Antiangiogenic activity of photobiomodulation in experimental model using chorioallantoic embryonic membrane of chicken eggs / Atividade antiangiogênica da fotobiomodulação em modelo experimental usando membrana embrionária corioalantóide de ovos de galinhas

ABSTRACT Purpose: The purpose of this study was to investigate the vascular effects of photobiomodulation using a light-emitting diode on the chorioallantoic embryonic membrane of chicken eggs grouped into different times of exposure and to detect the morphological changes induced by the light on the vascular network architecture using quantitative metrics. Methods: We used a phototherapy device with light-emitting diode (670 nm wavelength) as the source of photobiomodulation. We applied the red light at a distance of 2.5 cm to the surface of the chorioallantoic embryonic membrane of chicken eggs in 2, 4, or 8 sessions for 90 s and analyzed the vascular network architecture using AngioTool software (National Cancer Institute, USA). We treated the negative control group with 50 μl phosphate-buffered-saline (pH 7.4) and the positive control group (Beva) with 50 μl bevacizumab solution (Avastin, Produtos Roche Químicos e Farmacêuticos, S.A., Brazil). Results: We found a decrease in total vessel length in the Beva group (24.96% ± 12.85%) and in all the groups that received 670 nm red light therapy (2× group, 34.66% ± 8.66%; 4× group, 42.42% ± 5.26%; 8× group, 38.48% ± 6.96%), compared with the negative control group. The fluence of 5.4 J/cm2 in 4 sessions (4×) showed more regular vessels. The number of junctions in the groups that received a higher incidence of 670 nm red light (4× and 8×) significantly decreased (p<0.0001). Conclusion: Photo-biomodulation helps reduce vascularization in chorioallantoic embryonic membrane of chicken eggs and changes in the network architecture. Our results open the possibility of future clinical studies on using this therapy in patients with retinal diseases with neovascular components, especially age-related macular degeneration.

RESUMO Objetivo: investigar os efeitos vasculares da foto-biomodulação com diodo emissor de luz utilizando membrana embrionária corioalantóide de ovos de galinhas em grupos com diferentes tempos de exposição e detectar as alterações morfológicas por meio de métricas quantitativas promovidas pela luz na arquitetura da rede vascular. Métodos: Um aparelho de fototerapia com diodo emissor de luz no comprimento de onda de 670 nm foi usado como fonte de fotobiomodulação. A luz vermelha foi aplicada a uma distância de 2,5 cm da superfície da membrana embrionária corioalantóide em 2, 4 ou 8 sessões de 90 s a arquitetura da rede vascular foi analisada por meio do software AngioTool (National Cancer Institute, USA). Usamos um grupo controle negativo tratado com 50 µL de solução salina tamponada com fosfato (PBS) pH 7,4 e um grupo controle positivo (Beva) tratado com 50 µL de solução de bevacizumabe (Avastin, Produtos Roche Químicos e Farmacêuticos S.A., Brasil). Resultados: Uma diminuição no comprimento total do vaso foi detectada para o grupo Beva (24,96 ± 12,85%), e para todos os grupos que receberam terapia de luz vermelha de 670 nm, 34,66 ± 8,66% (2x), 42,42 ± 5,26% (4x) e 38,48 ± 6,96% (8x) em comparação ao grupo controle. A incidência de 5,4 J/cm2 em 4 sessões (4x) mostrou vasos mais regulares. A redução foi mais intensa nos grupos que receberam maior incidência de luz vermelha de 670 nm (4x e 8x). Conclusão: A fotobiomodulação contribui para a redução da vascularização nos vasos da membrana embrionária corioalantóide de ovos de galinhas e mudanças na arquitetura da rede. Os achados deste experimento abrem a possibilidade de considerar um estudo clínico usando esta terapia em pacientes com doenças retinais com componentes neovasculares, especialmente degeneração macular relacionada à idade.

