Effects of AFP-172 on COX-2-induced angiogenic activities on human umbilical vein endothelial cells

Purpose: To investigate the angiogenic effect of the free acid of tafluprost (AFP-172) on human umbilical vascular endothelial cells (HUVECs).

Methods: HUVECs cultured in the presence or absence of FP receptor antagonist (10 nM AL-8810) were exposed to escalating concentrations of 10(-7), 10(-6), 10(-5), 10(-4) and 10(-3) M AFP-172 (the free acid of tafluprost). For cell proliferation assays, the numbers of cells were derived from a CellTiter96® Aqueous One Solution Cell Proliferation Assay (Promega) by Microplate reader (Bio-Rad, Benchmark). Endothelial cell migration was evaluated by a BD Biocoat™ Angiogenesis System using FluoroBlok ™ 24-well inserts (BD Biosciences, Bedford, MA). BioTek FLx800 fluorescence plate reader was used for quantitative measurement of fluorescently-labeled invasive vascular endothelial cells. Endothelial capillary-like tube formation was evaluated by BD Biocoat Angiogenesis System using Matrigel Matrix 96-well plate. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the gene expression of vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2) and endothelial nitric oxide synthase (eNOS). COX-2 protein was detected by immunofluorescent staining and Western blot assay. Student's t-test was used for statistical analysis.

Results: 10(-4) M AFP-172 treated cells stimulated the proliferation, migration and tube formation of HUVECs as compared to 10(-5), 10(-6,) 10(-7) M AFP-172 treated cells and control (P < 0.01). RT-PCR showed that incubation of HUVECs with 10(-4) M AFP-172 stimulated the expression of COX-2 mRNA (P < 0.05). Western blot assay revealed that AFP-172 caused cells to increase in COX-2 protein at the concentrations of 10(-4) M.

Conclusions: >AFP-172 showed the angiogenic effects on HUVECs at the concentrations of 10(-4) M by inducing COX-2 protein.