Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-ß2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-ß2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-ß2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-ß2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

Time Course of Lens Epithelial Cell Behavior in Rabbit Eyes following Lens Extraction and Implantation of Intraocular Lens.

BACKGROUND: After cataract surgery, some lens epithelial cells (LECs) transdifferentiate into myofibroblast-like cells, which causes fibric posterior capsule opacification (PCO). Residual LECs differentiate into lens fiber cells, forming Elschnig pearls with PCO. This study was carried out to identify the time course of both types of LEC behavior in rabbit eyes following lens extraction and implantation of an intraocular lens (IOL). METHODS: Phacoemulsification and implantation of posterior chamber IOLs were performed in rabbit eyes. Following enucleation, immunohistochemical methods were used to detect α-smooth muscle actin (α-SMA), a marker for myofibroblast-like cells, in the pseudophakic rabbit eyes. A mouse monoclonal antibody against α-SMA was used. RESULTS: Soon after the operation, the LECs migrated and covered the lens capsule. Thereafter, the LECs around the anterior capsular margin were always positive for α-SMA. However, the distributions of these cells were not consistent. In some specimens, α-SMA-positive LECs were present around the IOL optic early after surgery, but most of them had disappeared several weeks after the surgery. The residual cells induced fibrotic PCO. In the other specimens, most LECs around the IOL optic except the anterior capsular margin were negative for α-SMA. In the peripheral region covered by the peripheral anterior and posterior capsules, LECs on the posterior capsule always differentiated into lens fiber cells and formed a Soemmering ring. Thereafter, migration of lens fiber cells from the Soemmering ring and differentiation of LECs in situ on the central posterior capsule consisted of Elschnig pearls type of PCO. CONCLUSIONS: Although postoperative LEC behavior is not consistent, residual α-SMA-positive LECs induced fibrotic PCO. The lens fiber cells that migrated from the peripheral capsular bag or that were differentiated in situ covered the central posterior capsule, forming Elschnig pearls with PCO.

Topical administration of a ROCK inhibitor prevents anterior subcapsular cataract induced by UV-B irradiation.

The deposition of extracellular matrix (ECM)-which is mainly composed of type I collagen-in anterior subcapsular cataracts (ASCs) during epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs) decreases visual function. Transforming growth factor (TGF)-ß is a key factor in the induction of EMT in LECs. Although Rho kinase (ROCK) plays an important role in EMT induced by TGF-ß, it is unknown whether ROCK inhibition affects type I collagen expression in TGF-ß-stimulated LECs and ASC formation. This was investigated in the present study both in vitro using human lens epithelium (HLE)-B3 cells and in vivo using mice with ultraviolet radiation (UVR)-B-induced cataracts. We found that TGF-ß2 increased type I collagen mRNA expression in HLE-B3 cells; this was inhibited in a dose-dependent manner by treatment with the ROCK inhibitor Y-27632. UVR-B exposure caused ASC formation in mice. A histopathological examination revealed that LECs in the anterior subcapsular area were flattened and multi-layered, and had a spindle shape in cross section. Immunohistochemical analysis revealed the presence of α-smooth muscle actin and type I collagen around these flattened LECs; these opacities were reduced by topical instillation of Y-27632. These findings suggest that suppression of TGF-ß signaling in LECs by topical application of a ROCK inhibitor can prevent the formation of ASCs.

Effect of Geranylgeranylacetone on Ultraviolet Radiation Type B-Induced Cataract in Heat-Shock Transcription Factor 1 Heterozygous Mouse.

PURPOSE: We investigated whether heat-shock transcription factor 1 (HSF1) was involved in ultraviolet radiation type B (UVR-B)-induced lens opacity (cataract) using HSF1 heterozygous mice. We also examined the effects of geranylgeranylacetone (GGA), an inducer of heat-shock proteins via activation of HSF, on the UVR-B-induced cataract. MATERIAL AND METHODS: Male HSF1+/- and WT mice were unilaterally exposed to UVR-B (total: 1200mJ) at 16 weeks of age. At 48 h after the last UVR-B irradiation, the lens was isolated and the induction of the cataract was quantified as the cataract area ratio (opacity area/anterior capsule). GGA was orally administered at a dosage of 500 mg/kg once a day for two days before the first UVR-B exposure until the end of the experiment (21days in total). RESULTS: The HSF1 expression was more greatly decreased in the lens from HSF1+/- mice than in that from WT mice (p < 0.01). UVR-B exposure could mainly induce cataracts in the anterior capsule in both HSF1+/- and WT mice, while the opacity of the lens was markedly enhanced in HSF1+/- mice compared to that in WT mice(p (0.01). GGA treatment could prevent the induction of lens opacity by UVR-B exposure in both WT and HSF1+/- mice as compared with the non-administration group (p < 0.01). No obvious alteration by the UVR-B radiation was seen in lens protein levels of αA-crystallin, αB-crystallin, or γ-crystallin with or without GGA administration among all groups of mice. In contrast to the crystallins, the lens protein level of HSP25 was decreased by UVR-B exposure in both HSF1+/- and WT mice, and was significantly recovered in WT mice by the GGA treatment (p < 0.01). The induction of HSP25 was suppressed in HSF1+/- mice compared with that in WT mice. CONCLUSIONS: These data suggest that HSF1 plays an important role in the occurrence of UVR-B-induced cataracts, possibly via regulation of HSPs such as HSP25.