Establishment of patient-derived xenografts of retinoblastoma and choroidal melanoma on the avian chorioallantoic membrane

Purpose: To develop a viable in vivo chorioallantoic membrane (CAM) model to study the growth and invasion of patient?derived retinoblastoma (RB) and choroidal melanoma (CM) xenografts (PDXs). The study utilizes primary tumor samples instead of cancer cell lines, which provides a more authentic representation of tumors due to conserved morphology and heterogeneity. Methods: Fertilized chicken eggs were procured, windowed, and their CAM layers were dropped. On embryonic development day (EDD) 10, freshly cut patient?derived CM and RB tumors were implanted on the CAM layer and the setup was incubated for 7 days. The tumor?embedded CAM layer was harvested on EDD 17, and the extracted tumor samples were subjected to hematoxylin and eosin staining and immunohistochemical analysis to evaluate the extent of tumor invasion. Results: Significant changes in the vascularity around the RB and CM PDXs were observed, indicating an angiogenic environment. The cross?sectional histological view of the tumor implant site revealed the invasion of both the tumors into the CAM mesoderm. Invasion of CM into CAM mesoderm was visualized in the form of pigmented nodules, and that of RB was indicated by synaptophysin and Ki?67 positivity in Immunohistochemistry (IHC). Conclusion: The CAM xenograft model was successfully able to support the growth of CM and RB PDXs and their invasion in CAM, thus presenting as a feasible alternative to mammalian models for studying tumorigenicity and invasiveness of ocular tumors. Moreover, this model can further be utilized to develop personalized medicine by inoculating patient?specific tumors for preclinical drug screening.

The role of enzymes in the angiogenic activity of Hancornia speciosa latex

The Hancornia speciosa latex has shown angiogenic activity. Angiogenesis plays a major role in wound healing, and materials that stimulate this process could be used to develop drugs. This study aimed to explain the role of proteins in the H. speciosa serum fraction latex in angiogenesis. Hence, this material was treated with proteinase K and the proteins were inactivated. After protein inactivation, angiogenic activity was assessed with the chick chorioallantoic membrane assay. The result showed that the proteins in the serum fraction are responsible for angiogenic activity. Then, the total protein content in the serum fraction and its enzymatic activity were investigated. The low protein content observed in the H. speciosa serum fraction latex suggests that this biomaterial could be used to develop new drugs with a hypoallergenic response. Despite the low protein content, there was a significant enzymatic activity of at least three enzymes in the serum fraction latex: ß-1,3 glucanase, ß-glucosidase, and proteases. These enzymes seem to influence the healing process, assisting debridement, extracellular matrix remodeling, and collagen deposition, and decreasing the chances of contamination by microorganisms. In conclusion, the enzymes in the H. speciosa serum latex are associated with the angiogenic activity of this biomaterial and may be used to assist the wound healing process.

Pharmacologically targeting molecular motor promotes mitochondrial fission for anti-cancer

Mitochondrial shape rapidly changes by dynamic balance of fusion and fission to adjust to constantly changing energy demands of cancer cells. Mitochondrial dynamics balance is exactly regulated by molecular motor consisted of myosin and actin cytoskeleton proteins. Thus, targeting myosin-actin molecular motor is considered as a promising strategy for anti-cancer. In this study, we performed a proof-of-concept study with a natural-derived small-molecule J13 to test the feasibility of anti-cancer therapeutics

In vivo evidence of angiogenesis inhibition by ß2-glycoprotein I subfractions in the chorioallantoic membrane of chicken embryos

The vascular network expansion and functioning are important factors affecting normal intra-uterine fetal development. This study addressed the previously reported antiangiogenic potential of beta-2-glycoprotein I (β2GPI) in vivo in the chick embryo model of angiogenesis. The effects of two naturally occurring β2GPI forms on the development of the chorioallantoic membrane (CAM) vessels and the chicken embryo were investigated. β2GPI monomers and dimers were obtained by fractioned purification and characterized using SDS-PAGE, immunoblot, and ELISA. The egg exposure was performed by injection of small volumes of 2.5 µg/mL solutions of the β2GPI subfractions. Angiogenesis was evaluated through quantitative measurements of vascular architecture parameters in the captured CAM images, using computational analysis of texture contrasts and computer vision techniques. Quantitative information was assigned to the CAM vasculature modifications. In vivo, the β2GPI dimer completely halted the formation of CAM vessels and led to embryo death after 48 h of exposure. The β2GPI monomer allowed the embryo to develop up to the 10th day, despite early changes of CAM vessels. The impaired normal vessel growth proceeded as a self-limited effect. The β2GPI monomer-exposed eggs showed reduced vascularization on the 6th day of incubation, but embryos were viable on the 10th day of incubation, with ingurgitated CAM vessels implying sequelae of the angiogenesis inhibition. Both subfractions impaired CAM vasculature development. The β2GPI dimer proved to be largely more harmful than the β2GPI monomer. β2GPI modification by cleavage or dimerization may play a role in angiogenesis control in vivo.

