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Andrew Kumar, Piyush C Kothary, Benjamin Rossi, Chintan Mehta, Monte A Del Monte; Valproic Acid Induced Inhibition of Fibroblast Growth Factor 2 Synthesis in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):364.
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© ARVO (1962-2015); The Authors (2016-present)
Human retinal pigment epithelial cells (hRPE) have been implicated in the pathogenesis of age related macular degeneration. Valproic acid (VPA), an epigenetic factor, reduces apoptosis in immortalized ARPE19 cells that were subjected to oxidative injury. In addition, VPA has also been shown to inhibit cancer cell growth and angiogenesis in various animal models. Since very little is known about the effect VPA on primary cultured hRPE cells, we examined the effect of VPA on hRPE cell viability, as well as synthesis of the angiogenic factor, fibroblast growth factor 2 (FGF2) and its receptor (FGFR1) in hRPE cells.
Primary hRPE cell cultures were established from donor non-pathologic human eyes obtained from the Michigan Eye Bank. Cell viability was assessed by the trypan blue exclusion method (T) and the cell proliferation was measured by 3H-thymidine incorporation (3H-thy). 14C-methionine labeled FGF2 (14C-FGF2) and 14C-FGFR1 synthesis were quantitated by immunoprecipitation using FGF2, and FGFR1 specific antibody. Phase contrast microscopy and nuclear staining studies were performed by DAPI. Data were tabulated on Excel spreadsheets, mean ± SEM were determined and statistical significance was established by student’s t-test.
Fetal Bovine Serum (FBS) and FGF2 stimulated hRPE cell proliferation and viability in a dose-dependent manner as shown with trypan blue exclusion and 3H-Thymidine incorporation. Valproic acid (0.5 mM), in contrast, reduced the cell viability (1.75 ± 0.37 vs 3.25 ± 0.87 cells x 10000 ± SEM, n=8, p<0.05). Increasing concentrations of valproic acid decreased the production of immunoprecipitated 14C-FGF2, and 14C-FGFR1 in hRPE cells in a dose dependent manner. Nuclear staining studies confirmed a reduction in hRPE cell number when compared with controls.
Valproic acid reduces the synthesis of the angiogenic factor FGF2, and its receptor FGFR1 in cultured hRPE cells. It also reduces overall cultured hRPE cell proliferation and viability. Since others have shown an anti-apoptotic effect of VPA in injured ARPE19 cells and we show an inhibitory effect of VPA on primary cultured normal hRPE cells, it appears that additional research into the mechanism of VPA anti- apoptotic and anti-proliferative action is required to support its use as a possible therapeutic agent in retinal degenerative and proliferative diseases.
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