The novel secretome ST266 promotes survival of oxidant-stressed human RPE by a mechanism involving the Akt/mTOR/p70S6k/GSK-3b pathway

Abstract

Purpose : Oxidative stress contributes to dysfunction and loss of retinal pigment epithelial (RPE) cells observed with age-related macular degeneration (AMD). ST266 is the biological secretome produced by a novel population of amnion-derived multipotent progenitor cells. We have previously shown that ST266 increases RPE cell survival and mitochondrial function after cells were treated with hydroquinone (HQ), hydrogen peroxide (H₂O₂), oxidants related to cigarette smoke, or all-trans Retinal (atRal), a pro-oxidant component of the retinoid cycle. Herein, we aim to determine the mechanism by which ST266 confers protection.

Methods : Cultured human RPE cells were pre-treated for 1h in the presence or absence of MK-2206, a known protein kinase B (Akt) inhibitor, treated with varying concentrations of H₂O₂, HQ, or atRal alone in serum-free media (SF-MEM) for 1.5h at 37^oC for WST-1 assay, followed by 1h or 24h treatment with STM100 (the media in which the amnion progenitor cells are grown) or ST266 (20% in STM100) for Western blot or WST-1 assay, respectively. Western blot analysis was performed to determine phosphorylation of Akt and its downstream targets p70 ribosomal S6 kinase (p70S6K), mammalian target of rapamycin (mTOR), and glycogen synthase kinase 3 beta (GSK-3B). Cell viability was measured with WST-1 reagent.

Results : ST266 significantly increased levels of phosphorylated Akt as well as its downstream targets p70S6k, mTOR, and GSK-3B (p<0.05). Addition of MK-2206 to STM100-treated cells virtually eliminated Akt phosphorylation, while the addition of MK-2206 to ST266-treated cells reduced Akt phosphorylation to near basal levels. ST266 significantly improved cell viability on average by 38%, 20%, and 29% in HQ-, H₂O₂-, and atRal-treated cells respectively compared to STM100 treated cells. This improved viability with ST266 was significantly reduced when MK-2206 was added to 17%, 7%, and 10% in HQ-, H₂O₂-, and atRal-treated cells respectively compared to STM100 treated cells (p<0.05).

Conclusions : When RPE cells were exposed to ST266, Akt phosphorylation increased as well as its downstream targets. ST266 improved cell viability after exposure to various oxidants, an effect that was significantly reduced when Akt activation was blocked. These data suggest that Akt is involved in the mechanism by which ST266 improves cell survival in oxidant-treated human RPE cells.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.