European Association for the Study of Diabetic Eye Complications

Annual Meeting Munich 21-23rd May 2004

Paper Abstracts



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


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.


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.


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


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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