In ovo administration of human recombinant leptin shows dose dependent angiogenic effect on chicken chorioallantoic membrane

BACKGROUND: Leptin, the cytokine produced by white adipose tissue is known to regulate food energy homeostasis through its hypothalamic receptor. In vitro studies have demonstrated that leptin plays a major role in angiogenesis through binding to the receptor Ob-R present on ECs by stimulating and initiating new capillary like structures from ECs. Various in vivo studies indicate that leptin has diverse effect on angiogenesis. A few reports have showed that leptin exerts pro angiogenic effects while some suggested that it has antiangiogenic potential. It is theoretically highly important to understand the effect of leptin on angiogenesis to use as a therapeutic molecule in various angiogenesis related pathological conditions. Chicken chorio allantoic membrane (CAM) on 9th day of incubation was incubated with 1, 3 and 5 µg concentration of HRL for 72 h using gelatin sponge. Images where taken after every 24 h of incubation and analysed with Angioguant software. The treated area was observed under microscope and histological evaluation was performed for the same. Tissue thickness was calculated morphometrically from haematoxylin and eosin stained cross sections. Reverse transcriptase PCR and immunohistochemistry were also performed to study the gene and protein level expression of angiogenic molecules. RESULTS: HRL has the ability to induce new vessel formation at the treated area and growth of the newly formed vessels and cellular morphological changes occur in a dose dependent manner. Increase in the tissue thickness at the treated area is suggestive of initiation of new capillary like structures. Elevated mRNA and protein level expression of VEGF165 and MMP2 along with the activation of ECs as demonstrated by the presence of CD34 expression supports the neovascularization potential of HRL. CONCLUSION: Angiogenic potential of HRL depends on the concentration and time of incubation and is involved in the activation of ECs along with the major interaction of VEGF 165 and MMP2. It is also observed that 3 µg of HRL exhibits maximum angiogenic potential at 72 h of incubation. Thus our data suggest that dose dependent angiogenic potential HRL could provide a novel role in angiogenic dependent therapeutics such as ischemia and wound healing conditions.

Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.

Angiogenic potential of the cerebrospinal fluid (CSF) of patients with high-grade gliomas measured with the chick embryo chorioallantoic membrane assay (CAM)

High-grade gliomas are highly vascularized tumors. Neo-angiogenesis plays a key role in tumor growth and resistance to therapy. A cerebrospinal fluid (CSF) sample could be a useful way to obtain pro-angiogenic predictive or prognostic markers at different stages of the disease. As a first step we looked for pro-angiogenic activity in the CSF of patients with high-grade gliomas. We performed the chicken embryo chorio-allantoic membrane (CAM) assay to study the angiogenic potential of the cerebrospinal fluid (CSF), obtained either by lumbar puncture (LP) or craniotomy from six patients with high-grade brain tumors (three glioblastoma (WHO grade IV), one anaplastic oligodendroglioma (WHO grade III), two anaplastic ganglioglioma (WHO grade III)), and four healthy controls. Significantly increased neo-angiogenesis was observed on the surface of the growing CAM in the 6 patients with high-grade gliomas compared to controls (3.69 ± 1.23 versus 2.16 ± 0.97 capillaries per area (mean ± SD), p<0.005). There was no statistical difference related to the hystological grade of the tumor (WHO grade III or IV), previous treatment (radio-chemotherapy plus temozolomide, temozolomide alone or no treatment), or the site of CSF sample (surgery or lumbar puncture). Our results suggest a pro-angiogenic potential in the CSF of patients with high-grade gliomas.

Angiogenic activity of Calendula officinalis flowers L. in rats / Atividade angiogênica das flores da Calendula officinalis L. em ratos

Purpose: In this work, angiogenic activity of Calendula officinalis L. (Asteraceae) ethanolic extract and dichloromethane and hexanic fractions were evaluated, considering medicinal properties, especially healing activity, are attributed to this plant. Methods: Models using 36 rats and 90 embryonated eggs were used to evaluate healing and angiogenic activities of extracts and fractions of the plant, through the induction of skin wounds and the chorioallantoic membrane, respectively. The effect of vascular proliferation was also tested from the study to verify the intensity of expression of vascular endothelial growth factor (VEGF) in cutaneous wounds in rats. Results: The angiogenic activity of the extract and the fractions was evidenced in both experimental models. It was verified that this effect is not directly related to the expression of VEGF and it could be associated to other pro-angiogenic factors. Conclusion: The healing activity referred to C. officinalis is related, among other factors, to its positive effect on angiogenesis, characterized by the induction of neovascularization.

