Methods

Human induced pluripotent stem cell derived RPE (iPSC-RPE) cells were cultured in 96-well plates. Oxidative stress was induced with NaIO3 (0-4 mM) for 24 h. Cell viability was assessed by using two different methods: resazurin cell viability assay and lactate dehydrogenase (LDH) release. Extracellular vascular endothelial growth factor A (VEGFA) levels were measured using ELISA. ROS production was assessed using a general ROS indicator. Cells were lysed in TRIzol, RNA was purified and converted to cDNA. RT-qPCR analysis included VEGFA, hypoxia inducible factor 1 subunit alpha (HIF1A), TRX, and TRX interacting protein (TXNIP) genes normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. qPCR data was analyzed with 2-ΔΔCT method for relative gene expression analysis.

Results

ROS levels significantly increased with low concentrations of NaIO3 (0-2 mM), peaking at 1 mM (7.7-fold higher compared to 0 mM), while viability indicators did not show decreased cell viability. ROS formation decreased at 1-4 mM, with a significant reduction (P < 0.001) at 4 mM compared to 1 mM. VEGFA release showed a significant increase at 1 mM (P = 0.02) and 4 mM (P < 0.001) compared to 0 mM. LDH release was significantly elevated (P < 0.001) at 3-4 mM compared to 0 mM. VEGFA, HIF1A, and TRX genes were significantly upregulated (P < 0.003) at 2-4 mM, and TXNIP gene was significantly downregulated (P < 0.05) at 2-4 mM.

Conclusions

NaIO3 significantly increased ROS formation in iPSC-RPE cells at low concentrations indicating intracellular oxidative stress damage without signs of decreased cell viability. Oxidative stress activated TRX system and TRX-HIF1α pathway, mitigating ROS formation and enhancing VEGFA release as part of the cellular response.

Robertas Cesna

ARVO 2025

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Authors

Robertas Cesna, Olga Vergun, Anni Kolehmainen, Dovilė Litvinavičiūtė, Giedrius Kalesnykas, Jenni J. Hakkarainen