Transcriptome-based insights into the calcium transport mechanism of chick chorioallantoic membrane

Chorioallantoic membrane(CAM)is responsible for respiratory gas exchange,eggshell calcium transport,embryonic acid-base equilibrium,allantoic ion,and water reabsorption during avian embryonic development.To further understand the timing of CAM gene expression during chick embryonic development,especially the calcium absorption mechanism,transcriptome quantitative comparative analysis was conducted on chick CAM during the embryonic period(E)of 9,13,17,and 20 days.A total of 6378 differentially expressed genes(DEGs)were identified.Functional enrichment analysis of DEGs showed that CAM DEGs were mainly involved in biological processes such as"ion transport regulation","immune response"and"cell cycle".Time series analysis of the differential genes showed that the functional cells of CAM began to proliferate and differentiate at E9 and the calcium content of egg embryo increased significantly at E13.Simultaneously,the observation of the ultrastructure of the eggshell showed that the interstice of the fiber layer was enlarged at E13,and the mastoid layer was partly exposed.Therefore,it is preliminarily inferred that CAM calcium transport starts at E13,and genes such as TRPV6,S100 A10,and RANKL cooperate to regulate calcium release and transport.

Establishment of xenografts of urological cancers on chicken chorioallantoic membrane(CAM)to study metastasis

Cancer of the urological system commonly occurs in the kidney,bladder,and prostate gland.The clear cell subtype of renal cell carcinoma(ccRCC)constitutes the great majority of kidney cancer.Metastatic ccRCC portends a very poor outcome with no effective treatment available.Prostate cancer is the most common cancer in males in the US.Despite recent advances in selective kinase inhibitors and immunotherapies,the rate of developing new treatment from bench to bedside is slow.A time-consuming step is at the animal drug testing stage,in which the mouse model is the gold standard.In the pursuit to streamline the in vivo cancer biology research and drug development,we explored the feasibility of the chicken chorioallantoic membrane(CAM)model to establish xenografts.The CAM model greatly shortens the time of tumor growth and lowers the cost comparing to immunocompromised mice.We generated CAM xenografts from ccRCC,bladder and prostate cancer,with established cancer cell lines and freshly isolated patient-derived tissues,either as primary tumor cells or small pieces of tumors.The successful CAM engraftment rate from the different tumor sources is 70%or above.Using our previously established metastatic ccRCC mouse model,we showed that the CAM xenograft maintains the same tumor growth pattern and metastatic behavior as observed in mice.Taken together,CAM can serve as a valuable platform to establish new patient-derived xenografts(PDXs)to study tumor biology,thus accelerating the development of individualized treatment to halt the deadly metastatic stage of cancer.

Antisense oligonucleotide targeting midkine suppresses in vivo angiogenesis

AIM: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) and in situ human hepatocellular carcinoma (HCC). METHODS: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylin- eosin (HE) staining. RESULTS: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues. CONCLUSION: MK-AS is an effective antiangiogenesis agent in vivo.

Expression of Soluble Vascular Endothelial Growth Factor Receptor-2 and Its Effect on Proliferation of Vascular Endothelial Cells

Vascular endothelial growth factors（VEGFs） respectively bind to each of three receptor tyrosine kinases（RTKs）,known as Flt-1,KDR and Flt-4.Since VEGFs and their respective families of receptor tyrosine kinases（VEGFRs） are critical proteins which can regulate vascular development during angiogenesis,we decided to explore the inhibitory effects of soluble kinase insert domain-containing receptor（sKDR） on endothelial cells and angiogenesis.Total RNA was extracted from human umbilical vein endothelial cells（HUVEC）,and cDNA of extracellular domains 1―4 was amplified and recombined with pQE40 vector.After being expressed,affinity purified,renatured and analyzed by Western blot,the sKDR was assayed for its effects on endothelial cells by [3-（4,5-dimethylthiazol-2-yl）2,5-diphenyltetrazolium bromide]（MTT）,and on angiogenesis by chick chorioallantoic membrane（CAM） experiment.sKDR cDNA of 1150 bp was obtained via real-time polymerase chain reaction（RT-PCR）,and sKDR was expressed by pQE40 procaryotic expression system,purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） analysis with only one band and proved by Western blot.MTT assay demonstrateds that sKDR could inhibit the VEGF-stimulated HUVEC from proliferation,and CAM experiment showed sKDR could block the VEGF-induced angiogenesis.sKDR has the biological activity to bind with VEGF ligands and is a potential target for tumor anti-angiogenesis therapy.

