Internalization of Angiotensin-(1-12) in Adult Retinal Pigment Epithelial-19 Cells.

Purpose: Angiotensin-(1-12) [Ang-(1-12)] serves as a primary substrate to generate angiotensin II (Ang II) by angiotensin-converting enzyme and/or chymase suggests it may be an unrecognized source of Ang II-mediated microvascular complication in hypertension-mediated retinopathy. We investigated Ang-(1-12) expression and internalization in adult retinal pigment epithelial-19 (ARPE-19) cultured cells. We performed the internalization of Ang-(1-12) in ARPE-19 cells in the presence of a highly specific monoclonal antibody (mAb) developed against the C-terminal end of the Ang-(1-12) sequence. Methods: All experiments were performed in confluent ARPE-19 cells (passage 28-35). We employed high-performance liquid chromatography to purify radiolabeled, 125I-Ang-(1-12) and immuno-neutralization with Ang-(1-12) mAb to demonstrate Ang-(1-12)'s internalization in ARPE-19 cells. Internalization was also demonstrated by immunofluorescence (IF) method. Results: These procedures revealed internalization of an intact 125I-Ang-(1-12) in ARPE-19 cells. A significant reduction (∼53%, P < 0.0001) in 125I-Ang-(1-12) internalization was detected in APRE-19 cells in the presence of the mAb. IF staining experiments further confirms internalization of Ang-(1-12) into the cells from the extracellular culture medium. No endogenous expression was detected in the ARPE-19 cells. An increased intensity of IF staining was detected in cells exposed to 1.0 µM Ang-(1-12) compared with 0.1 µM. Furthermore, we found hydrolysis of Ang-(1-12) into Ang II by ARPE-19 cells' plasma membranes. Conclusions: Intact Ang-(1-12) peptide is internalized from the extracellular spaces in ARPE-19 cells and metabolized into Ang II. The finding that a selective mAb blocks cellular internalization of Ang-(1-12) suggests alternate therapeutic approaches to prevent/reduce the RPE cells Ang II burden.

Implications of Diabetes-Induced Altered Metabolites on Retinal Neurodegeneration.

Diabetic retinopathy (DR) is one of the major complications of diabetic eye diseases, causing vision loss and blindness worldwide. The concept of diabetic retinopathy has evolved from microvascular disease into more complex neurovascular disorders. Early in the disease progression of diabetes, the neuronal and glial cells are compromised before any microvascular abnormalities clinically detected by the ophthalmoscopic examination. This implies understanding the pathophysiological mechanisms at the early stage of disease progression especially due to diabetes-induced metabolic alterations to damage the neural retina so that early intervention and treatments options can be identified to prevent and inhibit the progression of DR. Hyperglycemia has been widely considered the major contributor to the progression of the retinal damage, even though tight control of glucose does not seem to have a bigger effect on the incidence or progression of retinal damage that leads to DR. Emerging evidence suggests that besides diabetes-induced hyperglycemia, dyslipidemia and amino acid defects might be a major contributor to the progression of early neurovascular retinal damage. In this review, we have discussed recent advances in the alterations of key metabolites of carbohydrate, lipid, and amino acids and their implications for neurovascular damage in DR.

Flavonoid Naringenin Attenuates Oxidative Stress, Apoptosis and Improves Neurotrophic Effects in the Diabetic Rat Retina.

Diabetic retinopathy (DR) is one of the leading causes of decreased vision and blindness worldwide. Diabetes-induced oxidative stress is believed to be the key factor that initiates neuronal damage in the diabetic retina leading to DR. Experimental approaches to utilize dietary flavonoids, which possess both antidiabetic and antioxidant activities, might protect the retinal damage in diabetes. The aim of this study was to investigate the potential protective effects of naringenin in the retina of streptozotocin-induced diabetic rats. Diabetic rats were orally treated and untreated with naringenin (50 mg/kg/day) for five weeks and retinas were analyzed for markers of oxidative stress, apoptosis and neurotrophic factors. Systemic effects of naringenin treatments were also analyzed and compared with untreated groups. The results showed that elevated levels of thiobarbituric acid reactive substances (TBARs) and decreased level of glutathione (GSH) in diabetic rats were ameliorated with naringenin treatments. Moreover, decreased levels of neuroprotective factors (Brain derived neurotrophic factor (BDNF)), tropomyosin related kinase B (TrkB) and synaptophysin in diabetic retina were augmented with naringenin treatments. In addition, naringenin treatment ameliorated the levels of apoptosis regulatory proteins; B cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax) and caspase-3 in the diabetic retina. Thus, the study demonstrates the beneficial effects of naringenin that possesses anti-diabetic, antioxidant and antiapoptotic properties, which may limit neurodegeneration by providing neurotrophic support to prevent retinal damage in diabetic retinopathy.

Neuroprotective effects of quercetin in diabetic rat retina.

