Angiogenesis In Chick Chorioallantoic Membrane Research Articles

Evaluation of anti-angiogenic, anti-inflammatory and antinociceptive activity of coenzyme Q10 in experimental animals

Abstract Objectives This work aimed to assess some pharmacological activities of coenzyme Q10 (CoQ10) in animal experimental models. Methods The chick chorioallantoic membrane assay was used to evaluate anti-angiogenic activity of CoQ10. Anti-inflammatory activity of CoQ10 was confirmed using two animal models of inflammation. These were the vascular permeability and air pouch models, models of acute and sub-acute inflammation, respectively. Antinociceptive activity was assessed by the acetic acid-induced abdominal constriction response. Key findings CoQ10 dose-dependently displayed inhibition of chick chorioallantoic membrane angiogenesis. In the acetic acid-induced vascular permeability model in mice, CoQ10 at 50, 100 and 200 mg/kg reduced vascular permeability from 0.74 ± 0.01 (A590) to 0.67 ± 0.01 (P < 0.01), 0.46 ± 0.02 (P < 0.01) and 0.30 ± 0.01 (P < 0.01), respectively. In the carrageenan-induced inflammation in the air pouch, CoQ10 was able to diminish exudate volume, the number of polymorphonulcear leucocytes and nitrite content in the air pouches. CoQ10 at 25, 50 and 100 mg/kg significantly reduced acetic acid-induced abdominal constriction in mice from 27.0 ± 2.00 (number of abdominal constrictions) to 17.7 ± 0.33 (P < 0.01), 9.3 ± 0.67 (P < 0.01) and 1.3 ± 0.33 (P < 0.01), respectively, suggesting a strong antinociceptive activity. Conclusions CoQ10 possessed considerable anti-angiogenic, anti-inflammatory and antinociceptive activity, possibly via down-regulating the level of nitric oxide, which partly supported its use as a dietary supplement and in combination therapy.

Anti-inflammatory, anti-angiogenic and anti-nociceptive activities of an ethanol extract of Salvia plebeia R. Brown

Ethnopharmacological relevance Salvia plebeia R. Brown has been used for the treatment of a variety of inflammatory diseases, cold and tumors in many countries, including Korea and China. Aim of the study This study aimed to assess anti-inflammatory and related activities of an ethanol extract (SPEE) prepared from the dried whole parts of Salvia plebeia. Materials and methods Anti-angiogenic and anti-nociceptive activities of SPEE were analyzed using the chick chorioallantoic membrane (CAM) assay and acetic acid-induced writhing response, respectively. Anti-inflammatory activity of SPEE was evaluated using acetic acid-induced vascular permeability, carrageenan-induced inflammation in the air pouch and analyses of nitrite content and induced nitric oxide synthase (iNOS) level in the macrophage cells. Results SPEE gave rise to a significant inhibition in chick chorioallantoic membrane angiogenesis. SPEE exhibited anti-inflammatory activities in vascular permeability and air-pouch models. In the air-pouch model, SPEE was able to diminish exudate volume, number of polymorphonulcear leukocytes and nitrite content. SPEE also displayed anti-nociceptive activity in the writhing response model in mice. SPEE significantly decreased nitrite content and induced nitric oxide synthase (iNOS) in the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, while it could not modulate cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 levels in the stimulated phages. SPEE decreased reactive oxygen species (ROS) level in the stimulated macrophages. Conclusion The ethanol extract (SPEE) of Salvia plebeia possesses anti-inflammatory and related anti-angiogenic, anti-nociceptive and antioxidant activities, which offers partial support to its folkloric use.

Fluocinolone Inhibits VEGF Expression via Glucocorticoid Receptor in Human Retinal Pigment Epithelial (ARPE-19) Cells and TNF-α–Induced Angiogenesis in Chick Chorioallantoic Membrane (CAM)

