Premium
Exogenous regulation of the HIF pathway in RPE cells
Author(s) -
ANDRE H,
EKSTROM M,
TAKEI A,
MA Y,
KVANTA A
Publication year - 2014
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2014.t068.x
Subject(s) - transfection , western blot , vascular endothelial growth factor , pathogenesis , hypoxia (environmental) , microbiology and biotechnology , hypoxia inducible factors , regulator , retinal , immunoprecipitation , medicine , cancer research , cell culture , biology , chemistry , immunology , vegf receptors , gene , ophthalmology , biochemistry , genetics , organic chemistry , oxygen
Purpose To investigate the effects of a series of hypoxia‐inducible factors (HIF)‐regulating molecules (HRM) on the hypoxia pathway in retinal pigment epithelial (RPE) cells, critically involved in neovascular age‐related macular degeneration (nAMD) pathogenesis. Methods ARPE‐19 cells were transfected with plasmids encoding FLAG‐tagged HRM, as well as HIF‐1α and HIF‐2α. Effects of HMR on ARPE‐19 were analyzed by a luciferase reporter assay (DLR), Western blot, and soluble vascular endothelial factor (VEGF)‐capture assay. Effects on endothelial cells (EC) were studied by exposing HUVEC cells to ARPE‐19 pre‐conditioned media after transfection with particular HRM. Results The DLR denoted a marked negative regulation of HIF‐1α activity in ARPE‐19 in the presence of all PHDs. Analysis of HIF‐1α protein showed a very considerable decrease in the presence of PHD2, together with the most dramatic reduction of HIF‐1α protein life‐time. The immunoprecipitated level of VEGF from the media of ARPE‐19 cells transfected with PHD2 was considerably lower than that of an empty control. HUVEC proliferation was decreased in conditioned media from ARPE‐19 transfected with PHD2. Conclusion PHD2 seems to be the most potent negative‐regulator of the HIF pathway. Furthermore, the negative effects of PHD2 were clearly associated with decrease in secreted VEGF and reduced EC proliferation. These results may have implications for the clinical treatment of patients with nAMD, particularly regarding the use of gene therapy to negatively regulate the neoangiogenesis present in these patients.