RT-PCR of Up-Regulated Factors in Abnormally Proliferated Vascular Endothelial Cells by 1,2- Dimethylhydrazine

Many studies for verifying angiogenesis have been in progress, especially in the field of abnormal vascular proliferation to explain the pathogenesis and to develop a treatment of several diseases. In our previous experiments, endothelial cell proliferations were induced by DMH stimulation in vitro, and the 177 factors(142 up- regulated and 35 down-regulated factors) were identified. Among the up-regulated factors, 9 substances (EFEMP1, CTGF, CYR61, ITGbeta1, FHL2, SERPINE1, MYC, PTTG1 and MSH6) were selected, which were related to cell proliferation and showed high signal intensities. The RNA was isolated from HUVECs at the time of 0, 6, 12, 24 hours after the DMH treatment, and RNA of control group HUVECs was also isolated. Genetic information of selected molecules was used to make primer for each, and RT-PCR was performed to analyze both groups. In control and treatment groups, each substance presented variety of manifestation degree according to time differences. EFEMP1, CTGF, CYR61, ITGbeta1, FHL2 and MYC were related to abnormal vascular proliferation steadily and SERPINE1, PTTG1 and MSH6 were related secondarily. CTGF was related to both normal and abnormal proliferation, but it played a more significant role in abnormal proliferation from earlier stage. EFEMP1, CYR61, ITGbeta1, FHL2 and MYC were similar to CTGF, although the relation appeared lately. Further study should be performed to analyze the expressions and the interactions of growth factors, which could be utilized in the new therapeutic development.

Gene Expression Profiling of 1,2-Dimethylhydrazine-Stimulated Human Umbilical Vein Endothelial Cells

The purposes of this study are to establish standard model in which endothelial cell proliferations are induced by DMH stimulation in vitro, and to analyze the gene expressions of proliferative HUVECs using DNA chip technique which could evaluate the mechanisms of angiogenesis, and the development of vascular tumors. To perform the MTT assay in 96-well microplates, 104 cells were seeded in each well which were cultured in medium. On the third day, the cells were treated with 5 different concentrations of diluted DMH from 10 to 10-3 ng/ml. Five DMH-treated groups were compared with the control group which was not treated with DMH. The optical densities in each group were measured at the time of 0, 6, 12, 24, 36, 48, and 72 hours after DMH treatment. The same experiment was repeated 9 times. Statistically significant cell proliferations were observed in 1 and 10-1ng/ml group. The RNAs were isolated from HUVECs of control group and 1ng/ml DMH-treated group, and they were used to analyze the gene expressions using DNA chip technique. One hundred and seventy-seven genes(142 of up-retulated genes and 35 down-regulated genes) were identified, and several genes were associated with VEGF and FGF production. Also DMH could affect expression of genes that involve oncogenesis. Further study should be performed to evaluate the processes of angiogenesis and morphogenesis of vascular tumors, which could be utilized in the development of new therapeutic approaches.