Effects And Mechanisms Of Melatonin On ARPE-19 And Human Umbilical Vein Endothelial Cells(HUVEC)Co-culture System Under Hypoxia

Purpose:To study the proliferation and tube formation of HUVEC and its associated mechanisms through melatonin on ARPE-19 and Human Umbilical Vein Endothelial Cells (HUVEC) co-culture system under hypoxia.Methods:ARPE-19 cell line and HUVEC primary cells were contacting and non-contacting co-cultured using Transwell. CoCl2 was used to imitate the hypoxia condition. Different concentration of melatonin was added in the melatonin group. Cell proliferation assay and tube formation assay were used to evaluate the functions of HUVEC. supernatant of culture medium was collected and tested the level of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) by ELISA. ARPE-19 and HUVEC was collected separately and examined the level of VEGF and PEDF by Western Blot and immune cell fluorescence chemistry. The level of mRNA of VEGF and PEDF in both types of cells were tested using qPCR.Results:Part 1:Two co-culture models were built using Transwell. The concentration and time of exposure to CoCl2 were determined. The capabilities of HUVEC co-cultured with ARPE-19 in proliferation and tube formation were increased under hypoxia compared to control. There is no statistically significant between contacting co-culture group and non-contacting co-culture group.Part 2:The capabilities of HUVEC co-cultured with ARPE-19 in proliferation and tube formation were decreased when treated with melatonin compared to hypoxia group. VEGF level in the supernatant were tested, showing an increase underhypoxia and a decrease in the melatonin group. PEDF level showed an decrease in hypoxia group and increased after melatonin was added. The ratio VEGF to PEDF demonstrated an increase in hypoxia group and a decrease in melatonin group. VEGF protein in both ARPE-19 and HUVEC increased when CoCl2 was added, and decreased after treated with melatonin. PEDF protein in both ARPE-19 and HUVEC decreased when CoCl2 was added, and increased after treated with melatonin. mRNA of VEGF and PEDF in both cell types was consistent with the change of protein level.Part 3:The effects and mechanisms of melatonin on HUVEC in a direct way and through ARPE-19 was studied separately. In the HUVEC monoculture model, proliferation and tube formation was decreased when treated with melatonin compared to hypoxia group. VEGF level in the supernatant showed an increase under hypoxia and a decrease in the melatonin group. PEDF level showed a decrease in hypoxia group and an increase after melatonin was added. The ratio VEGF to PEDF demonstrated an increase in hypoxia group and a decrease in melatonin group. VEGF protein in HUVEC increased when CoCl2 was added, and decreased after treated with melatonin. PEDF protein in HUVEC decreased when CoCl2 was added, and increased after treated with melatonin. mRNA of VEGF and PEDF was consistent with the change of protein level.After co-cultured with ARPE-19 intervened with CoCl2, proliferation and tube formation of HUVEC were increased. When co-cultured with ARPE-19 treated with melatonin, those capabilities decreased. VEGF level in the supernatant showed an increase under hypoxia and a decrease in the melatonin group. PEDF level showed a decrease in hypoxia group and an increase after melatonin was added. The ratio VEGF to PEDF demonstrated an increase in hypoxia group and a decrease in melatonin group. VEGF protein in ARPE-19 increased when CoCl2 was added, and decreased after treated with melatonin. PEDF protein in ARPE-19 decreased when CoCl2 was added, and increased after treated with melatonin. mRNA of VEGF and PEDF was consistent with the change of protein level.Conclusion:Melatonin can inhibit proliferation and tube formation of HUVEC induced by ARPE-19 under hypoxia. It modulated the level of VEGF, PEDF and VEGF/PEDF, and changed the production and mRNA expression of VEGF and PEDF in both ARPE-19 and HUVEC. Melatonin has direct effect on the proliferation and tube formation by modulating mRNA expression and protein production of VEGF and PEDF. Melatonin can also decrease the proliferation and tube formation of HUVEC through ARPE-19 by regulating mRNA expression and protein production of VEGF and PEDF.