Novel Nargenicin A1 Analog Inhibits Angiogenesis by Downregulating the Endothelial VEGF/VEGFR2 Signaling and Tumoral HIF-1α/VEGF Pathway

Targeting angiogenesis is an attractive strategy for the treatment of angiogenesis-related diseases, including cancer. We previously identified 23-demethyl 8,13-deoxynargenicin (compound 9) as a novel nargenicin A1 analog with potential anticancer activity. In this study, we investigated the antiangiogenic activity and mode of action of compound 9. This compound was found to effectively inhibit in vitro angiogenic characteristics, including the proliferation, invasion, capillary tube formation, and adhesion of human umbilical vein endothelial cells (HUVECs) stimulated by vascular endothelial growth factor (VEGF). Furthermore, compound 9 suppressed the neovascularization of the chorioallantoic membrane of growing chick embryos in vivo. Notably, the antiangiogenic properties of compound 9 were related to the downregulation of VEGF/VEGFR2-mediated downstream signaling pathways, as well as matrix metalloproteinase (MMP)-2 and MMP-9 expression in HUVECs. In addition, compound 9 was found to decrease the in vitro AGS gastric cancer cell-induced angiogenesis of HUVECs by blocking hypoxia-inducible factor-1α (HIF-1α) and VEGF expression in AGS cells. Collectively, our findings demonstrate for the first time that compound 9 is a promising antiangiogenic agent targeting both VEGF/VEGFR2 signaling in ECs and HIF-1α/VEGF pathway in tumor cells.

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Apigenin Suppresses Angiogenesis by Inhibiting Tube Formation and Inducing Apoptosis

Natural Product Communications ◽

10.1177/1934578x1601101005 ◽

2016 ◽

Vol 11 (10) ◽

pp. 1934578X1601101

Author(s):

Hyun Ju Kim ◽

Mok-Ryeon Ahn

Keyword(s):

Umbilical Vein ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Anticancer Activities ◽

Apoptotic Pathway ◽

Inhibition Of Angiogenesis ◽

Induced Apoptosis ◽

Umbilical Vein Endothelial Cells

Apigenin has been reported to exert angiogenic and anticancer activities in vitro. The mechanism of inhibition of angiogenesis by apigenin, however, has not been well-established. In this study, we investigated whether apigenin not only inhibited tube formation but also induced apoptosis in human umbilical vein endothelial cells (HUVECs). Furthermore, strong antiangiogenic activity of apigenin was observed in the in vivo assay using chick embryo chorioallantoic membrane (CAM). We also analyzed changes in survival signals and the apoptotic pathway through Western blotting. The results indicate that apigenin exerts its antiangiogenic effects through induction of endothelial apoptosis.

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Aspergillus fumigatus inhibits angiogenesis through the production of gliotoxin and other secondary metabolites

Blood ◽

10.1182/blood-2009-07-231209 ◽

2009 ◽

Vol 114 (26) ◽

pp. 5393-5399 ◽

Cited By ~ 78

Author(s):

Ronen Ben-Ami ◽

Russell E. Lewis ◽

Konstantinos Leventakos ◽

Dimitrios P. Kontoyiannis

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Capillary Tube ◽

Umbilical Vein ◽

Tube Formation ◽

Dependent Manner ◽

Antiangiogenic Effect ◽

Umbilical Vein Endothelial Cells

AbstractIn susceptible hosts, angioinvasion by Aspergillus fumigatus triggers thrombosis, hypoxia, and proinflammatory cytokine release, all of which are stimuli for angiogenesis. We sought to determine whether A fumigatus directly modulates angiogenesis. A fumigatus culture filtrates profoundly inhibited the differentiation, migration, and capillary tube formation of human umbilical vein endothelial cells in vitro. To measure angiogenesis at the site of infection, we devised an in vivo Matrigel assay in cyclophosphamide-treated BALB/c mice with cutaneous invasive aspergillosis. Angiogenesis was significantly suppressed in Matrigel plugs implanted in A fumigatus–infected mice compared with plugs from uninfected control mice. The antiangiogenic effect of A fumigatus was completely abolished by deletion of the global regulator of secondary metabolism, laeA, and to a lesser extent by deletion of gliP, which controls gliotoxin production. Moreover, pure gliotoxin potently inhibited angiogenesis in vitro in a dose-dependent manner. Finally, overexpression of multiple angiogenesis mediator–encoding genes was observed in the lungs of cortisone-treated mice during early invasive aspergillosis, whereas gene expression returned rapidly to baseline levels in cyclophosphamide/cortisone-treated mice. Taken together, these results indicate that suppression of angiogenesis by A fumigatus both in vitro and in a neutropenic mouse model is mediated through secondary metabolite production.

