Diabetic retinopathy remains a major cause of blindness, and despite cutting edge research in the field, the molecular mechanism of its pathogenesis remains unclear. Our studies have documented a critical role of matrix metalloproteinase 9 (MMP-9) in diabetic retinopathy, and have demonstrated that cytosolic MMP-9 activation is an early event, that is followed by its mitochondrial accumulation, mitochondrial dysfunction and mtDNA damage, initiating a vicious cycle of free radicals. Epigenetic modifications play a critical role in MMP-9 transcription, and in diabetes, MMP-9 promoter DNA undergoes dynamic methylation-hydroxymenthlation and histone modifications. MMP-9 is also regulated by homocysteine, a thiol-containing non-protein amino acid, and diabetic patients have elevated plasma homocysteine levels. Increased homocysteine is implicated in cellular and metabolic abnormalities including mitochondrial damage and epigenetic modifications. Homocysteine is also a precursor of hydrogen sulfide (H2S), and due to impaired homocysteine metabolism, plasma levels of H2S are decreased in diabetic patients. Based on these, our central hypothesis is that in diabetes, high homocysteine activates MMP-9 and disturbs mitochondrial dynamics, and the damaged mitochondria accelerates apoptosis resulting in the development of diabetic retinopathy.
Aim 1 will investigate the mechanism(s) by which homocysteine activates MMP-9 in diabetes, and the model predicts that high homocysteine activates MMP-9 by (i) damaging interactions between MMP-9 and its tissue inhibitor, Timp1, and (ii) inducing epigenetic modifications and increasing the ratio of MMP-9-Timp1.
Aim 2 will determine the mechanism(s) by which homocysteine impairs mitochondrial dynamics, and will test the hypothesis that homocysteine increases mitochondrial fragmentation, and dysfunctional mitophagy in diabetes fails to properly remove the fragmented mitochondria.
Aim 3 will examine the therapeutic potential of regulating homocysteine-H2S metabolic balance on inhibition of diabetic retinopathy. The plan will employ in vitro (retinal endothelial cells) and in vivo (retinal microvessels from rodents) models of diabetic retinopathy, and will utilize fully optimized molecular biological and pharmacological approaches. Our overall goal is to identify novel regulatory mechanisms involved in the pathogenesis of diabetic retinopathy, specifically at the level of regulation of homocysteine-H2S. The proposal is based on a testable central hypothesis, and our proposed studies are innovative and carry a significant translational impact as they are expected to identify novel therapeutic targets to prevent the development and progression of diabetic retinopathy. This will offer patients additional therapeutic means to prevent/halt this sight-threatening complication of diabetes.

Public Health Relevance

Diabetic retinopathy remains the most frequent cause of blindness among young adults, and underlying and the underlying molecular and cellular mechanisms remain obscure. Activation of matrix metalloproteinase (MMP-9) in hyperglycemic milieu damages the mitochondria, activating the apoptotic machinery. MMP-9 is shown to be activated by homocysteine, and diabetic patients also have higher homocysteine levels. This proposal is focused on understanding the molecular mechanism by which homocysteine activates MMP-9 in diabetes, and how it damages mitochondria. The application represents our continued effort in trying to understand the role of MMP-9-Mitochondria and epigenetic modifications in the pathogenesis of diabetic retinopathy. The results obtained from our studies have strong potential to be translated into identifying therapies to treat/retard this sight threatening complication of diabetes.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Diseases and Pathophysiology of the Visual System Study Section (DPVS)
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Shen, Grace L
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Wayne State University
Schools of Medicine
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Kowluru, Renu A; Mishra, Manish (2018) Therapeutic targets for altering mitochondrial dysfunction associated with diabetic retinopathy. Expert Opin Ther Targets 22:233-245
Mishra, Manish; Duraisamy, Arul J; Kowluru, Renu A (2018) Sirt1: A Guardian of the Development of Diabetic Retinopathy. Diabetes 67:745-754
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
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
Mishra, Manish; Kowluru, Renu A (2017) Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy. Biochim Biophys Acta Mol Basis Dis 1863:1761-1769
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 (2017) Epigenetic regulation of redox signaling in diabetic retinopathy: Role of Nrf2. Free Radic Biol Med 103:155-164
Kowluru, Renu A; Mishra, Manish; Kumar, Binit (2016) Diabetic retinopathy and transcriptional regulation of a small molecular weight G-Protein, Rac1. Exp Eye Res 147:72-77
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
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

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