The purpose of this study was to determine whether fluocinolone inhibits vascular endothelial growth factor (VEGF) expression in a retinal pigment epithelial cell line (ARPE-19) and TNF-alpha-induced angiogenesis in chick chorioallantoic membrane (CAM) assay. The dose-dependent effect of fluocinolone (0.0001-1 microM) on VEGF secretion, VEGF mRNA expression, and cytotoxicity was determined in confluent monolayers of ARPE-19 cells using ELISA, RT-PCR, and MTT assay, respectively. The effect of a glucocorticoid receptor antagonist (RU486) on fluocinolone-mediated VEGF expression was determined. The effect of fluocinolone in inhibiting TNF-alpha-induced angiogenesis was determined using chick chorioallantoic membrane (CAM) assay. The dose-dependent effect of fluocinolone (0.0001-1 microM) in inhibiting 1% serum-stimulated ARPE-19 cell proliferation was determined using BrdU labeling assay. At concentrations devoid of cytotoxicity, fluocinolone inhibited VEGF secretion as well as mRNA expression in ARPE-19 cells. RU486 (1 microM) treatment prevented inhibition of VEGF secretion and VEGF mRNA expression by fluocinolone (0.1 microM). Fluocinolone (50 ng/egg) inhibited angiogenesis induced by TNF-alpha. The ARPE-19 cell proliferation was inhibited by fluocinolone in a dose-dependent manner. Fluocinolone inhibited VEGF expression in ARPE-19 cells via its glucocorticoid receptor activity. In addition, fluocinolone inhibited proliferation of ARPE-19 cells and TNF-alpha-induced angiogenesis in chorioallantoic membranes.

Anti-inflammatory, anti-angiogenic and anti-nociceptive activities of Saururus chinensis extract

Ethnopharmacolgical relevance: The aerial parts of Saururus chinensis Baill. are used for the treatment of edema and inflammatory diseases in the Oriental folk medicine. Aim of the study This study aimed to elucidate anti-inflammatory and related activities of the ethanol extract (SC) of the dried aerial parts of Saururus chinensis Baill. Materials and methods Anti-angiogenic and anti-nociceptive activities of SC were analyzed using the chick chorioallantoic membrane (CAM) assay and acetic acid-induced writhing response, respectively. Anti-inflammatory activity of SC was evaluated using acetic acid-induced vascular permeability, carrageenan-induced air pouch formation and analyses of nitrite content and induced nitric oxide synthase (iNOS) level in the macrophage cells. Results SC dose-dependently displayed a strong inhibition in the CAM angiogenesis. SC showed significant anti-nociceptive activity in the writhing model. The anti-inflammatory activity of SC was also assessed in the two in vivo models, such as vascular permeability and air pouch models in mice. SC suppressed production of nitric oxide and induction of iNOS and cyclooxygenase-2 in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Conclusion The aerial parts of Saururus chinensis possess potent anti-angiogenic and anti-nociceptive activities in addition to anti-inflammatory activity, which partly supports its therapeutic efficacy.

Effect of β-escin sodium on endothelial cells proliferation, migration and apoptosis

β-Escin, the major active compound in extracts of the horse chestnut Aesculus hippocastanum seed, has shown clinically significant activity in chronic venous insufficiency (CVI). Our previous studies had shown that β-escin sodium inhibited angiogenesis in chick chorioallantoic membrane (CAM) and in aortic disk assay. In this study, we explored the direct effect of β-escin sodium on proliferation, migration and apoptosis in human umbilical vein endothelial cells (HUVECs) and ECV304 cells. Sulforhodamine B (SRB) assay showed that β-escin sodium (10, 20, 40 μg/ml) inhibited endothelial cells (ECs) proliferation dose-dependently. β-escin sodium also induced ECs apoptosis at 40 μg/ml. Cell migration was evaluated by an improved wound assay: barren spot assay. And the direct effect on cell motility excluding influence of cell proliferation was examined by High Content Screening (HCS, Cellomics) assay. The data indicated that β-escin sodium suppressed ECs migration and cell motility. Western blot results suggested that β-escin sodium acts on ECs possibly by increasing expression of thrombospondin-1 (TSP-1), and decreasing expression of PKC-α and activation of p44/42 mitogen-activated protein kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK). Our findings give the evidence that β-escin sodium might have potential anti-angiogenic activity via its direct effects on ECs.