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Nymphaeol-A Isolated from Okinawan Propolis Suppresses Angiogenesis and Induces Caspase-Dependent Apoptosis via Inactivation of Survival Signals

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/826245 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 11

Author(s):

Ikumi Tsuchiya ◽

Takahiro Hosoya ◽

Motoko Ushida ◽

Kazuhiro Kunimasa ◽

Toshiro Ohta ◽

...

Keyword(s):

Screening Program ◽

Biological Activities ◽

Umbilical Vein ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Mitogen Activated Protein Kinase ◽

Cam Assay ◽

Prenylated Flavonoids

Propolis, a resinous substance that honeybees collect to protect their beehive from enemies, is reported to have various biological activities. In our screening program to search for antiangiogenic compounds from propolis, the ethanol extracts of Okinawan propolis (EEOP) showed significant antiangiogenic activities in a tube formation assay with human umbilical vein endothelial cells (HUVECs)in vitroat 3.13 μg/mL and chorioallantoic membrane (CAM) assayin vivoat 25 μg/egg. To elucidate the active compounds of EEOP and their mode of action, we isolated some prenylated flavonoids from EEOP and found that nymphaeol-A had the strongest antiangiogenic activity among them. Nymphaeol-A significantly reducedin vivoneovessel formation in the CAM assay at 25 μg/egg. At the molecular level, nymphaeol-A markedly inactivated mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2), whose molecular activations signal new vessel formation in HUVECs. In addition, nymphaeol-A dose- and time-dependently induced caspase-dependent apoptosis in tube-forming HUVECs. Taken together, nymphaeol-A was shown to inhibit angiogenesis at least in part via inactivation of MEK1/2–ERK1/2 signaling and induction of caspase-dependent apoptosis. Okinawan propolis and its major component, nymphaeol-A, may be useful agents for preventing tumor-induced angiogenesis.

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Korean Propolis Suppresses Angiogenesis through Inhibition of Tube Formation and Endothelial Cell Proliferation

Natural Product Communications ◽

10.1177/1934578x1400900434 ◽

2014 ◽

Vol 9 (4) ◽

pp. 1934578X1400900 ◽

Cited By ~ 4

Author(s):

Seon-Il Park ◽

Toshiro Ohta ◽

Shigenori Kumazawa ◽

Mira Jun ◽

Mok-Ryeon Ahn

Keyword(s):

Umbilical Vein ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

In Vitro Models ◽

Endothelial Cell Proliferation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Korean Propolis

Propolis, a sticky material that honeybees collect from living plants, has been used for its pharmaceutical properties since ancient times. In this study, we examined the effects of ethanol extracts of Korean propolis (EEKP) from various geographic regions on the inhibition of angiogenesis, both in vitro and in vivo. The effects of EEKP were tested on in vitro models of angiogenesis, that is, tube formation and proliferation of human umbilical vein endothelial cells (HUVECs). All EEKP samples exhibited significant inhibitory effects on tube formation of HUVECs in a concentration-dependent manner (6.25-25 μg/mL). In addition, two EEKP samples, prepared from Uijeongbu and Pyoseon propolis, significantly suppressed the proliferation of HUVECs in a concentration-dependent manner (3.13-25 μg/mL). Furthermore, in an in vivo angiogenesis assay using the chick embryo chorioallantoic membrane (CAM) system, we found that the two EEKP samples significantly reduced the number of newly formed vessels. These results indicate that Korean propolis may have potential applications in the prevention and treatment of angiogenesis-related diseases such as cancer.

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In Vitro and In Vivo Antiangiogenic Activity of Crowberry (Empetrum nigrum var. japonicum)

Natural Product Communications ◽

10.1177/1934578x1601100422 ◽

2016 ◽

Vol 11 (4) ◽

pp. 1934578X1601100 ◽

Cited By ~ 2

Author(s):

Hong-Sook Bae ◽

Hyun Ju Kim ◽

Da Hye Jeong ◽

Takahiro Hosoya ◽

Shigenori Kumazawa ◽

...