Ocular Tolerability And Efficacy Of Ebastine Colloidal Formulation In Allergic Conjunctivitis

Allergic conjunctivitis (AC), defined by ocular itching, hyperemia, lacrimation and edema, impairs the quality of life across the globe. Ebastine is available as an oral antihistamine formula, such as tablets and syrup, for allergic disorders. Topical antihistamines are preferred over oral agents since their direct application at the site of action results in rapid onset and superior efficacy with less systemic side effects. The objective of the present work was to evaluate the antiallergic potential of optimized ebastine (1% w/v) colloidal ocular formulation by performing in vitro study like hen's egg chorioallantoic membrane test (HET-CAM) for tolerability and in vivo efficacy study in ovalbumin (OA)-induced allergic conjunctivitis (AC) with acute ocular irritation study. Eye scratching behavior and edema were evaluated after topical antigen challenge. Edema was scored at periodic interval after the instillation of ovalbumin followed by histopathology. The results showed that ebastine (1% w/v) colloidal ocular formulation was effective in inhibiting symptoms of eye inflammation induced by ovalbumin. Further, the study indicated that said formulation has a quick onset and the duration of effect sufficient to provide relief from symptoms for 24 hr. Ocular irritation by HET-CAM assay showed that the developed formulation does not cause any irritation to the blood vessels. Acute ocular irritation test was performed using rabbits and results showed that developed formulation was non-irritant to the eye. The present study revealed that the ocular ebastine formulation could offer a novel therapeutic opportunity against IgE-mediated allergic conjunctivitis.

Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying

Monoclonal antibodies (MAbs) are widely applied in disease diagnoses. Herein, we report a MAb, WF-4, against Influenza A virus nucleoprotein (NP), its broad response with Influenza A virus, and its application in an immunohistochemistry (IHC) assay. WF-4 was screened by immunofluorescence assay (IFA). The results showed that its reactivity with baculovirus-expressed full-length recombinant NP (rNP) in Western blot (WB), indicating its IHC applicability. Fifteen Influenza A virus (reference subtypes H1 to H15) infected chicken embryonated chorioallantoic membranes (CAM), fixed by formalin, were all detectable in the WF-4-based IHC assay. Also, the reactivity of the IHC test with NP from experimentally inoculated H6N1 and from all recent outbreaks of H5 subtype avian Influenza A virus (AIV) field cases in Taiwan showed positive results. Our data indicate that CAM, a by-product of Influenza A virus preparation, is helpful for Influenza A virus-specific MAb characterization, and that the WF-4 MAb recognizes conserved and linear epitopes of Influenza A virus NP. Therefore, WF-4 is capable of detecting NP antigens via IHC and may be suitable for developing various tests for diagnosis of Influenza A virus and, especially, AIV infection.

Chorioallantoic urothelial tumor avatar. A clinical tool for phenotype-based therapy

Abstract In the muscle invasive bladder cancer (MIBC) standard of care treatment only patients presenting a major pathological tumor response are more likely to show the established modest 5% absolute survival benefit at 5 years after cisplatin-based neoadjuvant chemotherapy (NAC). To overcome the drawbacks of a blind NAC (i.e. late cystectomy with unnecessary NAC adverse events) with potential to survival improvements, preclinical models of urothelial carcinoma have arisen in this generation as a way to pre-determine drug resistance even before therapy is targeted. The implantation of tumor specimens in the chorioallantoic membrane (MCA) of the chicken embryo results in a high-efficiency graft, thus allowing large-scale studies of patient-derived "tumor avatar". This article discusses a novel approach that exploits cancer multidrug resistance to provide personalized phenotype-based therapy utilizing the MIBC NAC dilemma.

