Increased endocytosis in retinal vascular endothelial cells grown in high glucose medium is modulated by inhibitors of nonenzymatic glycosylation

Abstract

SummaryWe sought to determine if hyperglycaemia is responsible for increased retinal vascular endothelial-cell (RVEC) endocytosis in diabetes and to assess the role of nonenzymatic glycosylation in mediation of this novel endothelial-cell pathology. RVECs were propagated in media containing either 5 or 25 mmol/1 glucose for up to 10 days after which they were exposed to the protein tracer horseradish peroxidase for 30 min. The level of RVEC endocytosis was quantified in intact cell monolayers by electron microscopic stereology, and in cell lysates by a simple spectrophotometric method. The effect of the nonenzymatic glycosylation inhibitors, aminoguanidine and D-lysine, on high-glucose medium induced changes in RVEC endocytosis was tested by inclusion of these agents in the culture medium. RVECs exposed to 25 mmol/1 glucose showed a stepwise increase in endocytosis of horseradish peroxidase culminating in a two-to threefold increase after 10 days. Endocytosis returned to normal levels after a further 10 days in 5 mmol/1 glucose medium. The increase in RVEC endocytosis was markedly reduced, but not completely normalised, by aminoguanidine and D-lysine. Exposure of cultured RVECs to 25 mmol/1 glucose causes an increase in endocytosis of similar magnitude to that experienced by RVEC in early diabetes, and implicates hyperglycaemia in the latter situation. A significant component of the increase in RVEC endocytosis appears to be mediated by nonenzymatic glycosylation. [Diabetologia (1995[Diabetologia ( ) 38: 1271[Diabetologia ( -1275 Key words Endocytosis, high glucose, nonenzymatic glycosylation, endothelium.A recent study has shown a more than sixfold increase in endocytosis of horseradish peroxidase (HRP) by the endothelium of the retinal microvessels in 6-week diabetic rats, without an associated increase in capillary permeability [1]. This finding is in accord with many previous studies which have demonstrated an increased clearance of intravascular protein tracers by the retina of streptozotocin diabetic rats after similar durations of diabetes [2,3]. The

Impaired Retinal Angiogenesis in Diabetes

“…The in vitro assay used was a novel threedimensional model of angiogenesis that used retinal endothelial cells in an extracellular matrix gel (Matrigel; Becton Dickinson). Bovine retinal microvascular endothelial cells (RMECs) were isolated and cultured between passages 3 and 6 in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) with 20% porcine serum (Sigma) as previously described (16). RMECs were resuspended in DMEM mixed with an equal volume of Matrigel at 4°C and porcine serum to a final concentration of 20%.…”

Involvement of advanced glycation end products in the pathogenesis of diabetic complications: the protective role of regular physical activity

“…An increase in vascular permeability was associated in vitro with endothelial endocytosis. An increase in endocytosis in vascular endothelial cells of the retina has been observed with elevated concentrations of glucose, and this could be reduced by aminoguanidine, suggesting also that this alteration is mediated by the AGEs [125].…”

Diabetic retinopathy: Some cellular, molecular and therapeutic considerations

“…Short-term diabetic animals also demonstrate a similar increased endocytosis in the retinal vasculature.159 This exaggerated endocytotic effect can be ameliorated by anti-glycation agents such as D-lysine and aminoguanidine, suggesting that the increased endocytosis is an attempt by the cell to replenish its plasma membrane components and receptors which are perceived by the cell as damaged or senescent due to glycation. 160 It is conceivable that in diabetes, glycation of cell membranes interferes with normal plasma membrane recycling and receptor mediated transport of macromolecules.…”

“…Glycational processes culminating in the intra-and extracellular accumulation of reactive end-products and cross-linked proteins can affect the form and function of vascular cells and their extracellular matrices in established diabetes. There is a positive relationship between the incidence and progression of diabetic retinopathy and levels of HbA 1 e and it has been demonstrated that some of the pathological glycational processes can be prevented or minimised by anti glycation drugs in diabetic animal models, 160,173,215,216 suggesting therapeutic applications in the clinical setting. Anti-glycation agents such as aminoguanidine may also inhibit NO synthesis and reduce dysfunction induced by elevated NO levels in diabetes}17 and Stitt et al 178 have used aminoguanidine to reverse vitreous cross-linking mediated by AGEs.…”