Keyword(s):

Umbilical Vein ◽

Folk Medicine ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Empetrum Nigrum ◽

Antiangiogenic Activity

Crowberry, Empetrum nigrum var. japonicum, is widely used in folk medicine and grows naturally in Korea. Although some constituents and biological activity of Korean crowberry have been examined, there is little detailed information available. In this study, we investigated the effects of ethanol extracts of crowberry (EECB) on the inhibition of angiogenesis, both in vitro and in vivo. The effects of EECB were tested on in vitro models of angiogenesis, that is, tube formation and proliferation of human umbilical vein endothelial cells (HUVECs). EECB exhibited significant inhibitory effects on tube formation of HUVECs in a concentration-dependent manner. In addition, crowberry significantly suppressed the proliferation of HUVECs in a concentration-dependent manner. Furthermore, strong antiangiogenic activity of EECB samples was observed in the in vivo assay using chick embryo chorioallantoic membrane (CAM). These results indicate that crowberry may have potential applications in the prevention and treatment of angiogenesis-dependent human diseases.

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SiRNA Directed Against Annexin II Receptor Inhibits Angiogenesis via Suppressing MMP2 and MMP9 Expression

Cellular Physiology and Biochemistry ◽

10.1159/000369745 ◽

2015 ◽

Vol 35 (3) ◽

pp. 875-884 ◽

Cited By ~ 16

Author(s):

Hongyuan Song ◽

Dongyan Pan ◽

Weifeng Sun ◽

Cao Gu ◽

Yuelu Zhang ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Umbilical Vein ◽

Tube Formation ◽

Annexin Ii ◽

Mmp9 Expression ◽

Cell Arrest ◽

Umbilical Vein Endothelial Cells

Background/Aims: Annexin II receptor (AXIIR) is able to mediate Annexin II signal and induce apoptosis, but its role in angiogenesis remains unclear. This study tries to investigate the role of AXIIR in angiogenesis and the plausible molecular mechanism. Methods/Results: RNA interference technology was used to silence AXIIR, and the subsequent effects in vitro and in vivo were evaluated thereafter. Our data indicated that human umbilical vein endothelial cells (HUVECs) expressed AXIIR and knockdown of AXIIR significantly inhibited HUVECs proliferation, adhesion, migration, and tube formation in vitro and suppressed angiogenesis in vivo. Furthermore, AXIIR siRNA induced cell arrest in the S/G2 phase while had no effect on cell apoptosis. We found that these subsequent effects might be via suppressing the expression of matrix metalloproteinase 2and matrix metalloproteinase 9. Conclusion: AXIIR participates in angiogenesis, and may be a potential therapeutic target for angiogenesis related diseases.

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A novel vasculo-angiogenic effect of cilostazol mediated by cross-talk between multiple signalling pathways including the ERK/p38 MAPK signalling transduction cascade

Clinical Science ◽

10.1042/cs20110432 ◽

2012 ◽

Vol 123 (3) ◽

pp. 147-159 ◽

Cited By ~ 15

Author(s):

Ting-Hsing Chao ◽

Shih-Ya Tseng ◽

Yi-Heng Li ◽

Ping-Yen Liu ◽

Chung-Lung Cho ◽

...

Keyword(s):

