formation of angiodysplastic lesions, which continue to remain a major challenge. Understanding the role of VWF in angiogenesis could reveal new therapeutic targets that may lead to improved patient management strategies. Aim: To examine the impact and mechanisms of localized VWF replacement on in vivo angiogenesis in a VWF-deficient mouse model. Methods: Three separate experiments were carried out in 10 to 12-week old C57Bl/6 VWF-/- mice of both sexes. In all experiments plugs were excised after seven days of incubation and processed for hematoxylin and eosin (H&E) and immunohistochemical staining (IHC) to assess vessel formation and vascular permeability/maturity, except for the directed in vivo angiogenesis assay (DIVAA) where there was technical difficulty in obtaining histological sections. In the first experiment, DIVAA was employed to decrease inter-assay variability. Angioreactors containing Matrigel mixed with fibroblast growth factor (FGF), VEGF and varying doses of recombinant VWF (rVWF, VONVENDI®) were implanted subcutaneously in the right back flank of mice. Similar angioreactors without rVWF were implanted in the left back flank of mice as a control (N=15, 3 per dose group). In the second experiment the in vivo Matrigel plug assay was used to enable histological evaluation of plugs. 300 μL of Matrigel mixed with FGF, VEGF and rVWF at 0.5IU/ml and 10.0IU/ml, was injected subcutaneously in the right back flank of each mouse based on dose group. In the left back flank, an equal volume of Matrigel without rVWF was injected as a control (N=12, 6 per dose group of rVWF). The setup of the last experiment was similar to the second, however, Matrigel was supplemented with normal endothelial-colony-forming cells (ECFCs) as the source of localized VWF in place of rVWF, in a titration curve of cell numbers (1 x 105, 5 x 105, 1 x 106 cells; N=15, 3 per dose group). Results: Generally, there was a dose response gradient across all three experiments, with higher doses of rVWF or ECFC titres yielding more pronounced effects. In comparison to the lightly coloured angioreactors from the experimental group (supplemented with rVWF), those from the control group were dark red in colour indicating the presence of more vessels and erythrocytes in the Matrigel. No clear differences were observed between plugs that received rVWF at 0.5 IU/ml and those that did not, both grossly and histologically. Most plugs with 10 IU/ml of rVWF appeared lighter in colour compared to those without rVWF. A similar pattern was observed on H&E staining with sections from plugs supplemented with rVWF showing fewer capillaries and red cells, compared with plugs that had only growth factors (Figure 1A). In line with these observations, IHC quantitation of endothelial cells (ECs), pericytes and smooth muscle cells showed a decrease in capillary formation and vessel maturity in rVWF supplemented plugs, although this did not reach significance (Figure 1B). Compared to plugs that had ECFCs, those without ECFCs were dark red in colour, indicating the presence of more mature vessels with red cells. H&E staining of control plugs showed increased cellular infiltrate and capillary formation compared to those supplemented with ECFCs. Immunostaining of control Matrigel plugs also showed increased vascularization and maturity of vessels as evidenced by higher signals for neural/glial antigen 2 (NG2) stain for pericytes and alpha-smooth muscle actin (ASMA) which stains smooth muscle, compared to plugs with ECFCs across all groups. Conclusion: Despite clear differences observed in the gross images of plugs, no significant differences were seen following microscopic quantitation. This was possibly due to the small sample size per group and the marked variability between data points. However, considering the data reported above, the findings appear to highlight a potential inhibitory role of VWF in angiogenesis. Further experimentation is required to establish whether localized VWF can reverse the proangiogenic phenotype observed in VWF-deficient mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Drynaria fortunei Promoted Angiogenesis Associated With Modified MMP-2/TIMP-2 Balance and Activation of VEGF Ligand/Receptors Expression

