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Joseph Arboleda-Velasquez, Dhanesh Amarnani, Leslie Ramos, Santiago Delgado-Tirado, Leo A. Kim; An Explant Model of Human Proliferative Vitreoretinopathy Uncovers Synergistic Effects of Methotrexate and RUNX1 inhibition. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6419.
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© ARVO (1962-2015); The Authors (2016-present)
The aim of this study is to develop an explant model of proliferative vitreoretinopathy (PVR) and evaluate the utility of this model for drug validation. Here, we test two potential drug treatments on our established, primary culture model and a novel human patient-derived explant model of PVR.
PVR membranes were obtained from human donors who had grade C PVR. Specimens were cultured as explants (Ex-PVR) or digested with enzymes to establish primary cultures (C-PVR). The effect of methotrexate (MTX) and RUNX1 inhibition, a transcription factor that we recently found to be critical in PVR, was tested in ARPE-19, C-PVR cultures, and in Ex-PVR. We examined the response of these cells to MTX (10mM, 100μM and 400μM) and Ro5-3335 a small molecule inhibitor of RUNX1 (25μM, 50μM and 100μM) and their combination on proliferation using CyQuant kit at 24 and 48 hours. The effect of MTX (400μM), and Ro5-3335 (150μM) and their combination was tested on Ex-PVR. Phase contrast images were taken up to 14 days and outgrowths quantified using ImageJ.
At 24 hours, 400mM MTX revealed a slight reduction in proliferation in ARPE-19 and C-PVR cells, Ro5-3335 and the combination treatments showed a 20-30% reduction only in the C-PVR model. At the 48 hours, 400μM MTX showed a slight but non-significant reduction in ARPE-19 cells but a significant 25% reduction in C-PVR cells. Ro5-3335 and the combination treatments showed a significant 20-30% reduction in ARPE-19 cells and a 55-60% inhibition of proliferation of C-PVR cells. No significant additive or synergistic effects were observed in any of these conditions between MTX and Ro5-3335 in ARPE-19 or C-PVR cells. In Ex-PVR, Ro5-3335 (150μM) reduced number of outgrowths (35) compared to vehicle (90). Ex-PVR treated with a combination of MTX (400μM) and Ro5-3335 (150μM) showed an almost complete inhibition of any growth (13) suggesting synergistic effect.
In our assays, the Ex-PVR model was used to uncover a synergistic interaction between MTX and Ro5-3335, which suggests that this model may be superior to cell cultures models such as ARPE-19 cells or our primary C-PVR cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Less branching and a much faster regression of the branches were observed in human PVR explants treated with Ro5-3335 (150μM) or a combination of Ro5-3335 (150μM) and methotrexate (400μM).
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