scholarly journals Evaluation of the Anti-angiogenic Activity of Saponins from Maesa lanceolata by Different Assays

2012 ◽  
Vol 7 (9) ◽  
pp. 1934578X1200700
Author(s):  
Kenn Foubert ◽  
Annelies Breynaert ◽  
Mart Theunis ◽  
Rita Van Den Bossche ◽  
Guido R.Y. De Meyer ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Chorioallantoic Membrane ◽  
Rat Aorta ◽  
Cam Assay ◽  
Plant Metabolites ◽  
Angiogenic Activity ◽  
Test Systems ◽  
Aorta Ring

Angiogenesis, in which a vascular network is established from pre-existing vessels, is a complex multistep process. Mechanisms underlying angiogenesis can be investigated using a variety of in vitro, ex vivo and in vivo approaches. Evaluation of several promising plants and plant metabolites, including terpenoids, revealed promising anti-angiogenic activity. Since the maesasaponins displayed anti-angiogenic activity in the chick chorioallantoic membrane (CAM) assay, their activity was further investigated in several test systems. The rat aorta ring assay was compared with the placental vein assay and then selected for the ex vivo investigation of the saponins. Besides their effect on the viability of HUVEC, the anti-angiogenic capacity of the compounds was also investigated in an in vivo zebrafish assay. The activity of the saponins in the viability assay was more pronounced than in the rat aorta ring assay and similar to the effect observed in the CAM assay. The use of different test systems, however, implies different results in the case of saponins.

Effects Of Pentoxifylline, Penbutolol, Prenylamine, Clofibric Acid And Nicotinic Acid On The Release Of Prostacyclin-(PGI2-) Like Activity From Rat Aorta In Vivo And In Vitro

1981 ◽  
Author(s):  
K U Weithmann ◽  
A G Hoechst
Keyword(s):  
Cyclic Amp ◽  
Nicotinic Acid ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Rat Aorta ◽  
Clofibric Acid ◽  
Vessel Walls ◽  
Induced Aggregation

Aortas from rats, treated with 5-20 mg/kg of pentoxifylline (pof), penbutolol, prenylamine, clofibric acid or nicotinic acid showed, ex vivo, a significantly higher release of acid labile PGI2-like anti-aggregatory activity compared to controls. This activity could be suppressed by pre-treatment with 2 mg/kg Indomethacin. When incubated with rat aortas in vitro, pof showed a similar stimulatory effect on PGI2-like release, whereas clofibric-and nicotinic acid had no significant effect in this system. Pof and all other drugs mentioned above in therapeutical concentrations had virtually no effect on induced aggregation of human platelets in vitro. However, in the presence of small amounts PGI2 in vitro, inhibition of aggregation and platelet cyclic AMP are enhanced synergistically above the effects of PGI2 and pof individually.We conclude from these experiments that therapeutic doses of all drugs in our study stimulate in vivo the release of PGI2-like activity from vessel walls, thus inhibiting platelet aggregation in vivo. The primary site of action of pof seems to be the vessel wall, whereas the effect of clofibric acid and nicotinic acid on the vessel walls seem to be secondary. The elevation of platelet cyclic AMP levels which generally parallels PGI2-induced inhibition of aggregation might be further enhanced by pof known as an inhibitor of platelet cyclic AMP phosphodiesterase, thus explaining the observed synergistic effects between PGI2 and pof.

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

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
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.

scholarly journals Antiangiogenic Activity of Flavonoids: A Systematic Review and Meta-Analysis

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4712
Author(s):  
Mai Khater ◽  
Francesca Greco ◽  
Helen M. I. Osborn
Keyword(s):  
Systematic Review ◽  
Ex Vivo ◽  
Meta Analysis ◽  
Chorioallantoic Membrane ◽  
Structural Features ◽  
Antiangiogenic Agents ◽  
Antiangiogenic Activity ◽  
Wide Range

