Gene therapeutic enhancement of acrolein detoxification mechanisms for the treatment of diabetic retinopathy (DR)

B. M. Karan, J. Augustine, P. Barabas, M. Chen, A. W. Stitt, T. M. Curtis

Research output: Contribution to journalMeeting abstractpeer-review

Abstract

DESIGN. In vitro investigation of ALDH1A1 gene therapy as an effective treatment for DR.

PURPOSE. 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 during 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. In this study, we have set up a panel of in vitro assays to begin 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 Muller cells were treated with varying concentrations of ACR and cell viability, inflammatory gene expression and markers of Müller cell dysfunction and oxidative stress were evaluated using the Alamar Blue assay, qRT-PCR and Western blotting, respectively.QMMuC-1 cells were also exposed to increasing concentrations of AAV2.ALDH1A1(2800-50000 MOI) and the level of ALDH1A1 overexpression and inflammatory gene expression determined by qRT-PCR.

RESULTS. ACR triggered cell death in QMMuC-1 cells with an IC50 of 47.4 μM. Low concentrations of ACR (nM range) upregulated the mRNA expression of several inflammatory cytokines (IL-18, IL-6 and IP-10) and caused FDP-lysine accumulation on QMMuC-1 proteins. 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 the K+ channel, Kir4.1, which plays an important role in Müller cell K+ homeostasis. A concentration-dependent increase in ALDH1A1 mRNA 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.

CONCLUSIONS. Exposure of QMMuC-1 cells to ACR induces cellular damage similar to that observed in Müller cells in the diabetic retina. Studies are ongoing to study the feasibility of using ALDH1A1 gene therapy for preventing ACR-induced Müller cell dysfunction and death during diabetes.

Original languageEnglish
Pages (from-to)23
Number of pages1
JournalEuropean Journal of Ophthalmology
Volume32
Issue number1_suppl
DOIs
Publication statusPublished - 28 May 2022

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