056 Activation of Sterol Regulatory Element-Binding Proteins ((SREBPs) is Critical in IL-8-Induced Angigiogenesi

Angiogenesis is an essential process during wound healing. The chemokine IL-8 is a well-known inflammatory and angiogenic factor. We found that IL-8 stimulates activation of SREBPs, molecules that are critical for cholesterol and fatty acid metabolism, and that this activation is markedly increased during IL-8-induced angiogenesis. Two important processes in the early stages of angiogenesis are increase in proliferation and migration of endothelial cells. Therefore, we tested whether IL-8 stimulates these processes in a SREBP-dependent manner both in vivo and in culture. Our data show that IL-8 stimulates SREBP-1 and -2 gene expression which leads to an increase in both the precursor and the mature forms of these proteins in human microvascular endothelial cells (hMVEC). In addition, several genes downstream of SREBPs are also up-regulated by IL-8 stimulation, in particular 3-hydroxy -3-methylglutaryl coenzyme A reductase (HMGCA). Furthermore, SREBP-1 was abundant in the neovasculature of wound healing skin, and this expression could be suppressed by local application of antibodies to IL-8 or to its receptors, CXCR1 and CXCR2. To test whether SREBP activation is critical for the proliferation and migration of endothelial cells induced by IL-8 during angiogenesis, we used either RNA interference (RNAi) to inhibit the SREBP cleavage-activating protein (SCAP) that escorts SREBPs from the ER to the Golgi for maturation (activation), or treated endothelial cells with 25-hydroxycholesterol (25-HC), an inhibitor of SREBP activation. Both SCAP RNAi and 25-HC significantly inhibited IL-8-induced SREBP activation during endothelial cell migration and proliferation indicating that SREBPs are critical components of the signaling pathways leading to IL8-induced angiogenesis. In addition, in the chick chorioallantoic membrane (CAM) model, 25-HC markedly inhibited both developmental- and IL-8-induced angiogenesis in 7-day and 10-day CAMs, respectively. In conclusion, our results indicate a novel regulatory mechanism for IL-8-induced angiogenesis that is mediated by SREBPs.

Anti-angiogenic, antinociceptive and anti-inflammatory activities of Lonicera japonica extract

This study aimed to elucidate some novel pharmacological activities of Lonicera japonica (Caprifoliaceae), which is widely used in Oriental folk medicine. The ethanolic extract of L. japonica (LJ) dose dependently inhibited chick chorioallantoic membrane angiogenesis. The antinociceptive activity of LJ was assessed using the acetic acid-induced constriction model in mice. LJ showed anti-inflammatory activity in two in-vivo models: the vascular permeability and air pouch models. LJ suppressed the production of nitric oxide via down-regulation of inducible nitric oxide synthase in lipopolysaccharide-stimulated RAW264.7 macrophage cells. However, LJ was unable to suppress induction of cyclooxygenase-2 in the stimulated macrophage cells. LJ decreased the reactive oxygen species level in the stimulated macrophage cells. In brief, the flowers of L. japonica possess potent anti-angiogenic and antinociceptive activities, in addition to anti-inflammatory activity, which partly supports its therapeutic efficacy.

Inhibitory effects of propenone derivatives on angiogenesis in vitro and in vivo

Previously, 1‐furan‐2‐yl‐3‐pyridin‐2‐yl‐propenone (FPP‐3) has shown an anti‐invasive activity through inhibitory effect on matrix metalloproteinase (MMP)‐2 and MMP‐9 that are important enzymes involved in angiogenesis. In this study, the propenone derivatives, 1,3‐diphenyl‐propenone (DPhP), 3‐phenyl‐1‐thiophen‐2‐yl‐propenone (PhT2P), 3‐phenyl‐1‐thiophen‐3‐yl‐propenone (PhT3P) and 1‐furan‐2‐yl‐3‐phenyl‐propenone (FPhP‐3) were investigated for an antiangiogenic activity. Among the propenone derivatives, 1,3‐diphenyl‐propenone (DPhP) and 1‐furan‐2‐yl‐3‐phenyl‐propenone (FPhP‐3) showed strong inhibitory effects on both VEGF‐ and interleukin (IL‐8)‐induced angiogenesis in chick chorioallantoic membrane (CAM) assay in vivo. We also found that DPhP and FPhP‐3 concentration‐dependently inhibited VEGF‐induced endothelial cell invasion, one of the important criteria for angiogenesis in vitro, by using matirgel‐coated transwell assay. In addition, DPhP and FPhP‐3 significantly inhibited VEGF‐induced proliferation and tube formation of endothelial cells. Taken together, our results suggest that DPhP and FPhP‐3, propenone derivatives, may be valuable drug candidates as an angiogenesis inhibitor. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. R11‐2007‐040‐02004‐0).]