Background and Purpose: Drynaria fortunei J. Sm (D. fortunei), known as Gu-Sui-Bu, is used in traditional Chinese medicine to treat common injuries, including bone fractures and bruising. The specific functional mechanisms of the angiogenic and endothelial cell migration properties of D. fortunei are currently unclear. Thus, the purpose of this study is to validate the potential angiogenic and cellular migration properties and related mechanisms by D. fortunei both in vivo and in vitro.Experimental Approach: The present study investigates, both in vivo and in vitro, the wound healing effects of D. fortunei as associated with angiogenesis, specifically by the modulation of matrix metalloproteinases (MMPs) and upregulation of vascular endothelial growth factor (VEGF) ligand/receptors. In order to determine the potential angiogenic effects of D. fortunei, in vivo neovascularization of chick chorioallantoic membranes (CAMs) assay, and directed in vivo angiogenesis assay (DIVVA) were performed, while in vitro scratch wound healing, migration, and matrix-induced tube formation assays were performed by using human umbilical vascular endothelial cells (HUVECs). Furthermore, we used qPCR to analyze the gene expressions and Western blot to observe protein expressions of MMP-2, MMP-14, TIMP-2, RECK, and VEGF/VEGFRs.Results: This study identified five major compounds from the water extract of D. fortunei: protocatechuic acid, caffeic acid 4-O-β-D-glucopyranoside, 5,7-dihydroxychromone-7-O-rutinoside, neoeriocitrin, and naringin. D. fortunei was confirmed to activate in vivo angiogenesis by CAM and DIVVA assays. D. fortunei further exhibited in vitro angiogenic effects associated with cell migration, as demonstrated by the tube formation assay, transwell migration assay, and scratch wound healing assay. The extracellular MMP-2 activity was found to be dose-dependently augmented both in vitro and in vivo by D. fortunei. The mRNA and protein expressions of MMP-2, and MMP-14 were increased; while the tissue inhibitor metalloproteinase-2 (TIMP-2), and reversion-inducing cysteine-rich protein with kazal motifs (RECK) were both decreased. Furthermore, D. fortunei activated the gene and protein expressions of VEGF-A, -B, and VEGFR-2, -3.Conclusion: D. fortunei increased MMP-2 activity, thereby stimulating angiogenesis and cell migration, both in vivo and in vitro, as a result of MMP-2 and TIMP-2 balance modulation and the activation of VEGF/VEGFRs expression.

Correlation between Therapeutic Efficacy of CD34+ Cell Treatment and Directed In Vivo Angiogenesis in Patients with End-Stage Diffuse Coronary Artery Disease.

Background This study was aimed at testing the association between the therapeutic efficacy of CD34+ cell treatment in patients with end-stage diffuse coronary artery disease as reflected in angiographic grading and results of directed in vivo angiogenesis assay (DIVAA) on their isolated peripheral blood mononuclear cell- (PBMC-) derived endothelial progenitor cells (EPCs). Methods Angiographic grades (0: <5%; 1: 5–35%; 2: 35–75%; 3: >75%) which presented the improvement of vessel density pre- and post-CD34+ treatment were given to 30 patients with end-stage diffuse coronary artery disease having received CD34+ cell treatment. The patients were categorized into low-score group (angiographic grade 0 or 1, n = 12) and high-score group (angiographic grade 2 or 3, n = 18). The percentages of circulating EPCs with KDR+/CD34+/CD45−, CD133+/CD34+/CD45−, and CD34+ were determined in each patient using flow cytometry. PBMC-derived EPCs from all patients were subjected to DIVAA through a 14-day implantation in nude mice. The DIVAA ratio (i.e., mean fluorescent units in angioreactors with EPCs/mean fluorescent units in angioreactors without EPCs) was obtained for each animal with implanted EPCs from each patient. Results and Conclusions The number of EPCs showed no significant difference among the two groups. The DIVAA ratio in the high-score group was significantly higher than that in the low-score group (p = 0.0178). Logistic regression revealed a significant association between the DIVAA ratio and angiographic grading (OR 3.12, 95% CI: 1.14–8.55, p = 0.027). The area under the ROC curve (AUC) was 0.8519 (p = 0.0013). We proposed that DIVAA may be a reliable tool for assessing coronary vascularization after CD34+ cell treatment.

Characterization of the Effects of Semaphorin 4D Signaling on Angiogenesis.

The semaphorins and plexins comprise a family of cysteine-rich cell surface and secreted proteins originally shown to control nerve growth and the immune response, but that have recently been implicated in a wide variety of developmental and pathological processes that are influenced by cell adhesion and migration. Along those lines, our group and others have found that Semaphorin 4D (SEMA4D) plays an important role in angiogenesis by promoting chemotaxis of endothelial cells, which express its receptor, Plexin-B1. Indeed, some neoplasms produce SEMA4D along with other pro-angiogenic proteins for the purpose of enhancing blood vessel growth into a developing neoplasm. Here we describe the application of in vitro migration and tubulogenesis assays and the directed in vivo angiogenesis assay (DIVAA) in the measurement of the angiogenic potential of cell-derived and soluble SEMA4D.

