PRGF enhances RPE cell survival

Abstract

Purpose : To investigate the influence of PRGF (Plasma Rich in Growth Factors or platelet-enriched plasma) on the survival of RPE cells in situ and in culture.

Methods : ARPE19 cells were cultured in the dark in DMEM-F12 solution, supplemented with 10% FBS. Cells were initially exposed to PRGF for 60min followed by exposed either to blue light (400 lux, 18w/m²) or addition of rotenone (1µM) for 18 hours. Cell viability was then assessed by a MTT reduction assay. Mitochondrial status of cells were viewed using JC-1 dye and DHE was used for the analysis of ROS. For immunocytochemistry, ARPE19 cell cultures were fixed in cold 4% paraformaldehyde and stained for HO-1 and caspase-3. In addition, cultures were stained using the TUNEL method. For ex vivo studies, eyecups obtained from male Wistar rats were exposed to blue light (900 lux, 29.4 w/m²) for 180 minutes in 100% PRGF or DMEM-F12 and 10% FBS, and immediately fixed in 100% methanol and processed for the localization of ZO-1 and HO-1 immunoreactivities.

Results : Blue light and rotenone caused a loss of ARPE19 cell viability, stimulation of ROS production, mitochondrial dysfunction as indicated by use of JC-1 and resulted in some cells staining positively for TUNEL. In addition, HO-1 immunoreactivity was more intense for cells exposed to blue light than rotenone. PRGF dose-dependently blunted the negative effects of blue light and rotenone on cell viability. Moreover, 10% PRGF significantly counteracted the negative effects of blue light and rotenone on ARPE19 cells. ZO-1 staining of gap junctions separating RPE cells in situ were similar whether rat eyecups were immediately stained or processed after incubation DMEM-F12 and 10% FBS for 180 minutes. However, RPE cells in eyecups exposed to blue light showed ZO-1 staining to be irregular in appearance with some cells displaced in position. Moreover, HO-1 immunoreactivity was significantly upregulated in RPE cells in situ following exposure to blue light. Such eyecup RPE cells were clearly significantly less affected by blue light when the insult occurred in the presence of 100% PRGF. The RPE cells were more rigidly maintained in situ with the ZO-1 staining more regular in appearance and a reduced upregulation of HO-1 immunoreactivity.

Conclusions : PRGF protects RPE cells in situ and in culture from insults like rotenone and blue light which cause oxidative stress. Autologous PRGF might be considered for its use in clinical surgeries related to the RPE.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.