Abstract
Purpose :
Carboxylesterases (CES1, CES2) are expressed differently in various ocular tissues across species. Its activity increases with age and various stress factors such as ROS and nutrient deprivation. This study investigates the association of CES with mitochondrial dysfunction associated with retinal cell damage
Methods :
We used four groups of cells: wild type ARPE-19, Rho0 ARPE-19, differentiated ARPE-19 and Cybrids (Normal & AMD). The Rho0 ARPE-19 were depleted of their mitochondrial DNA (mtDNA) by serial passage with low concentration of ethidium bromide. ARPE-19 cells were differentiated by culturing in MEM-Nic media for 7 days. Patient-derived cybrids were made by fusing platelets from age-matched Normal and AMD patients with Rho0 cell lines. Changes in expression levels of CES1, CES2, IL6, IL8, IL1-β, and PGC-1α genes were measured using qRT-PCR from ARPE-19 cells treated with tert-butyl hydroperoxide (tBH,100μM) and serum-free (sf), Rho0 cells. Levels of CES1, CES2, and IL6 expressed in AMD cybrids treated with PU-91 (200μM) and quercetin (20μM) (carboxylesterase inhibitor) were measured using qRT-PCR. One-Way ANOVA and Unpaired t-test were performed to analyse the data
Results :
Differentiated ARPE-19 cells had a 15-fold increase (p=0.32) in CES1 mRNA expression, but no change in CES2. PGC-1α expression was lower in differentiated ARPE19 cells compared to undifferentiated ones. ARPE19-Rho0 cells showed increased CES1 (p=0.004), along with elevated mRNA expression of IL-6 (p=0.08), IL-8 (p=0.002), and IL-1β (p=0.004) compared to wildtype cells. ARPE19 cells cultured in tBH and sf media for 48 hours showed an increase in CES1 (sf p=0.41, tBH p=0.13) and CES2 (sf p=0.27; tBH p=0.014) expression. There was no change in the expression of CES1 or CES2 in the AMD cybrids after PU-91 treatment. In contrast, after quercetin treatment, the expression levels of CES1 were reduced by 50% (p=0.20). AMD cybrids treated with PU-91 plus quercetin showed 16-fold higher CES1 expression (p=0.018) while CES2 expression increased 3-fold compared to untreated
Conclusions :
These findings suggest that upregulation of CES1 is not dependent on the presence of mitochondria but may be more directly related to inflammation. CES1 and CES2 are upregulated in response to oxidative stress and serum deprivation. Quercetin is not a specific inhibitor of CES enzymes. We believe that CES could be a potential therapeutic target in treating AMD
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.