Abstract B3: Anti-angiogenic properties of FAK-VEGFR3 scaffold inhibitors, C9 and C10.

Abstract Introduction: Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine kinases, which function as key modulators of survival and metastasis signals in cancer cells. We have previously shown that VEGFR3 binds to the FAT domain of FAK and this interaction promotes the survival of tumor cells. We developed novel small molecule compounds C9 and C10 which specifically bind and inhibit the FAK-VEGFR3 interaction. Both these compounds inhibit cancer cell proliferation, induce apoptosis, disrupt the FAK-VEGFR3 signaling axis, and significantly inhibit tumor growth in xenograft mouse models of breast and pancreatic cancer. Since FAK and VEGFR3 have been implicated in tumor angiogenesis, we investigated the potential anti-angiogenic effects of C9 and C10. Methods: Binding of C9 and C10 to the FAT domain of FAK was tested using fluorescence polarization. Effects of C9 and C10 were tested in vitro by MTS assay (viability) and tube formation assay using HUVEC cells. Directed in vivo angiogenesis assay (DIVAA) kit was used to determine anti-angiogenic effects of both compounds. Single agent efficacy of C9 and C10 was tested in a pancreatic xenograft mouse model for 30 days. Mouse tumor tissues were analyzed for markers of proliferation (Ki67), endothelial vessels (CD31), and lymphangiogenic vessels (LYVE1) using immunohistochemistry. Results: Fluorescence polarization confirmed that C9 and C10 effectively bind to the FAT domain of FAK. C9 and C10 reduced cell viability of HUVEC cells and dramatically decreased tube formation by reducing total branching points, total loops and total tube length in a dose and time dependent manner. DIVAA assay confirmed that C9 and C10 significantly reduced the formation of endothelial blood vessels as compared to untreated groups. In vivo, 30 days treatment with low doses of C9 and C10 as single agents led to 40% to 45% reduction in tumor volume (P&amp;lt;0.05). Further analyses of these mouse tumors confirmed the anti-angiogenic effects of C9 and C10. Conclusions: We have shown that targeting the FAK-VEGFR3 protein protein interaction site allows for dual inhibition of tumor proliferation as well as tumor angiogenesis. This study highlights the importance of oral-based small molecule inhibitors that might impact the therapeutic management of solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B3. Citation Format: Priyanka A. Agharkar, Manivannan Ethirajan, Jianqun Liao, Michael Yemma, Brian Buckley, Mikhail Chernov, William Cance, Ravindra Pandey, Elena Kurenova. Anti-angiogenic properties of FAK-VEGFR3 scaffold inhibitors, C9 and C10. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B3.

Abstract C3: Tumor-derived soluble uPAR regulates tumor angiogenesis via PTEN.

Abstract The urokinase receptor uPAR (CD87), a GPI-anchored protein, is frequently over-expressed in tumor cells. uPAR provokes numerous cellular functions by focusing proteolytic activity on the cell surface, but also by inducing intracellular signal transduction via transmembrane interaction partners, thus inducing cell migration and invasion as well as cell survival. Soluble tumor-derived uPAR has recently been described to activate endothelial cells and was suggested to promote angiogenesis. Consistently, high uPAR expression in tumor cells or high serum levels of circulating uPAR predict worse prognosis of breast, lung or colorectal cancer patients. In this study, we found that tumor-derived soluble uPAR is a key regulator of PTEN expression in endothelial cells. HEK cells deficient in endogenous uPAR or uPAR deficient endothelial cells derived from uPAR -/- mice had high PTEN mRNA as well as protein levels, while reconstitution of endogenous as well as addition of soluble exogenous uPAR decreased PTEN levels. uPAR, thereby, led to an integrin-dependent activation of the NFkB pathway, a known transcription pathway inhibitor of PTEN. As a consequence of PTEN downregulation, the downstream PKB/Akt signalling pathway became activated, which led to an enhanced migratory as well as cell survival activity in endothelial cells. Cross-breeding of uPAR -/- mice with endothelial cell-specific PTEN +/- mice, thereby led to a rescue of the high migratory phenotype of PTEN heterozygous endothelial cells in vitro and angiogenesis formation in a directed in vivo angiogenesis assay (DIVAA). From our date we conclude that tumor-derived uPAR is a major regulator of PTEN expression in endothelial cells, which leads to the induction of the PI3K/Akt-pathway and an enhanced angiogenic phenotype. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C3. Citation Format: Gerald W. Prager, Matthias Unseld, Marina Poettler, Christoph Zielinski. Tumor-derived soluble uPAR regulates tumor angiogenesis via PTEN. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C3.

