Retinal capillary endothelial cells prefer different substrates for growth and migration

McIntosh, L. C.; Muckersie, L.; Forrester, John V.

doi:10.1016/0040-8166(88)90041-9

Cited by 37 publications

(17 citation statements)

References 28 publications

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“…Bovine retinal pericytes (BRP) were obtained from pools of 15 to 20 bovine retinas, with a partial modification of two precedent methods [15,16,17]. BRP were characterized by 3G5 (a specific membrane ganglioside) fluorescence immunostaining [17] and were grown in DMEM 5.6 mmol/l glucose with 20% FCS in primary cultures and 10% FCS in secondary cultures.…”

Section: Methodsmentioning

confidence: 99%

A study of capillary pericyte viability on extracellular matrix produced by endothelial cells in high glucose

et al. 2003

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Aims/hypothesis. Thickening of the basement membrane and selective loss of pericytes occur early in diabetic retinopathy. As we showed previously that pericyte adhesion is impaired on extracellular matrix produced by endothelial cells in high hexose concentrations, we aimed to verify if altered adhesion could influence pericyte viability and replication. Methods. Conditioned extracellular matrices were obtained by growing human umbilical vein endothelial cells in media containing 28 mmol/l D-glucose, with or without the inhibitors of protein glycation thiamine or aminoguanidine, and D-galactose or L-glucose up to 28 mmol/l. Having removed the endothelium, bovine retinal pericytes were grown on these matrices and, in separate experiments, on laminin, fibronectin or type IV collagen. Pericyte viability and replication were measured by cell counts and DNA synthesis after 7 days, cell cycle traversal after 2 days and apoptosis after 18 h, 2 days and 7 days.Results. Pericyte counts and DNA synthesis were reduced on matrices produced in high D-glucose and Dgalactose, whilst matrix obtained in L-glucose reduced DNA synthesis but not counts. Both thiamine and aminoguanidine corrected reduced pericyte viability when added to high D-glucose. Cell cycle and apoptosis were not affected by growing pericytes on different conditioned matrices. Laminin, fibronectin and type IV collagen did not modify pericyte replication. Conclusions/interpretations. Reduced pericyte counts could depend on impaired initial adhesion to the extracellular matrix produced by endothelium in high hexose concentrations, rather than impaired replication or viability. Altered cell-matrix interactions might facilitate pericyte dropout in diabetic retinopathy, independently of the effects of high glucose on pericyte replication. [Diabetologia (2003) 46:409-415] Keywords Diabetic retinopathy, extracellular matrix, pericytes, endothelium, glucose, galactose, aminoguanidine, thiamine, glycation. and is observed in other forms of hyperhexosaemia, such as galactose-fed rats, which also develop a diabetic-like retinopathy [3,4]. The evolution of diabetic retinopathy is strongly influenced by qualitative and quantitative changes in the extracellular matrix (ECM) of the capillary basement membrane. In particular, alterations of some ECM components were shown to influence the characteristics of endothelium [5] and ECM-associated growth factors [6], although the mechanisms are still unclear.Endothelium is likely to represent the major contributor to the synthesis of capillary ECM material. In particular, endothelial cells were found to increase their synthesis of type IV collagen and fibronectin Thickening of the basement membrane is an early event in the course of diabetic microangiopathy [1,2]

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Section: Methodsmentioning

confidence: 99%

A study of capillary pericyte viability on extracellular matrix produced by endothelial cells in high glucose

et al. 2003

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“…Bovine retinal pericytes (BRP) were obtained from pools of 15-20 bovine retinas with a partial modification of the method of Wong et al (26) and McIntosh et al (27) as described previously (28). BRP were characterized by 3G5 (a specific membrane ganglioside) fluorescence immunostaining (29).…”

Section: Methodsmentioning

confidence: 99%

Regulation of Intracellular Glucose and Polyol Pathway by Thiamine and Benfotiamine in Vascular Cells Cultured in High Glucose