Saikosaponins-b suppresses tumor growth and angiogenesis of hepatocellular carcinoma by regulating VEGF/ERK/HIF-1α signal pathway

OBJECTIVE Angiogenesis therapy has attracted interest as a potential treatment for hepatocellular carcinoma(HCC).In this study,we investigated the anti-proliferative activities and antiangiogenesis effects of saikosaponins(SS)-b on hepatocellular carcinoma(HCC)and its regulation on VEGF/ERK/HIF-1 αsignal 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 Westernblot.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-1group 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 downregulation of the expression of MMP2/9 and suppression of VEGF/ERK/HIF-1αsignaling in H22 mice and Hep-G2 cells.CONCLUSION Our findings showed that SS-b exerts anti-tumor effects by inhibiting tumor angiogenesis via regulating VEGF/ERK/HIF-1α signal pathway in vivo and in vitro.

Angiogenic Effect of Intercellular Adhesion Molecule-1

In order to investigate the angiogenic effect of intercellular adhesion molecule-1 （ICAM-1）, two parts of experiment were performed. Chick embryo chorioallantoic membrane （CAM） assay was used for in vivo angiogenic research. The chick embryos were divided into 4 groups： ICAM-1 group （divided into 3 subgroups, Ⅰ, Ⅱ and Ⅲ） for screening the angiogenic effect of ICAM-1 by adding different concentrations of ICAM-1 （0.1, 0.2 and 0.3 μg/μL） 5 μL into the chick embryo CAMs on the day 10 after incubation for every subgroup; Anti-ICAM-1 group A （divided into 2 subgroups, Ⅰ and Ⅱ） by adding different concentrations of Anti-ICAM-1 （1：100, 1：50） 5 μL into the chick embryo CAMs on the day 10 after incubation for every subgroup to evaluate the effect of ICAM-1 on the survival of microvessels through observing whether Anti-ICAM-1 could induce involution of the microvessels on CAMs; Anti-ICAM-1 group B （divided into 2 subgroups, Ⅰ and Ⅱ ） by adding different concentrations of Anti-ICAM-1 （1：100, 1：50） 5 μL into the chick embryo CAMs on the day 6 after incubation for every subgroup to evaluate whether ICAM-1 involved in embryonic angiogenesis through observing the growth of microvessels on CAMs; Control group： ICAM-1 or Anti-ICAM-1 was substituted by PBS 5 μL on the day 10 or day 6 after incubation. Three days later, the CAMs were photographed in vivo, excised, sectioned and the number of microvessels was counted. In ICAM-1 group, there was increased number of microvessels arranged radially with ＂spoked-wheel＂ pattern around the gelatin sponges. The new microvessels growing perpendicularly to gelatin sponges were observed. The number of the microvessels growing in the CAM mesenchymes around the sponges in 3 subgroups was higher than that in control group （P〈0.01）, however, there was no significant difference among the 3 subgroups （P〉0.05）. In anti-ICAM-1 group A, the radially arranged microvessels were very unclear around the sponges contrast to that of ICAM-1 group. Few new microvessels were detected in the center of the sponges. The number of the microvessels growing in the CAM mesenchymes around the sponges in subgroup Ⅱ was lower than that in control group （P〈0.01）. There was no significant difference in the number of the microvessels around the sponges between subgroup I and control group （P〉0.05）. In anti-ICAM-1 group B, the radially arranged microvessels were very unclear around the sponges contrast to that of control grouμ New microvessels were very scarce in the center of the sponges. The number of the microvessels growing in the CAM mesenchymes around the sponges in the 2 subgroups were less than that in control group （P〈0.01）, and there was significant difference between the 2 subgroups （P〈0.05）. It was suggested that ICAM-1 could induce angiogenesis and support the survival of microvessels, and ICAM-1 was involved in embryonic angiogenesis.

