Abstract
Purpose :
Diabetic retinopathy (DR) is a common neurovascular complication of diabetes. Acrolein (ACR) is a toxic lipid aldehyde which can lead to cellular dysfunction and death through the formation of FDP-lysine protein adducts. Studies from our lab have shown that increased FDP-lysine levels occur in Müller cells after just a few months of experimental diabetes and that this adduct contributes to the dysfunction of these cells during DR. Aldehyde dehydrogenase 1A1 (ALDH1A1) represents one of the major enzymes through which ACR is detoxified in vivo. It has been shown to be downregulated in the retina during diabetes. Here, we set up a panel of in vitro assays to investigate whether ALDH1A1 gene therapy is effective in preventing ACR-induced Müller cell dysfunction and death.
Methods :
To mimic Müller cell dysfunction in the diabetic retina, QMMuC-1 murine Müller cells were treated with varying concentrations of ACR. Cell viability, proliferation, and migration were evaluated using Alamar Blue, BrdU, and scratch wound assays, respectively. Quantitative RT-PCR was used to determine the effects of ACR on inflammatory gene expression. Markers of Müller cell dysfunction were assessed by Western blotting. The ability of AAV2.ALDH1A1 to prevent ACR-induced Müller cell changes was examined, with AAV2.blank and AAV2.eGFP used as negative controls.
Results :
ACR triggered cell migration and death in QMMuC-1 Müller cells with an EC50 of 47µM. Low concentrations of ACR (0.1-400µM) caused FDP-lysine accumulation on QMMuC-1 proteins and upregulated the mRNA expression of several inflammatory cytokines. These changes were paralleled by an increase in the protein expression of the oxidative stress marker, HO-1, and a decrease in the protein expression of Kir4.1. An MOI-dependent increase in ALDH1A1 mRNA and protein was observed by exposing the cells to varying levels of AAV2.ALDH1A1. Exposure of the cells to AAV2.ALDH1A1 did not evoke an inflammatory response in QMMuC-1 cells even at high MOIs. AAV2.ALDH1A1 gene therapy ameliorated ACR-induced Müller cell changes when compared with AAV2-untreated, AAV2.blank- and AAV2.eGFP-treated cells.
Conclusions :
Exposure of QMMuC-1 cells to ACR induces cellular damage similar to that observed in Müller cells in the diabetic retina. ALDH1A1 gene therapy may represent an effective approach for preventing ACR-induced Müller cell dysfunction and death during diabetes.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.