Abstract
Purpose: :
Corneal fibrosis as a contributor to "corneal blindness" is the third leading cause of blindness world-wide. The transformation of corneal keratocytes-to-myofibroblasts is characterized by the expression of α-smooth muscle actin (SMA), the hallmark of corneal fibrosis. The purpose of this study was to elucidate the in vivo role of specific members of cytoskeleton regulators in the transformation of corneal stroma keratocytes-to-myofibroblasts.
Methods: :
Corneal fibrosis was induced in the corneas of six-to-eight week-old female mice (C57BL/6) after an anterior corneal keratectomy under ketamine/xylazine anesthesia with corneal anesthesia and the topical application of TGF-β1 (1μg/ml). All animal procedures were approved by the SingHeakth IACUC. Cytoskeleton regulators RT² Profiler™ PCR Array was used to assay changes in the levels of specific cytoskeleton regulators involved in the development of corneal fibrosis. Real-time PCR was performed to validate the PCR array data using the same set of samples. In vivo studies evaluating effects of moesin on corneal fibrosis was performed by transfection of moesin siRNA into the injured corneal stroma by iontophoresis. The transformation of corneal keratocyte-to-myofibroblast, as defined by the expression of α-SMA, was determined by western blot. Dual immunofluorescence staining was performed to show the co-localization of moesin and α-SMA in the injured corneal stroma.
Results: :
Moesin was the most highly up-regulated (20.96-fold) gene among 84 cytoskeleton regulatory genes after corneal stroma injury with the topical application of TGF-β1. The successful in vivo delivery of moesin siRNA into the injured corneal stroma was confirmed by observing the localization of fluorescein-labeled moesin siRNA in the injured corneal stroma keratocytes. Moesin siRNA downregulated the expression of α-SMA in the injured corneal stroma to 82%, 83% and 70% of control, at PO days 1, 3, 5 respectively. Also in the injured corneal stroma, the up-regulation of phospho-Smad2 induced by TGF-β1 was reduced by moesin siRNA to 83%, 94% and 65% of control at PO days 1, 3, and 5 respectively and the expression of phospho-Smad3 was reduced to 83%, 92% and 81% of control for the same PO days.
Conclusions: :
Moesin has been shown to be a new cytoskeleton regulator and an important component in the development of corneal fibrosis. Additionally we have shown that the level of expression of moesin is amenable to therapeutic manipulation leading to the down regulation of other key biomarkers of fibrosis.
Keywords: cornea: stroma and keratocytes • cytoskeleton • cornea: basic science