Inhibition of the Human Choroidal Microvascular Endothelial Cells Proliferation by Endostatin

Abstract

Abstract: : Purpose: To develop an in vitro model for cultivation of human choroidal microvascular endothelial cells (HCMECs) and to observe the effect of endostatin on the inhibition of the HCMECs. Methods:Using the method of two–step digestion with trypsin and collagenase for human choroidal tissue samples, we obtained a cell suspension. Then cells were separated and purified by CD31 Dynabeads. Human choroidal microvascular endothelial cells were cultured. The HCMECs were identified by morphological observation, ultrastructural observation with transmission electron microscopy and immunohistochemical staining of endothelial specific marker, the FVIII–Ag, CD31, and CD34. Inhibition experiment of HCMECs proliferation by endostatin was performed. HCMECs were cultured under different conditions (without serum, without serum but with VEGF). Each group was studied by adding different concentrations of ES. The proliferation rate of endothelial cells was assayed with MTT after 24, 48, and 72 hours. The outcomes were analyzed by A values and the I values to demonstrate the inhibition ratio of cell proliferation. I=(A_control–A_test)/A_control×100%. The correlation between the quantity of cells and the A value were judged by correlation analysis(Excel 2003). Differences of A value among groups were determined by one–factor ANOVA and t–test. P<0.05 was considered statistically significant. All data were analyzed by SPSS 11.0 software. Results: We observed no statistically significant inhibition in the no–serum group (P>0.05) 24 hours after adding ES. In the group that did not have serum but had VEGF, statistically significant inhibition was observed in 24 hours if the concentration of ES was more than 313ng/ml (P<0.05). 48 Hours after adding ES, statistically significant inhibition was clearly noticed in the no–serum group if the concentration of ES was 1250ng/ml or more, and in the group that had no serum but had VEGF if the concentration of ES was 157ng/ml (P<0.05). 72 Hr after adding ES, statistically significant inhibition was clearly noticed in the no–serum group if the concentration of ES was 2500ng/ml or more. In the group that did not have serum but had VEGF if the concentration of ES was 10000ng/ml, the inhibition was statistically significant (P<0.05). Conclusions: ES could inhibit the proliferation of HCMECs in culture. Especially the proliferation induced by VEGF can be inhibited at early stages. ES may be a potent antiangiogenesis treatment for CNV.

Keywords: choroid • vascular cells • proliferation