Establishment of a transplantation tumor model of human osteosarcoma in chick embryo

Objective: We established a transplantation tumor model of human osteosarcoma in chick embryo, studied its morphological and biological characteristics, and observed its dynamic process of angiogenesis induction so that a simple and practical model can be provided for studying osteosarcoma. Methods: Human osteosarcoma cells at different concentrations were inoculated in chorioallantoic membrane (CAM) of chick embryos at different embryonic ages to observe the factors affecting the survival of the transplanted osteosarcoma in chick embryo, growth characteristics of the transplantation tumor, and the morphological characteristics and biological characteristics of the transplantation tumor. Results: The transplantation tumor model of human osteosarcoma in chick embryo was successfully established. It was found that the transplantation tumor was easy to grow and it showed strong angiogenesis-inducing effects. Under the light microscope, the transplantation tumor showed a similar tissue structure to human osteosarcoma. Conclusion: It is feasible to establish a transplantation tumor model of human osteosarcoma in chick embryo. The model can be easily duplicated with a simple operation, which provides a useful animal model for studying osteosarcoma.

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) and4-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 10-5M and 10-4M 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 40 m M of 4-AP and7 m M 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.

Anti-Angiogenesis and Anti-Tumor Effect of Shark Cartilage Extract

The effect of shark cartilage extract (SCE), purified in this laboratory, on angiogenesis in chick chorioallantoic membrane (CAM), on the activity of collagenase IV and on human umbilical vein endothelial cell (ECV 304) proliferation and apoptosis was investigated in vitro. The results showed that SCE caused a decline in CAM blood vessels and significantly prevented collagenase induced collagenolysis. Moreover, SCE produced a dose dependent decline in ECV 304 proliferation and altered its normal cell cycle. These results suggest that the anti angiogenesis and anti tumor effects of shark cartilage may be due to inhibition of endothelial cells as well as collagenolysis.

Evaluation,partial characterization and purification of acetylcholine esterase enzyme and antiangiogenic activity from marine sponges

Objective:To test three marine sponges Halichondria glabrata Keller,1891;Spirastrellapachyspira(S.pachyspira)Levi,1958 and Cliona lobata Hancock,1849 for the presence of the acetylcholinesterase(AChE)in both young and developed samples from western coastal area of India.S.pachyspira methanolic extract was selected for anti/pro angiogenic activity.Methods:They were evaluated for AChE activity using Ellman’s assay based on production of yellow colored 5-thio-2-nitrobenzoate.Purification of the enzyme was planned using ammonium sulphate precipitation and characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis.Chorioallantoic membrane(ChAM)assay model was used for angiogenic/antiangiogenic testing.Results:All the three sponges showed good specific enzyme activity and S.pachyspira contained maximum specific enzyme activity.Sixty percent of ammonium sulphate precipitation of crude protein sample gave single band at 66 kDa corresponding to the true AChE.ChAM assay was performed at 62.5,125.0 and 250.0μg/mL.Dosage beyond 250μg/mL extract showed toxic response with anti angiogenic activity at all the concentrations.Conclusions:AChE activity was detected in all samples.Extract showed good anti-angiogenic response at 62.5μg/mL.Extract was highly toxic affecting microvasculature of ChAM as well as normal growth and development of the embryo at 500μg/mL.With further characterization of bioactive compounds from the extract of S.pachyspira,the compounds can be developed for anti tumor activity.

The anti-angiogenic and antibacterial effect of Tinomiscium philippinense Miers.(Menispermaceae)leaf extract

Objective:To determine the toxicity profile,anti-angiogenic and antibacterial activity of the crude and semi-crude leaf extracts of Tinomiscium philippinense(T.philippinense).Methods:The leaves of T.philippinense were extracted with methanol and partitioned with solvents of different polarities,namely,hexane,dichloromethane and butanol.The extracts were subjected to duck chorioallantoic membrane assay to establish its anti-angiogenic property.Microwell assay was utilized to determine the minimum inhibitory concentration and minimum bactericidal concentration of the different extracts of the plant.Results:The dichloromethane leaf extract of T.philippinense at 1000μg/disc showed the highest anti-angiogenic activity with 37.46%inhibition.All the fractions exhibited a bacteriostatic and bactericidal effect on the three bacterial strains with Pseudomonas aeruginosa,a Gram negative lactose fermenter exhibiting a higher sensitivity to dichloromethane semi-crude extract among the treatment groups.For the toxicity test,no mortality and no change in behavior were observed in the Sprague-Dawley rats 14 days after the oral administration of the plant extracts.The methanolic leaf extract of T.philippinense is non-toxic at a maximum dose of 5000 mg/kg.Conclusions:The dichloromethane leaf extract of T.philippinense is a potential antiangiogenic endemic plant species.This plant extract is also a potential antibacterial candidate as determined by microwell assay.The anti-angiogenic and antibacterial activity of the plant may be attributed to the essential oil,steroid,flavonoid,sterol and triterpene content of the plant.