Iku Ishida

TGFβ-Smad signalling in postoperative human lens epithelial cells

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 β (TGFβ) cell surface receptors and gene promoters. Nuclear localisation of Smads indicates achievement of endogenous TGFβ 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 TGFβ2 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 TGFβ induced nuclear translocation of Smad3 in LECs of anterior capsule specimens in explant culture. Conclusions: This is consistent with TGFβ induced Smad signalling being involved in regulating the behaviour of LECs during wound healing after cataract surgery.

Latent TGFβ binding protein-1 and fibrillin-1 in human capsular opacification and in cultured lens epithelial cells

BACKGROUND/AIM It was previously reported that collagenous extracellular matrix (ECM) in human capsular opacification contained isoforms of transforming growth factor β (TGFβ). In the present study, the authors performed immunohistochemistry to examine whether ECM in human capsular opacification and in cultures of bovine lens epithelial cells (LECs) contained latent TGFβ binding protein-1 (LTBP-1), TGFβ1 latency associated peptide (β1-LAP), and fibrillin-1, a suspected ligand of LTBP-1 as well as a component of the extracellular microfibrillar apparatus. The aim of the study was to further clarify the mechanism of TGFβ1 deposition in ECM of capsular opacification. METHODS Human capsular opacification specimens and uninjured lens capsules, as well as cultured bovine LECs, were processed for immunohistochemistry using antibodies against LTBP-1, β1-LAP, fibrillin-1, and collagen type I. RESULTS LTBP-1, β1-LAP, and fibrillin-1 all were localised to the ECM in human capsular opacification. Uninjured lens epithelium stained for β1-LAP, but not for LTBP-1 and fibrillin-1. ECM deposited in confluent LEC cultures stained for LTBP-1, β1-LAP, and fibrillin-1, while cultures with only sparse cellularity were unstained for LTBP-1 or fibrillin-1. CONCLUSIONS LECs upregulate LTBP-1 and fibrillin-1 during postoperative healing. LTBP-1, β1-LAP, and fibrillin-1 colocalised to the ECM in capsular opacification and in confluent LEC cultures. TGFβ1 is considered to deposit in ECM in the large latent form. ECM secreted by LEC may function as a scavenger or repository of TGFβ.

Growth factor deposition in anterior subcapsular cataract

Purpose: To characterize immunohistochemically the distribution of growth factors and extracellular matrix (ECM) components in an anterior subcapsular cataract (ASC) and to determine the role of growth factors in the development of ASC. Setting: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. Methods: During cataract surgery in 22 patients, anterior capsules with an ASC were obtained. Sections of each specimen were immunostained with a panel of antibodies against ECM components, growth factors, cytoskeletal components, and signal transduction‐related molecules. Results: Collagen types I, V, and VI; fibronectin; fibrillin‐1; and latent transforming growth factor β binding protein‐1 (LTBP‐1) were localized to the ECM in ASC tissues. Collagen IV was localized to the ECM and the capsule. Lens epithelial cells (LECs) were positive for α‐smooth muscle actin (αSMA). Lens epithelial cells and ECM stained for transforming growth factor β2 (TGFβ2) and TGFβ3 in all samples, but TGFβ1 latency‐associated peptide (TGFβ1‐LAP) were detected in some samples. Fibroblast growth factor‐2 (FGF‐2) and hepatocyte growth factor‐α (HGF‐α) were localized to the ECM. Lens epithelial cells with nuclear staining for Erk‐1, the mitogen‐activated protein kinase (MAP kinase) cascade‐related molecule, and Smad3, 1 of the Smad family members involving TGFβ signaling, were detected. Conclusions: Matrix components (ie, collagen types, fibronectin, fibrillin‐1), as well as growth factors such as TGFβ1‐LAP, TGFβ2, TGFβ3, FGF‐2, and HGF‐α, were detected in ASC. Fibrillin‐1 might serve as a repository for TGFβs. These growth factors may modulate the phenotypic alteration and behavior of LECs. The MAP kinase cascade and TGFβ signaling are both activated in LECs in ASC.