Diabetic retinopathy (DR) is a severe complication of diabetes and the leading cause of blindness among working adults worldwide. DR is being widely recognized as a neurodegenerative disease of the retina, since, retinal neurons are damaged soon after diabetes onset. Diabetes-induced oxidative stress is considered as central factor that dysregulates neurotrophic factors and activates apoptosis, thereby damages neurons in the diabetic retina. Flavonoids being a powerful antioxidant have been considered to protect neurons in diabetic retina. The purpose of this study was to analyze the beneficial effects of flavonoid, quercetin to protect neurons in the diabetic rat retina. We quantitated the expression levels of BDNF, NGF, TrkB, synaptophysin, Akt, Bcl-2, cytochrome c and caspase-3 using Western blotting techniques in the diabetic retina with and without quercetin treatments and compared with non-diabetic rats. In addition, we employed ELISA techniques to determine the level of BDNF. Caspase-3 activity and the level of glutathione were analyzed by biochemical methods. Our results indicate that quercetin treatment to diabetic rats caused a significant increase in the level of neurotrophic factors and inhibited the level of cytochrome c and caspase-3 activity in the diabetic retina. Furthermore, the level of an anti-apoptotic protein Bcl-2 was augmented in quercetin treated diabetic retina. Thus, quercetin, may protect the neuronal damage in diabetic retina by ameliorating the levels of neurotrophic factors and also by inhibiting the apoptosis of neurons. Therefore, this study suggests that quercetin can be a suitable therapeutic agent to prevent neurodegeneration in diabetic retinopathy.

Protective Effects of Dietary Flavonoids in Diabetic Induced Retinal Neurodegeneration.

BACKGROUND: Diabetic retinopathy is one of the serious complications of diabetes and the leading cause of decreased vision and blindness worldwide. Neurodegeneration has been recognized as initiating factor in causing the retinal damage, which leads to micro-vascular damage in diabetic retinopathy. Diabetes-induced oxidative stress is believed to be the key factor that damages neurons in the diabetic retina. Various therapeutic approaches for effective attenuation of increased oxidative stress by antioxidants have emerged. One such approach is to utilize dietary flavonoids, which have been found to possess powerful antioxidant activity. Some of the naturally occurring flavones possess anti-diabetic effects by enhancing insulin sensitivity and reducing plasma glucose levels in diabetic animal models. OBJECTIVE: Considering the importance of developing new antioxidant compounds and the relevance of their applications in the treatment of diabetes and its complications, in this review article, we discuss and highlight various neuroprotective mechanisms of flavonoids in the diabetic retina. RESULTS: Dietary supplementation of flavonoids to diabetics may reduce oxidative stress, which in turn might ameliorate apoptosis and the levels of neurotrophic factors in the diabetic retina. CONCLUSION: This approach will elucidate a novel strategy for preventing and treating diabetic retinoneuropathy the leading cause of low vision and blindness.

Naringenin neutralises oxidative stress and nerve growth factor discrepancy in experimental diabetic neuropathy.

OBJECTIVES: Present study aims to investigate the ameliorative effects of naringenin (NG) on experimentally induced diabetic neuropathy (DN) in rats. METHODS: Diabetes was induced by single intraperitoneal injection of streptozotocin (STZ, 60  g/kg). Naringenin (25 and 50 mg/kg/day) treatment was started 2 weeks after the diabetes induction and continued for five consecutive weeks. Pain threshold behaviour tests were performed at the end of the treatment. Serum levels of glucose, insulin and pro-inflammatory cytokines were assessed. In sciatic tissues, markers oxidative stress, cytokines and neurotrophic factors were measured. RESULTS: NG treatments showed significant decrease in paw-withdrawal (P < 0.01) and tail-flick latency (P < 0.01). The drug attenuated the diabetic-induced changes in serum glucose, insulin and pro-inflammatory cytokines including tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6). In sciatic nerve, the diabetic-induced alterations in interleukins and oxidative stress biomarkers were significantly attenuated by NG. Decreased sciatic expressions of insulin growth factor (IGF) and nerve growth factor (NGF) in diabetic rats were also ameliorated by NG. Diabetes-induced dysregulated levels of nitric oxide (NO), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were ameliorated by NG. Histological analysis showed that NG corrected the altered sciatic changes in diabetic animals. DISCUSSION: We suggest that neuro-protective effect of NG molecules in sciatic nerve of diabetic rats, through its anti-diabetic as well as antioxidant and anti-inflammatory properties.

Flavonoid, morin inhibits oxidative stress, inflammation and enhances neurotrophic support in the brain of streptozotocin-induced diabetic rats.

Diabetes-induced damages in brain are known as diabetic encephalopathy, which is well characterized by cellular, molecular and functional changes in the brain of diabetic subjects and rodents. However, little is known about the mechanism of damages and the therapeutic strategies in ameliorating those damages in the diabetic brain. In this study, we utilized a flavonoid, morin which is emerging as a potent drug against a wide range of free radical-mediated as well as neurodegenerative diseases. Morin (15 and 30 mg/kg body weight/day) was orally administered to two different groups of rats after 1 week of diabetes induction, and continued for five consecutive weeks. Two other untreated groups of diabetic and non-diabetic rats were used to compare with drug-treated groups. After drug treatments, cerebral cortex of the brain harvested and analyzed for different factors. Morin supplementation especially at high dose increased the levels of insulin, reduced glutathione, superoxide dismutase and catalase activities, and decreased fasting glucose and thiobarbituric acid reactive substances in the diabetic brain compared to untreated diabetic rats (P < 0.05). Morin also significantly decreased the level of inflammatory markers (TNFα, IL1ß, IL-6) in the diabetic brain compared to untreated diabetic rats. Furthermore, the drug influenced an increase in the level of neurotrophic factors (BDNF, NGF and IGF-1) in the diabetic brain compared to untreated diabetic rats (P < 0.05). Thus, our results indicate a beneficial effect of morin by decreasing oxidative stress, inflammation and increasing the neurotrophic support in the diabetic brain, which may ameliorate diabetic encephalopathy.