Antiangiogenic potential of Jatropha curcas latex in the chick chorioallantoic membrane model / Potencial antiangiogênico do látex de Jatropha curcas em modelo de membrana corioalantoica de embrião de galinha

AIMS: To perform a physicochemical and phytochemical characterization of Jatropha curcas latex and to investigate its antiangiogenic potential. METHODS: We performed an initial physicochemical characterization of J. curcas latex using thermal gravimetric analyses and Fourier Transform Infrared spectroscopy. After that, phenols, tannins and flavonoids were quantified. Finally, the potential of J. curcas latex to inhibit angiogenesis was evaluated using the chick chorioallantoic membrane model. Five groups of 20 fertilized chicken eggs each had the chorioallantoic membrane exposed to the following solutions: (1) water, negative control; (2) dexamethasone, angiogenesis inhibitor; (3) Regederm®, positive control; (4) 25% J. curcas latex diluted in water; (5) 50% J. curcas latex diluted in water; and (6) J. curcas crude latex. Analysis of the newly-formed vascular net was made through captured images and quantification of the number of pixels. Histological analyses were performed to evaluate the inflammation, neovascularization, and hyperemia parameters. The results were statically analyzed with a significance level set at p<0.05. RESULTS: Physicochemical characterization showed that J. curcas latex presented a low amount of cis-1.4-polyisoprene, which reduced its elasticity and thermal stability. Phytochemical analyses of J. curcas latex identified a substantial amount of phenols, tannins, and flavonoids (51.9%, 11.8%, and 0.07% respectively). Using a chick chorioallantoic membrane assay, we demonstrated the antiangiogenic potential of J. curcas latex. The latex induced a decrease in the vascularization of the membranes when compared with neutral and positive controls (water and Regederm®). However, when compared with the negative control (dexamethasone), higher J. curcas latex concentrations showed no significant differences. CONCLUSIONS: J. curcas latex showed low thermal stability, and consisted of phenols, tannins, and flavonoids, but little or no rubber. Moreover, this latex demonstrated a significant antiangiogenic activity on a chick chorioallantoic membrane model. The combination of antimutagenic, cytotoxic, antioxidant and antiangiogenic properties makes J. curcas latex a potential target for the development of new drugs.

OBJETIVOS: Realizar uma caracterização físico-química e fitoquímica do látex de Jatropha curcas e investigar o seu potencial antiangiogênico. MÉTODOS: foi realizada uma caracterização físico-química inicial do látex de J. curcas utilizando as análises termogravimétricas e a espectroscopia com a Transformada de Fourier. Depois disso, fenóis, taninos e flavonoides foram quantificados. Finalmente, o potencial do látex de J. curcas em inibir a angiogênese foi avaliado através do uso de modelo de membrana corioalantoica de embrião de galinha. Cinco grupos, cada um com 20 ovos de galinha fertilizados, tiveram a membrana corioalantoica exposta às seguintes soluções: (1) água, controle negativo; (2) dexametasona, inibidor da angiogênese; (3) Regederm®, controle positivo; (4) 25% de látex de J. curcas diluído em água; (5) 50% de látex de J. curcas diluído em água; e (6) látex bruto de J. curcas. A análise da rede vascular recém-formada foi feita por meio de imagens capturadas e quantificação do número de pixels. Análises histológicas foram realizadas para avaliar os parâmetros de inflamação, neovascularização e hiperemia. Os resultados foram analisados estaticamente com nível de significância estabelecido em p<0,05. RESULTADOS: A caracterização físico-química mostrou que o látex de J. curcas apresenta uma baixa quantidade de cis-1,4-poliisopreno, o que reduz sua elasticidade e estabilidade térmica. Análises fitoquímicas do látex de J. curcas identificaram uma quantidade significativa de fenóis, taninos e flavonoides (51,9%, 11,8% e 0,07% respectivamente). Usando o modelo de membrana corioalantoica de ovo de galinha embrionado, demonstrou-se o potencial antiangiogênico do látex de J. curcas. O látex induziu a diminuição da vascularização das membranas, em comparação aos grupos controle neutro e positivo (água e Regederm®). CONCLUSÕES: O látex de J. curcas apresentou baixa estabilidade térmica, ausência ou pouca quantidade de borracha e presença de fenóis, taninos e flavonoides em sua composição. Além disso, apresentou alta atividade antiangiogênica no modelo de membrana corioalantoica de embrião de galinha. A combinação de propriedades antimutagênicas, citotóxicas, anti-inflamatórias, antioxidantes e antiangiogênicas faz com que o látex de J. curcas seja um alvo potencial para o desenvolvimento de novos medicamentos.

