Atm inhibition decreases lens opacity in a rat model of galactose-induced cataract

Abstract

Cataract causes vision loss and blindness due to formation of opacities of the lens. The regulatory mechanisms of cataract formation and progression remain unclear, and no effective drug treatments are clinically available. In the present study, we tested the effect of ataxia telangiectasia mutated (Atm) inhibitors using an ex vivo model in which rat lenses were cultured in galactose-containing medium to induce opacity formation. After lens opacities were induced by galactose, the lenses were further incubated with the Atm inhibitors AZD0156 or KU55933, which decreased lens opacity. Subsequently, we used microarray analysis to investigate the underlying molecular mechanisms of action, and extracted genes that were upregulated by galactose-induced opacity, but not by inhibitor treatment. Quantitative measurement of mRNA levels and subsequent STRING analysis revealed that a functional network consisting primarily of actin family and actin-binding proteins was upregulated by galactose treatment and downregulated by both Atm inhibitors. In particular, Acta2 is a known marker of epithelial-mesenchymal transition (EMT) in epithelial cells, and other genes connected in this functional network (Actn1, Tagln, Thbs1, and Angptl4) also suggested involvement of EMT. Abnormal differentiation of lens epithelial cells via EMT could contribute to formation of opacities; therefore, suppression of these genes by Atm inhibition is a potential therapeutic target for reducing opacities and alleviating cataract-related visual impairment.

HIF-1 inhibition reverses opacity in a rat model of galactose-induced cataract

“…In a previous study, we reported that the ATM inhibitors, AZD0156 and KU55933, decreased opacity in a rat lens model of galactose-induced cataract [ 17 ]. ATM is reported to stabilize HIF-1α by phosphorylation [ 18 ].…”

“…We tested 10 μM, 20 μM, and 40 μM of 2ME2 and observed that only 20 μM and 40 μM resulted in a decrease in lens opacity ( Fig 1B and S2 Fig ). Next, we quantified the opacity change before and after 2ME2 administration ( Fig 1C ), as previously established [ 17 ]. The quantification is shown in Fig 1B and S2 Fig .…”

“…The number of days indicated in each panel represents the total days of incubation, “q” indicates samples used for RT-qPCR, and “M” indicates samples used for microarray analysis. (C) The level of lens opacity with and without 2ME2, as well as the change in opacity before and after addition of the inhibitor, were calculated [ 17 ]. Data are expressed as the mean ± SE.…”

“…Six-week-old male SD rats were purchased from Sankyo Laboratory Service (Japan) and used for the experiments. All experiments were approved by the Animal Research Committee of the University of Fukui (approval number: 28091) and conducted in accordance with the University of Fukui regulations on animal experiments and Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research as described previously [ 17 ]. This study adhered to the ARRIVE guidelines in its reporting.…”

“…Samples were imaged in 35-mm Petri dishes containing 7 mL PBS. The lens-cortex opacity was measured in ImageJ as the luminance (0–255) of the opaque lens area, and a weighted average was calculated [ 17 ].…”