Abstract
Purpose :
Paraoxonase(PON) is established as an important enzyme in mitigating oxidative stress and inflammation. We have already identified that CML can modulate PON expression and induce mitochondrial dysfunction in retinal endothelial and retinal pigment epithelial cells. Retinal pericyte loss is the hallmark of diabetic retinopathy and we observed an increased expression of PON with CML treatment. Therefore, in this study we have tried to evaluate the expression of PON in retinal endothelial-pericytes co-culture and to understand its differential methylation status.
Methods :
We performed the Infinium bead chip methylation EPIC Array (935K array) to identify the methylation status of PON. DNA methyltransferases (DNMTs) (1,3a,3b) and PONs (1,2,3) expressions were quantified in 3.8nM CML treated Human Retinal Endothelial Cells (HREC) and Human Retinal Pericyte (HRP) monoculture and in co culture model with or without 5 Aza 2-Deoxycytidine (Methylation inhibitor). All the experiments were done in triplicates. Statistical analysis was done in comparison to the respective controls using two-way ANOVA.
Results :
In monoculture of HREC DNMTs and PONs expression were not altered with CML treatment when compared to control but when methylation was inhibited using 5 AZA significant decrease in DNMT1 expression was observed and PON genes showed insignificant change. In case of HRP cells when treated with CML all three PON genes and DNMTs expression was increased and when treated with 5 AZA, DNMTs and PON was significantly decreased when compared to CML. Indicating PON expression is influenced by methylation in monocultured HRP cells. whereas in co culture model CML treatment increases PON1 and 3 expressions in HREC and PON 1 in HRP but decreased PON3 unlike monoculture. Further on treatment with AZA in co-culture PON1 and 3 showed significant decrease in HRP and HREC. Methyl sequencing in co culture model revealed differential methylation of PON genes in both the cells.
Conclusions :
This study has identified for the first time that PON expression is modidied upon CML treatment in co-cultured HRP cells. Interestingly PON was found to be differentially methylayed using epic array.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.