Abstract
Purpose :
Increased oxidative/nitrative stress and chronic inflammatory responses have been implicated in the pathogenesis of diabetic retinopathy (DR). Xanthine oxidoreductase (XOD) is an enzyme involved in purine metabolism which generates superoxide anions and, as catabolic by-product, uric acid (UA). Recently we have shown that XOD is activated in the diabetic retina leading to increased production of UA. The mechanisms by which hyperglycemia stimulates XOD expression and activity as well as its pathogenic consequences in DR are poorly understood. Here, we have investigated the effects of in vivo blockade of XOD in the diabetic retina and the contribution of different toll-like receptors (TLRs) to hyperglycemia-induced up-regulation of XOD in human retinal endothelial cells (HuREC).
Methods :
Male streptozotocin-induced diabetic rats (STZ-rats) were kept diabetic for 8 weeks. Some of the STZ-rats received a dose (55 mg/Kg) of the XOD inhibitor allopurinol (ALL) administered orally every other day. At the time of sacrifice, the retinas were excised and analyzed for oxidative/nitrative stress parameters [4-hydroxynonenal (4-HNE) and nitrotyrosine (NY)] and expression of inflammatory markers such as ICAM-1, IL-1beta and IL-6. TLR4, 2 and 7/8 were silenced in HuREC by transfection with specific siRNAs. XOD expression at the mRNA and protein level was measured by qPCR and Western blotting analyses, respectively. XOD activity was assessed by spectrophotometric enzymatic analysis measuring UA production.
Results :
Treatments with ALL resulted in significant decreases of 4-HNE and NY and in blunted expression of inflammatory markers, such as IL-1beta, IL-6 and ICAM-1, in the diabetic rat retina. XOD expression and activity in HuREC exposed to glycidic stress (HG = 25 mM D-glucose for 48 hours) were significantly up-regulated in comparison to cells cultured in normal glucose levels (NG = 5 mM D-glucose) or with the osmotic control (LG = 25 mM L-glucose).Transfection of the cells with siRNA specific for TLR4, 2 or 7/8 significantly reduced HG-induced up-regulation of XOD expression and activity and showed highest inhibitory effects by TLR7/8 signaling.
Conclusions :
The data obtained suggest that TLR-mediated up-regulation of XOD plays a central role in mediating pro-oxidative/nitrative and pro-inflammatory challenges in the diabetic retina.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.