Hypoxia/reoxygenation and TGF-β increase αB-crystallin expression in human optic nerve head astrocytes

Abstract

Reactive astrocytes in glaucomatous optic nerve changes are characterized by an increased expression of αB-crystallin and transforming growth factor-beta (TGF-β). In the pathogenesis of glaucomatous optic nerve damage, ischemia/reperfusion injury may play an important role. The goal of the present study was to determine the influence of hypoxia/reoxygenation and TGF-β on the expression of αB-crystallin in cultured human astrocytes of the optic nerve head (ONH). Cultured human astrocytes were incubated under hypoxic conditions (1% O 2 for 4–12 h) with subsequent reoxygenation (12–24 h). Additionally, cells were treated with 1.0 ng/ml TGF-β1 and TGF-β2 for 12–48 h. Expression of αB-crystallin was examined by Northern- and Western-blotting. Levels of TGF-β1 and TGF-β2 were analyzed by RT-PCR analysis and ELISA. The effect of TGF-β blocking on the hypoxia/reoxygenation modulated expression of αB-crystallin was investigated by simultaneous incubation with neutralizing antibodies against TGF-β during the reoxygenation phase. Hypoxia/reoxygenation increased the expression of αB-crystallin at the mRNA (2.8- to 3.1-fold) and protein level (1.8- to 2.1-fold). Treatment with 1.0 ng/ml TGF-β1 and TGF-β2 for 12–48 h markedly enhanced αB-crystallin mRNA expression approximately three- to fourfold. Using Western blot analysis, this increase ranged from 2 to 3 times. Both cytokines showed a twofold increase after 12 and 24 h of reoxygenation at the mRNA and a two- to threefold increase at the protein level. Simultaneous treatment with neutralizing antibodies against both TGF-β isoforms prevented the hypoxia/reoxygenation-mediated elevation of αB-crystallin. The process of hypoxia/reoxygenation is capable of inducing the expression of αB-crystallin and TGF-ß in cultured ONH astrocytes. Therefore, optimization of conditions leading to hypoxia/reoxygenation in the ONH of glaucomatous patients may help to lower the incidence of characteristic changes in the optic nerve.