Efecto antiangiogénico de la triamcinolona asociada a lidocaína con epinefrina de uso odontológico en la membrana alantocoriónica de pollo / Antiangiogenic effect of triamcinolone associated with lidocaine with epinephrine for dental use in chicken chorioallantoic membrane

RESUMEN: El proceso angiogénico se define como el proceso en el que los vasos sanguíneos generan brotes dando como resultado neovascularidad. Un desbalance en el proceso angiogénico contribuye a numerosos desórdenes inflamatorios, infecciosos, isquémicos, inmunológicos y malignos. En el territorio maxilofacial se pueden encontrar patologías neoplásicas benignas de desarrollo local con un marcado componente angiogénico que determinan su crecimiento y agresividad. Sin embargo, existe escasa evidencia de cómo tratarlas en base al control de la angiogénesis. Terry & Jacoway (1994) desarrollaron un protocolo de tratamiento para lesiones neoplásicas benignas con un importante componente vascular que se utiliza actualmente. Este protocolo consiste en la infiltración intralesional de una suspensión de triamcinolona 10 mg/ml más una solución de anestésico local de uso odontológico como la lidocaína al 2 % asociada a epinefrina en una concentración de 1:200.000. Sin embargo, el uso de epinefrina podría disminuir la acción antiangiogénica de la triamcinolona al ser un vasoconstrictor. El objetivo de este estudio es comparar el efecto antiangiogénico, en la membrana alantocoriónica de pollo (MAC), de esta suspensión versus el efecto de la triamcinolona sin asociar a anestésicos locales. Los resultados del efecto antiangiogénico en la MAC de pollo, obtenidos en la investigación concluyeron que la suspensión de triamcinolona asociada a lidocaína con epinefrina es similar al de la suspensión de triamcinolona sin asociar a anestésicos locales. Además, se logró determinar que las suspensiones de triamcinolona sin asociar a anestésicos locales y las asociadas a anestésicos locales con o sin vasoconstrictor poseen un marcado efecto antiangiogénico, en la MAC de pollo, en comparación al grupo control.

SUMMARY: Angiogenesis is defined as the process through which new blood vessels form from previously existing vessels. Several inflammatory, infectious, ischemic, immunological and malignant disorders are caused by the lack of adequate angiogenesis balance. In the maxillofacial area, there are invasive benign neoplastic pathologies with a strong angiogenic component, which determines aggressive behavior and growth. Studies in the literature are scarce regarding treatment of these conditions based on angiogenesis control. Currently, the protocol used to treat these maxillofacial benign neoplastic lesions, was developed in 1994 by Terry & Jacoway and has a strong angiogenic component. Consequently lesions are treated via intra-lesion administration of triamcinolone 10 mg / mL, a solution used in dental local anesthetic, such as lidocaine 2 %, in conjunction with epinephrine at a concentration of 1:200,000. The objective of this study was to compare the antiangiogenic effect of this protocol in chicken chorioallantoic membrane (CAM) without the use of local anesthetic. The results of the antiangiogenic effect in the CAM obtained in this study concluded that the effect of the suspension of triamcinolone associated to lidocaine with epinephrine, is similar to the suspension of triamcinolone without associating local anesthetics. Furthermore, it was determined that suspensions of triamcinolone without local anesthetic, and those associated to local anesthetic with, and without vasoconstrictor have a strong antiangiogenic effect in CAM compared to the control group.

Study of antiangiogenic activity of “aqueous extract of Nigella sativa seeds” in chick chorioallantoic membrane (CAM) model

Background: Angiogenesis is important for the typical physiological activities such as cure from injury, menstrual cycle and embryo growth. It is also plays a crucial role in several pathological conditions in cancer. Antiangiogenesis, e.g., inhibition of blood vessel growth, is being investigated as a way to prevent the growth of tumors and other angiogenesis-dependent diseases. The chick embryo chorioallantoic membrane (CAM) is commonly used as an experimental in vivo assay to study both angiogenesis and antiangiogenesis in response to tissues, cells or soluble factors. Given the high occurrence of cancer worldwide and the major source of the discovery of new lead molecules are medicinal plants. The objective of the present research was to study the antiangiogenic property of “aqueous extract of Nigella sativa seeds” using chick chorioallantoic membrane (CAM) assayMethods: The chick chorioallantoic membrane (CAM) assay for screening the effect of Nigella sativa on anti-angiogenesis was performed according to the method given by Ribatti and co-workers.Results: The results of present study significantly increased the antiangiogenic effect on CAM by decreasing the proliferation of capillary networks in a dose (50 to 300 µg/egg) dependent manner which is probably related to the inhibition of neovascularization.Conclusions: It is concluded that aqueous extract of N. sativa seeds possesses significant antiangiogenic activity, and this is a possible rationale for its folkloric use as an anticancer agent.

