Abstract
Purpose :
Proliferative vitreoretinopathy (PVR) is characterized by fibrotic membranes that form on the retina surfaces that lead to retinal re-detachment. One key aspect of PVR pathogenesis is retinal pigment epithelial (RPE) cell epithelial mesenchymal transition (EMT). Compounds that inhibit EMT are promising candidates for treating PVR. Here we show that the polyether ionophore compound salinomycin (SNC) effectively inhibits TGFβ-induced EMT of RPE cells.
Methods :
Human ARPE-19 cells and human primary RPE cells (hRPE) were treated with 10ng/ml TGFβ to induce EMT, and simultaneously with DMSO (vehicle) or 100-500nM SNC. Inhibition of EMT was measured by Western blotting and/or immunofluorescence for early markers of EMT, including αSMA and Col1a1. Relative RNA levels of EMT transcripts were analyzed by RT-qPCR. Salinomycin’s effect on the TAK1/p38 and Smad signaling pathways was examined by Western blotting and immunofluorescence. All experiments were performed 2-3 times on different passages of RPE cells; similar trends were seen between experimental replicates.
Results :
Immunofluorescence microscopy showed αSMA expression was strongly induced by TGFβ and inhibited by SNC. Similarly, Western blotting analysis for αSMA and Col1a1 showed a 2- and 32-fold induction, respectively, upon TGFβ treatment, which was potently inhibited to pre-TGFβ treatment levels with 250nM SNC. RT-qPCR showed mRNA levels for αSMA and Col1a1 were inhibited 1.6- and 3-fold, respectively, with TGFβ+250nM SNC.
After 60-120 minutes of TGFβ stimulation, SNC-treated cells showed a decrease in phospho-p38 expression compared to control treatment. After the first two hours of TGFβ+SNC exposure, there was no significant difference in phospho-Smad2 expression levels, however, expression levels were significantly lower after 24 hours. Additionally, immunofluorescence in ARPE-19 cells at 48 hours after TGFβ treatment with antibodies for both pTAK1 and pSmad2/3 showed a decrease in expression in cells treated with SNC compared to those treated with DMSO. Together, these results show that SNC is effective at inhibiting TGFβ-induced RPE cell EMT, and both canonical and non-canonical TGFβ signaling pathways are involved.
Conclusions :
We show that SNC is effective at inhibiting TGFβ-induced EMT in RPE cells, a major process involved in PVR pathogenesis. Further in vivo studies will be done to examine SNC as a potential treatment for PVR.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.