Retinal capillary loss, due to hyperglycemia-induced vascular cell death, is a prominent feature of diabetic retinopathy (DR), a common complication of diabetes and the leading cause of blindness in adults. Hyperglycemia-induced oxidative stress (OxS) is thought to result in retinal vascular cell death and consequent capillary loss. Along with increased formation of reactive oxygen species (ROS), oxidative stress in the retinal hyperglycemic milieu can result from the impairment of endogenous anti-oxidant defense mechanisms. The overall goal of this proposal is to investigate the effects of diabetes on the activity of the endogenous anti-oxidant system of the thioredoxin, a central regulator of cellular redox homeostasis, and to develop strategies to prevent its malfunction in the diabetic retina. The thioredoxin system (TrxS) consists of thioredoxin (Trx1), Trx reductase (TrxR1), thioredoxin inhibitory protein (TXNIP) and NAD(P)H. Trx1 reduces oxidized molecules with its redox active center and is reduced back to its "active" form by the selenocysteine enzyme TrxR1, which utilizes NAD(P)H as an electron donor. The binding of TXNIP to Trx1 represents a stop signal for the system, thereby serving as a physiologic negative regulator of the system. We have recently found that hyperglycemia promotes TXNIP serine phosphorylation (SerP). Sequence analysis shows that this molecular event is dependent on Protein Kinase C delta (PKC?), a ROS sensitive PKC isoform up-regulated in the diabetic retina and in retinal endothelial cells (RECs) exposed to high glucose concentrations (HG). We have also found that hyperglycemia inhibits the activity of TrxR1 with no changes in its expression. Oxidative and nitrative post-translational modifications are known to affect the enzymatic activity of TrxR1. Protein nitration has been shown to be up-regulated in the diabetic retina and may also contribute to impairment of TrxR1. In this research proposal, we are aiming at determining the molecular mechanisms involved in hyperglycemia-induced changes in the TrxS by focusing on the events associated with TXNIP SerP and TrxR1 impairment. We hypothesized that hyperglycemia-induced impairment of the TrxS involves PKC?-dependent serine phosphorylation of TXNIP and TrxR1 protein nitration. We designed experiments to be conducted in vitro in RECs (a major target of diabetes) exposed to HG, and in vivo by using streptozotocin-induced diabetic rats (STZ-rats, a model of Type 1 diabetes).
Aim1)) Test the hypothesis that HG/diabetes promotes PKC-dependent serine phosphorylation (SerP) of TXNIP and this effect contributes to oxidative retinal endothelial cell injury.
Aim 2)) Test the hypothesis that HG/diabetes induces TrxR1 protein nitration and this effect contributes to oxidative retinal endothelial cell injury.
Oxidative stress (OxS) plays a key role in the pathogenesis of diabetic retinopathy, a leading cause of blindness worldwide. Hyperglycemia-induced dysfunction of the endogenous antioxidant system of the thioredoxins (TrxS) has been implicated in diabetes-induced OxS and retinal tissue injury. This research proposal focuses on determining diabetes-induced molecular modifications of the TrxS, as these contribute to hyperglycemia-induced OxS, retinal tissue injury and development of diabetic retinopathy.