Abstract
Purpose :
Diabetes-induced upregulation of lysyl oxidase (LOX) expression contributes to the pathogenesis of diabetic retinopathy (DR). However, the mechanism by which high glucose (HG) induces increased LOX expression is unknown. Since mature LOX partially translocates back from the extracellular environment (internalize) into the intracellular environment, in this study, we examined whether HG compromises this process through excess binding of LOX with extracellular matrix (ECM) proteins, thereby decreasing LOX internalization, attenuating negative feedback, and promoting LOX overexpression.
Methods :
To determine binding of LOX with ECM proteins, total protein (cell proteins + ECM proteins) or ECM-only proteins such as fibronectin (FN) or collagen IV (Coll IV) from rat retinal endothelial cells (RRECs) grown in normal (N; 5 mM) or HG (30 mM) medium for 7 days were subjected to co-immunoprecipitation (co-IP) with LOX antibody followed by Western blot (WB) analysis using Coll IV or FN antibodies. In parallel, to confirm binding of LOX with Coll IV or FN, co-immunostaining was performed with LOX/Coll IV and LOX/FN antibodies on ECM laid by RRECs grown in N or HG medium and Z-stack images using confocal microscopy were collected and analyzed.
Results :
Results from co-IP derived from total protein showed significant increase in LOX bound to Coll IV and FN in cells grown in HG medium compared to those of cells grown in N medium (144±12% of control, p<0.05; 168±11% of control, p<0.05, respectively). In parallel, WB analysis showed significant increase in LOX bound to Coll IV and FN ECM-only protein of cells grown in HG medium compared to those of cells grown in N medium (138±20% of control, p<0.05; 156±21% of control, p<0.05, respectively). Importantly, after removal of cells, immunostaining of LOX to ECM layer analyzed by Z-stack imaging revealed significant increase in bound LOX/Coll IV and LOX/FN in ECM of cells grown in HG medium compared to those of cells grown in N medium.
Conclusions :
Findings from this study indicate that HG promotes binding of LOX with ECM proteins extracellularly that could result in reduced LOX internalization, attenuation of negative feedback, and contribute to LOX overexpression associated with DR.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.