Anti-angiogenic activity of iridoids from Galium tunetanum

ABSTRACT The phytochemical study of Galium tunetanum Lam., Rubiaceae, leaves led to the isolation of 13 compounds from the chloroform-methanol and the methanol extracts, including six iridoid glycosides, one non-glycoside iridoid, two p-coumaroyl iridoid glycosides, two phenolic acids, and two flavonoid glycosides. The structural determination of the isolated compounds was performed by mono- and bidimensional NMR spectroscopic data, as well as ESI-MS experiments. All compounds were isolated from this species for the first time. The anti-angiogenic effects of the isolated iridoids were also reported on new blood vessels formation using the chick embryo chorioallantoic membrane as in vivo model. Results showed that among the isolated iridoids tested at the dose of 2 µg/egg, asperuloside (1), geniposidic acid (2), and iridoid V1 (3) reduced microvessel formation of the chorioallantoic membrane on morphological observations using a stereomicroscope. The anti-angiogenic effects of the active compounds, expressed as percentages of inhibition versus control, were 67% (1), 59% (2), and 54% (3), respectively. In addition, the active compounds were able to inhibit angiogenesis in the chorioallantoic membrane assay, in a dose-dependent manner (0.5-2 µg/egg) as compared to the standard retinoic acid.

Hexon and fiber gene changes in an attenuated fowl adenovirus isolate from Malaysia in embryonated chicken eggs and its infectivity in chickens

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 10(8.7)TCID₅₀/mL (TCID₅₀, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity.

Anti-proliferative and anti-angiogenic activities of ion-channel modulators: In-ovo, in-vitro and in-vivo study

Objective: Angiogenesis is the development of new blood vessels. The ion channels on endothelium play a vital action in cell proliferation and so in the related angiogenesis. We aimed to investigate the anti-angiogenic effects of Mefloquine (Cl?channel blocker) and 4-Aminopyridine (K+channel blocker). Methods: The anti-angiogenic activities of Mefloquine and 4-Aminopyridine (4-AP) were investigated by in-vivo (sponge implantation method), in-vitro (aortic ring assay) and in-ovo (CAM, Chick Chorioallantoic membrane) methods. The standard anti-angiogenic drug used was Bevacizumab. Results: In the CAM assay, both the ion channel blockers exhibited noticeable anti-angiogenic activity at the concentrations of 10?5 M and 10?4 M where they significantly exhibited ant proliferative activity by inhibiting the new blood vessel formation. For the further confirmation anti-angiogenic activity was evaluated in vitro and in vivo. In Rat aortic ring assay reduction in the area of sprouts were observed with 40μM of 4-AP and 7 μM of Mefloquine. A significant reduction in weight of sponges, number of blood vessels formed and hemoglobin content were observed at 4.2 mg/kg of 4-AP and 20 mg/kg and 30 mg/kg of Mefloquine. Conclusions: These scientific findings indicate the use of Mefloquine and 4-Aminopyridine in pathological situations involving excessive angiogenesis. Negative regulation of cell volume, cell migration and proliferation of blood vessels may be the underlying molecular mechanisms.

Contrastive Analysis of three transplanted tumor models in chick embryo chorioallantoic membrane / 中国比较医学杂志

Objective To compare the growth of three different cancer cell lines on chick chorioallantoic membrane (CAM), to select the best transplanted cancer cell line for establishing a transplanted tumor model and to observe the biological characteristics.Methods The human lung cancer cell line A549, human tongue cancer cell line TCA8113 and human liver cancer cell line QGY7703 were respectively inoculated into CAM at the 7th day of age.The chick embryo survival rate, tumor survival rate, tumor formation rate and induced angiogenesis were detected and the growth characteristics of the transplanted tumor model were observed.Results Compared with the groups inoculated with A549 cells and QGY7703 cells, the tumor formation rate of TCA8113 cells was the highest (P < 0.05), to be the best cancer cell line for transplanted tumor.The optimal inoculated number of cells was 8.0×106/chick embryo, the optimal growth period of the tumor was 4~8 d, and the best experiment time was 7 d after inoculation.Conclusion The TCA-CAM transplanted tumor model of tongue squamous cell cancer is successfully established for further study of the biological characteristics and mechanisms of tumor growth, angiogenesis, invasion and metastasis, and provide a good experimental animal model for anti-tumor drug screening.