An imbalance of angiogenesis contributes to many pathologies such as cancer, arthritis and retinopathy, hence molecules that can modulate angiogenesis are of considerable therapeutic importance. Despite many reports on the promising antiangiogenic properties of naturally occurring flavonoids, no flavonoids have progressed to the clinic for this application. This systematic review and meta-analysis therefore evaluates the antiangiogenic activities of a wide range of flavonoids and is presented in two sections. The first part of the study (Systematic overview) included 402 articles identified by searching articles published before May 2020 using ScienceDirect, PubMed and Web of Science databases. From this initial search, different classes of flavonoids with antiangiogenic activities, related pathologies and use of in vitro and/or in/ex vivo angiogenesis assays were identified. In the second part (Meta-analysis), 25 studies concerning the antiangiogenic evaluation of flavonoids using the in vivo chick chorioallantoic membrane (CAM) assay were included, following a targeted search on articles published prior to June 2020. Meta-analysis of 15 out of the 25 eligible studies showed concentration dependent antiangiogenic activity of six compared subclasses of flavonoids with isoflavones, flavonols and flavones being the most active (64 to 80% reduction of blood vessels at 100 µM). Furthermore, the key structural features required for the antiangiogenic activity of flavonoids were derived from the pooled data in a structure activity relationship (SAR) study. All in all, flavonoids are promising candidates for the development of antiangiogenic agents, however further investigations are needed to determine the key structural features responsible for their activity.

scholarly journals The integrin-binding defective FGF2 mutants potently suppress FGF2 signalling and angiogenesis

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Seiji Mori ◽  
Nobuaki Hatori ◽  
Naomasa Kawaguchi ◽  
Yoshinosuke Hamada ◽  
Tsung-Chieh Shih ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Ex Vivo ◽  
Integrin Αvβ3 ◽  
Tube Formation ◽  
Nih3t3 Cells ◽  
Aorta Ring ◽  
Binding Interface

We recently found that integrin αvβ3 binds to fibroblast growth factor (FGF)-αvβ31 (FGF1), and that the integrin-binding defective FGF1 mutant (Arg-50 to glutamic acid, R50E) is defective in signalling and antagonistic to FGF1 signalling. R50E suppressed angiogenesis and tumour growth, suggesting that R50E has potential as a therapeutic. However, FGF1 is unstable, and we had to express R50E in cancer cells for xenograft study, since injected R50E may rapidly disappear from circulation. We studied if we can develop antagonist of more stable FGF2. FGF2 is widely involved in important biological processes such as stem cell proliferation and angiogenesis. Previous studies found that FGF2 bound to αvβ3 and antagonists to αvβ3 suppressed FGF2-induced angiogenesis. However, it is unclear how FGF2 interacts with integrins. Here, we describe that substituting Lys-119/Arg-120 and Lys-125 residues in the predicted integrin-binding interface of FGF2 to glutamic acid (the K119E/R120E and K125E mutations) effectively reduced integrin binding to FGF2. These FGF2 mutants were defective in signalling functions (ERK1/2 activation and DNA synthesis) in NIH3T3 cells. Notably they suppressed, FGF2 signalling induced by WT FGF2 in endothelial cells, suggesting that the FGF2 mutants are antagonists. The FGF2 mutants effectively suppressed tube formation in vitro, sprouting in aorta ring assays ex vivo and angiogenesis in vivo. The positions of amino acids critical for integrin binding are different between FGF1 and FGF2, suggesting that they do not interact with integrins in the same manner. The newly developed FGF2 mutants have potential as anti-angiogenic agents and useful tools for studying the role of integrins in FGF2 signalling.

In vivo angiogenic activity of urokinase: role of endogenous fibroblast growth factor-2

1999 ◽  
Vol 112 (23) ◽  
pp. 4213-4221
Author(s):  
D. Ribatti ◽  
D. Leali ◽  
A. Vacca ◽  
R. Giuliani ◽  
A. Gualandris ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Chorioallantoic Membrane ◽  
Fibroblast Growth Factor 2 ◽  
Dependent Manner ◽  
Fibroblast Growth ◽  
Angiogenic Activity ◽  
Amino Terminal

