Glutathione and catalase suppress TGFbeta-induced cataract-related changes in cultured rat lenses and lens epithelial explants

Abstract

Purpose: The damaging effects of oxidative stress and transforming growth factor-beta (TGFbeta)-induced transdifferentiation of lens epithelial cells have both been implicated independently in the etiology of cataract. The aim of this study was to investigate whether the presence of antioxidant systems in the lens influences the ability of lens epithelial cells to respond to TGFbeta.

Methods: Whole lenses from young rats were cultured with or without TGFbeta in the presence or absence of reduced glutathione (GSH). Lens epithelial explants from weanling rats were used to investigate the effects of GSH and catalase on TGFbeta-induced cataract-related changes. Lenses were monitored for opacification for three to four days, photographed, and then processed for routine histology. Explants were assessed by phase contrast microscopy, enzyme-linked immunosorbent assay (ELISA) of alpha-smooth muscle actin (alphaSMA), and/or immunolocalization of alphaSMA and Pax6, markers for transdifferentiation and normal lens epithelial phenotype, respectively.

Results: In cultured lenses, GSH strongly suppressed TGFbeta-induced opacification and subcapsular plaque formation. In explants, both GSH and catalase suppressed changes typically associated with TGFbeta-induced transdifferentiation including wrinkling of the lens capsule, cell-surface blebbing, apoptotic cell loss, induction of alphaSMA, and loss of Pax6 expression.

Conclusions: This study suggests that antioxidant systems present in the normal lens, which protect the epithelium against the damaging effects of reactive oxygen species, may also serve to protect it against the potentially cataractogenic effects of TGFbeta. Taken together with other recent studies, it also raises the possibility that TGFbeta may induce cataract-related changes in lens epithelial cells via release of hydrogen peroxide.

Figure 1

Suppression of TGFβ-induced lens opacification by GSH. A,B: Dark field microscopy. Lenses were cultured for three days with 1.75 ng/ml TGFβ2 in the absence (A) or presence (B) of 10 mM GSH and then photographed. Lenses cultured with TGFβ alone typically exhibited numerous discrete opacities (A) whereas many lenses cultured with TGFβ and GSH remained transparent (B). The crescent-shaped haziness in each image is an artifact of the light source. C,D: Hematoxylin-eosin stained lens sections. In lenses cultured with TGFβ alone (C), the epithelium typically became multilayered (arrowhead) and contained large anterior subcapsular plaques of cells (arrow). In the lens shown in D, which retained transparency during the four days of culture with 0.3 ng/ml TGFβ2 and GSH, the epithelium remained cuboidal and monolayered as it is in the normal lens. The scale bar represents 120 μm (C) and 60 μm (D). GSH, glutathione.

Figure 2

Suppression of TGFβ-induced lens opacification by GSH: statistical analysis. Lenses were cultured with TGFβ with or without the addition of 10 mM GSH. The concentration of TGFβ was 0.3 ng/ml (A) or 1.75 ng/ml (B). Photomicrographs recorded on day 4 (A) or day 3 (B) as illustrated in Figure 1 were coded and ranked according to increasing severity of the opacification response. Box and whiskers plots are shown with arrows indicating the median value. The numbers inside the boxes indicate the number of lenses assessed in each case, and the p values indicate the significance of the difference between the two treatment groups (Mann–Whitney test). GSH, glutathione; T, TGFβ.

Figure 3

Suppression of TGFβ-induced cataractous changes in lens epithelial explants by GSH and catalase: phase contrast microscopy. A-C: Precultured explants were cultured for two days in the control medium (A) or with 75 pg/ml TGFβ2 in the absence (B) or presence (C) of 10 mM GSH and then photographed. In controls (A), the epithelial cells were present in the cobblestone array, typical of the normal lens epithelium. Explants cultured only with TGFβ (B) exhibited extensive wrinkling of the lens capsule (arrows) and loss of cells, leaving regions of the capsule exposed (asterisks). Many cells showed dark stippling indicative of blebbing of the cellular surface (arrowheads). Including GSH with TGFβ (C) suppressed these TGFβ-induced changes, resulting in slight wrinkling only (arrows). D,E: Explants were cultured for two days with 75 pg/ml TGFβ2 in the absence (D) or presence (E) of catalase at a concentration of 300 units/ml. Explants cultured with TGFβ alone were extensively wrinkled (D) whereas explants cultured with TGFβ and catalase retained normal cobblestone morphology (E). The scale bar represents 180 μm (A-E). Con, control; GSH, glutathione; Cat, catalase.

Figure 4

Suppression of TGFβ-induced αSMA expression by GSH and catalase: ELISA data. Explants were cultured in the control medium or with 75 pg/ml TGFβ2 in the absence or presence of 10 mM GSH (A) or 300 units/ml catalase (B). The culture period was 2 days (A) or 3 days (B). Explants were then lysed for determination of αSMA and DNA. Each value represents the mean±SEM, n=8–14 (A) and n=6–8 (B). In each case, the p value indicates the significance of the difference between the value for explants cultured with TGFβ alone and those cultured with TGFβ plus the test substance. Con, control; GSH, glutathione; T, TGFβ2; Cat, catalase.

Figure 5

Suppression of TGFβ-induced changes in αSMA and Pax6 expression by catalase. Explants were cultured for two days with catalase alone at a concentration of 300 units/ml (A-C) or with 75 pg/ml TGFβ2 in the absence (D-F) or presence (G-I) of catalase and then fixed as whole mounts and processed for immunolocalization of Pax6 (red; B,E,H) and αSMA (green; C,F,I) by a double labeling technique. Nuclei were counterstained using Hoechst dye (blue; A,D,G). Images of the same explant region are shown in each row. Explants cultured with catalase alone were indistinguishable from the controls in that they exhibited strong nuclear expression of the lens epithelial cell marker, Pax6 (B), and lacked reactivity for the transdifferentiation marker, αSMA (C). Explants cultured with TGFβ lost reactivity for Pax6 (E) and contained many αSMA-positive cells (F). Catalase protected against TGFβ-induced loss of Pax6 expression (H) and and strongly suppressed the induction of αSMA by TGFβ (I). The scale bar represents 60 μm (A-I). Cat, catalase.