Shedding of Vascular Adhesion Protein-1 by VEGF Stimulation under High Glucose Condition

Abstract

Purpose: Vascular adhesion protein (VAP)-1 is an adhesion molecule regulating leukocyte extravasation during leukocyte recruitment cascade, and it also possesses enzymatic activity as semicarbazide-sensitive amine oxidase (SSAO). SSAO is known to cause oxidative stress and generates advanced glycation end products (AGEs), both of which are involved in the pathogenesis of diabetic complications, by its enzymatic function. Soluble form of VAP-1 (sVAP-1) is reportedly increased in the serum of patients with diabetes, and we also showed that sVAP-1 was elevated in the vitreous fluids of diabetic retinopathy (DR). In this study, we investigate the molecular mechanism to increase sVAP-1 protein in DR using cultured retinal endothelial cells.

Methods: Rat retinal capillary endothelial cells (TR-iBRB2) were cultured with media containing either low glucose (5.5mM) or high glucose (25mM). Subsequently, the cells were stimulated with a rat recombinant vascular endothelial growth factor (VEGF, 100ng/ml), and 3 hours later the supernatants were harvested. Immunoprecipitation/western blot was performed to evaluate the levels of sVAP-1 after VEGF stimulation.

Results: Under high glucose condition, the level of sVAP-1 showed approximately 1.8-fold increase in the supernatants of TR-iBRB2 cells when stimulated with VEGF (P<0.05). By contrast, sVAP-1 was not altered with VEGF stimulation under low glucose condition.

Conclusions: The current data indicate that VEGF regulates the production and/or release of sVAP-1 via activation of intracellular signaling in retinal capillary endothelial cells under diabetic condition, leading to deterioration of DR through increase of oxidative stress and AGEs formation by sVAP-1/SSAO.

Keywords: 499 diabetic retinopathy