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.

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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
3R01EY022230-01A1S1
Application #
8534341
Study Section
Biology and Diseases of the Posterior Eye Study Section (BDPE)
Program Officer
Shen, Grace L
Project Start
2012-04-01
Project End
2016-02-29
Budget Start
2012-04-01
Budget End
2013-02-28
Support Year
1
Fiscal Year
2012
Total Cost
$50,500
Indirect Cost
Name
Wayne State University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
Devi, Takhellambam Swornalata; Somayajulu, Mallika; Kowluru, Renu Anjan et al. (2017) TXNIP regulates mitophagy in retinal Müller cells under high-glucose conditions: implications for diabetic retinopathy. Cell Death Dis 8:e2777
Kowluru, Renu A; Mishra, Manish (2017) Epigenetic regulation of redox signaling in diabetic retinopathy: Role of Nrf2. Free Radic Biol Med 103:155-164
Kowluru, Anjaneyulu (2017) Role of G-proteins in islet function in health and diabetes. Diabetes Obes Metab 19 Suppl 1:63-75
Mishra, Manish; Kowluru, Renu A (2017) Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy. Biochim Biophys Acta 1863:1761-1769
Kowluru, Anjaneyulu (2017) Tiam1/Vav2-Rac1 axis: A tug-of-war between islet function and dysfunction. Biochem Pharmacol 132:9-17
Baidwan, Sartaj; Chekuri, Anil; Hynds, DiAnna L et al. (2017) Glucotoxicity promotes aberrant activation and mislocalization of Ras-related C3 botulinum toxin substrate 1 [Rac1] and metabolic dysfunction in pancreatic islet ?-cells: reversal of such metabolic defects by metformin. Apoptosis 22:1380-1393
Duraisamy, Arul J; Mishra, Manish; Kowluru, Renu A (2017) Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes. Invest Ophthalmol Vis Sci 58:6440-6448
Kowluru, Renu A; Shan, Yang (2017) Role of oxidative stress in epigenetic modification of MMP-9 promoter in the development of diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol 255:955-962
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
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

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