Diabetic retinopathy remains the leading cause of blindness among young adults. We have shown that in the pathogenesis of diabetic retinopathy, matrixmetalloproteinase-9 (MMP-9) is activated in the retina via a small molecular weight G-protein, H-Ras, activated MMP-9 damages mitochondria and accelerates retinal capillary cell apoptosis, and MMP-9 gene knockout diabetic mice are protected from the development of retinopathy. Diabetes alters histone modifications, and MMP-9 is shown to be regulated by histone modifications in its promoter region. Our preliminary data show that in diabetes, the recruitment of p65 subunit of NF-kB is increased at retinal MMP-9 promoter and that of dimethylated lysine 9 of histone 3 (H3K9me2) is decreased, lysine-specific demethylase (LSD1) is activated and histone deacetylase (Sirt1) is inhibited, and overexpression of MnSOD protects diabetes-induced increase in MMP-9, histone modifications and alterations in the histone modifying enzymes. Furthermore, re-institution of normal glycemic control after a period of poor glycemic control in rodents fails to provide any benefit to MMP-9 activation, histone methylation and Sirt1 inhibition. Thus, our overall hypothesis is that in diabetes, retinal MMP-9 is regulated by histone modifications, and the enzyme remains elevated after termination of hyperglycemic insult and the retinopathy continues to progress. We propose to test this hypothesis methodically by addressing complementary questions proposed under three specific aims.
The first aim will investigate the molecular mechanism by which diabetes activates retinal MMP-9, and will test the hypothesis that due to histone modifications, the recruitment of p65 at MMP-9 promoter is increased resulting in its activation'. Since oxidative stress is considered to play an important role in the development of diabetic retinopathy, in the second aim, we will investigate the role of oxidative stress in the regulation of MMP-9. Our working hypothesis predicts that due to increased oxidative stress, Sirt1 is inhibited and deacetylation is decreased, and this facilitates the binding of p65 at the MMP-9 promoter.
The third aim will determine the role of MMP-9 in the metabolic memory phenomenon associated with the progression of diabetic retinopathy, and will test the hypothesis that due to continued histone modifications, MMP-9 remains active after reversal of hyperglycemia, and the dysfunctional mitochondria continues to accelerate apoptosis of capillary cells. These proposed studies are based on compelling preliminary data generated using valid in vitro and in vivo model systems. The central hypothesis will be tested using active plasmids, siRNAs and pharmacological inhibitors in isolated cells, and in vitro findings will be validated in in vivo models using genetically manipulated mice and in retinal microvessels from human donors with diabetic retinopathy. The results are expected to demonstrate the role of histone modifications in retinal MMP-9 activation in diabetes, and in the metabolic memory phenomenon associated with the progression of diabetic retinopathy. This should reveal novel targets for therapies, including regulators of histone modifying enzymes, to prevent retinopathy in the early stages of its development, and in its progression after termination of hyperglycemia. This will offer patients additional therapeutic means to prevent/retard the 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 exact 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 MMP-9 mediated damage of retinal capillary cells. We will test the role of histone modifications in the activationof MMP-9, leading to mitochondrial dysfunction, and the development of diabetic retinopathy. The application will use biochemical, molecular and epigenetic techniques, and represents the first attempt to examine the potential role of epigenetic modifications of MMP-9 in its activation in diabetes. 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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Special Emphasis Panel (DPVS)
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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, 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
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
Kowluru, Renu A; Kowluru, Anjan; Mishra, Manish et al. (2015) Oxidative stress and epigenetic modifications in the pathogenesis of diabetic retinopathy. Prog Retin Eye Res 48:40-61
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
Kowluru, Renu A; Zhong, Qing; Santos, Julia M et al. (2014) Beneficial effects of the nutritional supplements on the development of diabetic retinopathy. Nutr Metab (Lond) 11:8

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