In vitro experimental evidences suggest that the proteolytic degradation of the extracellular matrix (ECM) by activation of the urokinase-type plasminogen activator (uPA)/plasmin system may affect growth factor activity and bioavailability. However, no direct in vivo observations were available to support this hypothesis. Here we demonstrate that endothelial GM 7373 cells overexpressing human uPA (uPA-R5 cells) cause the release of (125)I-labeled fibroblast growth factor-2 (FGF2) from endothelial ECM in a plasmin-dependent manner. Accordingly, uPA-R5 cells are angiogenic in vivo when applied on the top of the chorioallantoic membrane (CAM) of the chick embryo. In contrast, mock-transfected Neo2 cells are unable to release ECM-bound (125)I-FGF2 and are poorly angiogenic. Neovascularization elicited by uPA-R5 cells is significantly reduced by neutralizing anti-FGF2 antibodies to values similar to those observed in Neo2 cell-treated CAMs. Accordingly, purified human uPA stimulates neovascularization of the CAM in the absence of an inflammatory response. The angiogenic activity of uPA is significantly inhibited by neutralizing anti-FGF2 antibodies or by pretreatment with phenylmethylsulfonyl fluoride. The non-catalytic, receptor-binding amino-terminal fragment of uPA is instead non angiogenic. Taken together, the data indicate that uPA is able to induce angiogenesis in vivo via a plasmin-dependent degradation of ECM that causes the mobilization of stored endogenous FGF2.

scholarly journals Cathepsin L promotes angiogenesis by regulating the CDP/Cux/VEGF-D pathway in human gastric cancer

2020 ◽  
Vol 23 (6) ◽  
pp. 974-987
Author(s):  
Tao Pan ◽  
Zhijian Jin ◽  
Zhenjia Yu ◽  
Xiongyan Wu ◽  
Xinyu Chang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cathepsin L ◽  
Chorioallantoic Membrane ◽  
Luciferase Reporter ◽  
Antibody Array ◽  
Human Gastric Cancer ◽  
Cam Assay ◽  
Migration Assay

Abstract Background Increasing evidence indicates that angiogenesis plays an important role in tumor progression. The function of cathepsin L (CTSL), an endosomal proteolytic enzyme, in promoting tumor metastasis is well recognized. The mechanisms by which CTSL has promoted the angiogenesis of gastric cancer (GC), however, remains unclear. Methods The nuclear expression levels of CTSL were assessed in GC samples. The effects of CTSL on GC angiogenesis were determined by endothelial tube formation analysis, HUVEC migration assay, and chick embryo chorioallantoic membrane (CAM) assay. The involvement of the CDP/Cux/VEGF-D pathway was analyzed by angiogenesis antibody array, Western blot, co-immunoprecipitation (Co-IP) and dual-luciferase reporter assay. Results In this study, we found that the nuclear CTSL expression level in GC was significantly higher than that in adjacent nontumor gastric tissues and was a potential important clinical prognostic factor. Loss- and gain-of-function assays indicated that CTSL promotes the tubular formation and migration of HUVEC cells in vitro. The CAM assay also showed that CTSL promotes angiogenesis of GC in vivo. Mechanistic analysis demonstrated that CTSL can proteolytically process CDP/Cux and produce the physiologically relevant p110 isoform, which stably binds to VEGF-D and promotes the transcription of VEGF-D, thus contributing to the angiogenesis of GC. Conclusion The findings of the present study suggested that CTSL plays a constructive role in the regulation of angiogenesis in human GC and could be a potential therapeutic target for GC.

scholarly journals Microvascular Experimentation in the Chick Chorioallantoic Membrane as a Model for Screening Angiogenic Agents including from Gene-Modified Cells

2021 ◽  
Vol 23 (1) ◽  
pp. 452
Author(s):  
Donna C. Kennedy ◽  
Barbara Coen ◽  
Antony M. Wheatley ◽  
Karl J. A. McCullagh
Keyword(s):  
Low Cost ◽  
Chorioallantoic Membrane ◽  
Visual Assessment ◽  
Cam Assay ◽  
Chick Chorioallantoic Membrane ◽  
Assay Procedure ◽  
Angiogenic Effect ◽  
Cultivation Techniques