Taspine isolated from Radix et Rhizoma Leonticis inhibits proliferation and migration of endothelial cells as well as chicken chorioallantoic membrane neovascularisation

The aim of the present study was to investigate an anti-angiogenic effect of taspine isolated from Radix et Rhizoma Leonticsi. Taspine was screened for the first time, using cell membrane chromatography (CMC). The anti-angiogeneic activity of taspine was tested by using the chicken chorioallantoic membrane (CAM) neovascularisation model in vivo and the HUVEC proliferation and migration models in vitro, respectively. The results showed that taspine could inhibit CAM angiogenesis significantly within the concentration range of 0.5–2 μg/egg, proliferation and migration of endothelial cells in a dose-dependent manner. The CAM histomorphology results indicated that taspine could inhibit blood vessels sprouts and proliferation of vascular endothelial cell. These findings suggest that taspine is a promising candidate for use as an angiogenesis inhibitor.

Suppression of tumor-induced angiogenesis by taspine isolated from Radix et Rhizoma Leonticis and its mechanism of action in vitro

The present study was to demonstrate the effect of taspine isolated from Radix et Rhizoma Leonticis on tumor angiogenesis and its mechanism of action. The anti-angiogenic effect in vivo was evaluated on chicken chorioallantoic membrane (CAM) neovascularisation model and CAM transplantation tumor model. Taspine exerted inhibitory influence on CAM angiogenesis and the growth and microvessel density (MVD) of CAM transplantation tumor at concentrations of 0.5–2 μg/egg. The mechanism was demonstrated through detecting vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) protein secretion by enzyme-linked immunosorbent assay (ELISA), as well as mRNA expression of VEGF, Flt-1 and Flk-1/KDR by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that taspine down-regulated the VEGF and bFGF secretion in human non-small cell lung cancer cell (A549 cell) and human umbilical vein endothelial cell (HUVEC), and the VEGF and Flk-1/KDR mRNA expression in HUVEC. Additionally, the effect of taspine on HUVEC migration was detected with the method of cell scrape. The result indicated that taspine inhibited HUVEC migration in a dose-dependent manner. These findings suggest that taspine might be a promising candidate as angiogenesis inhibitors.

Bilberry inhibits angiogenesis in chick chorioallontoic membrane

Angiogenesis is the process of generating new blood vessels from preexisting vessels and is considered essential in many pathological conditions. Bilberry has been traditionally used as a folk medicine for some countries. The purpose of the present study was to evaluate the effect of bilberry in chick chorioallantoic membrane angiogenesis model in vivo. In this well characterized model, bilberry inhibited angiogenesis in a concentration-dependent manner. Compared with the normal group, bilberry group has significant decreased vessels proliferation. These results provide evidence that bilberry inhibits angiogenesis and may be useful for treating angiogenesis-dependent human diseases.

Anti-angiogenic activities of chitooligosaccharides

Chitooligosaccharides (COS) was obtained from chitosan by depolymerization with enzyme and analyzed by HPLC and TOF-MS, and the results indicated that the polymerization degree of COS was 2–18. In order to explore the anti-angiogenic activities of COS, the effect of COS on chicken chorioallantoic membrane (CAM) angiogenesis and on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) induced by human hepatoma carcinoma cells (HCC) culture fluid was measured. The results showed COS had no toxicity to normal HUVECs, but could inhibit the CAM angiogenesis and the proliferation, migration and tube formation of induced HUVECs. All of these results indicate that COS have potential anti-angiogenic activities and can counteract the stimulation of HCC-culture-fluid on endothelial cells at a certain level.

Angiogenesis Inhibition and Choroidal Neovascularization Suppression by Sustained Delivery of an Integrin Antagonist, EMD478761

