Kun-Che Chang1, J Mark Petrash1. 1. Department of Ophthalmology School of Medicine, University of Colorado, Anschutz Medical Campus, Aurora, Colorado, United States Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz.
Abstract
PURPOSE: Cataract surgery involves removal of lens tissue, but is associated with a high complication rate due to regrowth of residual lens epithelial cells to produce posterior capsule opacification (PCO) and diminished visual acuity. As inhibitors of aldose reductase (AR) have been shown to suppress markers of PCO, our studies were designed to identify a role for AR in the pathogenesis of PCO. METHODS: Sorbinil-mediated AR inhibition was determined by measuring sorbitol accumulation. Cell migration was measured using both transwell and scratch assays. Proteins in the SMAD signaling pathway were measured by Western blotting. The interactions of AR and SMADs were demonstrated by co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA). Epithelial-to-mesenchymal transition (EMT) expression was measured by Western blot and quantitative PCR (q-PCR). Matrix metalloproteinase (MMP)-2 and MMP-9 activities were measured in conditioned medium by zymography. RESULTS: We observed that either Sorbinil-mediated AR inhibition or siRNA-mediated AR gene knockdown prevented migration of lens epithelial cells following exposure to TGF-β2. AR inhibition or AR knockdown reduced SMAD and MMP activation triggered by TGF-β2. In addition, we demonstrated AR inhibition or AR knockdown decreased TGF-β2-induced expression of EMT markers. Co-IP studies and PLA were used to demonstrate that AR and SMAD2 interact either directly or in close concert with additional factor(s) in a nonenzymatic manner. CONCLUSIONS: This study demonstrates that AR participates in the response of lens epithelial cells to TGF-β2. Our studies raise the possibility that AR inhibition may be effective in preventing development of PCO by disrupting the TGF-β2/SMAD pathway.
PURPOSE:Cataract surgery involves removal of lens tissue, but is associated with a high complication rate due to regrowth of residual lens epithelial cells to produce posterior capsule opacification (PCO) and diminished visual acuity. As inhibitors of aldose reductase (AR) have been shown to suppress markers of PCO, our studies were designed to identify a role for AR in the pathogenesis of PCO. METHODS:Sorbinil-mediated AR inhibition was determined by measuring sorbitol accumulation. Cell migration was measured using both transwell and scratch assays. Proteins in the SMAD signaling pathway were measured by Western blotting. The interactions of AR and SMADs were demonstrated by co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA). Epithelial-to-mesenchymal transition (EMT) expression was measured by Western blot and quantitative PCR (q-PCR). Matrix metalloproteinase (MMP)-2 and MMP-9 activities were measured in conditioned medium by zymography. RESULTS: We observed that either Sorbinil-mediated AR inhibition or siRNA-mediated AR gene knockdown prevented migration of lens epithelial cells following exposure to TGF-β2. AR inhibition or AR knockdown reduced SMAD and MMP activation triggered by TGF-β2. In addition, we demonstrated AR inhibition or AR knockdown decreased TGF-β2-induced expression of EMT markers. Co-IP studies and PLA were used to demonstrate that AR and SMAD2 interact either directly or in close concert with additional factor(s) in a nonenzymatic manner. CONCLUSIONS: This study demonstrates that AR participates in the response of lens epithelial cells to TGF-β2. Our studies raise the possibility that AR inhibition may be effective in preventing development of PCO by disrupting the TGF-β2/SMAD pathway.
Authors: Lucy Jean Dawes; Matthew Alexander Sleeman; Ian Keith Anderson; John R Reddan; Ian Michael Wormstone Journal: Invest Ophthalmol Vis Sci Date: 2009-06-10 Impact factor: 4.799
Authors: Leonid M Zukin; Michelle G Pedler; Kevin Chyung; Sarah Seiwald; Patricia Lenhart; Biehuoy Shieh; J Mark Petrash Journal: Chem Biol Interact Date: 2019-04-23 Impact factor: 5.192