European Association for the Study of Diabetic Eye Complications
Annual Meeting Munich 21-23rd May 2004
PATHOPHYSIOLOGY OF DIABETIC RETINOPATHY
NADPH OXIDASE PARTICIPTATES IN GLUCOSE-INDUCED TOXICITY IN CULTURED RETINAL CAPILLARY PERICYTES
Nik M. Mustapha, Bahaedin M. Ben-Mahmud, Giovanni E. Mann, Eva M. Kohner, Rakesh Chibber Centre for Cardiovascular Biology & Medicine, GKT School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL.
Purpose: Reactive oxygen species (ROS) is accepted as a unifying mechanism for the hyperglycemia-induced long-term diabetic complications, including retinopathy. The production of ROS can mediate changes in cellular growth, apoptosis, and survival. We hypothesized that increased intracellular production of ROS from NADPH oxidase is responsible for the loss of pericytes from retinal capillaries in early diabetic retinopathy.
Methods: Confluent cultures of bovine retinal capillary pericytes (BRP) in 3cm tissue culture dish were exposed to normal (5.8mM) and high (25mM) glucose for various times (0-96h). Total cell lysates were used for the measurement of intracellular glucose (Amplex ™ Red glucose assay kit, Molecular Probes), protein carbonyls as an indicator of oxidative protein damage (Oxyblot™ protein oxidation detection kit, Chemicon, UK), and the expression of NADPH oxidase (p47phox, gp91phox) using Western blot analysis.
Results: After 4-day incubation, there was a significant increase in the intracellular glucose concentration (71.9 ± 7.9 vs 8.8 ± 2.9 nmol/mg protein) in BRP exposed to high-glucose (25mM) compared to normal glucose (5.8mM). Glucose also caused increase in total carbonyls content as early as 2h (723.5 ± 343.5 % of normal glucose). The levels of p47phox and gp91phox proteins were also increased in BRP maintained in high-glucose condition.
Conclusion: Our present results suggest that activation of NADPH oxidase and intracellular protein oxidation in response to hyperglycemia is a possible mechanism for the early loss of retinal pericytes in diabetic retinopathy. Acknowledgements: Funded by Malaysian Government
DIABETIC RETINOPATHY: AGE-INDUCED NEURONAL DEGENERATION IN RETINAL EXPLANTS.
Amélie Lecleire-Collet, Luc-Henri Tessier, Pascale Massin, José A.Sahel, Serge Picaud. Laboratoire de Physiopathologie Cellulaire et Moléculaire de la Rétine, INSERM U 592, Hôpital Saint-Antoine, Bâtiment Kourilsky 6ème étage, 184 rue du Faubourg Saint- Antoine, 75571 Paris Cedex 12, France.
Purpose: Neuronal degeneration was reported to occur in diabetic retinopathy before the onset of detectable microvascular abnormalities. To investigate whether advanced glycation end products (AGE) generated during the disease are directly responsible for retinal degeneration, AGE were applied on retinal explants limiting the influence of retinal vasculature.
Methods: Retinal explants were obtained from non-diabetic adult rats by maintaining the retina attached to the retinal pigment epithelium (RPE). Retinal explants were incubated 4 days in the presence or absence of 200 µg/ml of AGE resulting from the glycation of bovine serum albumin with fructosamine (AGE-BSA). Neural apoptosis was quantified using in situ DNA terminal dUTP nick end fluorescein labeling (TUNEL-FITC) and immunostaining with antibodies to anti-cleaved caspase-3. Expressions of the glial fibrillary acidic protein (GFAP), the glutamate transporter (GLAST) and the glutamine synthetase were localized by immunofluorescence.
Results: Numbers of TUNEL-positive cells and cleaved caspase-3-positive cells were increased by 2.2 and 2.5 in AGE-incubated retinal explants with respect to controls (p<0.05). The ganglion cell layer was the most sensitive retinal layer to AGE-induced apoptosis, with a 4-fold increase of TUNEL-positive cells in AGE-incubated retinal explants with respect to controls (p<0.05). Neuronal degeneration was confirmed by the increased and extensive GFAP labeling in Müller glial cells from AGE-treated retinal explants, by contrast to the Müller cell end feet labeling in controls. No differences were observed for GLAST and glutamine synthetase between controls and AGE-treated retinal explants
Conclusions: These observations indicated that AGE can induce retinal neurodegeneration in the absence of blood perfusion. Neuronal degeneration was observed in the ganglion cell layer as previously reported in diabetic retinopathy and its animal models. These results validate retinal explants as a model for the study of diabetic retinopathy pathogenesis. They indicate further that AGE production could be an early pathological mechanism in the induction of diabetic retinopathy.
THIAMINE AND BENFOTIAMINE INHIBIT POLYOL PATHWAY AND RAISE TRANSKETOLASE EXPRESSION IN ENDOTHELIAL CELLS AND PERICYTES CULTURED IN HIGH GLUCOSE.
Elena Berrone, Elena Beltramo, Carmela Solimine, Alessandro Ubertalli, Massimo Porta. Dept. of Internal Medicine, University of Turin, Turin, Italy.
Background: The polyol pathway has been implicated in the pathogenesis of diabetic vascular complications. Aldose reductase (AR), the first enzyme of this pathway, trasforms D-glucose into D-sorbitol, leading to imbalances of intracellular homeostasis. We and others have shown previously that thiamine (T), a coenzyme for transketolase (TK), and its lipophilic analogue, benfotiamine (BT), correct a number of metabolic and viability abnormalities induced by high glucose in vascular cells in vitro and in animals with experimental diabetes.