To evaluate the angiogenic inhibitory effects of an alpha(v)beta(3)/alpha(v)beta(5) integrin antagonist, EMD478761, released from a polymeric implant in a chick chorioallantoic membrane (CAM) assay and laser-induced experimental choroidal neovascularization (CNV) in rats. Polyvinyl alcohol-based reservoir implants releasing EMD478761 were designed for placement onto a CAM or intravitreally in rats. In vitro release rates of the implants were measured using HPLC. Angiogenesis was induced on 10-day-old chick embryos by basic fibroblast growth factor (bFGF), and areas of neovascularization were measured. Experimental CNV was induced in the Brown-Norway rat with a diode laser. EMD478761 or sham microimplants were placed within the vitreous chamber of Brown-Norway rats. Two weeks later, areas of CNV were determined by FITC-dextran staining of choroidal flatmounts. Sustained delivery of EMD478761 significantly inhibited bFGF-induced angiogenesis in CAM, as determined by a reduction in angiogenesis areas, without drug toxicity to the normal CAM vasculature. In an experimental rat model, intravitreal EMD478761 implants significantly suppressed laser-induced CNV compared with intravitreal sham implants, with the mean area reduced by 63% (P < 0.05). Sustained delivery of EMD478761demonstrates potent antiangiogenic properties in vivo. These results suggest that an EMD478761 implant may be beneficial in the treatment of neovascular ocular diseases.

Antiangiogenic Effect of Deguelin on Choroidal Neovascularization

Age-related macular degeneration is the leading cause of blindness in the elderly. Choroidal neovascularization (CNV) leads to severe vision loss in patients of age-related macular degeneration. Previously, we have demonstrated that deguelin, isolated from plants in the Mundulea sericea family, is a chemopreventive agent. This study evaluates the antiangiogenic effect of deguelin on CNV. The toxicity of deguelin was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in human umbilical vein endothelial cells (HUVECs) as well as histological examination and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining in the deguelin-injected retina. Antiangiogenic activity of deguelin was evaluated by in vitro tube formation assay of HUVECs and in vivo angiogenesis of chick chorioallantoic membrane (CAM). In C57BL/6 mice with laser-induced CNV, deguelin or phosphate-buffered saline was injected intravitreously. CNV lesions were examined by fluorescence angiography and vessel counting in cross-sections. Deguelin showed no effect on cell viability of HUVECs and no retinal toxicity in a concentration range of 0.01 to 1 microM. Deguelin effectively inhibited in vitro tube formation of HUVECs and in vivo angiogenesis of CAM. Interestingly, deguelin significantly reduced CNV and its leakage in mouse model of laser photocoagulation-induced CNV. Our data suggests that deguelin is a potent inhibitor of CNV and may be applied in the treatment of other vasoproliferative retinopathies such as retinopathy of prematurity and diabetic retinopathy.

Inhibition of Tumor Angiogenesis by Targeting Endothelial Surface ATP Synthase with Sangivamycin

Sangivamycin, an antibiotic with anti-tumor and anti-herpes virus activities by inhibiting both DNA/RNA synthesis and protein kinase C activity, was reported to suppress selectively DNA synthesis and growth of human umbilical vein endothelial cells and their tube formation in vitro. Here, to address the potential clinical use of sangivamycin in future, we investigated its anti-angiogenic effect in in vivo chicken chorioallantoic membrane (CAM) and mouse dorsal air sac (DAS) assays, and investigated underlying mechanism. The effect of sangivamycin on blood vessel formation in CAM was observed under the microscope after treating for two days. For DAS assays, chambers fulfilled with tumor cells were implanted beneath mouse dorsal skin. After the mice were administered with sangivamycin, tumor-induced angiogenesis was observed under the microscope. The effect of sangivamycin on ATP synthesis on the endothelial cell surface was assayed by measuring ATP production with bioluminescence assay. Sangivamycin suppressed angiogenesis within CAM down to 94-71%, which was partially blocked by simultaneous addition of a 40-fold excess of adenosine. Sangivamycin also inhibited tumor-angiogenesis in the DAS assay by 61%, and suppressed ATP production on the endothelial cell surface by 75%. Sangivamycin inhibits the in vivo angiogenesis within CAM and tumor-induced angiogenesis within mouse dorsal skin, at least in part via inhibiting endothelial cell surface ATP metabolism in addition to inhibition of DNA/RNA synthesis and/or protein kinase C activity, suggesting a potential clinical use of sangivamycin as a novel anti-cancer reagent capable of targeting not only cancer cells but also endothelial cells.

