Retinopathy remains one of the most feared debilitating complications of diabetes. In the pathogenesis of diabetic retinopathy, superoxide levels are significantly elevated, mitochondria are dysfunctional and their DNA is damaged resulting in a vicious cycle of increased superoxide accumulation. Emerging evidence implicates NADPH oxidase (Nox) also as a potential source of reactive oxygen species (ROS) with detrimental effects on cell survival. Our preliminary data show that hyperglycemia activates Rac1/Nox2 signaling axis in the retina and its capillary cells prior to mitochondrial dysregulation suggesting that Nox2 activation represents an early event in diabetes-induced mitochondrial dysfunction and cell apoptosis. Thus, our overall hypothesis is that Nox2-derived ROS in diabetes damage retinal mitochondria leading to their dysfunction, and apoptosis of capillary cells is accelerated resulting in the development of diabetic retinopathy. We propose to test this hypothesis methodically by addressing complementary questions proposed under three specific aims.
The first aim will investigate the mechanism(s) by which hyperglycemia activates Nox2 in the retina, and will test the hypothesis that in hyperglycemia Rac1-mediated Nox2 activation and ROS generation initiate mitochondrial damage and cellular apoptosis. Since the severity of retinopathy is associated directly with hyperlipidemia, in the second aim, the mechanism(s) by which lipotoxic conditions promote the development of diabetic retinopathy will be investigated. Our working model predicts that lipotoxic conditions promote Rac1-mediated Nox2 activation and ROS generation to initiate mitochondrial damage and cellular apoptosis.
The third aim will determine the effect of regulation of Nox2 on the development of diabetic retinopathy, and will test the hypothesis that inhibition of Nox2 will attenuate mitochondrial damage and subsequent development of diabetic retinopathy. These proposed studies are based on compelling preliminary data generated via multi-disciplinary collaborative efforts between two PIs using valid in vitro and in vivo model systems. We propose to utilize known selective inhibitors of the Tiam1/Rac1/Nox2 signaling pathways;data from these studies will be confirmed via the use of inactive mutants and siRNAs for key signaling proteins in this pathway. In vitro findings will be further validated in vivo models (stz-induced diabetic rats and mice, and Zucker diabetic fatty rats), and also in the retina from human donors with diabetic retinopathy. We expect to demonstrate the role of Rac-1-mediated Nox2 derived ROS as the 'initiator'of mitochondrial dysfunction in the pathogenesis of retinopathy. This should reveal novel targets for therapies to prevent retinopathy in the early stages of its development, and offer patients additional therapeutic means to prevent/retard this sight-threatening complication of diabetes.
Diabetic retinopathy, a slow progressing, is the most frequent cause of blindness among young adults. Despite the cutting edge research to explore how the disease develops, the actual molecular and cellular mechanisms underlying this lesion remain elusive. This proposal is focused on understanding potential damaging mechanisms responsible for the development of diabetic retinopathy through systematic analysis of a specified signaling pathway, Tiam1-Rac1-Nox2, which increases oxidative stress and damages mitochondria. We will test the "initiator" roles for Nox2-derived ROS in mitochondrial dysfunction, and the development of diabetic retinopathy. The application represents a multi-disciplinary collaborative effort between two well-established laboratories actively focused on G-protein signaling pathways and mitochondrial dysfunction in the pathogenesis of diabetic retinopathy. Data from this study are expected to identify novel drug targets for halting the progression of this blinding disease in human diabetes.
|Mishra, Manish; Zhong, Qing; Kowluru, Renu A (2014) Epigenetic modifications of Nrf2-mediated glutamate-cysteine ligase: implications for the development of diabetic retinopathy and the metabolic memory phenomenon associated with its continued progression. Free Radic Biol Med 75:129-39|
|Khadija, Syeda; Veluthakal, Rajakrishnan; Sidarala, Vaibhav et al. (2014) Glucotoxic and diabetic conditions induce caspase 6-mediated degradation of nuclear lamin A in human islets, rodent islets and INS-1 832/13 cells. Apoptosis 19:1691-701|
|Kowluru, Anjaneyulu; Kowluru, Renu A (2014) Phagocyte-like NADPH oxidase [Nox2] in cellular dysfunction in models of glucolipotoxicity and diabetes. Biochem Pharmacol 88:275-83|
|Mishra, Manish; Zhong, Qing; Kowluru, Renu A (2014) Epigenetic modifications of Keap1 regulate its interaction with the protective factor Nrf2 in the development of diabetic retinopathy. Invest Ophthalmol Vis Sci 55:7256-65|
|Santos, Julia M; Mishra, Manish; Kowluru, Renu A (2014) Posttranslational modification of mitochondrial transcription factor A in impaired mitochondria biogenesis: implications in diabetic retinopathy and metabolic memory phenomenon. Exp Eye Res 121:168-77|
|Kowluru, Renu A; Kowluru, Anjaneyulu; Veluthakal, Rajakrishnan et al. (2014) TIAM1-RAC1 signalling axis-mediated activation of NADPH oxidase-2 initiates mitochondrial damage in the development of diabetic retinopathy. Diabetologia 57:1047-56|
|Santos, Julia M; Tewari, Shikha; Lin, Jonathan Y et al. (2013) Interrelationship between activation of matrix metalloproteinases and mitochondrial dysfunction in the development of diabetic retinopathy. Biochem Biophys Res Commun 438:760-4|
|Zhong, Qing; Kowluru, Renu A (2013) Regulation of matrix metalloproteinase-9 by epigenetic modifications and the development of diabetic retinopathy. Diabetes 62:2559-68|
|Mohammed, Abiy M; Syeda, Khadija; Hadden, Timothy et al. (2013) Upregulation of phagocyte-like NADPH oxidase by cytokines in pancreatic beta-cells: attenuation of oxidative and nitrosative stress by 2-bromopalmitate. Biochem Pharmacol 85:109-14|
|Santos, Julia M; Kowluru, Renu A (2013) Impaired transport of mitochondrial transcription factor A (TFAM) and the metabolic memory phenomenon associated with the progression of diabetic retinopathy. Diabetes Metab Res Rev 29:204-13|