[Show abstract][Hide abstract] ABSTRACT: This study compared the protective effects of three different anti-glycation compounds, aspirin, D-penicillamine and vitamin E, against high glucose and advanced glycation endproduct (AGE) mediated toxicity in cultured bovine aortic endothelial cells using two approaches. Their proliferation was assessed in culture in different concentrations of glucose (5.5-100 mmol/l) with and without these inhibitors. A monolayer of cultured endothelial cells was wounded and recovery at the wound site was measured following exposure to different concentrations of glucose with and without inhibitors. The ability of these compounds to protect cultured endothelial cells following exposure to bovine serum albumin-derived advanced glycation endproducts (BSA-AGE) was also studied. Addition of glucose to cultured endothelial cells inhibited their proliferation in a dose dependent manner. All three compounds protected against the anti-proliferative effects of high glucose, with vitamin E being the most effective. The migration of cultured endothelial cells following wounding was inhibited by increasing concentrations of glucose but was maintained in the presence of all three anti-glycation compounds with vitamin E, again giving the greatest protection. Vitamin E was also the most effective at protecting against the anti-proliferative effects of BSA-AGE. D-penicillamine was not as effective as vitamin E whereas aspirin offered no significant protection against AGE-induced cellular toxicity. Our studies suggest that compounds, such as vitamin E, with combined antiglycation and antioxidant properties offer maximum therapeutic potential in protection against high glucose and AGE-mediated cellular toxicity.
[Show abstract][Hide abstract] ABSTRACT: Hyperglycaemia reduces proliferation of bovine aortic endothelial cells in vitro. A similar effect in vivo may contribute to long-term complications of diabetes such as impaired wound-healing and retinopathy. We report the effect of increased glucose concentrations, glycated basic fibroblast growth factor (FGF-2) and bovine serum albumin-derived advanced glycation endproducts (BSA-AGE) on the proliferation of bovine aortic endothelial cells. Glucose (30 and 50 mmol/l) had an antiproliferative effect on endothelial cells. This effect may be mediated through reduced mitogenic activity of FGF-2. The glycation of FGF-2 with 250 mmol/l glucose-6-phosphate led to reduced mitogenic activity compared to native FGF-2. BSA-AGE at concentrations of 10, 50 and 250 microg/ml had an antiproliferative effect on cultured endothelial cells. Aminosalicylic acid at a concentration of 200 micromol/l proved to be more effective than equimolar concentrations of aminoguanidine in protecting endothelial cells against the antiproliferative effects of both high (30 mmol/l) glucose and 50 microg/ml BSA-AGE. FGF-2 glycated in the presence of 4 mmol/l aminosalicylic acid or aminoguanidine retained mitogenic activity compared to that glycated in their absence. Compounds like aminoguanidine and, in particular, aminosalicylic acid protect endothelial cells against glucose-mediated toxicity and may therefore have therapeutic potential.
[Show abstract][Hide abstract] ABSTRACT: Ineffectual wound healing in hyperglycaemic patients suffering from diabetes mellitus is characterised by a reduction in capillary reformation (angiogenesis). Basic fibroblast growth factor (FGF-2) is secreted by fibroblasts, macrophages and in particular endothelial cells (EC) in response to tissue injury and is important in promotion of neovascularisation. Recently, glycation of FGF-2 has been shown to significantly reduce its activity in vitro. We have examined the kinetics of FGF-2 glycation and compared its ability with that of native FGF-2 to activate mitogenesis, capillary formation and associated signal transduction in bovine aortic EC (BAEC). FGF-2 was exposed to 0.25 M glucose-6-phosphate (G-6-P) for 24-72 h and the degree of glycation determined by matrix assisted laser desorption ionisation mass spectrometry. Native FGF-2 was heterogeneous with Mw in the range 15,153.6-17,903 Da. After 24 h incubation with G-6-P there was evidence of glycation, and the mass increase corresponded to addition of 2.7 mol of G-6-P residues; after 48 h, 4 mol sugar was added and this increased to 8.7 after 72 h. Dimerisation of FGF-2 was observed after 72 h of treatment. Induction of mitogenesis in BAEC was significantly reduced by 25%-40% after treatment for 48-96 h with glycated (24 h) FGF-2 (gFGF-2; 100 pg/ml-5 ng/ml; P < 0.05), whilst capillary tubule formation was significantly reduced by between 60% and 90% (100 pg/ml-1 ng/ml; P < 0.05) after 5 days compared to native FGF-2. Subsequent investigation of the signal transduction molecules associated with mitogenesis showed a reduction in FGF-2 induced tyrosine phosphorylated proteins of approximate Mw 20-150 kDa between 10 min and 24 h, in particular, mitogen activated protein kinase (MAPK)/early response kinase (ERK-1, ERK-2), after glycation. To determine the reason for reduced angiogenic activity of gFGF-2, we compared its binding characteristics to that of native FGF-2. Total binding of gFGF-2 to the cell surface was significantly reduced in BAEC analysed by FACS compared to native FGF-2 (P < 0.05). Further investigation using 125I-labelled differentially washed samples, demonstrated a significant reduction in gFGF-2 binding to the high affinity tyrosine kinase receptor (46%) compared to native FGF-2. In summary, glycation of FGF-2 in vitro occurs rapidly within 24 h in the presence of elevated levels of G-6-P. Glycation caused a significant reduction in the ability of FGF-2 to bind to the tyrosine kinase receptor and activate signal transduction pathways responsible for both mitogenesis and capillary formation in BAEC. These results could help to explain the mechanism behind impaired wound healing in patients with diabetes mellitus.