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.

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.

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.