Objetivo: Neste trabalho a atividade sobre a angiogênese do extrato etanólico (EEC) e das frações diclorometano e hexânica das flores de Calendula officinalis L. (Asteraceae) cultivada no Brasil foram avaliados, visto que propriedades medicinais têm sido atribuídas às flores da planta, destacando-se a atividade cicatrizante. Métodos: Modelos utilizando 36 ratos e 90 ovos embrionados foram usados para avaliar as atividades cicatrizante e angiogênica dos extratos e frações da planta, por meio da indução de feridas cutâneas e da membrana corioalantóide, respectivamente. O efeito proliferativo vascular foi também testado a partir do estudo imunoistoquímico, realizado para verificar a intensidade da expressão do fator de crescimento endotelial vascular (VEGF) na derme de ratos. Resultados: A atividade angiogênica do extrato e das frações foi evidenciada nos dois modelos experimentais empregados. Foi evidenciado que este efeito não estava diretamente relacionado à expressão do VEGF, podendo estar associado a outros fatores pró-angiogênicos. Conclusão: A atividade cicatrizante referida a C. officinalis está relacionada ao seu efeito positivo sobre a angiogênese, e este foi caracterizado pela indução de neovascularização.

In vivo anti-angiogenic effects further support the promise of the antineoplasic activity of methyl jasmonate / Efeitos antiangiogênicos in vivo convalidam a atividade antineoplásica potencial do metiljasmonato

Molecular plant components have long been aimed at the angiogenesis and anti-angiogenesis pathways, and have been tested as sources for antineoplasic drugs with promising success. The present work deals with the anti-angiogenic effects of Methyl Jasmonate. Jasmonate derivatives were demonstrated to selectively damage the mitochondria of cancer cells. In vitro, 1-10 mM Methyl Jasmonate induced the cell death of the human umbilical vein endothelial cells (HUVEC) and the Murine melanoma cells (B16F10), while micromolar concentrations were ineffective. In vivo, comparable concentrations were toxic and reduced the vessel density of the Chorioallantoic Membrane of the Chicken Embryo (CAM). However, 1-10 µM concentrations produced a complex effect. There was increased capillary budding, but the new vessels were leakier and less organised than corresponding controls. It is suggested that not only direct toxicity, but also the drug effects upon angiogenesis are relevant to the antineoplasic effects of Methyl Jasmonate.

Moléculas de origem vegetal são, há muito, conhecidas como substâncias ativas sobre as vias de angiogênese e antiangiogênese e foram testadas como fonte de drogas antineoplásicas com sucesso promissor. Este trabalho trata dos efeitos antiangiogênicos do Metiljasmonato, um protótipo da família dos derivados do ácido jasmônico, que danificam seletivamente a mitocôndria de células neoplásicas. In vitro, metiljasmonato 1-10 mM promoveu a morte celular de células endoteliais humanas de cordão umbilical (HUVEC) e de melanoma murino (B16F10); concentrações micromolares foram inócuas. In vivo, concentrações equivalentes foram tóxicas e reduziram a densidade de vasos em membranas corioalantoicas de embrião de galinha (CAM). Entretanto, concentrações entre 1-10 µM produziram um efeito complexo. Ocorreu aumento no brotamento capilar, mas os novos vasos apresentaram-se frágeis e menos organizados que os controles correspondentes. Sugere-se que, além da toxicidade direta contra as células tumorais, a ação do metiljasmonato sobre a angiogênese seja relevante para seu efeito antineoplásico.

Friend erythroleukemia cells induce angiogenesis in chick embryo chorioallantoic membrane and in human umbilical vein endothelial cells

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias.