**3.1. Biomarkers of oxidative stress**

NPDR, severe NPDR, and proliferative diabetic retinopathy (PDR) [1–3]. The major risk factors for developing DR are the duration of diabetes, hyperglycemia, hypertension, and dyslipidemia [4]. Glucose concentration increases in retinal cells leading to saccular capillary microaneurysms, pericyte deficient capillaries, and degenerate capillaries that decrease the retinal perfusion and contribute to the progression of DR [4]. Several types of evidence prove the benefits of tight glycemic and blood pressure control in decelerating the progression of DR. Nevertheless, the numbers of DR patients and the development of DR complications are

For the last several decades, many studies have been performed in order to better understand DR progression from a molecular viewpoint. The biochemical mechanisms implicated in DR progression have been shown in various animal models and patients with diabetes [1]. It is believed that the involvement of hyperglycemia and hormonal factors in diabetic patients could disturb hemostasis in the retina and change the balance of some mediators including growth factors, cytokines, inflammatory, and adhesion molecules [5]. These changes result in altered capillary permeability, apoptosis of capillary cells, and angiogenesis, leading to DR complications [3]. With improved clarity of molecular pathways in DR pathophysiology, the advancement of selective therapeutic approaches could be discovered and the management of DR could be more effective [1, 5]. This chapter focuses on the inflammatory molecules and

The immune system protects the body from both exogenous pathogens called pathogenassociated molecular patterns (PAMPs) and endogenous harmful molecules known as damage-associated molecular patterns (DAMPs). DAMPs include oxidized or glycated proteins, mislocated proteins/antigens, and intracellular contents released by necrotic cells. In normal conditions, the immune system regulates the inflammatory process and prevents uncontrolled inflammation that damages cells. In hyperglycemic conditions, the accumulation of DAMPs induces chronic inflammation in various tissues, which in turn manifests into the

The retina is one of few tissues in the human body that has immune privilege. It is protected from the attack of the systemic immune system by a series of complex defense mechanisms. This protection is afforded by a physical barrier formed between endothelial cells of retinal vasculature as the inner blood-retinal barrier (BRB) and retinal pigmented epithelial cells as the outer BRB. This barrier limits the movement of cells and molecules from the systemic circulation into the retinal parenchyma. The BRB also separates retinal antigens within the intraocular compartment, avoiding activation of T cells. This phenomenon is known as immunological ignorance. In addition, there is no lymphatic system in the retina. This inhibits systemic immune cells from detecting damage-associated molecular patterns in the retina thus preventing an overt systemic inflammatory response. Retinal cells (retinal neurons and RPE cells) express immune modulators that can suppress immune cells and complement system activation. The retina is protected by the local innate immune system (microglia, perivascular

macrophages, and the complement system) whose activation is tightly controlled [6].

still increasing, while therapeutic approaches are limited [1, 2].

72 Early Events in Diabetic Retinopathy and Intervention Strategies

biomarkers involved in the pathophysiology of DR.

**2. The immune system in proliferative diabetic retinopathy**

various complications of diabetes, including diabetic retinopathy [6].

The presence of oxidative stress biomarkers indicate an imbalance of reactive oxygen species (ROS) and the functional capabilities of cellular antioxidants [18, 22]. This imbalance can cause

**Figure 1.** Immune system role in progression of diabetic retinopathy.

cells and forms molecules called advanced lipoxidation end-products (ALE). MDA levels in specific tissues can be measured to represent oxidative damage induced by physical or chemical oxidative stress in the corresponding tissues [24, 25, 27]. Brzović-Šarić et al. found a significant difference between vitreous MDA values in non-diabetic patients with an eye disorder and PDR patients [25]. On the other hand, several studies found an increase in MDA serum of diabetic patients compared to control patients, but there was no significant difference in MDA serum level between non-proliferative DR and proliferative DR patients [24, 27]. Our study found a mean activity level of MDA in the vitreous of patients with PDR at 1.661 ± 1.21 nmol/mL [26]. Another study about oxidative stress levels with PDR by Mancino et al. found a mean activity level of MDA in vitreous of patients with PDR at 520 ± 210 nmol/mL [24]. What causes

Proliferative Diabetic Retinopathy: An Overview of Vitreous Immune and Biomarkers