Protein Kinase ◽

P38 Mapk ◽

Umbilical Vein ◽

Tube Formation ◽

Mitogen Activated Protein Kinase ◽

Signalling Pathways ◽

No Production ◽

Angiogenic Effect

Cilostazol is an anti-platelet agent with vasodilatory activity that acts by increasing intracellular concentrations of cAMP. Recent reports have suggested that cilostazol may promote angiogenesis. In the present study, we have investigated the effect of cilostazol in promoting angiogenesis and vasculogenesis in a hindlimb ischaemia model and have also examined its potential mechanism of action in vitro and in vivo. We found that cilostazol treatment significantly increased colony formation by human early EPCs (endothelial progenitor cells) through a mechanism involving the activation of cAMP/PKA (protein kinase A), PI3K (phosphoinositide 3-kinase)/Akt/eNOS (endothelial NO synthase) and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) signalling pathways. Cilostazol also enhanced proliferation, chemotaxis, NO production and vascular tube formation in HUVECs (human umbilical vein endothelial cells) through activation of multiple signalling pathways downstream of PI3K/Akt/eNOS. Cilostazol up-regulated VEGF (vascular endothelial growth factor)-A165 expression and secretion of VEGF-A in HUVECs through activation of the PI3K/Akt/eNOS pathway. In a mouse hindlimb ischaemia model, recovery of blood flow ratio (ipsilateral/contralateral) 14 days after surgery was significantly improved in cilostazol-treated mice (10 mg/kg of body weight) compared with vehicle-treated controls (0.63±0.07 and 0.43±0.05 respectively, P<0.05). Circulating CD34+ cells were also increased in cilostazol-treated mice (3614±670 compared with 2151±608 cells/ml, P<0.05). Expression of VEGF and phosphorylation of PI3K/Akt/eNOS and ERK/p38 MAPK in ischaemic muscles were significantly enhanced by cilostazol. Our data suggest that cilostazol produces a vasculo-angiogenic effect by up-regulating a broad signalling network that includes the ERK/p38 MAPK, VEGF-A165, PI3K/Akt/eNOS and cAMP/PKA pathways.

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Cilengitide Inhibits Neovascularization in a Rabbit Abdominal Aortic Plaque Model by Impairing the VEGF Signaling

BioMed Research International ◽

10.1155/2021/5954757 ◽

2021 ◽

Vol 2021 ◽

pp. 1-18

Author(s):

Fawang Zhu ◽

Shuai Yuan ◽

Jing Li ◽

Yun Mou ◽

Zhiqiang Hu ◽

...

Keyword(s):

Umbilical Vein ◽

Tube Formation ◽

Control Group ◽

In Vitro Experiments ◽

Aortic Plaque ◽

Abdominal Aortic ◽

Aortic Plaques ◽

Different Doses

Background. Cilengitide is a selective αvβ3 and αvβ5 integrin inhibitor. We sought to investigate the effect of cilengitide on the neovascularization of abdominal aortic plaques in rabbits and explore its underlying antiangiogenic mechanism on human umbilical vein endothelial cells (HUVECs). Materials and Methods. For the in vivo experiment, the abdominal aortic plaque model of rabbits was established and injected with different doses of cilengitide or saline for 14 consecutive days. Conventional ultrasound (CUS) and contrast-enhanced ultrasound (CEUS) were applied to measure the vascular structure and blood flow parameters. CD31 immunofluorescence staining was performed for examining neovascularization. Relative expressions of vascular endothelial growth factor (VEGF) and integrin of the plaque were determined. For in vitro experiments, HUVECs were tested for proliferation, migration, apoptosis, and tube formation in the presence of different doses of cilengitide. Relative expressions of VEGF, integrin, and Ras/ERK/AKT signaling pathways were determined for the exploration of underlying mechanism. Results. CEUS showed modestly increased size and eccentricity index (EI) of plaques in the control group. Different degrees of reduced size and EI of plaques were observed in two cilengitide treatment groups. The expressions of VEGF and integrin in the plaque were inhibited after 14 days of cilengitide treatment. The neovascularization and apoptosis of the abdominal aorta were also significantly alleviated by cilengitide treatment. For in vitro experiments, cilengitide treatment was found to inhibit the proliferation, migration, and tube formation of HUVECs. However, cilengitide did not induce the apoptosis of HUVECs. A higher dose of cilengitide inhibited the mRNA expression of VEGF-A, β3, and β5, but not αV. Lastly, cilengitide treatment significantly inhibited the Ras/ERK/AKT pathway in the HUVECs. Conclusions. This study showed that cilengitide effectively inhibited the growth of plaque size by inhibiting the angiogenesis of the abdominal aortic plaques and blocking the VEGF-mediated angiogenic effect on HUVECs.

