Effects of AFP-172 on COX-2-induced angiogenic activities on human umbilical vein endothelial cells.
OBJECTIVE
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.
Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
Young Jung
YJ
Roh
Young Gun
YG
Park
Seungbum
S
Kang
Soo Young
SY
Kim
Jung Il
JI
Moon
D043925:Angiogenesis Inducing Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002196:Capillaries / Q000473:pathology; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism; D015237:Dinoprost / Q000031:analogs & derivatives / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000235:genetics; D011460:Prostaglandins F / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D011982:Receptors, Prostaglandin / Q000037:antagonists & inhibitors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
C485334:(5Z)-7-((1R,2R, 3R,5S)-2-((1E)-3,3-difluoro-4-phenoxy -1-butenyl)-3,5-dihydroxycyclopentyl)-5-heptenoic acid; D043925:Angiogenesis Inducing Agents; D011460:Prostaglandins F; D012333:RNA, Messenger; D011982:Receptors, Prostaglandin; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C121227:AL 8810; D015237:Dinoprost; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human