Identification of first proadrenomedullin N-terminal 20 peptide (PAMP) modulator by means of virtual screening and NMR interaction experiments

PAMP (proadrenomedullin N-terminal 20 peptide) is a regulatory peptide that is detected in a large variety of cell types and exerts important biological activities. PAMP acts as a potent angiogenic factor and decorates microtubules in cells from different origins, controlling tubulin polymerization. A high-throughput docking-based virtual screening was performed, followed by a competitive monoclonal antibody assay on selected compounds, and a detailed 1H, 15N NMR spectroscopy study. This procedure has allowed us to describe the first small molecule capable of interacting with PAMP and potentially modulate its biological activity. Molecular modeling methods such as docking and molecular dynamics were carried out to obtain a theoretical model of binding mode. Finally a directed in vivo angiogenesis assay (DIVAA) showed that the small molecule by itself has pro-angiogenic properties.

Abstract 4360: CD87 is essential for mediating growth-factor induced angiogenesis via distribution of integrin beta-1

Abstract CD87 (urokinase-type plasminogen activator receptor, uPAR) expression has been correlated with tumor progression most likely via its functional ole as a central receptor for extracellular proteolysis, but also via its role in initiating intracellular signal transduction. The role of CD87 in angiogenesis has recently been elucidated. Thus, we observed that in VEGF-stimulated ndothelial cells CD87 is essential for endothelial cell survival as well as for n efficient growth factor- induced endothelial cell migration. Thereby, the PI-anchored protein CD87 formed a complex with its ligand urokinase uPA) as well as the inhibitor plasminogen activator inhibitor-1 (PAI-1), which became redistributed to the leading edge via low density lipoprotein receptor- (LDLR-) like proteins. In this study, we analyzed the functional role of CD87 in integrin redistribution during growth factor-induced endothelial cell migration. CD87, thereby, directly interacted with integrin adhesion receptors (a5b1) as well as with LDLR- proteins. Either by interfering with CD87/integrin nteraction or by interfering with CD87/LDLR-protein interaction by using specific interfering peptides, we observed disruption of integrin redistribution to the leading edge in migrating cells. As a consequence diminished focal adhesion formations were observed and endothelial cell migration was diminished. In CD87-deficient HEK 293 cells as well as in endothelial cells derived from CD87 -/- mice, expression of CD87 mutants (mutL3/uPAR), which lacked the binding motif for LDLR, led to an impaired cell spreading as well as a diminished migratory response towards VEGF, while full length CD87 (wt/CD87) reconstituted this phenotype. That CD87 plays a central role in growth factor induced endothelial cell invasion and capillary-like tube formation in vivo was demonstrated by a directed in vivo angiogenesis assay (DIVAA), which was performed either in CD87 deficient or wild type animals in the presence or absence of interfering peptides. Our data give novel insights into the central role of CD87 in pro- angiogenic endothelial cell behavior as well as in its role in angiogenesis. Thus, CD87 might lead to a novel therapeutic strategies in tumor- angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4360. doi:1538-7445.AM2012-4360

An indirubin derivative, E804, exhibits potent angiosuppressive activity

Angiogenesis, the development of neovessels from pre-existing vessels, is obligatory for solid tumors survival, growth, invasion, and metastasis. Many anti-angiogenic agents are small molecules originated from natural sources. Recently, angiosuppressive effects of indirubin and its derivatives, the active components in indigo-producing herbs, have been shown to possess anti-viral and anti-inflammatory potentials. In this study, we identified another indirubin derivative, indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804), could exhibit potent angiosuppressive effects. In vitro study showed that E804 could significantly inhibit human umbilical vein endothelial cells proliferation, migration, and tube formation in a concentration-dependent manner (0.4–40 μM); at the concentration of 1 μmol or above, angiosuppressive potency of E804 was found to be more significant than indirubin-3′-oxime. Using in vivo Matrigel plug model and directed- in vivo-angiogenesis-assay (DIVAA), E804 was shown more effective to attenuate the VEGF/bFGF-induced neovessel formation. The hemoglobin content and the invaded endothelial cells in the implants were also greatly reduced. Results from the aortic ring assay indicated E804 (4 μM) could completely suppress ex vivo sprouting of endothelial cells from the rat aorta fragments; with concomitant reduction of gelatinolytic activities of matrix metalloproteinase-2 and -9. E804 also concentration-dependently (0.04–10 μM) inhibited the subintestinal vessels formation in zebrafish embryos. This study provides the first evidence that E804, a novel indirubin derivative, could more effectively inhibit angiogenesis. With the improved anti-angiogenic potency when compared with indirubin-3′oxime, E804 would be a new potential candidate in the treatment of angiogenesis-dependent diseases.

