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. 2002 Dec;86(12):1428-33.
doi: 10.1136/bjo.86.12.1428.

TGFbeta-Smad signalling in postoperative human lens epithelial cells

S Saika  1 , T Miyamoto, I Ishida, K Shirai, Y Ohnishi, A Ooshima, J W McAvoy
Affiliations

TGFbeta-Smad signalling in postoperative human lens epithelial cells

S Saika et al. Br J Ophthalmol. 2002 Dec.

Abstract

Aims: To localise Smads3/4 proteins in lens epithelial cells (LECs) of fresh and postoperative human specimens. Smads3/4 are involved in signal transduction between transforming growth factor beta (TGFbeta) cell surface receptors and gene promoters. Nuclear localisation of Smads indicates achievement of endogenous TGFbeta signalling in cells.

Methods: Three circular sections of the anterior capsule, one lens, and 17 capsules undergoing postoperative healing were studied. Immunohistochemistry was performed for Smads3/4 in paraffin sections of the specimens. The effect of exogenous TGFbeta2 on Smad3 subcellular localisation was examined in explant cultures of extracted human anterior lens epithelium.

Results: The cytoplasm, but not the nuclei, of LECs of uninjured lenses was immunoreactive for Smads3/4. In contrast, nuclear immunoreactivity for Smads3/4 was detected in LECs during capsular healing. Nuclei positive for Smads3/4 were observed in monolayered LECs adjacent to the regenerated lens fibres of Sommerring's ring. Interestingly, the nuclei of LECs that were somewhat elongated, and appeared to be differentiating into fibre-like cells, were negative for Smads3/4. Fibroblast-like, spindle-shaped lens cells with nuclear immunoreactivity for nuclear Smads3/4 were occasionally observed in the extracellular matrix accumulated in capsular opacification. Exogenous TGFbeta induced nuclear translocation of Smad3 in LECs of anterior capsule specimens in explant culture.

Conclusions: This is consistent with TGFbeta induced Smad signalling being involved in regulating the behaviour of LECs during wound healing after cataract surgery.

