Oxidative stress and epigenetic modifications in the pathogenesis of diabetic retinopathy

Abstract

Diabetic retinopathy remains the major cause of blindness among working age adults. Although a number of metabolic abnormalities have been associated with its development, due to complex nature of this multi-factorial disease, a link between any specific abnormality and diabetic retinopathy remains largely speculative. Diabetes increases oxidative stress in the retina and its capillary cells, and overwhelming evidence suggests a bidirectional relationship between oxidative stress and other major metabolic abnormalities implicated in the development of diabetic retinopathy. Due to increased production of cytosolic reactive oxygen species, mitochondrial membranes are damaged and their membrane potentials are impaired, and complex III of the electron transport system is compromised. Suboptimal enzymatic and nonenzymatic antioxidant defense system further aids in the accumulation of free radicals. As the duration of the disease progresses, mitochondrial DNA (mtDNA) is damaged and the DNA repair system is compromised, and due to impaired transcription of mtDNA-encoded proteins, the integrity of the electron transport system is encumbered. Due to decreased mtDNA biogenesis and impaired transcription, superoxide accumulation is further increased, and the vicious cycle of free radicals continues to self-propagate. Diabetic milieu also alters enzymes responsible for DNA and histone modifications, and various genes important for mitochondrial homeostasis, including mitochondrial biosynthesis, damage and antioxidant defense, undergo epigenetic modifications. Although antioxidant administration in animal models has yielded encouraging results in preventing diabetic retinopathy, controlled longitudinal human studies remain to be conducted. Furthermore, the role of epigenetic in mitochondrial homeostasis suggests that regulation of such modifications also has potential to inhibit/retard the development of diabetic retinopathy.

Keywords: Diabetic retinopathy; Epigenetic modifications; Mitochondria; Oxidative stress; Reactive oxygen species; Transcriptional regulation.

Fig. 1.

Fundus photographs of diabetic patients with A. background retinopathy showing some white patches (cotton wool spots) above the optic disc, indicating blocked small blood vessels, and a small hemorrhage. B. Proliferative retinopathy with new vessels on the optic nerve head and numerous hemorrhages.

Fig. 2.

Chronic hyperglycemia can result in many acute and cumulative changes in cellular metabolism, and these can damage structure and function of many organs. Repeated acute changes in the metabolism can also produce cumulative changes in the macromolecules. In addition to hyperglycemia, genetic/environmental factors and other systemic factors (hyperlipidemia or/and hypertension) also influence the tissue damage.

Fig. 3.

Increased ROS damage macromolecules directly via by influencing gene expressions or by damaging membrane. Damage to nucleotides, oxidation of thiols in protein, and oxidation of lipids in the membranes can also alter enzyme activity, resulting in tissue damage.

Fig. 4.

Hyperglycemic environment activates a number of metabolic pathways in the retina, including PKC, AGEs, hexosamine pathway (Hexosam), polyol pathway (POP), oxidative stress (Oxid stress) and increases inflammatory mediators (inflam). These all lead to increase in mitochondrial ROS. Increased mitochondrial ROS, instead can also activate these metabolic pathways, suggesting two way interactions between various metabolic abnormalities and mitochondrial damage. Damaged mitochondria, via accelerating apoptosis of capillary cells, result in acellular capillaries and pericyte ghosts.

Fig. 5.

The transcription factor Nrf2 controls the catalytic subunit of GSH biosynthesis enzyme glutamate cysteine ligase (GCL), and glutamylcysteine formed from glutamate and cysteine, is converted to GSH by GSH synthetase.

Fig. 6.

Sustained high glucose produces mismatches in retinal mtDNA, and due to suboptimal sequence repair machinery, mtDNA is damaged.

Fig. 7.

Retinal MMPs are activated by cytosolic ROS, and via mitochondrial membrane transporters (TIM44), move into the mitochondria. Inside the mitochondria, MMPs act on connexin 43 (conx43), and damage mitochondrial membrane, cytochrome c leaks out into the cytosol, and apoptotic machinery is activated.