β-Hairpin Peptide That Targets Vascular Endothelial Growth Factor (VEGF) Receptors

VEGF receptors have been the target of intense research aimed to develop molecules able to inhibit or stimulate angiogenesis. Based on the x-ray structure of the complex placental growth factor-VEGF receptor 1(D2), we designed a VEGF receptor-binding peptide reproducing the placental growth factor β-hairpin region Gln(87)-Val(100) that is involved in receptor recognition. A conformational analysis showed that the designed peptide adopts the expected fold in pure water. Moreover, a combination of NMR interaction analysis and cell binding studies were used to demonstrate that the peptide targets VEGF receptors. The VEGF receptor 1(D2)-interacting residues were characterized at the molecular level, and they correspond to the residues recognizing the placental growth factor sequence Gln(87)-Val(100). Finally, the peptide biological activity was characterized in vitro and in vivo, and it showed a VEGF-like behavior. Indeed, the peptide activated VEGF-dependent intracellular pathways, induced endothelial cell proliferation and rescue from apoptosis, and promoted angiogenesis in vivo. This compound is one of the few peptides known with proangiogenic activity, which makes it a candidate for the development of a novel peptide-based drug for medical applications in therapeutic angiogenesis.

Directed in vivo angiogenesis assay and the study of systemic neoangiogenesis in cancer

Targeting neoangiogenesis is a well-established anticancer strategy, however, one of the major problems in angiogenesis research, both at the basic and applied levels, remains the development of suitable in vivo methods for assessing and quantifying the systemic angiogenic response. Therefore, there is an urgent need to adopt technically simple and reproducible methodologies which allow to easily quantify neoangiogenesis independently of morphological parameters. Recently, a reproducible and quantitative method was developed, the directed in vivo angiogenesis assay (DIVAA) consisting of the subcutaneous implantation of surgical grade silicone cylinders closed at one end, called angioreactors, into the dorsal flanks of nude mice. In the past few years, DIVAA has been successfully used in evaluating the inhibition and or enhancement of systemic perturbation of angiogenesis by several molecules. Thus, DIVAA studies systemic angiogenesis and its therapeutic modulation associated to cancer progression and metastasis.

Doxycycline's Effect on Ocular Angiogenesis: An In Vivo Analysis

To determine the in vivo effect of doxycycline on choroidal angiogenesis and pterygium growth by using a choroidal neovascular (CNV) murine model, a directed in vivo angiogenesis assay (DIVAA) and a pterygium murine model. Experimental study. Three murine models were investigated with 4 mice minimum per group and 22 maximum per group. Mice received water with or without doxycycline. For the CNV, the neovascular lesion volume was determined in choroid-retinal pigment epithelial flat mounts using confocal microscopy 7 days after laser induction. For DIVAA, silicone capsules containing 10,000 human pterygium epithelial cells were implanted in the flanks of mice subcutaneously. After 11 days, neovascularization (NV) was quantified using spectrofluorometry after murine tail-vein injection of fluorescein isothiocyanate-labeled dextran. A pterygium epithelial cell model was developed by injecting 10,000 human pterygium epithelial cells in the nasal subconjunctival space in athymic nude mice. Doxycycline was started on day 6 at 50 mg/kg per day; corneal lesions that resulted from the injections were compared at days 6 and 15. The Student t-test was used to evaluate the data for the CNV and DIVAA models and histologic preparations were used to evaluate pterygia lesions. There was significantly less NV and lesion volume with doxycycline taken in drinking water versus plain water. With doxycycline treatment, the laser-induced CNV showed a maximal 66% decrease in choroidal blood vessel volume (P< or =0.008) and the DIVAA showed a 30% reduction of blood vessel growth and migration (P<0.004). Histologic preparations demonstrated that pterygium cell lesions regressed when mice were administered doxycycline for 9 days. Doxycycline significantly inhibited angiogenesis in 3 murine models. The most dramatic effect was found in the CNV model followed by the pterygia epithelial cell DIVAA model. The anterior segment pterygium model also showed regression histologically. This suggests that doxycycline may be successful as an adjunctive treatment for CNV and pterygia in humans; clinical trials would be necessary to determine if there is a benefit.

Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model

BackgroundAngiogenesis is the formation of neovasculature from a pre-existing vascular network. Progression of solid tumors including lung cancer is angiogenesis-dependent. We previously introduced a bioinformatics-based methodology to identify endogenous anti-angiogenic peptide sequences, and validated these predictions in vitro in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays.MethodsOne family of peptides with high activity is derived from the α-fibrils of type IV collagen. Based on the results from the in vitro screening, we have evaluated the ability of a 20 amino acid peptide derived from the α5 fibril of type IV collagen, pentastatin-1, to suppress vessel growth in an angioreactor-based directed in vivo angiogenesis assay (DIVAA). In addition, pentastatin-1 suppressed tumor growth with intraperitoneal peptide administration in a small cell lung cancer (SCLC) xenograft model in nude mice using the NCI-H82 human cancer cell line.ResultsPentastatin-1 decreased the invasion of vessels into angioreactors in vivo in a dose dependent manner. The peptide also decreased the rate of tumor growth and microvascular density in vivo in a small cell lung cancer xenograft model.ConclusionsThe peptide treatment significantly decreased the invasion of microvessels in angioreactors and the rate of tumor growth in the xenograft model, indicating potential treatment for angiogenesis-dependent disease, and for translational development as a therapeutic agent for lung cancer.

CC-5079: A Small Molecule with MKP1, Antiangiogenic, and Antitumor Activity

CC-5079, a small molecule inhibitor oftubulin polymerization and phosphodiesterase-4 activity, was evaluated for antiangiogenic and antitumor activities. First, CC-5079 in vitro activity on human umbilical vein endothelial cells (HUVECs), fibroblasts, and MC38 were evaluated by proliferation, migration, and invasion assays. Second, CC-5079 effect on microvessel formation was evaluated ex vivo by chick chorioallantoic membrane (CAM), rat aortic rings assays, and with directed in vivo angiogenesis assay (DIVAA). Third, CC-5079 antitumor effect was determined in treatment of C57BL/6 mice with MC38 tumors. Finally, CC-5079 modulation of MKP1 in HUVECs, human fibroblast, and MC38 were determined by RNA isolation for qRT-PCR. At the 0.1 μM concentration CC-5079 significantly inhibited HUVEC, fibroblast, and MC38 proliferation and migration (all P < 0.001). At the 0.1 μM concentration, CC-5079 also inhibited HUVEC invasion (P < 0.05) but not fibroblast. In the CAM and rat aortic ring assays, CC-5079 at 0.1 μM inhibited microvessel formation (P < 0.05). By DIVAA, CC-5079 at 1 mg/kg/d continuous delivered inhibited microvessel formation (P < 0.05). Intraperitoneal CC-5079 was well tolerated and inhibited the growth of subcutaneous MC38 at 100 mg/kg/d (P < 0.01). By qRT-PCR, CC-5079 stimulated MKP1 expression in HUVEC and fibroblast. CC-5079 demonstrated stimulation ofMKP1, antiangiogenic, and antitumor properties.

CD97, an adhesion receptor on inflammatory cells, stimulates angiogenesis through binding integrin counterreceptors on endothelial cells

AbstractCD97, a membrane protein expressed at high levels on inflammatory cells and some carcinomas, is a member of the adhesion G protein–coupled receptor family, whose members have bipartite structures consisting of an extracellular peptide containing adhesion motifs noncovalently coupled to a class B 7-transmembrane domain. CD97α, the extracellular domain of CD97, contains 3 to 5 fibrillin class 1 epidermal growth factor (EGF)–like repeats, an Arg-Gly-Asp (RGD) tripeptide, and a mucin stalk. We show here that CD97α promotes angiogenesis in vivo as demonstrated with purified protein in a directed in vivo angiogenesis assay (DIVAA) and by enhanced vascularization of developing tumors expressing CD97. These data suggest that CD97 can contribute to angiogenesis associated with inflammation and tumor progression. Strong integrin α5β1 interactions with CD97 have been identified, but αvβ3 also contributes to cell attachment. Furthermore, soluble CD97 acts as a potent chemoattractant for migration and invasion of human umbilical vein endothelial cells (HUVECs), and this function is integrin dependent. CD97 EGF-like repeat 4 is known to bind chondroitin sulfate. It was found that coengagement of α5β1 and chondroitotin sulfate proteoglycan by CD97 synergistically initiates endothelial cell invasion. Integrin α5β1 is the first high-affinity cellular counterreceptor that has been identified for a member within this family of adhesion receptors.