Comparison of Scheimpflug images of posterior capsule opacification and histological findings in rabbits and humans

Purpose: To compare the posterior capsule opacification in Scheimpflug photographic images produced by an electronic anterior eye segment analysis system with the histopathological findings in rabbits and humans. Setting: Department of Ophthalmology, Wakayama Medical College, Japan. Methods: Opacified posterior capsules were photographed using the EAS‐1000 system (Nidek) and were then extracted during vitreous surgery for proliferative diabetic retinopathy or proliferative vitreoretinopathy in 2 patients. In rabbits, phacoemulsification and aspiration (PEA) with intraocular lens (IOL) implantation was performed. The IOL was implanted in the bag or in the sulcus. After intervals of healing, the posterior capsule was photographed with the EAS‐1000 and the animals were then killed. In both clinical and experimental specimens, the posterior capsule was processed for light microscopic histology and immunohistochemistry. Results: Opacified human capsules were well imaged by the EAS‐1000. Histology showed that lens epithelial cells proliferated with and without an accumulation of extracellular matrix. Details such as rolling of the capsulotomy edge were seen well. Regenerated lens fibers of Soemmerings ring were seen as a mass within the capsule. In the rabbit model, Scheimpflug images accurately represented the capsules as they appeared histologically. Conclusion: The EAS‐1000 system provided faithful, relatively high‐resolution images that corresponded to the histologic findings in the posterior capsules after PEA‐IOL surgery in humans and rabbits.

Collagens XII and XIV (FACITs) in capsular opacification and in cultured lens epithelial cells

Purpose. We previously reported that extracellular matrix (ECM) accumulation in human capsular opacification included collagen types I, III, IV, V, and VI. To further characterize the ECM in capsular opacification we performed immunohistochemistry to localize collagen types XII and XIV (fibril-associated collagens with interrupted triple helices, or FACITs) in specimens of human capsular opacification and in cultures of bovine lens epithelial cells (LECs). Methods. Cryosections and paraffin sections of human capsular opacification specimens or uninjured lens capsules, as well as cultured bovine LECs, were processed for immunohistochemistry using antibodies against collagen types I to VI, XII, and XIV. A rat crystalline lens was punctured through the central cornea and the eye was processed for immunohistochemistry for FACITs after healing intervals. Results. In the absence of injury human LECs were unstained for FACITs, but as early as 10 days after operation, LECs in healing capsules were immunoreactive. Collagen types I, III, IV, V, and VI were also detected. ECM deposited in confluent LEC cultures stained for FACITs. Normal rat LECs were not stained for FACITs, but ECM accumulated in injured lens stained for them. Conclusions. LECs up-regulate FACITs during post-opera-tive healing. FACITs, as well as other collagen types, are deposited in ECM in healing injured rat lens, in human capsular opacification and in LEC cultures. ECM components may regulate LEC behavior during postoperative healing.

Cellular Fibronectin, but not Collagens, Disappears in the Central Posterior Capsules During Healing After Lens Extraction and IOL Implantation in Rabbits

PURPOSE To investigate the nature of capsular opacification after cataract-intraocular lens (IOL) surgery in rabbit eyes, we immunohistochemically located extracellular matrix components in lens capsules after the surgery using light microscopy. The study was conducted also to compare the extracellular matrix components in rabbit capsules with those previously reported in the human eye. METHODS Twenty-seven eyes of 17 Japanese albino rabbits were lensectomized by phacoemulsification, and IOLs were implanted. Using immunohistochemical methods, the lens capsules were examined immediately after surgery, and 1, 2, 4, and 8 weeks after surgery. RESULTS In all cases at each time point, the edge of the anterior capsulotomy had contracted and was found to adhere to the inner surface of the posterior capsule, with both IOL haptics remaining in the capsular bag. Collagen types I and III were detected around the adhesion between the anterior capsulotomy edge and posterior capsule during all stages of healing and also observed on the central posterior capsules 1 or more weeks after surgery. Immunoreactivity for cellular fibronectin was seen around the adhesion between the anterior capsulotomy edge and posterior capsule during all stages of healing. It was also detected on the posterior capsules 2 and 4 weeks after surgery, but disappeared 8 weeks after surgery. CONCLUSION Extracellular matrix components such as collagen types I and III and cellular fibronectin were expressed inside the residual lens capsular bag. Cellular fibronectin may play a role in the early wound healing process in the postoperative posterior capsule because the immunoreactivity in the central posterior capsule disappears in the later phase of healing.

Lens epithelial cell regeneration of a capsule-like structure during postoperative healing in rabbits.

Purpose: To determine whether lens epithelial cells (LECs) can regenerate the lens capsule during healing after lens extraction and intraocular lens (IOL) implantation. Setting: Department of Ophthalmology, Wakayama Medical College, Japan. Methods: Extracapsular lens extraction and IOL implantation were performed in 5 adult albino rabbits. Lens capsules were examined histologically and immunohistochemically 3 and 5 months later. Results: Lens epithelial cells proliferated and regenerated lens fibers within the capsular bag. A multilayered homogenous capsule‐like structure was present in the equatorial region. The structures contained type IV collagen but not type I collagen. Conclusion: Lens epithelial cells can regenerate lens capsule‐like structures during healing after lens extraction. Postoperative LECs without phenotypic conversion to a fibroblastic type may produce this structure.