The chick embryo chorioallantoic membrane assay as a model for the study of angiogenesis / Ensaio da membrana corioalantóide de embrião de galinha como modelo para o estudo da angiogênese

Angiogenesis is a fundamental physiological process with strong implications in tissue homeostasis. Animal models helping to identify how angiogenesis is regulated are fundamental to answer many biological questions. Chick embryo chorioallantoic membrane (CAM) assay is one of the most employed methods to study angiogenesis. In this study we applied a scientometric approach to evaluate the employment of CAM assay in published articles. Temporal trends indicated that CAM assay was the preferred method to investigate angiogenesis over time. The publications had a significant number of citations and the impact factor of journals publishing articles is relevant for the scientific community. A total of 52 different research areas have articles published using this particular technique. Oncology is the research field in which CAM assay was mostly used. Accordingly, tumor-derived cell lines were the most frequent sample tested on CAM. We also identified that 73,6% of articles published used only CAM assay to answer questions concerning angiogenesis. We concluded that although the CAM assay is a classical approach, that does not need so much infrastructure and financial support to be performed, it is a well-accepted technique by the scientific community. In addition, this methodology has gain attention in scientific community because no pain is experienced by the chick and they are minor ethical concerns to employ this method. Moreover, this data can help researchers who are unfamiliar with the CAM assay to identify if this particular method is suitable for their research.

A angiogênese é um processo fisiológico fundamental com fortes implicações na homeostase tecidual. Modelos animais que ajudam a entender como a angiogênese é regulada, são fundamentais para responder a muitas questões biológicas. O ensaio de membrana corioalantóide de embrião de galinha (CAM) é um dos métodos mais empregados para estudar a angiogênese. Neste estudo foi aplicada uma abordagem cientométrica para avaliar o emprego do ensaio CAM em artigos científicos já publicados. Tendências temporais indicaram que o ensaio CAM foi o método mais usado para investigar a angiogênese ao longo do tempo. Os artigos científicos que usaram a metodologia CAM foram publicados em periódicos com significativos números de citações e fator de impacto. No total 52 diferentes áreas de conhecimento usaram a técnica CAM, sendo a oncologia o campo o qual produziu maior número de artigos usando essa metodologia. Consequentemente o material biológico mais testado foi as linhagens celulares tumorais. Também foi identificado que 73,6% dos artigos publicados utilizaram apenas o teste CAM para responder questões relacionadas à angiogênese. Pode se concluir que embora o ensaio CAM seja uma abordagem clássica, que não necessita de muita infraestrutura e apoio financeiro para ser realizado, é uma técnica bem aceita pela comunidade científica. Além disso, esta metodologia tem ganhado atenção na comunidade científica porque os animais testados não sofrem dor e por essa razão esse modelo experimental exige mínimas preocupações éticas. Além disso, esses dados podem ajudar os pesquisadores que não estão familiarizados com o ensaio CAM a identificar se esse método específico é adequado para sua pesquisa.

Cytotoxic and angiosuppressive potentials of Zehneria japonica (Thunb. ex Murray) S.K. Chen (Cucurbitaceae) crude leaf extracts

@#<p style="text-align: justify;"><strong>BACKGROUND AND OBJECTIVES: </strong>Zehneria japonica belongs to the Cucurbitaceae family which is one of the most important plant families. It is commonly known as "Pipinong-gubat," widely distributed in Central Luzon regions and in areas along streams and clearings at low and medium altitudes in the Philippines. This study aimed to evaluate the potential cytotoxic and angiosuppressive properties of Zehneria japonica (Thunb. ex Murray) S.K. Chen (Cucurbitaceae) leaf extracts.</p><p style="text-align: justify;"><strong>METHODOLOGY:</strong> The Z japonica semi-crude extracts were obtained by sequential extraction using hexane, ethyl acetate, and n-butanol. A modified duck egg chorioallantoic membrane (CAM) assay was aided by AngioQuant, a digital imaging software used to evaluate angiogenic activity. Inhibition of angiogenesis was evaluated by percent increase or decrease in mean length of blood vessels, mean size of blood vessels, and total number of blood vessel junctions. Moreover, the cytotoxic effects of the extracts were determined through MTT Assay. Osteosarcoma (U20s) and hepatocellular carcinoma (HepG2) cells were used as cancer representatives while human umbilical vein endothelial cells (HUVEC) were used as the normal cell control.</p><p style="text-align: justify;"><strong>RESULTS:</strong> Analysis with AngioQuant revealed that treatment of the duck egg CAM with Z. japonica semi-crude extracts suppressed angiogenesis with ICso values of 1,810.00 ug/mL, 192.50 ug/ml, and 147.70 ug/mL for hexane, ethyl acetate, and n-butanol, respectively, with Celecoxib (20 ug/mL) as the positive control. For MTT assay, Z. japonica extracts exhibited strong cytotoxic effect against U2Os with an ICso values of 19.65 ug/mL, 9.89 ug/ml, and 31.04 ve/mL for the hexane, ethyl acetate, and n-butanol extracts, and no cytotoxic effects against HepG2 with IC50 values of 770.90 ug/mL, 130.10 ug/mL and 231.60 ug/mL for the hexane, ethyl acetate, and n-butanol extracts. Doxorubicin (0.544 ug/mL) was used as the positive control. The extracts also inhibited the growth of the normal cells, with IC50 values of 69.46 ng/mL, 42.23 ug/mL and 63.44 ug/mL for the hexane, ethyl acetate, and n-butanol extracts. There were no mortality and toxic symptoms observed for 14 days after the administration of the crude butanolic extract of Z. japonica in six female Sprague Dawley rats.</p><p style="text-align: justify;"><strong>CONCLUSION:</strong> Z. japonica crude leaf extracts exhibited angio-suppressive activity through CAM assay. In MTT assay, the extracts exhibited strong cytotoxicity in U20s (IC50 S20 ug/mL), no cytotoxic effect in HepG2 (ICso >100 ug/mL) cells, and mild cytotoxic effect in HUVEC (IC50 40-60 ug/mL). Phytochemical screening through TLC revealed that the extracts contain alkaloids, anthrones, flavonoids, and sterols. </p>

