*6.1. DNA Methylation*

The most primitive epigenetic modification is DNA methylation and, according to various studies, it has a correlation with the progression of DR. In DNA methylation the methyl group is transferred from S-adenosylmethionine (SAM) to DNA molecules, this reaction is catalyzed by DNA methyltransferases. It was found that patients with DR have shown a significantly higher level of DNA methylation compared to those without DR, indicating that increased DNA methylation is a relevant component in the development of DR [237]. Another study revealed that methylation and activation of the matrix metalloproteinase 9 (MMP-9) gene associated with DR has an important role in accelerating apoptosis of retinal vascular endothelium [238]. This indicates that DNA methylation in DR is a highly dynamic process, which involves various epigenetic changes targeting extracellular matrix, manganese superoxide dismutase (MnSOD) also cross-linking with OS, and mitochondrial homeostasis causing mitochondrial dysfunction with subsequent capillary damage [238–241].

ROS generated from mitochondrial oxidative phosphorylation is the most representative source of OS in endothelial cells, which cause peroxidation of PUFAs, protein damage and consequently mitochondrial DNA (mtDNA) damage [239,242,243]. Methylation of mtDNA has recently been associated with the development of diseases and as a potential biomarker. Iacobazzi divided mtDNA methylation into global mtDNA methylation biomarkers, such as 5-methylcytosine (5mc) derived from the incorporation of a methyl group at position 5 of cytosine, and 5-hydroxymethylcytosine (5hmc), produced from 5mc through a hydroxymethylation reaction catalyzed by ten-eleven-translocation, which are related to aging and neurodegenerative disorders; and specific methylation biomarkers such as ND6 gene, which is suppressed under OS conditions by increased DNA methyltransferase (DNMT) [244]. As a general methylation biomarker, 5hmc can be seen at rac family small GTPase 1, which leads to increased binding of NF-kB linking this epigenetic modification with inflammation in DR [245]. In the retina, there is a more specific area where methylation is occurring called *D-loop* where transcription and replication of mtDNA is controlled [243,246] which has been used as a biomarker in a clinical study that showed significantly higher methylation rates in the serum of patients with PDR than patients without DR, these had higher methylation rates than non-diabetic patients [247] functioning as a novel diagnostic tool. Another potential biomarker related with DNA methylation in DR is homocysteine, of which high levels have been associated with increased risk of developing DR in diabetic patients, and with higher global DNA methylation probably linking it to altered metabolic memory phenomenon [248,249]. Interestingly, high homocysteine levels have been associated with increased inflammatory cytokines and activation of NF-kB [250].

Several therapeutic approaches have been assessed targeting mitochondrial dysfunction despite the low specificity of DNMT, synthetic DNA methylation inhibitors such as hydralazine and procainamide are being evaluated in clinical trials, or polyphenols like resveratrol have shown to directly inhibit DNMTs as well as being a powerful antioxidant which ultimately lead to epigenetic changes with altered gene expression [251–254].

#### *6.2. Modification of Histones*

Other relevant epigenetic alteration is the modification of histones, which has also been a key contributor to the pathophysiology of the DR. Histones are proteins in the nucleus related to DNA and play a role in regulation of gene expression and can be post-translational modified by methylation, acetylation, ubiquitination and phosphorylation regulating chromatin structure, and these histones can have an active cross-talk with other histone modifications [235,255]. Relevant examples of these epigenetic changes are histone acetyltransferase (HAT) and histone deacetylases are involved in regulating gene expression in the complications of diabetes [256]. For example, H3 acetylated histone expression was decreased in induced diabetic models. Furthermore, it was discovered that these changes were irreversible once the blood glucose of these rats was restored to a normal level, indicating that the development of DR is associated with the modification of histones and that it would also be participating in the phenomenon of metabolic memory [241,257]. When there is a high-glucose environment in the retina, histone acetylation is increased due to a decreased activity of histone deacetylase (HDAC) inducing an increased expression of pro-inflammatory cytokines in Müller cells [256].

During DR, an alteration of mitochondrial homeostasis and dynamics occurs, where a vicious circle is created between the alteration of mitochondrial enzymes that induce the formation of superoxide, which in turn alters the physiology of the organelles. Furthermore, dysfunction of the repair pathways generates even more mitochondrial damage [239]. However, regulation of the mtDNA replication/repair machinery has the ability to prevent mitochondrial dysfunction and the development of DR to some extent [239,258]. Decreased mitochondrial SOD2 and inhibition of Nrf2 (nuclear factor-(erythroid-derived 2-)like 2), a transcription factor affecting antioxidants, have been observed. What happens is that during the state of OS, Nrf2 translocate to the nucleus where it binds to the antioxidant response element (ARE). On the other hand, Keap1, an inhibitor of Nrf2, binds it in the cytosol and leads it to proteomic degradation through cullin-3-dependent degradation [2,252,259]. Moreover, in vitro studies have shown metabolic memory phenomenon through exposure of retinal capillary cells to high glucose concentrations, inducing epigenetic modification of SOD2; the gene that encodes for MnSOD, by decreasing methylation of H3K4 at Sod2 promoter predisposing to worsening of the oxidative stress damage cycle [255,260]. There have been different therapeutic approaches regarding histone acetylation/deacetylation in diabetic nephropathy; histone deacetylase

inhibitors like Vorinostat may have a protecting e ffect by decreasing OS, agents like sodium butyrate inhibited HDAC activity and thus elevated the expression of Nrf2, both protecting against renal injury [256]. These therapeutic approaches have ye<sup>t</sup> to be assessed in DR, but HDAC inhibition may have ameliorative e ffects in diabetic microvascular complications.

A study of the e ffects of diabetes on nuclear–mitochondrial communication in the retina revealed that the mitochondrial biogenesis of the retina is weakened in diabetes and is under the control of superoxide radicals. Therefore, this makes us think that the regulation of biogenesis by pharmaceutical or molecular means could provide a way to prevent the development/progression of DR [261]. Examples of these are approaches with intravitreal adeno associated viral MnSOD which showed to reduce retinal capillary basement membrane thickness, inhibit apoptosis of these capillaries, by e ffectively overexpressing MnSOD, suggesting an ameliorative e ffect on the metabolic memory phenomenon [254,262]. Moreover, di fferent approaches have been made with di fferent polyphenols, the most promising one is resveratrol that is associated with decreased phosphorylation of 5'-adenosine monophosphate activated protein kinase (AMPK) that regulates histone deacetylase Sirtuin 1 (Sirt1) which may ultimately suppress NF-kB activation [252].
