Role of NF-κB activation and VEGF gene polymorphisms in VEGF up regulation in non-proliferative and proliferative diabetic retinopathy.

The present study was aimed to investigate the relation between nuclear factor kappa beta (NFκB) activation and downstream up-regulation of vascular endothelial growth factor (VEGF) in diabetic retinopathy (DR). Moreover the study was intended to evaluate the role of VEGF gene single nucleotide polymorphisms (SNPs) in DR occurrence and to investigate the functional relevance of VEGF gene SNPs in terms of VEGF expression in DR. Serum level of VEGF, VEGF R1 (receptor 1), VEGF R 2 (receptor 2) and NFκB (p50/65) activity was measured by enzyme linked immune sorbent assay. Genotyping and allelic composition of different SNPs i.e., rs2010963, rs3025039, rs1570360 and rs 2071559 were investigated by Taqman SNP genotyping assay. VEGF, NFκB p50/p65, and VEGF R1 & R2 gene expressions were quantified by real time quantitative polymerase chain reaction. Increased NFκB p50/p65 activity and expressions were observed in non proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) subjects compared to type 2 diabetes mellitus without retinopathy (DNR) group. Significantly elevated levels of serum VEGF and highest VEGF expression were found among PDR subjects compared to DNR or NPDR subjects. CC genotype and C allele of rs2010963 and TT genotype and T allele of rs3025039 were significantly over represented among PDR subjects compared to DNR group. Increased activation of NFκß in NPDR and PDR subjects might involve increased up regulation of VEGF. VEGF SNPs i.e., rs2010963 C allele and rs3025039 T allele might be associated with PDR occurrence and in turn regulates VEGF expression among PDR subjects.

Oxidative stress-associated neuroretinal dysfunction and nitrosative stress in diabetic retinopathy.

OBJECTIVE: The present study was intended to investigate whether oxidative stress is the key regulator to alter neuroretinal biochemical homeostasis and in turn aggravate the process of diabetic retinopathy by inducing nitrosative stress in the retinal neurovascular unit. METHODS: Peripheral blood mononuclear cell reactive oxygen species level was measured by flow cytometry along with spectrophotometric detection of malondialdehyde (MDA) and glutamate from serum or plasma and a vitreous sample of study groups (i.e. subjects with proliferative diabetic retinopathy [PDR], type 2 diabetes without retinopathy [DNR] and healthy controls [HCs]). Further, nitrosative stress assessment was performed by spectrophotometric and enzyme-linked immunosorbent assay-based detection of serum and vitreous nitrite and nitrotyrosine concentrations, respectively. RESULTS: The plasma glutamate level remains insignificant between subjects with PDR and DNR (p=0.505) or in HC (p=0.1344) individuals. However, serum MDA (p=0.0004), nitrite (p=0.0147) and nitrotyrosine (p=0.0129) were found to be strikingly higher among PDR subjects compared with the DNR group. Significantly increased levels of peripheral blood mononuclear cell reactive oxygen species (p<0.0001), vitreous glutamate (p=0.0009, p<0.0001), MDA (p=0.0058, p=0.0003), nitrite (p=0.0014, p<0.0001) and nitrotyrosine (p=0.0008, p<0.0001) were found in PDR subjects compared with DNR and HC subjects, respectively. CONCLUSIONS: Our observation suggests that oxidative stress is associated with impairment in neuroretinal biochemical homeostasis among PDR subjects, which further augments retinal nitrosative stress and thus worsens the pathogenic process of retinopathy among PDR subjects.

Association of hyperglycemia mediated increased advanced glycation and erythrocyte antioxidant enzyme activity in different stages of diabetic retinopathy.

AIM: This study aimed to evaluate whether hyperglycemia mediated increased formation of advanced glycation end products (AGEs) was associated with erythrocyte antioxidant enzyme activity in subjects with different stages of diabetic retinopathy (DR). METHODS: Serum level of AGEs was determined by enzyme linked immunosorbent assay. Erythrocyte superoxide dismutase (SOD), glutathione reductase (GR) and catalase activity were estimated by enzymatic reaction based spectrophotometric assay in patients with type 2 diabetes with proliferative diabetic retinopathy (PDR), non-proliferative diabetic retinopathy (NPDR) and no retinopathy (DNR) and also in healthy non-diabetic controls (HC). RESULT: Erythrocyte SOD and GR activity was significantly lower among NPDR (p=0.024, 0.0017, respectively) and PDR (p=0.0003, 0.0001, respectively) subjects compared with DNR individuals. A significant inverse correlation was observed between serum AGEs and erythrocyte SOD or GR activity in DNR (p=0.0019; r=-0.3033, p=0.0021; r=-0.3015, respectively), NPDR (p=0.0001; r=-0.4602, p=0.0003; r=-0.4161, respectively), and PDR (p<0.0001; r=-0.6753, p<0.0001; r=-0.5854, respectively) individuals. CONCLUSION: Poor glycemia may be the key factor enhancing AGE formation, which may be associated with lower erythrocyte SOD and GR activity along with increased catalase activity in DR.

