*5.3. Apoptosis*

Apoptosis is the most studied type of cell death in diabetic retinopathy its characteristics are well defined and it is easily detected with techniques such as TUNEL (Terminal dUTP Nick End Labeling). Despite this, there are types of cell death that are difficult to detect due to the lack of defined markers and available techniques [208]. The hyperosmolar state has a relevant change in various tissues, but in tear film and cornea increases desquamation, loss of intercellular connections, disruption of cell membranes and decreases cytoplasmic density, it was found that proinflammatory stimulus in the corneal epithelium increases the expression of cytokines like IL-1ß, TNF-<sup>α</sup>, IL-8, MMP-9 activating the MAPK signaling pathway then the expression of proapoptotic markers (Fas, Fas ligand, APO2, CD40 and CD40 ligand) and cytochrome-c, this mediates an apoptotic pathway reported in vitro with an hyperosmotic state (≥450 mOsm) which converge on proteolytic activation of caspase-3 capable of cleaving various cellular proteinases to finally cause apoptotic death [130]. An increase in pericyte apoptosis in retinal tissue of diabetic patients compared to non-diabetic patients has been demonstrated with the use of TUNEL staining [208,209].

Lipocalin is a tear endonuclease which plays a role in the catalytic activity of DNA and its effect in the concentration of NaCl, Mg<sup>2</sup>+, Ca2+ and variation in pH facilitates lipocalin its role in prevention of viral infections and anti-inflammatory activity by regelation of tear viscosity, capture and release of lipids, inactivating endonucleases and pathogens binding sites on the ocular surface [210,211]. In diabetic patients with neurotrophic keratopathy, have been found increasing levels of AGE's products in cornea which activates a signal pathway mediated by NK-kB, generating an increase on oxidative stress, and anormal accumulation of MMP-9, this is correlated with ocular surface inflammation and induce a cycle of damage and apoptosis [212,213]. Cathepsins are a group of proteases key players in extracellular space, they have collagenase and elastase activity and participate in extracellular matrix (ECM) remodeling, but also are signaling molecules. Cathepsin C and B are present in tears, but its role in the ocular surface is not well explained. Cathepsin S is involved in initiating inflammatory responses by effecting degradation of lysosomal protein and ECM which is essential for homeostasis and cellular differentiation [127].

Matrix metalloproteinases (MMPs) are a family of calcium-activated zinc-containing enzymes that are involved in turnover and remodeling of the ECM and collectively are capable of breaking down most of their protein components, including collagens, laminin, fibronectin, elastin and other components. MMPs can also degrade a number of non-ECM proteins, including growth factors, chemokines, cytokines, and some surface receptors [214]. Metalloproteinases are involved in a number of both physiological and pathological processes, the enzymes may participate in pathological processes such as neovascularization [128]. MMP-2 and MMP-9 can degrade pigment epithelial derived factor (PEDF), which is the main antiangiogenic protein of the eye, specifically it is a 50 kDa glycoprotein highly expressed in the retinal pigment epithelium [215]. We know that one of the first characteristics of DR is the rupture of the blood–retina barrier. This results in vascular permeability in the retina and the development of retinal edema. These types of findings sugges<sup>t</sup> that elevated MMP expression in AH may facilitate an increase in vascular permeability [216]. MMP-9 belongs to the group of type IV collagenases which plays an important role in new vessel formation and were previously found to be upregulated by VEGF [217]. Increased levels of pro-MMP-9 and activated MMP-9 have previously been found in the vitreous of patients with PDR associated with vitreous hemorrhage [218]. MMPs can also act in the early stages of DR, in the breakdown of the BRB and in the destruction of the tight junctions of endothelial cells [216]. Among the di fferent MMPs examined in the vitreous samples, only the levels of MMP-2 and MMP-9 levels are significantly increased in the PDR eyes. In another study both metalloproteinases are present in the vitreous samples from PDR patients, being MMP-9 the only one elevated in PDR patients [128]. Noda et al. discovered that the vitreous proliferative membranes in diabetic retinopathy, in addition to having elevated levels of MMP-9, contain high levels of MMP-2. Furthermore, elevated levels of MMP-9 have been previously reported in the vitreous humor and fibrovascular membranes of patients with PDR [219].