The chick chorioallantoic membrane (CAM) assay model of angiogenesis has been highlighted as a relatively quick, low cost and effective model for the study of pro-angiogenic and anti-angiogenic factors. The chick CAM is a highly vascularised extraembryonic membrane which functions for gas exchange, nutrient exchange and waste removal for the growing chick embryo. It is beneficial as it can function as a treatment screening tool, which bridges the gap between cell based in vitro studies and in vivo animal experimentation. In this review, we explore the benefits and drawbacks of the CAM assay to study microcirculation, by the investigation of each distinct stage of the CAM assay procedure, including cultivation techniques, treatment applications and methods of determining an angiogenic response using this assay. We detail the angiogenic effect of treatments, including drugs, metabolites, genes and cells used in conjunction with the CAM assay, while also highlighting the testing of genetically modified cells. We also present a detailed exploration of the advantages and limitations of different CAM analysis techniques, including visual assessment, histological and molecular analysis along with vascular casting methods and live blood flow observations.

scholarly journals The avian chorioallantoic membrane as an alternative tool to study medullary thyroid cancer

2019 ◽  
Vol 8 (5) ◽  
pp. 462-467 ◽  
Author(s):  
Nassim Ghaffari-Tabrizi-Wizsy ◽  
Christina Angelika Passegger ◽  
Laura Nebel ◽  
Fabian Krismer ◽  
Gudrun Herzer-Schneidhofer ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Drug Testing ◽  
Medullary Thyroid Cancer ◽  
Chorioallantoic Membrane ◽  
Cost Effective ◽  
Suitable Model ◽  
Cam Assay ◽  
Medullary Thyroid

Preclinical trials of medullary thyroid cancer (MTC) therapeutics require both in vitro and in vivo analyses. Human tumour xenografted rodent models, which are considered the ‘gold standard’ to study and validate the efficacy and toxicity of lead compounds before translation to clinical trials, are very expensive, subject to organismal variability and ethical controversies. The avian chorioallantoic membrane (CAM) assay provides an alternative versatile, cost-effective and ethically less objectionable short-term, in vivo model for reliable screening of drugs. In this work, we grafted two MTC cell lines and patient-derived MTC tumour samples onto the avian CAM and characterised the resulted tumours histologically and immunohistochemically. Our findings provide the evidence that the CAM assay is a suitable model for studying the pathophysiology of MTC and can even be used as in vivo system for drug testing.

scholarly journals Neuroprotection in early stages of Alzheimer’s disease is promoted by transthyretin angiogenic properties

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Tiago Gião ◽  
Joana Saavedra ◽  
José Ricardo Vieira ◽  
Marta Teixeira Pinto ◽  
Gemma Arsequell ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Chorioallantoic Membrane ◽  
Tube Formation ◽  
Cam Assay ◽  
Vessel Length ◽  
Angiogenic Potential ◽  
Chick Chorioallantoic Membrane ◽  
Brain Microvessels

Abstract Background While still controversial, it has been demonstrated that vascular defects can precede the onset of other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. Methods We evaluated the thickness of the basement membrane and the length of brain microvessels, by immunohistochemistry, in AβPPswe/PS1A246E (AD) transgenic mice and non-transgenic mice (NT) bearing one (TTR+/−) or two (TTR+/+) copies of the TTR gene. The angiogenic potential of TTR was evaluated in vitro using the tube formation assay, and in vivo using the chick chorioallantoic membrane (CAM) assay. Results AD transgenic mice with TTR genetic reduction, AD/TTR+/−, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the CAM assay, revealed that TTR is a pro-angiogenic molecule, and the neovessels formed are functional. Also, TTR increased the expression of key angiogenic molecules such as proteins interleukins 6 and 8, angiopoietin 2, and vascular endothelial growth factor, by endothelial cells, in vitro, under tube formation conditions. We showed that while TTR reduction also leads to a thicker BM in NT mice, this effect is more pronounced in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with the TTR tetrameric stabilizer iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length, when compared to non-treated littermates. Conclusion Our in vivo results demonstrate the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.

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