Oxidation of Pericelullar Endothelial Protein Disulfide Isomerase by Urate Hydroperoxide: Implications on Vascular Homeostasis

Uric acid is associated with endothelial dysfunction and progression of cardiovascular disease. When oxidize by plasma peroxidases, uric acid is converted into urate free radical and urate hydroperoxide. Urate hydroperoxide oxidizes small antioxidants like glutathione and methionine and might target thiol groups from proteins in endothelial cell surface. The protein disulfide isomerase (PDI) is a redox sensitive protein that can be found (1 - 2 % of its total expression) at the external surface of the cell (PecPDI). PecPDI is involved in vascular remodeling, platelet adhesion and thrombus formation in a redox-dependent way. Our aim was to investigate whether PDI would be a target to urate hydroperoxide and if it would alter the endothelial cell function. When incubated with recombinant PDI, urate hydroperoxide was rapidly reduced to hydroxyisourate and the thiol groups from PDI were oxidized. The rate constant for this oxidation was 6 × 10 3 M -1 s -1 . After marking surface reduced proteins with the biotinylating reagent Na-(3-maleimidylpropionyl) biocytin, we observe that lower concentrations of urate hydroperoxide are already capable of increasing the extracellular pool of oxidized PDI in HUVECs. In addition, HUVECs exposed to increasing concentrations of urate hydroperoxide exhibited morphological changes - contracted and rounded - and became more sensitive to detachment from culture plates. Nevertheless, treatment with the oxidant did not result in severe cell death, as shown by the results of nuclear staining with specific dyes and detection of dehydrogenase lactate enzyme in supernatant. In conclusion, these results revealed that urate hydroperoxide is capable of causing disturbances in endothelial cells without leads them to death. The oxidation of PecPDI may be one of the mechanisms by which uric acid alters the vascular physiology and supports cardiovascular disease. Supported by: FAPESP CEPID-REDOXOMA 2013/07937-8 and 2011/18106-4, CNPq, CAPES, USP.