The release of cytochrome c (*cy<sup>t</sup> c*) from the mitochondria is a fundamental step for the beginning of the apoptotic process [220]. *Cyt c* is a small globular protein that contains iron porphyrin cofactor (heme c) that covalently binds to the unique polypeptide chain. Its main function is its participation in the electron transport chain of the inner mitochondrial membrane (IMM). *Cyt c* is reversibly reduced and oxidized as an electron is transferred from ubiquinol-cytochrome c reductase (complex III) to *cyt c* oxidase (complex IV) in the mitochondrial respiratory chain [221]. Premature death of retinal cells occurs prior to the development of other lesions characteristic of retinopathy, suggesting that it may play a critical role in the development of DR [222]. The rate of apoptosis of vascular and nonvascular retinal cells in diabetes is low, but statistically higher than normal, which is consistent with the slow development of DR [223]. *Cyt c* has the ability to perform a variety of functions depending on the site and conditions of the cell where it is located; These properties have allowed it to be identified as an "extremely multifunctional" protein (EMF) [224]. So the structural and biological properties of the complex (ferric) *Cyt c-CL* (cytochrome c—cardiolipin), promotes the transformation of proteins into a peroxidase in the early stages of cellular apoptosis [225]. Besides, *cyt c* in mammalian cells can activate caspases, a family of cysteine proteases that have within their functions that of cleaving crucial substrates to induce cell dismantling [225].

Caspase-3 activation is a slower process compared to increased OS in diabetes, or from another point of view, its activation may be occurring as a consequence of increased OS [226]. Caspases involved in the activation of proinflammatory cytokines and the initiation and execution of apoptosis [223]. Because apoptosis of retinal capillary cells probably contributes to capillary "dropout" and retinal ischemia in DR, there is an interest in caspases that may be involved in initiating and executing this apoptotic process [223]. Caspase-3 is the executioner caspase that plays a central role in the proteolytic cascade during apoptosis its immunoreactivity occurred in ganglion cells in diabetic retinas, following their apoptotic death induced by ischemia, excitotoxicity, axotomy, and chronic ocular hypertension, their inhibition reduces apoptotic cell death induced in retinal cells [227]. Nuclear factor-kβ (NF-kβ) is an important polyphenic nuclear factor which participates in apoptosis and cellular neovascularization, it can be activated by a variety of signals such as IL-1β, TNFα, and OS. Several studies have shown that is closely related to inflammation, the appearance of tumors, cellular apoptosis, among other pathological processes. However, the main pathological changes in the DR include retinal cell apoptosis and neovascularization. This highlights that NF-kβ plays a role in DR [228,229]. In another scenario when cells are stimulated by hypoxia, hyperglycemia, and some inflammatory cytokines, the NF-kβ-specific inhibitor protein (IkB-α) is phosphorylated and degraded just to be released and activated, entering the nucleus to regulate gene transcription [229].

The BRB function of endothelial cells is supported by surrounding cells, such as Müller cells, pericytes, and astrocytes. As the blood–retinal barrier depends to a large extent on this microenvironment where the function of a specific type of cell depends on the support of other types of cells, any cellular injury or cellular loss will have grea<sup>t</sup> effects on the proper function of the retinal barrier and, in fact, any retinal function [230,231]. The NF-kβ present in the subretinal membranes and micro vessels is activated in response to increased ROS and AGE, which in turn further activate the apoptosis process. The activated NF-kβ is then further bound to nuclear DNA and thereby overexpresses different genes that lead to free radical production and further cell death [232]. Activated NF-kβ also increases the expression of cytokines IL-1b, IL-6, and IL-8 and the proapoptotic molecule caspase-3 in vitreous fluid and serum, leading to inflammation-mediated cellular apoptosis [233]. Together, the activation of NF-kβ, TNF-<sup>α</sup>, and interleukins improve MMP-9 transcription leading to DNA alkylation and the development of DR [234].

#### **6. Metabolic Memory of Oxidative Stress in Diabetic Retinopathy**

Several studies maintain that epigenetic modification is a significant factor in the development of DR. The duration of hyperglycemia decides if better subsequent glycemic control would be effective in the DR, which implies that the sustained state of hyperglycemia produces a metabolic memory phenomenon and be attributed to epigenetics, which may be the reason for inter-individual differences in drug response and variation in the progression of diabetes complications [235–237].

There have been several epigenetic modifications studied in DR: methylation directly to DNA molecule, which can repress transcription of certain genes; chromatin remodeling and modifications to the DNA condensing proteins, histones, which can also activate or suppress DNA on its own; and non-coding RNA that post-transcriptionally regulates gene expression. Although there are more epigenetics modifications known involved in DM or cancer, these alterations are the most studied in DR functioning as potential prognostic, therapeutic, or diagnostic biomarkers.
