RESUMEN
PURPOSE: The epithelial-mesenchymal transition of human lens epithelial cells plays a role in posterior capsule opacification, a fibrotic process that leads to a common type of cataract. Hyaluronic acid has been implicated in this fibrosis. Studies have investigated the role of transforming growth factor (TGF)-ß2 in epithelial-mesenchymal transition. However, the role of TGF-ß2 in hyaluronic acid-mediated fibrosis of lens epithelial cell remains unknown. We here examined the role of TGF-ß2 in the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells. METHODS: Cultured human lens epithelial cells (HLEB3) were infected with CD44-siRNA by using the Lipofectamine 3000 transfection reagent. The CCK-8 kit was used to measure cell viability, and the scratch assay was used to determine cell migration. Cell oxidative stress was analyzed in a dichloro-dihydro-fluorescein diacetate assay and by using a flow cytometer. The TGF-ß2 level in HLEB3 cells was examined through immunohistochemical staining. The TGF-ß2 protein level was determined through western blotting. mRNA expression levels were determined through quantitative real-time polymerase chain reaction. RESULTS: Treatment with hyaluronic acid (1.0 µM, 24 h) increased the epithelial-mesenchymal transition of HLEB3 cells. The increase in TGF-ß2 levels corresponded to an increase in CD44 levels in the culture medium. However, blocking the CD44 function significantly reduced the TGF-ß2-mediated epithelial-mesenchymal transition response of HLEB3 cells. CONCLUSIONS: Our study showed that both CD44 and TGF-ß2 are critical contributors to the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells, and that TGF-ß2 in epithelial-mesenchymal transition is regulated by CD44. These results suggest that CD44 could be used as a target for preventing hyaluronic acid-induced posterior capsule opacification. Our findings suggest that CD44/TGF-ß2 is crucial for the hyaluronic acid-induced epithelial-mesenchymal transition of lens epithelial cells.
Asunto(s)
Movimiento Celular , Células Epiteliales , Transición Epitelial-Mesenquimal , Receptores de Hialuranos , Ácido Hialurónico , Cristalino , Factor de Crecimiento Transformador beta2 , Humanos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/fisiología , Ácido Hialurónico/farmacología , Receptores de Hialuranos/metabolismo , Factor de Crecimiento Transformador beta2/farmacología , Factor de Crecimiento Transformador beta2/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Cristalino/citología , Cristalino/efectos de los fármacos , Cristalino/metabolismo , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Western Blotting , Opacificación Capsular/metabolismo , Opacificación Capsular/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Citometría de Flujo , Inmunohistoquímica , Células CultivadasRESUMEN
In order to study the mechanisms involved in the development of posterior capsule opacification (PCO) we compared in vivo developed PCO with PCO formed in tissue culture with focus on the periphery of the lens capsule to evaluate lens regeneration potential. We studied three human tissue groups: Cultured lens capsules after mock cataract surgery (nâ¯=â¯6, 30 days), lens capsules from donors that had previously undergone cataract surgery (IOL capsules) (nâ¯=â¯12) and intact lenses (nâ¯=â¯6). All samples were stained with Vimentin, alpha Smooth Muscle Actin, Picro Sirius Red (for collagen) and Paired box protein (Pax6). We found that cultured capsules and less developed IOL capsules consisted mainly of monolayers of mesenchymal cells, while more developed IOL capsules, contained lens epithelial cells (LECs), globular cells and lens fiber cells. Many IOL capsule samples expressed collagen I and III in areas where cells were in contact with the IOL. Pax6 had a similar dispersed distribution in less developed IOL capsules and cultured capsules, while more developed IOL capsules and intact lenses, concentrated Pax6 in LECs at the equatorial lens bow. The similarities between cultured capsules and less developed IOL capsules indicate that our in vitro developed PCO is comparable to early in vivo developed PCO. The similar morphology of more developed IOL capsules and intact lenses seems to indicate an attempt at lens regeneration.