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.
Cataract, a disease that causes opacity of the lens, is the leading cause of blindness worldwide. Cataracts secondary to diabetes are common, even in young patients, so they are of significant clinical importance. Here, we used an ex vivo model of galactose-induced cataracts in the rat lens to investigate the therapeutic effects of histone acetyltransferase (HAT) inhibitors. Among the tested HAT inhibitors, TH1834 was the only one that could reverse most of the opacity once it had formed in the lens. Combination treatment with C646/CPTH2 and CBP30/CPTH2 also had therapeutic effects. In lens cross-sections, vacuoles were present in the tissue of the cortical equatorial region of untreated cataract samples. In treated cataract samples, lens tissue regenerated to fill the vacuoles. To identify the genes regulated by HAT inhibitors, qRT-PCR was performed on treated and untreated cataract samples to determine candidate genes. Expression of Acta1 and Stmn4, both of which are involved in the cytoskeleton, were altered significantly in C646+CPTH2 samples. Expression of Emd, a nuclear membrane protein, and Prtfdc1, which is involved in cancer cell proliferation, were altered significantly in CBP30+CPTH2 samples. Acta1, Acta2, Arrdc3, Hebp2, Hist2h2ab, Pmf1, Ppdpf, Rbm3, RGD1561694, Slc16a6, Slfn13, Tagln, Tgfb1i1, and Tuba1c in TH1834 samples were significantly altered. These genes were primarily related to regulation of cell proliferation, the cytoskeleton, and cell differentiation. Expression levels increased with the onset of cataracts and was suppressed in samples treated with HAT inhibitors.
Although cataracts affect almost all people at advanced age and carry a risk of blindness, the mechanisms of cataract development remain incompletely understood. Oxidative stress, which is a causative factor in cataract, results in DNA breakage, which suggests that DNA damage could contribute to the formation of cataracts. We developed an ex vivo experimental system to study changes in gene expression during the formation of opacities in the lens by culturing explanted rat lenses with Methylmethanesulfonate (MMS) or Bleomycin, which induce DNA damage. Lenses cultured using this experimental system developed cortical opacity, which increased in a concentration- and time-dependent manner. In addition, we compared expression profiles at the whole gene level using microarray analysis of lenses subjected to MMS or Bleomycin stress. Microarray findings in MMS-induced opacity were validated and gene expression was measured from Days 1–4 using RT-qPCR. Altered genes were classified into four groups based on the days of peak gene expression: Group 1, in which expression peaked on Day 1; Group 2, in which expression peaked on Day 2; Group 3, in which expression progressively increased from Days 1–4 or were upregulated on Day 1 and sustained through Day 4; and Group 4, in which expression level oscillated from Days 1–4. Genes involved in lipid metabolism were restricted to Group 1. DNA repair- and cell cycle-related genes were restricted to Groups 1 and 2. Genes associated with oxidative stress and drug efflux were restricted to Group 2. These findings suggest that in temporal changes of MMS-induced opacity formation, the activated pathways could occur in the following order: lipid metabolism, DNA repair and cell cycle, and oxidative stress and drug efflux.
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