Daniel Ruggiero

Modification of Enzymatic Antioxidants in Retinal Microvascular Cells by Glucose or Advanced Glycation End Products

Oxidative stress is one possible pathogenic mechanism to explain diabetic microangiopathy. In the present study, we determined the antioxidant enzyme activities in bovine retinal microvessels and cultured retinal microvascular cells: endothelial cells (BREC) and pericytes (BRP). We further investigated the effects of high glucose and advanced glycation end products (AGE) on these enzyme activities in BREC and BRP. Antioxidant enzyme activities in native retinal microvessels and BREC were quite similar but differed markedly from the BRP ones. High glucose decreased Se-GPx activity (about 20%) in BREC compared to mannitol. High concentrations of mannitol or NaCl increased Se-GPx activity (up to 40%) compared to control medium, suggesting that hyperosmolarity could regulate Se-GPx in BREC. No changes in antioxidant enzyme activities were observed when BRP were cultured with glucose or mannitol at high concentrations. AGE-BSA had no effect on enzyme activities in BREC, whereas 20 microM AGE-BSA increased catalase (40%) and superoxide dismutase (60%) activities in BRP. Differences in antioxidant enzyme activities observed between BREC and BRP, cultured with high concentrations of glucose or AGE, might help to explain their different behavior during the pathogenesis of diabetic retinopathy, i.e., early pericyte drop-out and late endothelial cell proliferation.

Vascular permeability increase as induced by histamine or bradykinin is enhanced by advanced glycation endproducts (AGEs).

Advanced glycation endproducts (AGEs) may enhance vascular permeability in diabetic subjects. To test this hypothesis, AGEs were prepared in the presence of albumin (AGE-Alb). Control albumin (Alb) and AGE-Alb were then labeled with FITC (fluoresceinisothiocyanate) and injected i.v. into anesthetized hamsters at a dose of 7 mg/100 g B.W. Normal hamsters were given FITC-Alb or FITC-AGE-Alb and FITC-dextran. Vascular permeability changes were measured by direct intravital microscopy of the hamster cheek pouch preparations in fluorescent light and recorded as number of sites (=leaks) with extravasation of FITC-labeled albumin in postcapillary venules. No changes were seen during 1 hour after i.v. injection of FITC-Alb or FITC-AGE-Alb. Repeated local application of histamine 5 x 10(6) M or bradykinin 5 x 10(7) M to the cheek pouch for 5 min with 30-min intervals induced reversible increases in vascular permeability in all hamsters. Maximal number of leaks/cm2 before and at 30 and 60 min after FITC-Alb-injection and histamine application was 257 +/- 6 (SEM), 243 +/- 6 and 231 +/- 6 leaks/cm2 in the FITC-Alb-group and 258 + 6 (SEM), 302 +/- 12 and 316 +/- 11 leaks/cm2 in the FITCAGE-Alb-group, respectively, (P < 0.05 at 30 and 60 min). Similar results were seen with bradykinin. Our conclusions showed that i.v.-injected AGEs augmented the histamine- and bradykinin-induced increase in vascular permeability by 34% and 46%.

Effects of advanced glycation end products on glycosphingolipids of retinal pericytes

Diabetic retinopathy is a chronic diabetic complication characterized by alterations of cell-cell and cell-matrix interactions in retinal microvessels. Advanced glycation end products (AGES) accumulate in microvessels during diabetes and they affect vascular cell biology. Glycosphingolipids are components of the cell plasma membrane and they have an important role in cell-cell interactions and cell growth regulation. The aim of this study was to investigate the effect of AGES on glycosphingolipid metabolism in retinal microvascular cells such as pericytes. Bovine retinal pericytes were cultured with either albumin or AGES. AGES were prepared by incubation of albumin with methylglyoxal at 37°C during 50h. Glycosphingolipids were metabolically radiolabelled with 14C-galactose, extracted and analysed by HPTLC and autoradiography. GM3 fatty acids of pericytes were analysed by GC-MS after derivatization with PFB and BSTFA. The results obtained show that AGES affect the glycosphingolipid composition of retinal pericytes in culture. AGES increased the three major neutral glycosphingolipidsdetected in these cells: globoside (+30%), CTH (+20%) and CMH (+80%). GM3 and GD3 were the main gangliosides detected. GM3 was increased by AGES (+30%) whereas GD3 was decreased (-35%), suggesting an alteration of the sialyltransferase II activity which is responsible for GD3 synthesis. GM3 fatty acid analysis suggests that AGES induced only quantitative but not qualitative changes on retinal pericyte glycosphingolipids. These results show a new metabolic effect of AGES modifying the composition of cell glycosphingolipids which play a main role in cell-cell interactions and cell growth. Further investigations will be needed to determine whether these modifications could be involved in the microvascular alterations observed in diabetes.