Saikosaponins-b suppresses tumor growth and angiogenesis of hepatocellular carcinoma by regulating VEGF/ERK/HIF-1α signal pathway / 中国药理学与毒理学杂志

OBJECTIVE Angiogenesis therapy has attracted interest as a potential treatment for hepatocellular carcinoma (HCC). In this study, we investigated the anti-proliferative activities and anti-angiogenesis effects of saikosaponins (SS)-b on hepatocellular carcinoma (HCC) and its regulation on VEGF/ERK/HIF-1α signal pathway. METHODS H22 hepatoma-bearing mice model and HepG-2 cells were used to study the anti-tumor and anti-angiogenesis effects of SS-b in vivo and in vitro. Pathological change of tumor tissue was observed by HE staining, the microvascular changes were detected by immunohistochemical method. The effects of SS-b on angiogenesis were examined by using the chick embryo chorioallantoic membrane (CAM) model. The effects of SS- b on proliferation, migration and invasion were investigated by MTT assay, scratch wound healing assay and transwell assay inhuman umbilical vein endothelial cell (HUVEC) and HepG2 cells in vitro. Vascular endothelial growth factor (VEGF), matrix metalloproteinase-2/9(MMP-2/9), hypoxia-inducible factor-1α (HIF-1α) expression and the phosphorylation of extracellular regulated kinase(ERK) were analyzed using RT-PCR and Western-blot. RESULTS SS-b effectively inhibited the tumor growth of H22 mice in vivo. The inhibitory rate of tumor was 49.1%, 50.7%, 66.1% in SS-b 5, 10 and 20 mg·kg-1 group respectively. HE staining results showed that SS-b induced tumor necrosis and nuclear dissolution in H22 mice. Moreover, SS-b also reduced the number of microvessels of tumor tissue in H22 mice significantly and suppressed the angiogenesis of CAM induced by b-FGF. SS-b had an obvious inhibitory effect on cell proliferation, migration and invasion of HUVEC cells and HepG-2 cells. These effects were associated with down-regulation of the expression of MMP2/9 and suppression of VEGF/ERK/HIF-1α signaling in H22 mice and Hep-G2 cells. CONCLUSION Our findings showed that SS-b exerts anti-tumor effects by inhibit?ing tumor angiogenesis via regulating VEGF/ERK/HIF-1α signal pathway in vivo and in vitro.

Anti-proliferative and anti-angiogenic activities of ion-channel modulators: In-ovo, in-vitro and in-vivo study

Objective Angiogenesis is the development of new blood vessels. The ion channels on endothelium play a vital action in cell proliferation and so in the related angiogenesis. We aimed to investigate the anti-angiogenic effects of Mefloquine (Cl− channel blocker) and 4-Aminopyridine (K+ channel blocker). Methods The anti-angiogenic activities of Mefloquine and 4-Aminopyridine (4-AP) were investigated by in-vivo (sponge implantation method), in-vitro (aortic ring assay) and in-ovo (CAM, Chick Chorioallantoic membrane) methods. The standard antiangiogenic drug used was Bevacizumab. Results In the CAM assay, both the ion channel blockers exhibited noticeable antiangiogenic activity at the concentrations of 10

Methanolic extract of Euchelus asper exhibits in-ovo anti-angiogenic and in vitro anti-proliferative activities

BACKGROUND: The marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME). METHODS: The anti-angiogenic activity of EAME (50-800 µg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histo-logical investigations. In vitro testing of EAME (5-20 µg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student's t test for two unpaired groups. RESULTS: Analysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9). CONCLUSION: Overall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.

