*3.3.1. Vascular endothelial growth factor*

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 (DME) [5]. Several studies have shown marked increases of VEGF in vitreous and vitreous compared to plasma concentration in DME and PDR patients [19, 36–46]. Treatments that target VEGF have been proven highly effective in treating DR. VEGF antibodies, which were originally used for cancer treatments, such as bevacizumab and its correlate ranibizumab have been used effectively. These have also been tested in several small trials which showed improved vision in DR patients, demonstrating the involvement of VEGF in the pathophysiology of PDR [5, 36].

*3.3.3. Erythropoietin*

the decrease of its neuroprotective function [54].

*3.3.4. Matrix metalloproteinases 9*

*3.3.5. Transforming growth factor β*

Erythropoietin (EPO) is a glycoprotein cytokine that acts as a major regulator of erythropoiesis. Besides erythropoiesis, several studies state that erythropoietin has a neuroprotective and angiogenic effect in brain and retina. Production of EPO in serum and vitreous is mainly caused by hypoxia [54–57]. EPO is found in many organs, including kidney, liver, brain, and retina [55]. The angiogenic effect of EPO is a potential equivalent to VEGF, and has been suspected as an important factor in the angiogenesis of PDR [56]. Watanabe et al. showed that vitreous EPO levels of PDR patients are significantly higher (464.0 mlU/mL) compared to non-diabetic patients (36.5 mlU/mL). They also found that EPO levels are higher with active as compared to quiescent PDR [54]. These are consistent with Katsura et al., who also reported increases of vitreous EPO levels in PDR patients compared to controls [55]. Cristina et al. found that EPO levels in vitreous fluid are significantly higher (326 mU/mL) compared to serum EPO (11.2 mU/mL) in PDR patients [56]. This shows that intraocular production is responsible for the high concentration of erythropoietin found in the vitreous fluid of retinal degeneration patients [54, 56, 57]. Garci et al. found increased vitreous EPO concentrations in DME patients (430 mU/mL) compared to control patients (25 mU/mL) [57]. Treatment involving the erythropoietin blockade is likely to be beneficial, but may worsen the disease due to

Proliferative Diabetic Retinopathy: An Overview of Vitreous Immune and Biomarkers

http://dx.doi.org/10.5772/intechopen.74366

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Matrix metalloproteinases (MMPs) are a family of zinc ion-binding endopeptidases that degrade most of the extracellular matrix (ECM). MMPs regulate many cellular functions including apoptosis, wound healing, and angiogenesis. In angiogenesis, MMPs increase VEGF production and remove physical barriers to new vessel growth [58, 59]. MMPs are produced as a response to increased oxidative stress. Diabetic patients often have increased MMP, mainly MMP-9 and MMP-2 in the retina and vitreous. These are controlled by endogenous tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 regulates MMP-9 and TIMP-2 regulates MMP-2 [59]. Several studies suggest that MMPs are responsible for many diabetic complications, including cardiomyopathy, nephropathy, and retinopathy. MMPs are suspected to facilitate apoptosis of retinal capillary cells during early stages leading to disruption of blood-retinal barrier integrity [58–60]. Kowluru et al. found an increase in MMP-9 and a decrease in TIMP-1 in the retina of DR patients [58]. Abu et al. found significant increases in vitreous zymography levels of MMP-9 in PDR patients (392.3 ± 253.6 scanning units) compared to non-diabetic control patients (168.2 ± 65.0 scanning units). However, the levels of vitreous MMP-2 in PDR patients (540.9 ± 185.6 scanning units) did not differ significantly from non-diabetic control patients (505.4 ± 216.1 scanning units) [60]. Inhibitors of MMPs have been used to treat several diseases,

however, there have been no studies using these inhibitors to treat DR patients [59].

Transforming growth factor β (TGF-β) is a polypeptide responsible for controlling cell proliferation and differentiation. It is usually secreted in a latent phase and must be transformed

Brzović-Šarić also demonstrated a significant difference between vitreous VEGF values in nondiabetic patients with eye disorders and PDR patients [25]. Loukovaara et al. state that VEGF is a major factor in PDR development and found significant increases of VEGF levels in the vitreous of DR patients (465.1 ± 1470.2 pg/mL) compared to control patients (40.3 ± 165.8 pg/mL) [37]. Our study found a mean level of VEGF in vitreous of patients with PDR of 0.356 + 0.60 pg/mL [26]. Yoshimura et al. found that there was significantly elevated VEGF in PDR patients, but not in DME patients [47]. The increased levels of VEGF expression in patients with diabetic retinopathy was mainly produced by Muller glial cells. Experiments in diabetic mice, demonstrated that conditional knockout of VEGF in Muller cells effectively blocked the increase in retinal VEGF expression [48]. Lange and co-workers suggest that oxygen tension levels were positively correlated with vitreous VEGF levels, and oxygen tension levels at the posterior pole were increased in PDR patients [49]. The vitreous levels of VEGF will decrease in the most severe stage of PDR, when there is a transition from angiogenesis to fibrosis [50].

### *3.3.2. Angiopoietin*

Angiopoietins are a group of proteins with the role of regulating vascular development and angiogenesis. Two types of angiopoietins, angiopoietin-1 and angiopoietin-2, contribute to the maintenance of retinal vasculature. The former exerts a stabilizing effect on vessels, organizing and limiting the angiogenesis response, while the latter exhibits angiogenic activity if VEGF is present, but promotes endothelial cell death and vascular regression in the absence of VEGF. The ratio between these two angiopoietins represents the inflammatory process in the cell. Fiedler et al. state that hypoxia/ischemia activates endothelial cells upregulating angiopoientin-2 thus lowering the angiopoientin-1/angiopoientin-2 ratio [37, 51]. A recent publication by Loukovaara et al. demonstrates significant correlation between intravitreal concentrations of Ang-2 with MMP-9, VEGF, EPO and TGFb1 levels in diabetic eyes undergoing vitrectomy, indicating its role in retinal tissue neovascularization in PDR patients. The study shows a slight increase in angiopoietin-1 from the control group (19.1 ± 25.4 pg/mL) to the study group (25.6 ± 27.1 pg/mL), and a great increase in angiopoietin-2 from the control group (43.0 ± 60.9 pg/mL) to the study group (317.1 ± 419.1 pg/mL), thus lowering the angiopoietin-1/angiopoietin-2 ratio in study group. The plasma value of angiopoietin-1 is similar in both groups, but the plasma value of angiopoietin-2 is increased from the control group (2623.4 ± 2142.0 pg/mL) compared to the study group (5690.4 ± 8064.7 pg/mL) [36, 37, 52]. Several studies state that angiopoietin-1 can be used for the prevention and treatment of diabetic retinopathy by its ability to suppress VEGF expression in diabetic retina [53].
