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 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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Biology and Diseases of the Posterior Eye Study Section (BDPE)
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Shen, Grace L
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Wayne State University
Schools of Medicine
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Mishra, Manish; Kowluru, Renu A (2016) The Role of DNA Methylation in the Metabolic Memory Phenomenon Associated With the Continued Progression of Diabetic Retinopathy. Invest Ophthalmol Vis Sci 57:5748-5757
Kowluru, Anjaneyulu (2016) A lack of 'glue' misplaces Rab27A to cause islet dysfunction in diabetes. J Pathol 238:375-7
Kowluru, Renu A; Mishra, Manish; Kumar, Binit (2016) Diabetic retinopathy and transcriptional regulation of a small molecular weight G-Protein, Rac1. Exp Eye Res 147:72-7
Kowluru, Renu A; Shan, Yang; Mishra, Manish (2016) Dynamic DNA methylation of matrix metalloproteinase-9 in the development of diabetic retinopathy. Lab Invest 96:1040-9
Mishra, Manish; Lillvis, John; Seyoum, Berhane et al. (2016) Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy. Invest Ophthalmol Vis Sci 57:4035-44
Mishra, Manish; Flaga, Jadwiga; Kowluru, Renu A (2016) Molecular Mechanism of Transcriptional Regulation of Matrix Metalloproteinase-9 in Diabetic Retinopathy. J Cell Physiol 231:1709-18
Zhang, Xiangmin; Damacharla, Divyasri; Ma, Danjun et al. (2016) Quantitative proteomics reveals novel protein interaction partners of PP2A catalytic subunit in pancreatic β-cells. Mol Cell Endocrinol 424:1-11
Kowluru, Renu A; Mishra, Manish; Kowluru, Anjaneyulu et al. (2016) Hyperlipidemia and the development of diabetic retinopathy: Comparison between type 1 and type 2 animal models. Metabolism 65:1570-81
Veluthakal, Rajakrishnan; Kumar, Binit; Mohammad, Ghulam et al. (2015) Tiam1-Rac1 Axis Promotes Activation of p38 MAP Kinase in the Development of Diabetic Retinopathy: Evidence for a Requisite Role for Protein Palmitoylation. Cell Physiol Biochem 36:208-20
Fang, Jingye; Liu, Ming; Zhang, Xuebao et al. (2015) COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis. Mol Endocrinol 29:1156-69

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