Aim: To verify if T and BT modify mRNA expression of TK and AR, along with AR activity and sorbitol concentrations, in human endothelial cells (HUVEC) and bovine retinal pericytes (BRP) cultured in high glucose.
Methods: HUVEC and BRP were cultured for 7 days in normal (5.6 mmol/l) or high (28 mmol/l) glucose concentrations, with or without T or BT (50 or 100 µmol/l). TK and AR mRNA expression was determined by relative quantitative RT-PCR, AR activity was measured spectrophotometrically by assessing the decrease of NADPH, and sorbitol concentrations were quantified by GC-MS.
Results: Both in HUVEC and BRP, high glucose did not modify TK mRNA and increased AR mRNA and activity along with sorbitol concentrations. Addition of T and BT 50-100 µmol/l to high glucose: 1) increased TK mRNA, 2) reduced AR activity and mRNA expression and, 3) reduced sorbitol concentrations.
Conclusions: T and BT correct polyol pathway activation induced by high glucose in vascular cells, possibly through activation of TK and consequent shift of metabolites into the pentose phosphate cycle. Polyol pathway inhibition, together with other beneficial effects reported for this vitamin in high glucose, could justify testing it as a potential approach to the prevention and/or treatment of diabetic complications.
TNF-ALPHA IN DIABETIC PLASMA INCREASES THE ACTIVITY OF CORE 2 GlcNAc-T AND ADHERENCE OF HUMAN LEUKOCYTES TO RETINAL ENDOTHELIAL CELLS
Rakesh Chibber1, Bahaedin M. Ben-Mahmud1, Giovanni E. Mann1, Alessandro Datti 2, Aldo Orlacchio2, Eva M. Kohner1
1Centre for Cardiovascular Biology & Medicine, GKT School of Biomedical Sciences, King's College London, London, UK. 2Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, Università degli Studi di Perugia, 06122 Perugia, Italy
A large body of evidence now implicates increased leukocyte-endothelial cell adhesion as a key early event in the development of diabetic retinopathy. We recently reported that raised activity of the glycosylating enzyme Core 2 -1, 6- N-acetylglucosaminyltransferase (Core 2 GlcNAc-T) through PKC b2-dependent phosphorylation, plays a fundamental role in increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy (Chibber et al. Diabetes 2003). In the present study, we demonstrate that following exposure to plasma from diabetic patients, human leukocytes (U937 cells) exhibits a significant 7-fold elevation in Core 2 GlcNAc-T activity, as well as 5-fold increased adherence to cultured retinal capillary endothelial cells. These effects of diabetic plasma on enzyme activity and cell adhesion, mediated by protein kinase Cb2 (PKCb2)-dependent phosphorylation of the Core 2 GlcNAc-T protein, were triggered by plasma levels of tumour necrosis factor-a (TNF-a). Levels of enzyme activity in plasma-treated U937 cells were closely dependent on the severity of retinopathy, with the highest values observed upon treatment with plasma of patients with proliferative retinopathy. Furthermore, we noted much higher correlation, as compared to controls, between increased values of Core 2 GlcNAc-T activity and cell adhesion properties. Based on the prominent role of TNF-_ in the development of diabetic retinopathy, these observations further validate the significance of Core 2 GlcNAc-T in the pathogenesis of capillary occlusion, thereby enhancing the therapeutic potential of specific enzyme inhibitors.
Supported by the JDF & Guy's, St Thomas' Charitable Foundation & Charities Advisory Board
EFFECT OF ACIDOSIS ON PORCINE RETINAL ARTERIOLES
Anders Hessellund1 , Christian Aalkjaer 2 and Toke Bek1
1. Dept of Ophthalmology, Aarhus University Hospital, Denmark
2. Dept of Physiology, Aarhus University, Denmark
Purpose: Disturbances in the regulation of retinal blood flow are a key element in the pathogenesis of diabetic retinopathy (DR). A major element in the regulation of the retinal blood flow is the vascular response to metabolites released from the ischaemic tissue such as H+ and CO2. To study this mechanism, isolated porcine retinal arterioles were exposed to normocapnic (NA) and hypercapnic acidosis (HA) during measurement of the tone, the intracellular calcium level, and the membrane potential of the smooth muscle cells.
Methods: Twenty-four porcine retinal arterioles were mounted in a wire myograph for isometric recording of wall tension. The vessels were precontracted with 0.3 µM U46619 and exposed to NA (pH = 7.0) and HA (pH = 7.0). Intracellular calcium was measured using the fluorophore Fura-2AM (n=12). In six vessels 0.1 mM NG-Nitroarginine Methyl Ester (L-NAME) was added to block NO synthesis. The membrane potential of the smooth muscles in situ was measured with sharp glass electrodes (n=12).
Results: NA and HA caused a similar significant decrease in wall tension (p<0.01), membrane potential (from -18 ± 0.7 mV during precontraction to -26 ± 1.9 mV in NA (p=0.002) and -24 ± 2.6 mV in HA (p=0.02)), and similar significant decrease in intracellular Ca2+ (p<0.01), which was unaffected by inhibition of NO synthesis.
Conclusions: Acidosis induced relaxation of the tone in isolated porcine retinal arterioles is associated with a decrease in intracellular calcium and a hyperpolarisation of the smooth muscle cells. The acidosis induced relaxation is independent of CO2, and is not mediated through NO. Possible implications for the pathophysiology of diabetic retinopathy will be presented.
Last Name: Mustapha
First Name: Nik Musaadah
Institution: King's College London
Address: Centre for Cardiovascular Biology & Medicine
GKT School of Biomedical Sciences
King's College London, Guy's Campus
London SE1 1UL
Phone: 0044-(020) 7848-6211
Fax: 0044-(020) 7848-6202
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