Role of hyperglycemia-mediated erythrocyte redox state alteration in the development of diabetic retinopathy.

PURPOSE: To evaluate erythrocyte redox state and its surrogates in patients with different stages of diabetic retinopathy and their association with cellular metabolic derangement developed in retinal microvascular cells. METHODS: Sixty type 2 diabetic patients with nonproliferative diabetic retinopathy (NPDR), 85 patients with proliferative diabetic retinopathy (PDR), and 70 patients with diabetes but without retinopathy were considered as diabetic control (DC) for the study. In addition, 65 normal individuals without diabetes were enrolled as healthy control in this study. Erythrocyte oxidized nicotinamide adenine dinucleotide phosphate / reduced nicotinamide adenine dinucleotide phosphate (NADP / NADPH), oxidized nicotinamide adenine dinucleotide / reduced nicotinamide adenine dinucleotide (NAD / NADH) glutathione, plasma and vitreous lactate, and pyruvate levels were determined by enzymatic reaction-based spectrophotometric assay for the patients and individuals. RESULT: Erythrocyte NADP+ to NADPH ratio to NADPH ratio was found to be significantly higher among NPDR and PDR patients compared with DC subjects (P < 0.0001). Erythrocyte-reduced glutathione was significantly decreased in patients of NPDR (P = 0.0004) and patients of PDR (P = 0.0157) compared to DC. Erythrocyte NAD to NADH ratio was also significantly decreased in patients of NPDR (P < 0.0001) and PDR (P < 0.0001) compared to DC subjects. Lactate to pyruvate ratio of plasma was elevated significantly in patients with NPDR compared with DC (P < 0.0001) and those having PDR (P = 0.0046). In the vitreous fluid, the lactate to pyruvate ratios were found to be significantly lower in normal individuals without diabetes compared with patients having PDR (P < 0.0001). CONCLUSION: Hyperglycemia-mediated erythrocyte redox state alterations might be a potential risk factor for the development of NPDR in poorly controlled diabetic subjects.

Assessment of gelatinase and tumor necrosis factor-α level in the vitreous and serum of patients with Eales disease: role of inflammation-mediated angiogenesis in the pathogenesis of Eales disease.

BACKGROUND: Eales disease (ED) is an idiopathic, inflammatory, venoocclusive disorder of peripheral retina resulting in retinal angiogenesis and vitreous hemorrhage. The objective of the present study is to investigate the expression and activation of gelatinase associated with the retinal neovascularization in ED and the relation between the levels of gelatinase and the cytokine tumor necrosis factor-α, known to upregulate matrix metalloproteinase (MMP) expression on various cells. METHODS: Vitreous and serum samples from 19 patients with ED who underwent retinal surgery were estimated for levels of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, and tumor necrosis factor-α by enzyme-linked immunosorbent assay method. Matrix metalloproteinase-2 and MMP-9 activities in serum and vitreous samples were evaluated by gelatin zymography method. Vitreous samples from 16 patients with macular hole undergoing vitrectomy were used as controls. RESULTS: Among the 2 gelatinase examined in vitreous and serum samples, only level and activity of MMP-9 were significantly higher in serum (P = 0.0001) and vitreous (P = 0.0002) samples of patients with ED than those of control subjects. Simultaneously, a positive correlation was found between intraocular tumor necrosis factor-α and MMP-9 concentration (Spearman correlation coefficient, r = 0.7040, P = 0.0023) in patients with ED. CONCLUSION: Increase in MMP-9 activity and its concentration in serum and vitreous of patients with ED compared with that of control subjects and correlation between intraocular levels of MMP-9 and tumor necrosis factor-α in patients with ED seem to provide a plausible explanation for inflammation-mediated angiogenesis during the development of this condition.