Inhibition of Src Family Kinases Blocks the Formation of Cortical Cataracts in Cultured Chick Embryo Lenses

Abstract

Abstract: : Purpose: The goal of this study was to determine the role of Src Family Kinases (SFKs) in the development of lens cataract. This question is particularly significant because these tyrosine kinases mediate the stress pathways known to lead to cataract formation. The experiments are focused on whether the inhibition of SFK activity suppresses the formation of lens opacities. Methods: We developed a whole lens culture system in which E10 lenses grown in Medium 199 containing 10% fetal bovine serum form cortical opacities within 5 days. SFK activity was blocked in the cultured lenses by growth in the presence of the SFK-specific inhibitor PP1. Control cultures were grown in medium without inhibitor or in the presence of PP3, the inactive analogue of PP1. Lenses were cultured for 10 days, observed and photographed daily. Opacification was quantified using image analysis software. Tissue architecture was determined following hematoxylin and eosin staining and cellular organization by fluorescent localization of filamentous actin with fluorescein-conjugated phalloidin. Results: Almost all lenses in the control cultures developed cortical opacities covering approximately 50% of the lens area by day 10. Like controls, PP1 treated lenses developed mild posterior opacities during the first 5 days in culture, but then became strikingly transparent. Only 7% of the PP1 treated lenses developed cortical cataract, and the average area of opacity was just 0.5% by culture day 10. In all cultured lenses, even in the presence of the PP1 inhibitor, the bow region of lens extended to the posterior pole and distribution of nuclei from the posterior pole towards the anterior aspects of the lens suggested that newly added fiber cells were misdirected. However, neither this feature, nor the presence of vacuoles appeared to correlate with the development of opacity in the cultured lenses. Instead, the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones, as observed by F-actin staining. Culturing the lenses in the presence of the SFK inhibitor prevented these lens cell aberrations as well as the development of lens opacity. Conclusion: We have shown that the formation of cataract can involve activation of Src Family Kinase-mediated pathway(s) leading to disorganization of developing lens fiber cells and that inhibiting these tyrosine kinases blocks cataract progression.

Keywords: 338 cataract • 580 signal transduction • 592 stress response