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HIF-1α is a DNA-binding protein complex that is continuously expressed and degraded by cells in the body. Under hypoxic conditions, the HIF-1α degradation rate decreases, causing increased concentration of HIF-1α which then translocates into the nucleus and dimerizes with HIF-1β. The HIF-1 complex then regulates the expression of genes responsible for the hypoxic response of the cell by binding into the hypoxia response element (HRE) [28]. The HIF-1 complex is known to cause angiogenic effects on these hypoxic tissues [29]. Previous studies by Arden et al. on patients with diabetic retinopathy shows that hypoxia is present in retinal tissues suffering from oxidative damage [30]. Accordingly, Wang and co-workers found increased levels of HIF-1α protein in vitreous samples of PDR patients compared to levels in non-diabetic subjects [28]. Furthermore, the vitreous levels of vascular endothelial growth factor (VEGF) and HIF-1α were highly correlated in PDR patients. Several studies demonstrated positive immunohistochemical staining for HIF-1α and VEGF proteins in epiretinal neurovascular membranes. This evidence shows that HIF might play an important role in regulating

Angiogenesis is a complex multistep process that involves angiogenic factors and is induced by various cytokines and growth factors [33]. These factors have been suggested to be correlated with the development of diabetic retinopathy [5, 33–35]. These are also known to be hypoxiaresponsive factors [5, 35]. Pro-angiogenic factors, like VEGF, angiopoientin, and erythropoietin are well-known factors contributing to neovascularization and whose levels increase in diabetic retinopathy patients [3, 5, 33–35]. Several therapies designed to target these factors have

Vascular endothelial growth factor (VEGF) is a signaling molecule that promotes development of new blood vessels. It is released by cells in response to hypoxic conditions. Abcouwer stated that VEGF increases vascular permeability by promoting the disassembly of junctions between endothelial cells. This leakage can cause diabetic macular edema

been proven effective in decreasing the progression of the disease [5].

these differences in vitreous MDA levels still needs to be explored.

**3.2. Hypoxia-inducible factors**

the neovascularization of retina in PDR [31, 32].

*3.3.1. Vascular endothelial growth factor*

**3.3. Angiogenic factors**

**Figure 2.** Vitreous biomarkers involved in proliferative diabetic retinopathy.

cell instability and contribute to the development of many diseases, including diabetic retinopathy [18, 23]. Oxidative stress will remain high even after the patient reaches a normoglycemic state. This phenomenon is called "metabolic memory" and can lead to the accumulation of ROS in diabetic patients [24]. The biological markers of oxidative stress can include changes in molecules of the antioxidant system and molecules modified by ROS. Antioxidant enzymes like the superoxide dismutases are an example of changes in molecules of the antioxidant system, and malondialdehyde is the best known oxidative stress marker [18].

### *3.1.1. Superoxide dismutases*

Superoxide dismutases (SODs) are a group of enzymes found in our cells, which function as major antioxidant defense systems against ROS in the body. SODs consist of three isoforms: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3), all of which require catalytic metal (Cu or Mn) to activate. SOD activities will increase due to the presence of oxidative stress in the body. Vitreous SOD activity can also be used to measure oxidative stress levels inside the eye, allowing it to be a viable biomarker of oxidative stress in patients with PDR. Brzović-Šarić et al. state that PDR patients serum oxidative stress markers were higher than non-diabetic patients with an eye disorders (NDED) serum. Brzović-Šarić et al. found a mean activity level of SODs in the vitreous of male diabetic patients at 30.5 ± 2.5 U/mL, and 28.5 ± 3.8 U/mL in vitreous of female patients with diabetes [25]. Our previous study found a mean activity level of SODs in vitreous of patients with PDR at 0.403 + 0.50 U/mL [26].

#### *3.1.2. Malondialdehyde*

Malondialdehyde (MDA) is a highly reactive compound produced by lipid peroxidation of polyunsaturated lipid found in cell membranes. MDA exerts its oxidative stress effect inside cells and forms molecules called advanced lipoxidation end-products (ALE). MDA levels in specific tissues can be measured to represent oxidative damage induced by physical or chemical oxidative stress in the corresponding tissues [24, 25, 27]. Brzović-Šarić et al. found a significant difference between vitreous MDA values in non-diabetic patients with an eye disorder and PDR patients [25]. On the other hand, several studies found an increase in MDA serum of diabetic patients compared to control patients, but there was no significant difference in MDA serum level between non-proliferative DR and proliferative DR patients [24, 27]. Our study found a mean activity level of MDA in the vitreous of patients with PDR at 1.661 ± 1.21 nmol/mL [26]. Another study about oxidative stress levels with PDR by Mancino et al. found a mean activity level of MDA in vitreous of patients with PDR at 520 ± 210 nmol/mL [24]. What causes these differences in vitreous MDA levels still needs to be explored.