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Figures

Figure 1
Figure 1
Intracellular immunolocalisation of Smad3 in human lens epithelial cells on the capsular specimen obtained during cataract surgery (A, case 2) or extracted 10 days after cataract surgery (B; case 6). Lens epithelial cells on the capsular specimen, obtained during first cataract surgery are immunoreactive for Smad3 in the cytoplasm, but not in the nuclei. On the other hand, lens cells 10 days postoperatively show nuclear immunoreactivity for Smad3 with a weak reaction in the cytoplasm. Indirect immunostaining. Bar, 10 μm.
Figure 2
Figure 2
Immunolocalisation of Smad3 in lens cells in Sommerring’s ring 2.7 years postoperatively (case 12). Lens epithelial cells beneath the capsule (arrowheads) are positive for nuclear Smad3. (A) and (B) Higher magnification pictures of boxed areas a and b, respectively, show the progressive disappearance of nuclear Smad3 in elongating lens cells (arrows) as they differentiate into the fibre-like cells. Indirect immunostaining. Bar, 20 μm.
Figure 3
Figure 3
Immunolocalisation of β-crystallin in Sommerring’s ring 2.7 years postoperatively (case 12). (A) Enlarged/elongated lens cells forming the Sommerring’s ring structure are immunostained for β-crystallin, whereas monolayered epithelial-shaped lens cells are unstained; (B) and (C) are the high magnification pictures of boxed areas b and c, respectively. Asterisks in (B) show immunostained enlarged/elongated cells reactive for fibre specific β-crystallin. Arrows in (C) show a monolayer of unstained lens cells. Indirect immunostaining. Bar, 60 μm (A); 20 μm (B and C).
Figure 4
Figure 4
Immunolocalisation of Smad3 in lens cells in Sommerring’s ring 5 years postoperatively (case 16). Lens epithelial cells beneath the anterior capsule (boxed area a) are positive for nuclear Smad3, but somewhat elongated lens cells in the equator (boxed area b) do not show nuclear immunoreactivity. The asterisk indicates the position of the haptic loop of the intraocular lens. Indirect immunostaining. Bar, 50 μm; inserts bar, 10 μm.
Figure 5
Figure 5
Intracellular immunolocalisation of Smad4 in lens epithelial cells following cataract surgery. (A) (Case 1), epithelial cells (arrows) in an uninjured lens show Smad4 immunoreactivity in the cytoplasm. In (B) a specimen obtained 6 days after cataract surgery (case 5), some of lens epithelial cells show Smad4 in the nuclei (arrowheads), while others do not. (C) Nuclei of the lens epithelial cells (arrowheads) in the closed capsular bag 1 year postoperatively exhibit nuclear Smad4 immunoreactivity (case 10). AC = anterior capsule. Indirect immunostaining. Bar, 20 μm (A) and (B), and 50 μm (C).
Figure 6
Figure 6
Reduction of nuclear immunoreactivity of Smad4 in elongating lens cells. In (A) (case 9), some of the lens cells are positive for nuclear Smad4 (arrowheads), whereas others are negative for nuclear Smad4 (arrows). In (B) (case 7) and (C) (case 20), the cells with strong nuclear immunoreactivity tend to be cuboidal (arrowheads) whereas in regions where the cells appear to be elongating, Smad4 immunoreactivity tends to be absent or reduced (arrows). Indirect immunostaining. Bar, 20 μm.
Figure 7
Figure 7
Intracellular immunolocalisation of Smads3/4 in lens cells amid ECM on the healing capsules. (A) Lens cells embedded in ECM exhibit nuclear immunoreactivity for Smad3 (arrowheads) in specimens from case 9 (0.75 years postoperatively). (B) Lens cells with nuclei positive for Smad4 (arrowheads) amid extracellular matrix accumulated inside the capsule of the specimen (case 9). (C) Some lens cells within the ECM show Smad4 immunoreactivity in nuclei (arrowheads), while others exhibit only faint cytoplasmic immunoreactivity. Lens cells attached to the intraocular lens (IOL) optic tend to exhibit the strongest nuclear Smad4 staining (case 13). (D) Nuclei of lens cells in the ECM that has accumulated around the optic portion of an IOL still stain for Smad4 2.7 years after surgery (arrowheads, case 12). No nuclear Smad4 positive cells are seen in the ECM (asterisk) that is not attached to the lens optic. (E) Shows increased magnification of the lens cells in the boxed area in (D). Broken line in (C) indicates the anterior surface of the anterior capsule. Indirect immunostaining. Bar, 50 μm for (A) and (B) and 20 μm for (C) to (E).
Figure 8
Figure 8
Expression of α-smooth muscle actin in elongated, (myo)fibroblast-like lens cells (case 15). Almost all the cells with such a configuration (arrows) are positive for α-smooth muscle actin in a filamentous pattern in the cytoplasm. Indirect immunostaining. Asterisk, extracellular matrix accumulation; Bar, 50 μm.
Figure 9
Figure 9
Nuclear translocation of Smad3 in the epithelium of human lens anterior capsule specimens cultured in the presence of exogenous TGFβ2. In controls, cultured in the absence of TGFβs, Smad3 immunoreactivity was weak, or absent, from the cytoplasm of lens epithelial cells after 30 minutes’ incubation (A). Cytoplasmic immunoreactivity was very strong after 1 hour (C) and weak after 4 hour (E) incubation periods. The cells in the presence of TGFβ2 at 30 minutes showed faint nuclear Smad3 (B), whereas those at 1 hour exhibited marked Smad3 nuclear immunoreactivity (arrowheads, D). After 4 hours, weak nuclear immunoreactivity was detected in some cells (arrowheads), but not in others (F). Indirect immunostaining. Bar, 10 μm.
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