Berrone

Beltramo

Solimine

et al. 2006

Journal of Biological Chemistry

132

110

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Hyperglycemia is a causal factor in the development of the vascular complications of diabetes. One of the biochemical mechanisms activated by excess glucose is the polyol pathway, the key enzyme of which, aldose reductase, transforms D-glucose into D-sorbitol, leading to imbalances of intracellular homeostasis. We aimed at verifying the effects of thiamine and benfotiamine on the polyol pathway, transketolase activity, and intracellular glucose in endothelial cells and pericytes under high ambient glucose. Human umbilical vein endothelial cells and bovine retinal pericytes were cultured in normal (5.6 mmol/liter) or high (28 mmol/liter) glucose, with or without thiamine or benfotiamine 50 or 100 mol/liter. Transketolase and aldose reductase mRNA expression was determined by reverse transcription-PCR, and their activity was measured spectrophotometrically; sorbitol concentrations were quantified by gas chromatographymass spectrometry and intracellular glucose concentrations by fluorescent enzyme-linked immunosorbent assay method. Thiamine and benfotiamine reduce aldose reductase mRNA expression, activity, sorbitol concentrations, and intracellular glucose while increasing the expression and activity of transketolase, for which it is a coenzyme, in human endothelial cells and bovine retinal pericytes cultured in high glucose. Thiamine and benfotiamine correct polyol pathway activation induced by high glucose in vascular cells. Activation of transketolase may shift excess glycolytic metabolites into the pentose phosphate cycle, accelerate the glycolytic flux, and reduce intracellular free glucose, thereby preventing its conversion to sorbitol. This effect on the polyol pathway, together with other beneficial effects reported for thiamine in high glucose, could justify testing thiamine as a potential approach to the prevention and/or treatment of diabetic complications.Diabetic retinopathy is one of the most serious complications in diabetic patients and a leading cause of blindness. Among its earliest steps is the loss of retinal microvascular pericytes.Hyperglycemia is a prerequisite for the development of the chronic complications of diabetes, but the precise mechanisms leading to vascular and tissue damage have not been fully elucidated. Biochemical mechanisms that have been hypothesized to account for the adverse effects of hyperglycemia include increased glucose flux through the polyol pathway (1), formation of advanced glycation end-products (AGE) 2 (2, 3), accelerated generation of reactive oxygen species (ROS) (4, 5), and activation of the diacylglycerol-protein kinase C pathway (6, 7). It has been suggested that excess production of ROS inside the endothelium, resulting from increased glucose flux through the Krebs cycle, may represent a possible common denominator ("unifying mechanism") of these apparently independent biochemical pathways (5). In fact, ROS can partially inhibit glyceraldehyde-phosphate dehydrogenase, resulting in the accumulation of glycolytic metabolites, among which glyceraldehyde 3-pho...

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“…HUVEC were obtained and cultured with a partial modification of Jaffe's method [9], as previously described [3]. BREC were obtained from bovine retinas with a partial modification of the methods of Wong et al [10] and McIntosh et al [11], as previously described [4].…”

Section: Methodsmentioning

confidence: 99%

Thiamine corrects delayed replication and decreases production of lactate and advanced glycation end-products in bovine retinal and human umbilical vein endothelial cells cultured under high glucose conditions

et al. 1996

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High glucose concentrations impair the replication of cultured human umbilical vein (HUVEC) [1][2][3] and bovine retinal endothelial cells (BREC) [4] and this may be secondary to enhanced glycolytic flux. Altered concentrations of intermediate phosphorylated metabolites could play a role in determining cell damage. In particular, increased availability of glyceraldehyde 3-phosphate (G3P), which is much more active than glucose in promoting protein glycation and AGE formation [5], might be one of the mechanism(s) involved.Thiamine (vitamin B 1 ) acts as a coenzyme for transketolase [6], pyruvate-dehydrogenase [7] and a -ketoglutarate-dehydrogenase [8]. The former shifts G3P from glycolysis to the pentose phosphate shunt, the second transforms pyruvate in acetyl-coenzyme A, which then enters the Krebs cycle, and the third catalyses the oxidation of ketoglutaric acid to succinyl-CoA within the Krebs cycle.The aim of this study was to ascertain whether thiamine would modify glycolysis, AGE production and replication in HUVEC and BREC cultured in high glucose concentrations. Diabetologia (1996) Summary This study aimed at verifying whether thiamine, a co-enzyme which decreases intracellular glycolysis metabolites by allowing pyruvate and glyceraldheyde 3-phosphate to enter the Krebs cycle and the pentose-phosphate shunt, respectively, corrects delayed replication caused by high glucose concentrations in cultured human umbilical vein (HU-VEC) and bovine retinal endothelial cells (BREC). After incubation in physiological (5.6 mmol/l) or high (28.0 mmol/l) glucose with or without 150 m mol/l thiamine, cells were counted and proliferation assessed by mitochondrial dehydrogenase activity. Lactate was measured in both cell types as an index of glycolytic activity and fluorescent advanced glycosylation end-products (AGE) concentration was determined in the HUVEC lysate. Both cell counts and proliferation assays in either of the cell types confirmed the impairment to cell replication induced by high glucose. When thiamine was added to cells kept under high glucose conditions, the number of surviving cells was significantly increased and the reduced cell proliferation appeared to be corrected. Lactate assays confirmed the increased production of this metabolite by BREC and HUVEC in high glucose, which was reduced by thiamine. Fluorescent AGE determination showed that thiamine may prevent non-enzymatic glycation in HUVEC. Thiamine restores cell replication, decreases the glycolytic flux and prevents fluorescent AGE formation in endothelial cells cultured in high glucose, suggesting that abnormal levels of glycolytic metabolite(s) may damage cells.

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Retinal capillary endothelial cells prefer different substrates for growth and migration

Cited by 37 publications

References 28 publications

A study of capillary pericyte viability on extracellular matrix produced by endothelial cells in high glucose

A study of capillary pericyte viability on extracellular matrix produced by endothelial cells in high glucose

Regulation of Intracellular Glucose and Polyol Pathway by Thiamine and Benfotiamine in Vascular Cells Cultured in High Glucose

Thiamine corrects delayed replication and decreases production of lactate and advanced glycation end-products in bovine retinal and human umbilical vein endothelial cells cultured under high glucose conditions

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