Alterations in prothrombin time and activated partial thromboplastin time in patients with acute myocardial infarction.

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) respectively measures the extrinsic and intrinsic pathways of coagulation and are used to determine the bleeding or clotting tendency of blood. We compared PT and aPTT levels in acute myocardial infarction (AMI) patients and normal subjects. There were significant increases in PT levels in patients with STEMI (15.98 ± 0.96 s), NSTEMI (16.03 ± 0.97 s) and chest pain (15.02 ± 0.54 s) as compared to control group (8.86 ± 0.08 s). The level of aPTT in control subjects was 31.35 ± 0.48 s. Patients with STEMI (40.79 ± 1.83 s), NSTEMI (41.33 ± 2.06) and chest pain (37.84 ± 1.66 s) showed significantly higher levels of aPTT. There was a significant correlation between PT and aPTT levels. Both PT and aPTT were significantly correlated with age however there was no correlation between these coagulation markers and gender or body mass index. In conclusion, both PT and aPTT are significantly increased in AMI patients on anticoagulation therapy. The elevations in PT values were more than 2.5-fold greater than aPTT suggesting a high potential of PT for predicting blood clotting tendency in patients receiving anticoagulation therapy.

Expression of thrombospondin-2 as a marker in proliferative diabetic retinopathy.

PURPOSE: To determine the expression of the endogenous anti-angiogenic and pro-fibrotic matricellular protein thrombospondin (TSP)-2 and its receptors CD36 and CD47 in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of TSP-2 in the retinas of diabetic rats. METHODS: Epiretinal membranes from 14 patients with PDR and nine patients with proliferative vitreoretinopathy were studied by immunohistochemistry. Vitreous samples from 30 PDR and 25 nondiabetic patients were studied by enzyme-linked immunosorbent assay. Vitreous samples and retinas of rats were examined by Western blotting. RESULTS: In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed TSP-2, CD36 and CD47. In PDR membranes, significant correlations were observed between numbers of blood vessels expressing the panendothelial cell marker CD34 and numbers of blood vessels and stromal cells expressing TSP-2, CD36 and CD47. The numbers of blood vessels and stromal cells expressing CD34, TSP-2, CD36 and CD47 were significantly higher in membranes with active neovascularization when compared with those with quiescent disease. Thrombospondin-2 levels in vitreous samples from PDR patients were significantly higher than those in control patients without diabetes (p < 0.001). Western blot analysis revealed a significant increase in the expression of intact and cleaved TSP-2 in vitreous samples from PDR patients and in the retinas of diabetic rats compared to nondiabetic controls. CONCLUSIONS: Upregulation of TSP-2 may be a protective mechanism against inflammation and angiogenesis associated with PDR.

The sigma receptor ligand (+)-pentazocine prevents apoptotic retinal ganglion cell death induced in vitro by homocysteine and glutamate.

Recent studies demonstrated that the excitotoxic amino acid homocysteine induces apoptotic death of retinal ganglion cells in vivo. In the present study, an in vitro rat retinal ganglion cell (RGC-5), culture system was used to analyze the toxicity of acute exposure to high levels of homocysteine, the mechanism of homocysteine-induced toxicity, and the usefulness of type 1 sigma receptor (sigmaR1) ligands as neuroprotectants. When cultured RGC-5 cells were subjected to treatment with 1 mM D,L-homocysteine, a significant increase in cell death was detected by terminal dUTP nick end labeling (TUNEL) analysis and analysis of activated caspase. When cells were treated with homocysteine- or glutamate in the presence of MK-801, an antagonist of the N-methyl-D-aspartate (NMDA) receptor, the cell death was inhibited significantly. In contrast, NBQX, an antagonist of the AMPA/Kainate receptor, and nifedipine, a calcium channel blocker, did not prevent the homocysteine- or glutamate-induced cell death. Semiquantitative RT-PCR and immunocytochemical analysis demonstrated that RGC-5 cells were exposed to homocysteine or glutamate express type 1 sigma receptor at levels similar to control cells. Treatment of RGC-5 cells with 3 or 10 microM concentrations of the sigmaR1-specific ligand (+)-pentazocine inhibited significantly the apoptotic cell death induced by homocysteine or glutamate. The results suggest that homocysteine is toxic to ganglion cells in vitro, that the toxicity is mediated via NMDA receptor activation, and that the sigmaR1-specific ligand (+)-pentazocine can block the RGC-5 cell death induced by homocysteine and glutamate.