**3.2. Hypoxia-inducible factors**

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

### **3.3. Angiogenic factors**

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

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

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

malondialdehyde is the best known oxidative stress marker [18].

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

74 Early Events in Diabetic Retinopathy and Intervention Strategies

*3.1.1. Superoxide dismutases*

with PDR at 0.403 + 0.50 U/mL [26].

*3.1.2. Malondialdehyde*

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