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Abstract 11: Thioredoxin-Interacting Protein Is Required for VEGF-Induced Angiogenesis Through Regulation of VEGFR2 Internalization

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a11 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Shin-Young Park ◽

Chen Yan ◽

Bradford C Berk

Keyword(s):

Umbilical Vein ◽

Fold Increase ◽

Tube Formation ◽

In Vivo Studies ◽

Vegf Receptor ◽

Cell Fractionation ◽

Interacting Protein ◽

Thioredoxin Interacting Protein

Introduction— Thioredoxin-interacting protein (TXNIP) is an arrestin-like scaffold protein. We have shown previously that it is necessary for the transactivation of the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) as well as promoting the migration and survival of endothelial cells (ECs). However, its roles in VEGF-induced angiogenesis and in vivo studies of TXNIP function have not been elucidated. Hypothesis— TXNIP regulates VEGF-mediated angiogenesis through modulation of angiogenic signaling pathways in ECs. Methods and Results— To determine the functions of TXNIP in ECs, we generated endothelial-specific TXNIP knockout (EC-TXNIP KO) mice (TXNIPflox/flox: Tie2-Cre/+). These mice displayed impaired capillary growth of the retinal vasculature compared to control mice. Furthermore, aortic rings from EC-TXNIP KO mice exhibited fewer and shorter vascular sprouts than those in control mice. To investigate the role of TXNIP in the regulation of VEGF-induced angiogenesis, we determined the subcellular localization of TXNIP in human umbilical vein EC (HUVEC). Immunofluorescence and cell fractionation studies revealed that upon VEGF stimulation (10ng/ml). TXNIP translocated from cytoplasm to the plasma membrane. There was a 9 fold increase of membrane associated TXNIP with a peak at 15 minutes compared to non-VEGF treatment cells. We hypothesized that membrane associated TXNIP may modulate VEGFR2 internalization and thereby affect VEGF-induced signaling and angiogenesis. To investigate this, we performed in vitro cell surface biotinylation assays in HUVEC. VEGFR2 internalization was decreased by 65% in TXNIP siRNA knockdown cells compared to control siRNA treated cells following VEGF stimulation. Consistent with this result, VEGF-induced phosphorylation of VEGFR2, PLCγ and ERK1/2 was decreased by knockdown of TXNIP. Significantly, TXNIP knockdown inhibited VEGF-induced proliferation and tube formation in vitro. Conclusion— Our results suggest that TXNIP can modulate VEGF-induced angiogenesis and signaling by regulation of VEGFR2 internalization.

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Topical Application of Hyaluronic Acid-RGD Peptide-Coated Gelatin/Epigallocatechin-3 Gallate (EGCG) Nanoparticles Inhibits Corneal Neovascularization via Inhibition of VEGF Production

Pharmaceutics ◽

10.3390/pharmaceutics12050404 ◽

2020 ◽

Vol 12 (5) ◽

pp. 404 ◽

Cited By ~ 1

Author(s):

Takuya Miyagawa ◽

Zhi-Yu Chen ◽

Che-Yi Chang ◽

Ko-Hua Chen ◽

Yang-Kao Wang ◽

...

Keyword(s):

Hyaluronic Acid ◽

Topical Application ◽

Umbilical Vein ◽

Corneal Neovascularization ◽

Tube Formation ◽

Antiangiogenic Effect ◽

Arginine Glycine Aspartic Acid ◽

Umbilical Vein Endothelial Cells

Neovascularization (NV) of the cornea disrupts vision which leads to blindness. Investigation of antiangiogenic, slow-release and biocompatible approaches for treating corneal NV is of great importance. We designed an eye drop formulation containing gelatin/epigallocatechin-3-gallate (EGCG) nanoparticles (NPs) for targeted therapy in corneal NV. Gelatin-EGCG self-assembled NPs with hyaluronic acid (HA) coating on its surface (named GEH) and hyaluronic acid conjugated with arginine-glycine-aspartic acid (RGD) (GEH-RGD) were synthesized. Human umbilical vein endothelial cells (HUVECs) were used to evaluate the antiangiogenic effect of GEH-RGD NPs in vitro. Moreover, a mouse model of chemical corneal cauterization was employed to evaluate the antiangiogenic effects of GEH-RGD NPs in vivo. GEH-RGD NP treatment significantly reduced endothelial cell tube formation and inhibited metalloproteinase (MMP)-2 and MMP-9 activity in HUVECs in vitro. Topical application of GEH-RGD NPs (once daily for a week) significantly attenuated the formation of pathological vessels in the mouse cornea after chemical cauterization. Reduction in both vascular endothelial growth factor (VEGF) and MMP-9 protein in the GEH-RGD NP-treated cauterized corneas was observed. These results confirm the molecular mechanism of the antiangiogenic effect of GEH-RGD NPs in suppressing pathological corneal NV.

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