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Brian Chirn, Dongjoon Kim, Philip C Trackman, Sayon Roy; Role of high glucose-induced lysyl oxidase overexpression on Ras activity in rat retinal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4040.
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© ARVO (1962-2015); The Authors (2016-present)
In diabetic retinopathy, cells in the retina undergo significant apoptosis resulting in the development of acellular capillaries and pericyte ghosts. In this study, we determined whether high glucose (HG)-induced lysyl oxidase (LOX) overexpression plays a role in contributing to increased apoptosis by lowering Ras activity, which is known to promote cell survival via the Ras-Raf–ERK pathway.
Rat retinal endothelial cells were grown in normal (N; 5 mM) or HG (30 mM) medium for 7 days, and in parallel, cells grown in HG were transfected with LOX siRNA, or scrambled siRNA as control. Total protein isolated from the cells was assessed for Ras protein activation using an affinity purification assay that utilizes the Ras Binding Domain. Subsequently, activated Ras derived through pull-down assay was evaluated by Western blot (WB) analysis using a Ras specific antibody. To determine if siRNA mediated LOX inhibition altered Ras activity and thereby promoted apoptosis under HG condition, differential dye staining assay was performed.
WB analysis showed significant LOX upregulation in cells grown in HG medium compared to those grown in N medium (132±11% of control, P<0.05). Ras pulldown assay and WB analysis revealed significant decreased Ras activity in cells grown in HG medium compared to those grown in N medium (59±18% of control, P<0.005). When HG-induced LOX overexpression was reduced by approximately 40% via LOX siRNA, Ras activity increased by approximately 20%, compared to those grown in HG medium, and those grown in HG medium transfected with scrambled siRNA. As expected, a significant increase in the number of apoptotic cells was observed under HG medium compared to those grown in N medium (210±37% of control). Interestingly, the number of apoptotic cells was significantly decreased when cells grown in HG exhibited increased Ras activity via LOX siRNA transfection (154±48% of control, P<0.05).
Findings from this study indicate that HG-induced LOX overexpression promotes apoptosis, at least in part, by inhibiting Ras signaling. Therefore, reducing LOX overexpression may protect retinal vascular cells from undergoing HG-induced apoptosis associated with the pathogenesis of diabetic retinopathy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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