Efecto del aliskireno sobre la angiogénesis en modelo de membrana alantocoriónica (MAC) de pollo / Effect of aliskiren on angiogenesis in chick embryo chorioallantoic membrane (CAM) assay

La acción antiangiogénica de los inhibidores del receptor de angiotensina II (ARA II), ha sido documentada previamente, sin embargo, no ha sido descrita la relación entre angiogénesis e inhibidores directos de la renina (DRIs), los cuales participan regulando el sistema renina-angiotensina-aldosterona (SRAA). El objetivo fue demostrar el efecto antiangiogénico de aliskireno, un DRI, en membranas alantocoriónicas (MAC) de pollo, para lo cual fueron instilados aliskireno y enalapril sobre MAC en distintas concentraciones para realizar su comparación posterior. En secciones histológicas seriadas se registró el número de vasos sanguíneos presentes en 9000 µm2 bajo microscopio de luz a máximo aumento, y se realizó análisis estadístico utilizando ANOVA y el test de Tukey para demostrar posibles diferencias. Los receptores tratados con aliskireno, en ambas concentraciones utilizadas, presentaron menor densidad vascular, en comparación con los controles, siendo ésta estadísticamente significativa a mayor concentración. Aliskireno en concentraciones altas tiene un efecto antiangiogénico en un modelo experimental de MAC. Este hallazgo plantea la necesidad de estudios posteriores, dada la proyección que podría tener el uso inhibidores directos de la renina. A partir de estos resultados, se podría pensar en la factibilidad del uso de aliskireno para la modulación de la angiogénesis en diversas enfermedades crónicas no transmisibles.

Angiogenesis is the formation of new blood vessels from pre-existing ones. Antiangiogenic effect of angiotensin receptor blockers has been reported, however, the relationship between direct renin inhibitors and angiogenesis has not been well described. To assess the antiangiogenic effect of aliskiren, a direct renin inhibitor, on chick embryo chorioallantoic membrane (CAM) assay. Aliskiren and enalapril were instilled in different concentrations and compared. In serial histological sections, the number of blood vessels per 9000 µm2 area under observation through optical microscope using maximum zoom, was registered. Statistical analysis using Anova and Tukey test in order to show possible differences, was performed. Receptors treated with aliskiren presented lower vascular density, which was statistically significant when a higher concentration was administered. High concentrations of aliskiren have an antiangiogenic effect on CAM assay. This finding means further studies are needed, because of the usefulness direct renin inhibitors could have. These results, also, might enhance the possibility of using aliskiren for regulating angiogenesis in the context of non-transmissible chronic diseases.

Anti-angiogenic effects of ethanolic extract of Artemisia sieberi compared to its active substance, artemisinin

Abstract Angiogenesis plays a key role in tumor growth, invasion and metastasis of cancer diseases and therefore, the inhibition of angiogenesis can provide an important therapeutic approach in cancer diseases. This study was designed to compare the anti-angiogenic activities of the ethanolic extract of Artemisia sieberi Besser, Asteraceae, and its active substance, artemisinin in both in vitro and in vivo models. To compare cytotoxicity level of ethanolic extract of A. sieberi with artemisinin, different concentrations (1–100 µg/ml) were tested using MTT assay on human umbilical vein endothelial cells. The anti-angiogenic properties of serial concentrations of ethanolic extract of A. sieberi and artemisinin were examined on human umbilical vein endothelial cells using a three-dimensional angiogenesis assay (in vitro model) and in the chick chorioallantoic membrane assay as in vivo model. The effects of ethanolic extract of A. sieberi and artemisinin were also tested on the expression of VEGFR-1, VEGFR-2 and CD34 genes using real-time PCR. Ethanolic extract of A. sieberi and artemisinin significantly (p < 0.001) inhibited the angiogenesis in the human umbilical vein endothelial cells culture whilst the ethanolic extract of A. sieberi showed higher effect in a concentration-dependent fashion (p < 0.001). The chick chorioallantoic membrane angiogenesis was also completely inhibited by ethanolic extract of A. sieberi at concentration of 33 ng/100 µl/egg. The gene expression analysis showed that the ethanolic extract of A. sieberi and artemisinin reduced the transcription of VEGFR-1, VEGFR-2 and CD34 genes in a concentration-dependent manner. This study demonstrated that the ethanolic extract of A. sieberi is strongly able to inhibit the angiogenesis in human umbilical vein endothelial cells and chick chorioallantoic membrane models compared to the artemisinin.