Effects of three compounds extracted from Tripterygium wilfordii Hook on angiogenesis in chick chorioallantoic membrane

To investigate the effects of three compounds extracted from Tripterygium wilfordii Hook (TW) on angiogenesis in the chick chorioallantoic membrane (CAM). Fifty fresh Hongkong Mahua chicken eggs were divided into five groups: PBS-treated group, TW1-, TW2- and TW3-treated groups and Rg3-treated group. After disinfection, the eggs were incubated for six days in a constant temperature box with the temperature being controlled within 37.8 degrees C, then exposed CAM, laid the filter papers with specimen on the CAM, and the eggs were incubated for another two days. CAM was fixed with the mixture of methyl alcohol and acetone at room temperature for about 15 min, and then cutting the CAM, taking photos and observing the angiogenesis in the CAM. There were many CAM vessels in the PBS-treated group and the blood vessel net could be seen clearly. The number of CAM vessels in the TW1-, TW2- and TW3-treated groups (10 microg/egg) was much less than that in the PBS-treated group. Furthermore, the frame of the vessels was not clear, and the color was obscure. Inhibition rates of angiogenesis in the TW1-, TW2- and TW3-treated groups were 80%, 60% and 100% respectively, while the inhibition rate of angiogenesis in the Rg3-treated group (10 microg/egg) was only 10%. TW1, TW2 and TW3 can obviously restrain the angiogenesis in CAM and still need further study.

Intra- and extracellular signaling by endothelial neuregulin-1

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.

Antisense oligonucleotide targeting midkine suppressesin vivoangiogenesis

To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) and in situ human hepatocellular carcinoma (HCC). An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylin-eosin (HE) staining. MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues. MK-AS is an effective antiangiogenesis agent in vivo.

Antiangiogenic activity of 4′-thio-β-d-arabinofuranosylcytosine

4'-Thio-beta-D-arabinofuranosylcytosine (T-araC), a new-generation deoxycytidine nucleoside analogue, showed significant efficacy against numerous solid tumors in preclinical studies and entered clinical development for cancer therapy. It is a structural analogue of cytarabine (araC), a clinically used drug in the treatment of acute myelogenous leukemia, which has no or very limited efficacy against solid tumors. In comparison with araC, the excellent in vivo activity of T-araC against solid tumors suggests that, in addition to inhibition of DNA synthesis, T-araC may target cellular signaling pathways, such as angiogenesis, in solid tumors. We studied T-araC and araC for their antiangiogenic activities in vitro and in vivo. Both compounds inhibited human endothelial cell proliferation with similar IC50s. However, only T-araC inhibited endothelial cell migration and differentiation into capillary tubules. T-araC also abrogated endothelial cell extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, a key signaling molecule involved in cellular processes of angiogenesis. Results from chick chorioallantoic membrane angiogenesis assays revealed that T-araC significantly inhibited the development of new blood vessels in vivo, whereas araC showed much less effect. The findings of this study show a role of T-araC in antiangiogenesis and suggest that T-araC combines antiproliferative and antiangiogenic activity in one molecule for a dual mechanism of drug action to achieve the excellent in vivo efficacy against several solid tumors. This study also provides important information for optimizing dosage and sequence of T-araC administration in clinical investigations by considering T-araC as both an antiproliferative and an antiangiogenic agent.

Chrysin Inhibits Lipopolysaccharide-Induced Angiogenesis via Down-Regulation of VEGF/VEGFR-2(KDR) and IL-6/IL-6R Pathways

The relationship between chrysin and inflammation-induced angiogenesis remains unclear. The aim of this study was to evaluate the suppressive effects of chrysin on lipopolysaccharide (LPS)-induced angiogenesis in chicken chorioallantoic membrane (CAM) as well as in human umbilical endothelial cells (HUVEC). The IN VIVO CAM model was applied to evaluate the percentage of new vessels formation, followed by measuring endothelial migration and tube formation in HUVEC cultures. The mechanisms of the suppressive effect of chrysin on LPS-induced angiogenesis, in terms of VEGF, VEGF receptors (VEGFR), interleukin 6 (IL-6) and IL-6 receptor gene expressions, were analyzed by Western blot, ELISA cytokine assay, and quantitative real time PCR. The results showed that chrysin (10(-8) - 10(-5) M) inhibited LPS-induced CAM neovascular density. There was a significant down-regulation of VEGF and VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) gene expression by chrysin in LPS-treated HUVEC cultures. Besides, chrysin concentration-dependently inhibited the auto-regulation loop of IL-6/IL-6R in LPS-treated HUVEC cells. We conclude that chrysin suppresses both IN VITRO and IN VIVO LPS-induced angiogenesis.