In vitro and in vivo evaluation of the antiangiogenic activities of Trigonella foenum-graecum extracts

Objective To assess the antiangiogenic activity of fenugreek. Methods Different fractions of fenugreek crude extracts were prepared and their antiangiogenic properties were assessed using the ex vivo rat aortic ring assay and in vivo chicken embryo chorioallantoic membrane (CAM) assay. They were investigated for their direct cytotoxic activity in the MCF7 cells using the MTT assay. Results The ethanol extract showed 100% inhibition of blood vessel outgrowth from primary tissue explants in the rat aortic ring assay at a concentration of 100 μg/mL while the other extracts did not show significant antiangiogenic activity. The ethanol extract was therefore investigated at varying concentrations and exhibited a significant dose dependent effect. The CAM assay coincided with the results of the aortic ring assay as ethanol extract showed a significant inhibition of formation of new blood vessels. The extracts only showed anti-proliferative activity at the highest concentration of 400 μg/mL towards MCF7 breast cancer cell lines in the MTT assay. Conclusions Findings of the both assays confirmed that the ethanol extract inhibited vascularization significantly. Further studies on the ethanol extract would be beneficial in isolating the active ingredient responsible for the inhibition.

In vitro and in vivo evaluation of the antiangiogenic activities of Trigonella foenum-graecum extracts

Objective: To assess the antiangiogenic activity of fenugreek. Methods: Different fractions of fenugreek crude extracts were prepared and their anti-angiogenic properties were assessed using the ex vivo rat aortic ring assay and in vivo chicken embryo chorioallantoic membrane (CAM) assay. They were investigated for their direct cytotoxic activity in the MCF7 cells using the MTT assay. Results: The ethanol extract showed 100％ inhibition of blood vessel outgrowth from primary tissue explants in the rat aortic ring assay at a concentration of 100μg/mL while the other extracts did not show significant antiangiogenic activity. The ethanol extract was therefore investigated at varying concentrations and exhibited a significant dose dependent effect. The CAM assay coincided with the results of the aortic ring assay as ethanol extract showed a significant inhibition of formation of new blood vessels. The extracts only showed anti-proliferative activity at the highest concentration of 400μg/mL towards MCF7 breast cancer cell lines in the MTT assay. Conclusions: Findings of the both assays confirmed that the ethanol extract inhibited vascularization significantly. Further studies on the ethanol extract would be beneficial in isolating the active ingredient responsible for the inhibition.

Angiogenesis inhibition by commercial soybean oil on duck chorioallantoic membrane in ovo

Angiogenesis inhibition is one of the fast developing approaches against tumor proliferation and metastasis. The angiogenesis-inhibition property of naturally-occuring peroxisome proliferator-activator receptor (PPAR) ligands, particularly of linoleic and linolenic acids that are present in commercially available soy bean oil, was investigated using chrorioallantoic membrane (CAM) assay. Human recombinant fibroblast growth factor (rhFGF) was utilized to stimulate human growth conditions on the CAM. Three groups consisting of 20 eggs each were treated with gelatin sponges containing: (1) rhFGF alone, the negative control; (2) soy bean oil and rhFGF, the treatment group; and. (3) rosiglitazone and rhFGF, the positive control. After incubation for 5 days, both macroscopic and microscopic methods of counting were employed. The treatment group demonstrated inhibition although it did not differ significantly from the negative control group (a=0.05, df=35, tcr=2.03, t=1.92). (Author)