**4.3 Activation of the protein kinase C (PKC) pathway**

PKC activation has been shown to induce retinal vascular abnormalities in diabetes. Diacylglycerol (DAG) is the primary activator of PKC in physiology [4]. Increased total levels of DAG in DR were found [71]. Augmentation of DAG levels in diabetes can occur by several pathways. Hyperglycemia results in an increase of glucose flux through the glycolysis pathway, which in turn leads to enhanced de novo synthesis of DAG from glycolytic intermediates [72–74]. DAG can be gained as well from the hydrolysis of phosphatidylinositides, from the metabolism of phosphatidylcholine by phospholipase C [75]. Increased generation of DAG and the subsequent activation of PKC isoforms affect retinal functions in multiple different ways. Activation of DAG-PKC pathway is associated with cellular and vascular abnormalities in the retina such as increased endothelial permeability, basement membrane thickening, leucocyte adhesion, cytokine activation, abnormal angiogenesis, and excessive apoptosis [72]. Activation of PKC regulates gene expression via of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Induced by phosphorylation, in response to extracellular signals, MAPK and PI3K regulate functions of a broad array of proteins involved in cell growth, proliferation, motility, adhesion, survival, apoptosis, and angiogenesis [76, 77]. Among the various PKC isoforms, the beta-isoform seems to be activated preferentially in the vasculatures of diabetic animals [78]. It was demonstrated that PKC beta-isoform plays a role in the VEGF-induced vascular permeability in the retina of diabetic animals and VEGF-induced proliferation of endothelial cells. Furthermore, PKC betaisoform-selective inhibitors decreased VEGF-induced vascular permeability and endothelial cell growth [77, 78]. VEGF is a dimeric glycoprotein and has a crucial role in the development and progression of DR. In mammals, the VEGF family comprises seven members where VEGF-A typically, and below, referred to as VEGF. VEGF regulates cell functions via vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2, which belongs to the receptor tyrosine kinase family and primarily implicated in angiogenesis [79]. Hypoxia is the major inducer of increased VEGF transcription in the retina via hypoxia inducible factor-1 (HIF-1) [44, 80]. In addition to hypoxia, a number of other factors can stimulate the overexpression of VEGF in DR, including oxidative stress and insulin-like growth factor [81]. The pathways by which these factors regulate upregulation of retinal VEGF transcription are not yet understood. However, it has been demonstrated that ROS can induce VEGF transcription by a mechanism involving the activity of signal transducer and activator of transcription factor 3 (STAT3) [82]. It was found that increased vessel permeability is correlated with increased ocular levels of VEGF [83]. It was suggested that VEGFinduced permeability results from triggering of a cascade of proteolytic activities on the endothelial cell surface. VEGF induces expression of urokinase plasminogen activator receptor (uPAR) that initiates cleavage of plasminogen by urokinase plasminogen activator (uPA). Subsequently, plasmin formation leads to activation of membrane-bound pro matrix metalloproteinase-9 pro (MMP-9) [84]. MMP-9 induces pericellular proteolysis affecting cell-cell and cell-BM attachment, producing leaky vessels and permitting to the endothelial cells to penetrate the underlying BM, migrate, and proliferate [85]. It was shown as well that VEGF increases microvascular permeability via increasing the intracellular calcium concentration in endothelial cells [86]. Growth of new blood vessels is induced by VEGF-VEGFRs mediated activation of MAPK cascades resulting in endothelium proliferation, migration, and tube formation [44]. Anti-vascular endothelial growth factor (anti-VEGF) drugs are

viable treatment option for patients with diabetic macular edema and proliferative diabetic retinopathy [87, 88].

A role of PKC activation in the thickening of capillary basement membrane that is the prominent structural abnormality in the retinal microvessels in early DR was demonstrated. Treatment with PKC agonists stimulated type IV collagen expression and fibronectin accumulation that may increase the BM thickness [89, 90]. It was reported that inhibition of Na+ -K+ -ATPase by hyperglycemia was due to consecutive activation of PKC and cytosolic phospholipase A2 (cPLA2), inducing release of arachidonic acid and increased production of PGE2, which are known inhibitors of Na+,K(+)-ATPase [91]. Na+ -K+ -ATPase is a component of sodium pump, and it takes part in regulation of cellular contractility, MAPK transduction pathways, ROS formation, intracellular calcium levels. PKC takes part in sustaining of chronic inflammation in DR. A row of studies were demonstrated that activation of PKC in endothelial cells triggered upregulation of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule (VCAM)-1 that increased adhesion of leukocytes to the vascular endothelium. PKC inhibitors prevented upregulation of ICAM -I and (VCAM)-1 and adherence of neutrophils to endothelial cells [92–96]. It was demonstrated that leukocytes trapped in retinal vasculature course capillary occlusion, vascular cell, and BM alterations in the animal diabetic model [97]. Being adhered to the vessel wall, leukocytes may release ROS, enzymes, and cytokines, which damage the endothelial cells and increase vascular permeability [98, 99]. Taking in account the significance of pathological events inducing by activation of PKC, inhibitors of PKC have been studied as potential therapeutic agents for the treatment of patients with microvascular complications associated with diabetes [100].

#### **4.4 Increased flux through the hexosamine pathway**

The hexosamine biosynthesis pathway (HBP) is a comparatively minor part of glycolysis. Hyperglycemic condition increases glucose flux through HBP. High availability of intracellular glucose leads to an excess amount of fructose-6-phosphate. The largest proportion of fructose-6-phosphate is utilized in the glycolytic pathway. Glutamine: fructose-6-phosphate aminotransferase (GFAT) regulates the entry of fructose-6-phosphate into the HBP. The major end product of the HBP is UDP-N-acetylglucosamine (UDP-GlcNAc) that catalyzes the addition of *O*-linked β-*N*acetylglucosamine (*O*-GlcNAc) to serine and threonine residues of proteins [101]. O-GlcNAcylation is an important protein posttranslational modification (PTM) that involves the addition of *O*-GlcNAc moiety to the hydroxyl groups of serine and/or threonine residues of proteins. Such as phosphorylation, protein O-GlcNAc modification can directly modify protein functions and also lead to the changes of gene expression [102]. Under conditions of sustained hyperglycemia that occur in diabetes, GFAT is upregulated, fructose-6-phosphate flux increases through the HBP and results in increase of *O*-GlcNAc-modified proteins. There are studies showing an association between elevated flux through HBP and insulin resistance [103]. It was demonstrated that high levels of *O*-GlcNAcylation of proteins in the retinal endothelial cells and pericytes correlated with glucose concentration levels, but the physiological consequences of this mainly remain unknown [104]. O-GlcNAc protein modification dysregulation under hyperglycemia and/or ischemia may contribute to the pathogenesis of the DR and retinal neovascularization [104, 105]. Decreasing glucose flux through the HBP by preventing the biosynthesis of UDP-GlcNAc would appear to reduce glucose toxicity, but would also induce adverse effects. A lot of proteins including kinases, phosphatases, transcriptional factors, and metabolic enzymes can be O-GlcNAc modified, but the functional consequences of this modification remain unknown for most of these proteins and need to be clarified.