1007 Accuracy of end of treatment 18F-FDG PET for predicting relapse in patients with Hodgkin's disease (HD) and Non-Hodgkin's lymphoma (NHL)

Angiogenesis, the development of neovessels from pre-existing vessels, is obligatory for solid tumors survival, growth, invasion, and metastasis. Many anti-angiogenic agents are small molecules originated from natural sources. Recently, angiosuppressive effects of indirubin and its derivatives, the active components in indigo-producing herbs, have been shown to possess anti-viral and anti-inflammatory potentials. In this study, we identified another indirubin derivative, indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804), could exhibit potent angiosuppressive effects. In vitro study showed that E804 could significantly inhibit human umbilical vein endothelial cells proliferation, migration, and tube formation in a concentration-dependent manner (0.4–40 μM); at the concentration of 1 μmol or above, angiosuppressive potency of E804 was found to be more significant than indirubin-3′-oxime. Using in vivo Matrigel plug model and directed-in vivo-angiogenesis-assay (DIVAA), E804 was shown more effective to attenuate the VEGF/bFGF-induced neovessel formation. The hemoglobin content and the invaded endothelial cells in the implants were also greatly reduced. Results from the aortic ring assay indicated E804 (4 μM) could completely suppress ex vivo sprouting of endothelial cells from the rat aorta fragments; with concomitant reduction of gelatinolytic activities of matrix metalloproteinase-2 and -9. E804 also concentration-dependently (0.04–10 μM) inhibited the subintestinal vessels formation in zebrafish embryos. This study provides the first evidence that E804, a novel indirubin derivative, could more effectively inhibit angiogenesis. With the improved anti-angiogenic potency when compared with indirubin-3′oxime, E804 would be a new potential candidate in the treatment of angiogenesis-dependent diseases.

Quantitative Assessment of Angiogenic Responses by the Directed in Vivo Angiogenesis Assay

One of the major problems in angiogenesis research remains the lack of suitable methods for quantifying the angiogenic response in vivo. We describe the development and application of the directed in vivo angiogenesis assay (DIVAA) and demonstrated that it is reproducible and quantitative. This assay consists of subcutaneous implantation of semiclosed silicone cylinders (angioreactors) into nude mice. Angioreactors are filled with only 18 micro l of extracellular matrix premixed with or without angiogenic factors. Vascularization within angioreactors is quantified by the intravenous injection of fluorescein isothiocyanate (FITC)-dextran before their recovery, followed by spectrofluorimetry. Angioreactors examined by immunofluorescence show cells and invading angiogenic vessels at different developmental stages. The minimally detectable angiogenic response requires 9 days after implantation and >/=50 ng/ml (P < 0.01) of either fibroblast growth factor-2 or vascular endothelial growth factor. Characterization of this assay system demonstrates that the FITC-labeled dextran quantitation is highly reproducible and that levels of FITC-dextran are not significantly influenced by vascular permeability. DIVAA allows accurate dose-response analysis and identification of effective doses of angiogenesis-modulating factors in vivo. TNP-470 potently inhibits angiogenesis (EC(50) = 88 pmol/L) induced by 500 ng/ml of fibroblast growth factor-2. This inhibition correlates with decreased endothelial cell invasion. DIVAA efficiently detects differences in anti-angiogenic potencies of thrombospondin-1 peptides (25 micro mol/L) and demonstrates a partial inhibition of angiogenesis ( approximately 40%) in a matrix metalloprotease (MMP)-2-deficient mouse compared with that in wild-type animals. Zymography of angioreactors from MMP-deficient and control animals reveals quantitative changes in MMP expression. These results support DIVAA as an assay to compare potencies of angiogenic factors or inhibitors, and for profiling molecular markers of angiogenesis in vivo.