Fig. 8.

ROS produced in the cytosol damage mitochondrial function, and the levels of ROS are increased in the mitochondria. Sustained increase in mitochondrial ROS damages mtDNA, and the transcription is impaired. This leads to a compromised the electron transport chain, which further fuels into increased ROS and mitochondrial dysfunction, and the vicious cycle of ROS continues.

Fig. 9.

Chromatin can either be open (active, allowing gene expression) or condensed (inactive, repressing gene expression). Active chromatin is maintained by H3K9, H3K27, and H4K20 demethylation and H3K4, H3K79, H3K6 methylation, and histone acetylation or ubiquitination. Conversely, selective histone methylation (e.g., H3K9, H3K20, H3K27) results in chromatin condensation and transcriptional repression. Histone acetyltransferases (HATs) add an acetyl group, while histone deacetylases (HDACs) remove an acetyl group.

Fig. 10.

A. Diabetes increases the levels of H3K4me1 and decreases H3K9me3, and the lysine on H3K9 becomes available for acetylation. Increased Ac-H3K9 facilitates the binding of p65 at MMP-9 promoter, and increases MMP-9 transcription. B. Decreased levels of Sirt1 deacetylase increase acetylation of p65, and this increases it’s binding at the MMP-9 promoter. Increased MMP-9 leads to mtDNA damage and cell apoptosis in diabetic retinopathy.

Fig. 11.

Due to epigenetic modifications at Keap1 promoter, the binding of transcriptional factor Sp1 is increased resulting in overexpression of Keap1. Increased Keap1 restrains Nrf2 from moving into the nucleus, and the binding of Nrf2 at Gclc-ARE4 promoter region is decreased resulting in decreased GSH biosynthesis and increased oxidative stress.

Fig. 12.

Diabetes increases translocation of Dnmt1 into the mitochondria, where it methylates mtDNA. Increased mtDNA methylation suppresses its transcription, and the electron transport system becomes compromised, further increasing ROS levels, leading to cells apoptosis.

Fig. 13.

Schematic representation of epigenetic modifications in diabetic retinopathy: diabetes induces oxidative stress, which alters the expression of genes involved in histone (LSD1, KDM5A, HDACs), and DNA (Dnmts) modifications. Histone methylation (H3K4me3, HeK4me1, H3K4me2, H3K9me2, H4K20me3) and acetylation (H3K9-Ac, p300) regulates the binding of transcription factor (Nrf2, Sp1, NF-kB-p65) and alter the gene expression (GCLC, Keap1, MMP-9, Sod2, TXNIP), and DNA methylation at POLG1 promoter suppresses its expression in diabetic retinopathy. MicroRNAs (miR-200b, miR-129b, miR-146) also regulate the transcript levels of various genes (Oxr1, VEGF, Rax, NF-kB) in diabetic retinopathy. Although this scheme represents a number of modifications, we cannot rule out the role of many, yet identified, miRNAs and other histone and DNA modifications in diabetic retinopathy.

Fig. 14.

Diabetes increases oxidative stress, and this could be either via Nox2 activation or abnormal glucose metabolism, or by auto-oxidation of glucose itself. Increased oxidative stress leads to AGEs formation, activation of PKC, hexosamine, polyol pathways, and increase in inflammatory mediators. Increased ROS attenuate the GAPDH, which further activates PKC, hexosamine and polyol pathways and AGEs formation, and these pathways also can produce ROS. Epigenetic modifications in the histones or DNA alter the gene expressions of proteins associated with the oxidative damage and antioxidant defense, miRNA levels are altered, and these further dysfunction mitochondria and impair mtDNA transcription. The vicious cycle of ROS continues to fuel in, resulting in cell apoptosis and the development of diabetic retinopathy.